# Can selenium-enriched spirulina supplementation ameliorate sepsis outcomes in selenium-deficient animals?

Thomas Castel, Michaël Theron, Karine Pichavant-Rafini, Anthony Guernec, Aurélie Joublin-delavat, Bleuenn Gueguen, Karelle Léon

► To cite this version:

Thomas Castel, Michaël Theron, Karine Pichavant-Rafini, Anthony Guernec, Aurélie Joublin-delavat, et al.. Can selenium-enriched spirulina supplementation ameliorate sepsis outcomes in selenium-deficient animals?. Physiological Reports, 2021, 9, pp.e14933. 10.14814/phy2.14933. hal-04438201

HAL Id: hal-04438201

https://hal.univ-brest.fr/hal-04438201

Submitted on 5 Feb 2024

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<figure><img src="image_1.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
# Can selenium-enriched spirulina supplementation ameliorate sepsis outcomes in selenium-deficient animals?

Thomas Castel, Michaël Theron, Karine Pichavant-Rafini, Anthony Guernec, Aurélie Joublin-delavat, Bleuenn Gueguen, Karelle Léon

► To cite this version:

Thomas Castel, Michaël Theron, Karine Pichavant-Rafini, Anthony Guernec, Aurélie Joublin-delavat, et al.. Can selenium-enriched spirulina supplementation ameliorate sepsis outcomes in selenium-deficient animals?. Physiological Reports, 2021, 9, pp.e14933. 10.14814/phy2.14933. hal-04438201

HAL Id: hal-04438201

https://hal.univ-brest.fr/hal-04438201v1

Submitted on 5 Feb 2024

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_1.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
# Can selenium-enriched spirulina supplementation ameliorate sepsis outcomes in selenium-deficient animals?

Thomas Castel<fnref n="1" /> | Michaël Theron<fnref n="1" /> | Karine Pichavant-Rafini<fnref n="1" /> | Anthony Guernec<fnref n="1" /> |
Aurélie Joublin-Delavat<fnref n="1" /> | Bleuenn Gueguen<fnref n="2" />,<fnref n="3" /> | Karelle Leon<fnref n="1" />

## Correspondence

Thomas Castel, Université de Brest, EA 4324 ORPHY, UFR Sciences et Techniques, 6 avenue Victor Le Gorgeu, F-29200 Brest, France.

E-mail: thomas.castel@univ-brest.fr

## Funding information

This research was supported by a grant from the Brittany Regional Council.

## Abstract

In intensive care units, sepsis is the first cause of death. In this pathology, inflammation and oxidative status play a crucial role in patient outcomes. Interestingly, 92% of septic patients exhibit low selenium plasma concentrations (a component of antioxidant enzymes). Moreover, *Spirulina platensis*, a blue-green algae, demonstrated anti-inflammatory effects. In this context, the main purpose of our study was to analyze the effect of a selenium-enriched spirulina after a selenium deficiency on sepsis outcome in rats. Sixty-four rats were fed 12 weeks with a selenium-deficient food. After 8 weeks, rats were supplemented (via drinking water) for 4 weeks with sodium selenite (Se), spirulina (Spi), or selenium-enriched spirulina (SeSp). Sepsis was then induced by cecal ligature and puncture, and survival duration was observed. The plasma selenium concentration was measured by ICPMS. Expression of GPx1 and GPx3 mRNA was measured by RT-PCR. Blood parameters (lactates and HCO₃⁻ concentrations, pH, PO₂, and PCO₂) were analyzed at 0, 1, and 2 h as well as inflammatory cytokines (IL-6, TNF-α, IL-10). Sodium selenite and SeSP supplementations restored plasma selenium concentration prior to sepsis. The survival duration of SeSP septic rats was significantly lower than that of selenium-supplemented ones. Gpx1 mRNA was increased after a selenium-enriched spirulina supplementation while Gpx3 mRNA levels remained unchanged. Furthermore, sodium selenite prevented sepsis-induced acidosis. Our results show that on a basis of a Se deficiency, selenium-enriched spirulina supplementations significantly worsen sepsis outcome when compared to Se supplementation. Furthermore, Se supplementation but not selenium-enriched spirulina supplementation decreased inflammation and restored acid-base equilibrium after a sepsis induction.

## KEYWORDS

GPx, lactates, selenium, sepsis, Spirulina, supplementation

<footnote n="1">¹Université de Brest, EA 4324 ORPHY, UFR Sciences et Techniques, Brest, France</footnote>

<footnote n="2">²CNRS, Univ Brest, UMS 3113, Plouzané, France</footnote>

<footnote n="3">³UMR 6538, Laboratoire Géosciences Océan, CNRS, Univ Brest, Plouzané, France</footnote>
# Can selenium-enriched spirulina supplementation ameliorate sepsis outcomes in selenium-deficient animals?

Thomas Castel<fnref n="1" /> | Michaël Theron<fnref n="1" /> | Karine Pichavant-Rafini<fnref n="1" /> | Anthony Guernec<fnref n="1" /> |
Aurélie Joublin-Delavat<fnref n="1" /> | Bleuenn Gueguen<fnref n="2" />,<fnref n="3" /> | Karelle Leon<fnref n="1" />

**Correspondence**

Thomas Castel, Université de Brest,
EA 4324 ORPHY, UFR Sciences et
Techniques, 6 avenue Victor Le Gorgeu,
F-29200 Brest, France.
E-mail: thomas.castel@univ-brest.fr

**Funding information**

This research was supported by a grant
from the Brittany Regional Council.

**Abstract**

In intensive care units, sepsis is the first cause of death. In this pathology, inflam-
mation and oxidative status play a crucial role in patient outcomes. Interestingly,
92% of septic patients exhibit low selenium plasma concentrations (a component of
antioxidant enzymes). Moreover, *Spirulina platensis*, a blue-green algae, demon-
strated anti-inflammatory effects. In this context, the main purpose of our study was
to analyze the effect of a selenium-enriched spirulina after a selenium deficiency on
sepsis outcome in rats. Sixty-four rats were fed 12 weeks with a selenium-deficient
food. After 8 weeks, rats were supplemented (via drinking water) for 4 weeks with
sodium selenite (Se), spirulina (Spi), or selenium-enriched spirulina (SeSp). Sepsis
was then induced by cecal ligature and puncture, and survival duration was observed.
The plasma selenium concentration was measured by ICPMS. Expression of GPx1
and GPx3 mRNA was measured by RT-PCR. Blood parameters (lactates and HCO₃⁻
concentrations, pH, PO₂, and PCO₂) were analyzed at 0, 1, and 2 h as well as inflam-
matory cytokines (IL-6, TNF-α, IL-10). Sodium selenite and SeSP supplementations
restored plasma selenium concentration prior to sepsis. The survival duration of
SeSP septic rats was significantly lower than that of selenium-supplemented ones.
Gpx1 mRNA was increased after a selenium-enriched spirulina supplementation
while Gpx3 mRNA levels remained unchanged. Furthermore, sodium selenite pre-
vented sepsis-induced acidosis. Our results show that on a basis of a Se deficiency,
selenium-enriched spirulina supplementations significantly worsen sepsis outcome
when compared to Se supplementation. Furthermore, Se supplementation but not
selenium-enriched spirulina supplementation decreased inflammation and restored
acid-base equilibrium after a sepsis induction.

**KEYWORDS**

GPx, lactates, selenium, sepsis, Spirulina, supplementation

<footnote n="1">¹Université de Brest, EA 4324 ORPHY, UFR Sciences et Techniques, Brest, France</footnote>

<footnote n="2">²CNRS, Univ Brest, UMS 3113, Plouzané, France</footnote>

<footnote n="3">³UMR 6538, Laboratoire Géosciences Océan, CNRS, Univ Brest, Plouzané, France</footnote>
<figure><img src="image_2.png" /></figure>

# 1 | INTRODUCTION

Sepsis is the first cause of mortality in intensive care units (Rudd et al., 2020), but despite numerous clinical trials and experimental studies, its pathogenesis is still unclear (Singer et al., 2016). Sepsis appears to be associated with systemic inflammation, cardiovascular system impairments, hemodynamic alterations, and acid-base imbalances (Bagshaw et al., 2009; Bateman et al., 2015; Boissier et al., 2017; Levraut et al., 1998; Razazi et al., 2019). In addition, the activation of immune cells leads to a release of pro-inflammatory mediators (such as tumor necrosis factor-α [TNF-α] or interleukin-6 [IL-6]) that triggers an overproduction of reactive oxygen species (ROS) (Blaser et al., 2016; Mantzarlis et al., 2017). Moreover, septic patients display macrocirculatory failure resulting in tissue hypoxia (Hallisey & Greenwood, 2019), decrease in cellular oxygen consumption and lactate production contributing to develop an oxidative stress and inflammation (Gattinoni et al., 2019; Pan et al., 2019).

Selenium, a trace element essential to human health, is a component of selenoproteins involved in several functions such as fertility (Pieczyńska & Grajeta, 2015), thyroid or cardiovascular function (Benstoem et al., 2015; Köhrle, 2015). Moreover, selenoproteins like glutathione peroxidase and thioredoxin reductase are involved in oxidative stress regulation (Hosnedlova et al., 2017). Furthermore, it has been shown that, in intensive care units 92% of septic patients are selenium deficient (Sakr et al., 2007) and this selenium deficiency could play a role in sepsis development (Cirino Ruocco et al., 2018; Forceville et al., 1998; Mertens et al., 2015; Weber et al., 2008). In fact, a selenium deficiency is associated with a worsening of clinical outcome, excess mortality (Alhazzani et al., 2013; Angstwurm et al., 2007; Huang et al., 2013; Landucci et al., 2014) and an increase of nosocomial infections (Forceville et al., 2007). Selenium supplementation has therefore been largely studied. Recent studies demonstrate that if selenium administration does not reduce 28-day mortality and duration of mechanical ventilation (Bloos et al., 2016; Kong et al., 2013; Li et al., 2019; Manzanares et al., 2016), it reduces the duration of vasopressor therapy (Chelkeba et al., 2015; Li et al., 2019). The beneficial effect of selenium supplementation in septic patients is still subject to debate and further investigations are needed to understand its involvement.

*Spirulina platensis* is a filamentous blue-green algae with high protein, vitamin, phycocyanin, antioxidant, and polysaturated fatty acid contents (Habib, 2008; Lamela & Rocha, 2000; Leema et al., 2010; Pereira et al., 2019). Spirulina has been reported to have antioxidant effects in many models (Abdelkhalek et al., 2015; Bashandy et al., 2016; Gargouri et al., 2018; Nasirian et al., 2018). Furthermore, spirulina

## New findings

### What is the central question of this study?

Sepsis, the first cause of death in intensive care units, is associated with systemic inflammation and oxidative stress and 92% of septic patients are selenium deficient. Spirulina and selenium have shown promising anti-inflammatory and antioxidant effects. We have therefore hypothesized that a selenium-enriched spirulina supplementation could provide beneficial effects on sepsis outcome in selenium-deficient animals.

### What is the main finding and its importance?

On a basis of a Se deficiency, selenium-enriched spirulina supplementations significantly worsen sepsis outcome when compared to Se supplementation. Furthermore, Se supplementation but not Spirulina nor selenium-enriched spirulina supplementations decreased inflammation and restored acid-base equilibrium after a sepsis induction.

showed promising anti-inflammatory properties, especially by reducing pro-inflammatory cytokine release (Abdel-Daim et al., 2018; Morsy et al., 2019; Pham et al., 2019; Zahran & Emam, 2018). Interestingly, spirulina can also be enriched in several essential elements such as selenium (Chen et al., 2006; Li et al., 2003).

In this context, we hypothesized that a selenium-enriched spirulina supplementation could provide beneficial effects on sepsis outcome in selenium-deficient animals. The effects of sodium selenite, *S. platensis*, and selenium-enriched spirulina supplementations were studied in rats after 8 weeks of selenium-deficient diet and 4 weeks of supplementation. Sepsis was induced by cecum ligature and puncture and the survival rates, acid-base equilibrium, blood pro-inflammatory cytokines, and Gpx expression were evaluated.

# 2 | MATERIAL AND METHODS

## 2.1 | *Spirulina platensis* powder

The *Spirulina* strain used in this study was *S. platensis*. Production and conditioning (as dried powder) of spirulina and spirulina enriched with selenium was carried out by TAM company (Plougastel). The selenium concentration in selenium-enriched *Spirulina* was 55 µg Se/g of dry weight of *S. platensis*.
<figure><img src="image_2.png" /></figure>

# 1 | INTRODUCTION

Sepsis is the first cause of mortality in intensive care units (Rudd et al., 2020), but despite numerous clinical trials and experimental studies, its pathogenesis is still unclear (Singer et al., 2016). Sepsis appears to be associated with systemic inflammation, cardiovascular system impairments, hemodynamic alterations, and acid-base imbalances (Bagshaw et al., 2009; Bateman et al., 2015; Boissier et al., 2017; Levraut et al., 1998; Razazi et al., 2019). In addition, the activation of immune cells leads to a release of pro-inflammatory mediators (such as tumor necrosis factor-α [TNF-α] or interleukin-6 [IL-6]) that triggers an overproduction of reactive oxygen species (ROS) (Blaser et al., 2016; Mantzarlis et al., 2017). Moreover, septic patients display macrocirculatory failure resulting in tissue hypoxia (Hallisey & Greenwood, 2019), decrease in cellular oxygen consumption and lactate production contributing to develop an oxidative stress and inflammation (Gattinoni et al., 2019; Pan et al., 2019).

Selenium, a trace element essential to human health, is a component of selenoproteins involved in several functions such as fertility (Pieczyńska & Grajeta, 2015), thyroid or cardiovascular function (Benstoem et al., 2015; Köhrle, 2015). Moreover, selenoproteins like glutathione peroxidase and thioredoxin reductase are involved in oxidative stress regulation (Hosnedlova et al., 2017). Furthermore, it has been shown that, in intensive care units 92% of septic patients are selenium deficient (Sakr et al., 2007) and this selenium deficiency could play a role in sepsis development (Cirino Ruocco et al., 2018; Forceville et al., 1998; Mertens et al., 2015; Weber et al., 2008). In fact, a selenium deficiency is associated with a worsening of clinical outcome, excess mortality (Alhazzani et al., 2013; Angstwurm et al., 2007; Huang et al., 2013; Landucci et al., 2014) and an increase of nosocomial infections (Forceville et al., 2007). Selenium supplementation has therefore been largely studied. Recent studies demonstrate that if selenium administration does not reduce 28-day mortality and duration of mechanical ventilation (Bloos et al., 2016; Kong et al., 2013; Li et al., 2019; Manzanares et al., 2016), it reduces the duration of vasopressor therapy (Chelkeba et al., 2015; Li et al., 2019). The beneficial effect of selenium supplementation in septic patients is still subject to debate and further investigations are needed to understand its involvement.

*Spirulina platensis* is a filamentous blue-green algae with high protein, vitamin, phycocyanin, antioxidant, and polyunsaturated fatty acid contents (Habib, 2008; Lamela & Rocha, 2000; Leema et al., 2010; Pereira et al., 2019). Spirulina has been reported to have antioxidant effects in many models (Abdelkhalek et al., 2015; Bashandy et al., 2016; Gargouri et al., 2018; Nasirian et al., 2018). Furthermore, spirulina

## New findings

### What is the central question of this study?

Sepsis, the first cause of death in intensive care units, is associated with systemic inflammation and oxidative stress and 92% of septic patients are selenium deficient. Spirulina and selenium have shown promising anti-inflammatory and antioxidant effects. We have therefore hypothesized that a selenium-enriched spirulina supplementation could provide beneficial effects on sepsis outcome in selenium-deficient animals.

### What is the main finding and its importance?

On a basis of a Se deficiency, selenium-enriched spirulina supplementations significantly worsen sepsis outcome when compared to Se supplementation. Furthermore, Se supplementation but not Spirulina nor selenium-enriched spirulina supplementations decreased inflammation and restored acid-base equilibrium after a sepsis induction.

showed promising anti-inflammatory properties, especially by reducing pro-inflammatory cytokine release (Abdel-Daim et al., 2018; Morsy et al., 2019; Pham et al., 2019; Zahran & Emam, 2018). Interestingly, spirulina can also be enriched in several essential elements such as selenium (Chen et al., 2006; Li et al., 2003).

In this context, we hypothesized that a selenium-enriched spirulina supplementation could provide beneficial effects on sepsis outcome in selenium-deficient animals. The effects of sodium selenite, *S. platensis*, and selenium-enriched spirulina supplementations were studied in rats after 8 weeks of selenium-deficient diet and 4 weeks of supplementation. Sepsis was induced by cecum ligature and puncture and the survival rates, acid-base equilibrium, blood pro-inflammatory cytokines, and Gpx expression were evaluated.

# 2 | MATERIAL AND METHODS

## 2.1 | *Spirulina platensis* powder

The *Spirulina* strain used in this study was *S. platensis*. Production and conditioning (as dried powder) of spirulina and spirulina enriched with selenium was carried out by TAM company (Plougastel). The selenium concentration in selenium-enriched *Spirulina* was 55 µg Se/g of dry weight of *S. platensis*.
## 2.2 | Ethical approval

This study was performed in accordance with the European recommendations (2010/63/EU), approved by the regional ethical committee (CEFEA B29-019-08) and the French Ministry (18325_2018123119211520). The authors understand the journal ethical principles and acknowledge that this study complies with this animal ethics checklist. Pain and suffering were minimized during the entire experiment.

## 2.3 | Experimental protocol

### 2.3.1 | Animals

Sixty-four 3-week-old female Wistar rats (Janvier, SAS-Le Genest St Isle) with an average weight of 97.5 ± 1.6 g were included in this study. Rats were raised under a 12 h light-12 h dark cycle at 21°C. Food and water were given ad libitum. Rats were allowed to adapt to environmental conditions for 1 week before the experiments.

### 2.3.2 | Diet and supplementation

During the first 8 weeks of the experiment, animals were provided with tap water and food devoid of selenium (U8959P v.0170, SAFE, France) as can be seen in Figure 1. From week 8 to week 12, the rats (randomly assigned to one of the four following experimental groups—16 rats/group) were still fed with a selenium-deficient diet but the beverage differed between the groups:

* D group (“D” for deficient) received tap water;

* Se group received selenium in water (Na₂SeO₃, 71950, Sigma Aldrich) at 20 µg Se/kg bw (body mass)/day;

* Spi group received *spirulina* in water at 3 g/kg bw/day;

* SeSP group received Se enriched *spirulina* in water at 3 g/kg bw/day, bringing 20 µg of selenium/kg bw/day.

All rats were weighted weekly, food, and drink intakes (given ad libitum) were measured twice a week during the 12 weeks of the experiment.

### 2.3.3 | Acute sepsis induction

Each group of 16 rats was randomly subdivided in two subgroups of 8 rats: sham and septic (described below).

In all cases, the rats were anesthetized by an intraperitoneal injection of a ketamine (100 mg/kg bw) and xylazine (10 mg/kg bw); a subcutaneous saline solution injection (3 ml/100 g bw) was performed and temperature was maintained at 38°C (±0.5°C). The anesthesia was controlled all along the procedure and if needed, a half dose of ketamine xylazine was administered. A heparinized catheter (250 UI/ml) was inserted into the carotid to allow the collection of blood samples.

In the sepsis subgroups, cecal ligature and puncture (CLP) was performed as previously described (Léon et al., 2012). Briefly, after a laparotomy, the cecum was exposed and ligated in its middle. Next, the cecum was punctured once from side to side by a catheter (8 Gauge) and replaced in the abdominal cavity.

In the Sham subgroups, only laparotomy was done.

Survival times were evaluated for 6 h, after this delay, the animals (still under anesthesia) were euthanized by intracardiac puncture and blood draw.

## 2.4 | Analytical procedure after acute sepsis induction

Arterial blood samples (300 µl) were collected via the carotid catheter immediately after the surgical procedure

<figure><img src="image_3.png" /><figcaption>FIGURE 1 Experimental protocol</figcaption></figure>
<figure><img src="image_3.png" /></figure>

<figure><img src="image_4.png" /></figure>

## 2.2 | Ethical approval

This study was performed in accordance with the European recommendations (2010/63/EU), approved by the regional ethical committee (CEFEA B29-019-08) and the French Ministry (18325_2018123119211520). The authors understand the journal ethical principles and acknowledge that this study complies with this animal ethics checklist. Pain and suffering were minimized during the entire experiment.

## 2.3 | Experimental protocol

### 2.3.1 | Animals

Sixty-four 3-week-old female Wistar rats (Janvier, SAS-Le Genest St Isle) with an average weight of 97.5 ± 1.6 g were included in this study. Rats were raised under a 12 h light-12 h dark cycle at 21°C. Food and water were given ad libitum. Rats were allowed to adapt to environmental conditions for 1 week before the experiments.

### 2.3.2 | Diet and supplementation

During the first 8 weeks of the experiment, animals were provided with tap water and food devoid of selenium (U8959P v.0170, SAFE, France) as can be seen in Figure 1. From week 8 to week 12, the rats (randomly assigned to one of the four following experimental groups—16 rats/group) were still fed with a selenium-deficient diet but the beverage differed between the groups:

* D group (“D” for deficient) received tap water;

* Se group received selenium in water (Na₂SeO₃, 71950, Sigma Aldrich) at 20 µg Se/kg bw (body mass)/day;

* Spi group received *spirulina* in water at 3 g/kg bw/day;

* SeSP group received Se enriched *spirulina* in water at 3 g/kg bw/day, bringing 20 µg of selenium/kg bw/day.

All rats were weighted weekly, food, and drink intakes (given ad libitum) were measured twice a week during the 12 weeks of the experiment.

### 2.3.3 | Acute sepsis induction

Each group of 16 rats was randomly subdivided in two subgroups of 8 rats: sham and septic (described below).

In all cases, the rats were anesthetized by an intraperitoneal injection of a ketamine (100 mg/kg bw) and xylazine (10 mg/kg bw); a subcutaneous saline solution injection (3 ml/100 g bw) was performed and temperature was maintained at 38°C (±0.5°C). The anesthesia was controlled all along the procedure and if needed, a half dose of ketamine xylazine was administered. A heparinized catheter (250 UI/ml) was inserted into the carotid to allow the collection of blood samples.

In the sepsis subgroups, cecal ligature and puncture (CLP) was performed as previously described (Léon et al., 2012). Briefly, after a laparotomy, the cecum was exposed and ligated in its middle. Next, the cecum was punctured once from side to side by a catheter (8 Gauge) and replaced in the abdominal cavity.

In the Sham subgroups, only laparotomy was done.

Survival times were evaluated for 6 h, after this delay, the animals (still under anesthesia) were euthanized by intracardiac puncture and blood draw.

## 2.4 | Analytical procedure after acute sepsis induction

Arterial blood samples (300 µl) were collected via the carotid catheter immediately after the surgical procedure

<figure><img src="image_5.png" /><figcaption>FIGURE 1 Experimental protocol</figcaption></figure>
<figure><img src="image_4.png" /></figure>

<figure><img src="image_5.png" /></figure>

(T0h), then at T1h, T2h, and T4h (when the survival duration of rats made it possible). Seventy-five microliters of blood was used for determination of selenoprotein expression by RT-PCR, 75 µl was used extemporaneously for blood parameter measurements and 150 µl was used for plasma collection (by centrifugation at 2000 g for 5 min at 4°C) and subsequent cytokines quantification. Furthermore, at the animal death, total blood was collected for plasma isolation. Frozen samples were kept at -80°C until assays.

## 2.5 | Measured parameters after sepsis induction

### 2.5.1 | Measurement of plasma selenium concentrations

One hundred microliters of plasma, collected at the animal death, was weighed in PTFE vials, mixed with 2 ml of distilled 14N HNO₃ and evaporated to dryness (on a hot plate overnight). One milliliter of 2.5% HNO₃ was then weighted and added to the samples for ICP-MS measurements. The concentrations of selenium were measured with a HR-ICP-MS Element XR (Thermofisher Scientific) at Pôle de Spectrométrie Océan (PSO) (IUEM/Ifremer) using indium as an internal standard for a drift signal correction. Selenium was measured at medium resolution to compensate for spectral interferences forming in the argon plasma. Calibration was performed with external standards; the detection limit was ~3 ng/g. Two procedural blanks were also processed following the above protocol and analyzed with the samples.

### 2.5.2 | Survival duration

Survival duration was determined in the four groups in septic and sham conditions. As mentioned earlier, 6 h after the beginning of the surgical procedure, surviving animals were euthanized.

### 2.5.3 | Selenoprotein expression

#### RNA isolation for RT-PCR

Total RNA was isolated from total blood using the Nucleospin RNA Blood (740200.50, Macherey-Nagel) according to a manufacturer's protocol adapted for total blood. Briefly, an enzymatic lysis was performed with 200 µl of Lysis Buffer and 5 µl of Proteinase K 15 min at room temperature. Then, 200 µl of 70% ethanol was added and the lysate was transferred into a Nucleospin column. After centrifugation at 11,000 g for 30 s, 350 µl of Membrane Desalting Buffer was added onto the column and centrifuged at 11,000 g for 30 s. The following RNA extraction steps (including DNase treatment) were performed according to the kit procedure. At the end, RNA was eluted with 40 µl of DNase/RNAse-free water and stored at -80°C. RNA concentrations were measured with a SimpliNano™ spectrophotometer (29-0617-12, GE Healthcare Life Sciences) and their purity was assessed using OD₂₆₀/OD₂₈₀ ratios. Their integrity was also checked by electrophoresis on a 1.5% agarose gel with ethidium bromide.

#### Quantification of Gene Expression by Real-Time Reverse Transcriptase -PCR (RT-PCR)

Blood mRNA levels of GPx3 and GPx1 were quantified by RT-PCR. Total RNA (1000 ng) was reverse transcribed with the qScript cDNA synthesis kit (733-1174, QUANTA BioSciences) containing a mix of oligo(dT) and random primers, dNTPs, Mg²⁺ and the reverse transcriptase. All cDNAs were then diluted 10-fold for PCR experiments, which were realized with a 7500 Fast Real-Time PCR (Applied Biosystems, Thermo Fisher Scientific). Target genes were amplified and quantified by SYBR® green incorporation (EurobioGreen® Mix qPCR 2x Lo-Rox, Eurobio Ingen) with the primers given in Table 1.

The cycling conditions consisted of a denaturing step at 95°C for 2 min, followed by 40–45 cycles of amplification (denaturation: 95°C for 5 s; annealing/extension step: 60°C for 30 s). Finally, a melting curve program was carried out from 60°C to 95°C with a heating rate of 0.1°C/s, showing a single product with a specific melting temperature for each gene and sample evaluated.

<table><caption>TABLE 1 Primer sequences used for Real-Time RT-PCR analysis</caption><thead><tr><td>Target gene</td><td>Abbreviation</td><td>Primer sequence (5' to 3')</td><td>Accession number</td><td>Database</td></tr></thead><tbody><tr><td>Glutathion Peroxidase 3</td><td>Gpx3</td><td>(F) CAAGAAGAACTTGGCCCATTC<br>(R) GCTGGAAATTAGGCACAAAGC</td><td>BC062227</td><td>GenBank</td></tr><tr><td>Glutathion Perixdase 1</td><td>Gpx1</td><td>(F) TGCAATCAGTTCGGACATCAG<br>(R) TTCACCTCGCACTTCTCAAAC</td><td>NM_030826.4</td><td>GenBank</td></tr><tr><td>Glyceraldehyde 3-phosphate deshydrogenase</td><td>Gapdh</td><td>(F) GTATCCGTTGTGGATCTGACA<br>(R) CTGCTTCACCACCTTCTTGAT</td><td>P04797</td><td>GenPept</td></tr></tbody></table>
<figure><img src="image_6.png" /></figure>

<figure><img src="image_7.png" /></figure>

(T0h), then at T1h, T2h, and T4h (when the survival duration of rats made it possible). Seventy-five microliters of blood was used for determination of selenoprotein expression by RT-PCR, 75 µl was used extemporaneously for blood parameter measurements and 150 µl was used for plasma collection (by centrifugation at 2000 g for 5 min at 4°C) and subsequent cytokines quantification. Furthermore, at the animal death, total blood was collected for plasma isolation. Frozen samples were kept at -80°C until assays.

## 2.5 | Measured parameters after sepsis induction

### 2.5.1 | Measurement of plasma selenium concentrations

One hundred microliters of plasma, collected at the animal death, was weighed in PTFE vials, mixed with 2 ml of distilled 14N HNO₃ and evaporated to dryness (on a hot plate overnight). One milliliter of 2.5% HNO₃ was then weighted and added to the samples for ICP-MS measurements. The concentrations of selenium were measured with a HR-ICP-MS Element XR (Thermofisher Scientific) at Pôle de Spectrométrie Océan (PSO) (IUEM/Ifremer) using indium as an internal standard for a drift signal correction. Selenium was measured at medium resolution to compensate for spectral interferences forming in the argon plasma. Calibration was performed with external standards; the detection limit was ~3 ng/g. Two procedural blanks were also processed following the above protocol and analyzed with the samples.

### 2.5.2 | Survival duration

Survival duration was determined in the four groups in specific and sham conditions. As mentioned earlier, 6 h after the beginning of the surgical procedure, surviving animals were euthanized.

### 2.5.3 | Selenoprotein expression

#### RNA isolation for RT-PCR

Total RNA was isolated from total blood using the Nucleospin RNA Blood (740200.50, Macherey-Nagel) according to a manufacturer's protocol adapted for total blood. Briefly, an enzymatic lysis was performed with 200 µl of Lysis Buffer and 5 µl of Proteinase K 15 min at room temperature. Then, 200 µl of 70% ethanol was added and the lysate was transferred into a Nucleospin column. After centrifugation at 11,000 g for 30 s, 350 µl of Membrane Desalting Buffer was added onto the column and centrifuged at 11,000 g for 30 s. The following RNA extraction steps (including DNase treatment) were performed according to the kit procedure. At the end, RNA was eluted with 40 µl of DNase/RNase-free water and stored at -80°C. RNA concentrations were measured with a SimpliNano™ spectrophotometer (29-0617-12, GE Healthcare Life Sciences) and their purity was assessed using OD₂₆₀/OD₂₈₀ ratios. Their integrity was also checked by electrophoresis on a 1.5% agarose gel with ethidium bromide.

#### Quantification of Gene Expression by Real-Time Reverse Transcriptase-PCR (RT-PCR)

Blood mRNA levels of GPx3 and GPx1 were quantified by RT-PCR. Total RNA (1000 ng) was reverse transcribed with the qScript cDNA synthesis kit (733-1174, QUANTA BioSciences) containing a mix of oligo(dT) and random primers, dNTPs, Mg²⁺ and the reverse transcriptase. All cDNAs were then diluted 10-fold for PCR experiments, which were realized with a 7500 Fast Real-Time PCR (Applied Biosystems, Thermo Fisher Scientific). Target genes were amplified and quantified by SYBR® green incorporation (EurobioGreen® Mix qPCR 2x Lo-Rox, Eurobio Ingen) with the primers given in Table 1.

The cycling conditions consisted of a denaturing step at 95°C for 2 min, followed by 40-45 cycles of amplification (denaturation: 95°C for 5 s; annealing/extension step: 60°C for 30 s). Finally, a melting curve program was carried out from 60°C to 95°C with a heating rate of 0.1°C/s, showing a single product with a specific melting temperature for each gene and sample evaluated.

<table><caption>TABLE 1 Primer sequences used for Real-Time RT-PCR analysis</caption><thead><tr><td>Target gene</td><td>Abbreviation</td><td>Primer sequence (5' to 3')</td><td>Accession number</td><td>Database</td></tr></thead><tbody><tr><td>Glutathion Peroxidase 3</td><td>Gpx3</td><td>(F) CAAGAAGAACTTGGCCCATTC<br>(R) GCTGGAAATTAGGCACAAAGC</td><td>BC062227</td><td>GenBank</td></tr><tr><td>Glutathion Perixdase 1</td><td>Gpx1</td><td>(F) TGCAATCAGTTCGGACATCAG<br>(R) TTCACCTCGCACTTCTCAAAC</td><td>NM_030826.4</td><td>GenBank</td></tr><tr><td>Glyceraldehyde 3-phosphate deshydrogenase</td><td>Gapdh</td><td>(F) GTATCCGTTGTGGATCTGACA<br>(R) CTGCTTCACCACCTTCTTGAT</td><td>P04797</td><td>GenPept</td></tr></tbody></table>
<figure><img src="image_6.png" /></figure>

<figure><img src="image_7.png" /></figure>

To obtain standard curves, all target genes were first ampli-
fied from a pool of RT products prepared with all rat samples.
PCR products obtained were purified after electrophoretic
separation on a 1.5% agarose gel using the Nucleospin gel and
PCR Clean-Up kit (Macherey-Nagel). PCR products were
then quantified using a SimpliNano™ spectrophotometer be-
fore proceeding to a serial dilution from 10 pg/µl to 0.001 fg/
µl. These seven-point standard curves were used to determine
the PCR efficiency of each primer pair.

The calculation of absolute mRNA level of a specific
target gene was based on the PCR efficiency value (E) and
the Threshold Cycle deviation (ΔCT) of an unknown cDNA
versus a control one (here, a pool of blood cDNA) according
to the equation proposed by Pfaffl (2001): absolute mRNA
level of a target gene = E<sub>target</sub><sup>ΔCT(control-sample)</sup>. For one gene,
four runs were required to quantify the mRNA levels in all
samples. Each run included no-template controls and tripli-
cates of the control cDNA. Inter-assays variations were found
to be <1.0%. To account for variations due to mRNA ex-
traction and reverse-transcription reaction, absolute mRNA
levels obtained were corrected by 18s rRNA levels, used as
a housekeeping gene. Thus, relative mRNA levels were ex-
pressed in arbitrary unit as ratios target genes/18S rRNA.

2.5.4 | Blood parameters measurement

Seventy-five microliters of blood sample was immediately
analyzed using a blood gas analyzer (ABL 80, Radiometer).
Arterial blood pH, carbon dioxide partial pressure (PCO2), oxy-
gen partial pressure (PO2), hematocrit, hemoglobin concentra-
tion, lactates, and bicarbonate concentrations were measured.

2.5.5 | Inflammation markers quantification

Plasma TNF-α, IL-10, and IL-6 concentrations were meas-
ured by microElisa method using rat immunoassay kits
(RTA00, R1000 and R6000B R&D Systems).

**2.6** | **Statistics**

All results are expressed as mean ± standard error of mean
(SEM) or median ± interquartile range (IQR). Survival
duration was analyzed using log-rank test and illustrated
in a Kaplan-Meier plot. Statistics were performed using
GraphPad Prism v8.0.1 software. Normality was tested using
the Shapiro-Wilk test. Adapted tests were then performed
(ANOVA for repeated measures, Kruskal-Wallis test, and
ANOVA 2 factors). ANOVA were followed by Tukey's post
hoc test while Kruskal-Wallis test was followed by Dunn's
post hoc test. A *p* value <0.05 was considered significant.

3 | RESULTS

3.1 | Effects of Se deficiency and
supplementations (Se/Spi/SeSp) on rat growth

The mean body weight gain during the experiment is presented
in Figure 2a. During the 8 weeks of selenium deficiency, no
significant difference between groups was observed. The
supplementations did not affect the weight gain regardless of
the supplementation type. As shown in Figure 2b, selenium
deficiency and supplementations had no impact on the food
consumption during the 12 weeks of experimentation.

However, water intake depended on the supplementation
(see Figure 2c). During the 4 weeks of supplementation, rats
supplemented with sodium selenite (Se) (23.00 ± 1.51 ml/
day) and Se-enriched *spirulina* (SeSP) (24.97 ± 1.60 ml/
day) showed a significant increase in the amount of
water consumed compared to the deficient (D) group
(20.39 ± 0.75 ml/day). The group supplemented with *spiru-
lina* (Spi) exhibited the highest daily water intake during this
period (30.43 ± 1.21 ml/day). In order to determine whether
the rise of supplementation consumption was linear during
the 4 weeks of supplementation, a presentation of water in-
take at weeks 1 and 4 is shown in Figure 2d. It appears that
Spi (35.59 ± 1.19 ml/day) and SeSP (31.36 ± 2.31 ml/day)
triggered an increase in water consumption at week 1. This
effect was reduced at week 4 and only Spi group maintains
a significantly higher water intake compared to the D group
(26.19 ± 1.33 vs 18.69 ± 1.39 ml/day).

**3.2** | **Plasma Selenium concentration**

Plasmatic Se concentration was measured from the blood
sample performed at the death of the animals. The plasma se-
lenium concentration in the D group was 252.27 ± 12.02 ppb
(see Figure 3). Supplementation with spirulina (Spi group)
did not induce any modification of this concentration. Se and
SeSP supplementations led to a strong and significant in-
crease in plasma selenium concentrations. Selenium concen-
tration in Se group (764.74 ± 37.53 ppb) was significantly
higher compared to SeSP group (636.33 ± 27.34 ppb).

**3.3 | Survival analysis**

In Sham subgroups, the animals still alive at 6h were sac-
rificed and rat survival was not different from one group to
the other (see Figure 4). However, at 6h the percentage of
surviving rats ranged from 88% in the Se group to 50 and
40% in the SeSp and Spi groups, respectively; the difference
between Se Sham and Spi Sham is at the limit of statistical
significance (p = 0.06).
<figure><img src="image_8.png" /></figure>

<figure><img src="image_9.png" /></figure>

To obtain standard curves, all target genes were first ampli-
fied from a pool of RT products prepared with all rat samples.
PCR products obtained were purified after electrophoretic
separation on a 1.5% agarose gel using the Nucleospin gel and
PCR Clean-Up kit (Macherey-Nagel). PCR products were
then quantified using a SimpliNano™ spectrophotometer be-
fore proceeding to a serial dilution from 10 pg/µl to 0.001 fg/
µl. These seven-point standard curves were used to determine
the PCR efficiency of each primer pair.

The calculation of absolute mRNA level of a specific
target gene was based on the PCR efficiency value (E) and
the Threshold Cycle deviation (ΔCT) of an unknown cDNA
versus a control one (here, a pool of blood cDNA) according
to the equation proposed by Pfaffl (2001): absolute mRNA
level of a target gene = E<sub>target</sub><sup>ΔCT(control-sample)</sup>. For one gene,
four runs were required to quantify the mRNA levels in all
samples. Each run included no-template controls and tripli-
cates of the control cDNA. Inter-assays variations were found
to be <1.0%. To account for variations due to mRNA ex-
traction and reverse-transcription reaction, absolute mRNA
levels obtained were corrected by 18s rRNA levels, used as
a housekeeping gene. Thus, relative mRNA levels were ex-
pressed in arbitrary unit as ratios target genes/18S rRNA.

2.5.4 | Blood parameters measurement

Seventy-five microliters of blood sample was immediately
analyzed using a blood gas analyzer (ABL 80, Radiometer).
Arterial blood pH, carbon dioxide partial pressure (PCO2), oxy-
gen partial pressure (PO2), hematocrit, hemoglobin concentra-
tion, lactates, and bicarbonate concentrations were measured.

2.5.5 | Inflammation markers quantification

Plasma TNF-α, IL-10, and IL-6 concentrations were meas-
ured by microElisa method using rat immunoassay kits
(RTA00, R1000 and R6000B R&D Systems).

**2.6 | Statistics**

All results are expressed as mean ± standard error of mean
(SEM) or median ± interquartile range (IQR). Survival
duration was analyzed using log-rank test and illustrated
in a Kaplan-Meier plot. Statistics were performed using
GraphPad Prism v8.0.1 software. Normality was tested using
the Shapiro-Wilk test. Adapted tests were then performed
(ANOVA for repeated measures, Kruskal-Wallis test, and
ANOVA 2 factors). ANOVA were followed by Tukey's post
hoc test while Kruskal-Wallis test was followed by Dunn's
post hoc test. A *p* value <0.05 was considered significant.

3 | RESULTS

3.1 | Effects of Se deficiency and
supplementations (Se/Spi/SeSp) on rat growth

The mean body weight gain during the experiment is presented
in Figure 2a. During the 8 weeks of selenium deficiency, no
significant difference between groups was observed. The
supplementations did not affect the weight gain regardless of
the supplementation type. As shown in Figure 2b, selenium
deficiency and supplementations had no impact on the food
consumption during the 12 weeks of experimentation.

However, water intake depended on the supplementation
(see Figure 2c). During the 4 weeks of supplementation, rats
supplemented with sodium selenite (Se) (23.00 ± 1.51 ml/
day) and Se-enriched *spirulina* (SeSP) (24.97 ± 1.60 ml/
day) showed a significant increase in the amount of
water consumed compared to the deficient (D) group
(20.39 ± 0.75 ml/day). The group supplemented with *spiru-
lina* (Spi) exhibited the highest daily water intake during this
period (30.43 ± 1.21 ml/day). In order to determine whether
the rise of supplementation consumption was linear during
the 4 weeks of supplementation, a presentation of water in-
take at weeks 1 and 4 is shown in Figure 2d. It appears that
Spi (35.59 ± 1.19 ml/day) and SeSP (31.36 ± 2.31 ml/day)
triggered an increase in water consumption at week 1. This
effect was reduced at week 4 and only Spi group maintains
a significantly higher water intake compared to the D group
(26.19 ± 1.33 vs 18.69 ± 1.39 ml/day).

**3.2 | Plasma Selenium concentration**

Plasmatic Se concentration was measured from the blood
sample performed at the death of the animals. The plasma se-
lenium concentration in the D group was 252.27 ± 12.02 ppb
(see Figure 3). Supplementation with spirulina (Spi group)
did not induce any modification of this concentration. Se and
SeSP supplementations led to a strong and significant in-
crease in plasma selenium concentrations. Selenium concen-
tration in Se group (764.74 ± 37.53 ppb) was significantly
higher compared to SeSP group (636.33 ± 27.34 ppb).

**3.3 | Survival analysis**

In Sham subgroups, the animals still alive at 6h were sac-
rificed and rat survival was not different from one group to
the other (see Figure 4). However, at 6h the percentage of
surviving rats ranged from 88% in the Se group to 50 and
40% in the SeSp and Spi groups, respectively; the difference
between Se Sham and Spi Sham is at the limit of statistical
significance (p = 0.06).
<figure><img src="image_8.png" /></figure>

<figure><img src="image_9.png" /></figure>

<figure><img src="image_10.png" /></figure>

<figure><img src="image_11.png" /></figure>

FIGURE 2 Effect of selenium-deficient diet and sodium selenite, spirulina, or selenium-enriched spirulina supplementations on rat growth. (a) Weight gain (g per day) in selenium-deficient diet for 8 weeks and supplementations for 4 weeks. (b) Food intake (g per day) in selenium-deficient diet for 8 weeks and supplementations for 4 weeks. (c) Water intake (ml per day) in selenium-deficient diet for 8 weeks and supplementations for 4 weeks. (d) Comparison of water intake in week 1 and week 4 of supplementation. D, deficient, non-supplemented group; Se, selenium-supplemented group; Spi, spirulin-supplemented group; SeSP, selenium-enriched spirulina-supplemented group. Data are expressed as mean ± SEM (n = 16 for each group). Different letters indicate significant difference between groups (*p* < 0.05)

Sepsis induction significantly decreased the survival duration with every type of supplementation compared to Sham rats. Within the septic rat groups, a difference of survival duration was observed between the Se (3 h 37 min ±50 min) and SeSP groups (1 h 52 min ±21 min).

## 3.4 | GPx1 and GPx3 mRNA levels

From T0 to T4h, no significant difference throughout time appeared within the conditions for GPx1 and GPx3 mRNA levels; results were hence grouped (see Figure 5). There was no significant difference in GPx1 mRNA expression levels between the D and Spi groups (respectively, at 0.42 ± 0.05 and 0.32 ± 0.02). Se-supplementation induced a slight increase of GPx1 mRNA (0.84 ± 0.06). In the SeSP group (2.06 ± 0.12), a fivefold increase appeared when compared to the D group. Concerning GPx3 mRNA

levels, no significant difference was observed between conditions.

## 3.5 | pH, P<sub>CO2</sub>, plasma HCO<sub>3</sub><sup>-</sup>, and lactate concentration

Davenport diagrams, linking P<sub>CO2</sub> with plasmatic HCO<sub>3</sub><sup>-</sup> concentration ([HCO<sub>3</sub><sup>-</sup>]<sub>p</sub>) and pH, are shown in Figure 6. At T0, no significant difference was observed between groups for [HCO<sub>3</sub><sup>-</sup>], P<sub>O2</sub>, P<sub>CO2</sub>, and pH (data not shown). Sham rats, at T1 and T2, displayed no significant difference with T0 in any condition. In the D group (Figure 6a), sepsis induced a significant acidosis at T2 (7.21 ± 0.02) compared to T0 (7.32 ± 0.01), T2 sham (7.30 ± 0.02), and T1 sepsis (7.30 ± 0.03). A decrease of [HCO<sub>3</sub><sup>-</sup>]<sub>p</sub> was also observed in D septic rats at T2 (18.5 ± 1.1 mmol/L) compared to T0 (23.83 ± 0.97 mmol/L). In the Se group (Figure 6b),
<figure><img src="image_10.png" /></figure>

<figure><img src="image_11.png" /></figure>

<figure><img src="image_12.png" /></figure>

<figure><img src="image_13.png" /></figure>

FIGURE 2 Effect of selenium-deficient diet and sodium selenite, spirulina, or selenium-enriched spirulina supplementations on rat growth. (a) Weight gain (g per day) in selenium-deficient diet for 8 weeks and supplementations for 4 weeks. (b) Food intake (g per day) in selenium-deficient diet for 8 weeks and supplementations for 4 weeks. (c) Water intake (ml per day) in selenium-deficient diet for 8 weeks and supplementations for 4 weeks. (d) Comparison of water intake in week 1 and week 4 of supplementation. D, deficient, non-supplemented group; Se, selenium-supplemented group; Spi, spirulin-supplemented group; SeSP, selenium-enriched spirulina-supplemented group. Data are expressed as mean ± SEM (n = 16 for each group). Different letters indicate significant difference between groups (*p* < 0.05)

Sepsis induction significantly decreased the survival duration with every type of supplementation compared to Sham rats. Within the septic rat groups, a difference of survival duration was observed between the Se (3 h 37 min ±50 min) and SeSP groups (1 h 52 min ±21 min).

## 3.4 | GPx1 and GPx3 mRNA levels

From T0 to T4h, no significant difference throughout time appeared within the conditions for GPx1 and GPx3 mRNA levels; results were hence grouped (see Figure 5). There was no significant difference in GPx1 mRNA expression levels between the D and Spi groups (respectively, at 0.42 ± 0.05 and 0.32 ± 0.02). Se-supplementation induced a slight increase of GPx1 mRNA (0.84 ± 0.06). In the SeSP group (2.06 ± 0.12), a fivefold increase appeared when compared to the D group. Concerning GPx3 mRNA

levels, no significant difference was observed between conditions.

## 3.5 | pH, $P_{CO2}$, plasma $HCO_3^-$, and lactate concentration

Davenport diagrams, linking $P_{CO2}$ with plasmatic $HCO_3^-$ concentration ($[HCO_3^-]_p$) and pH, are shown in Figure 6. At T0, no significant difference was observed between groups for $[HCO_3^-]$, $P_{O2}$, $P_{CO2}$, and pH (data not shown). Sham rats, at T1 and T2, displayed no significant difference with T0 in any condition. In the D group (Figure 6a), sepsis induced a significant acidosis at T2 (7.21 ± 0.02) compared to T0 (7.32 ± 0.01), T2 sham (7.30 ± 0.02), and T1 sepsis (7.30 ± 0.03). A decrease of $[HCO_3^-]_p$ was also observed in D septic rats at T2 (18.5 ± 1.1 mmol/L) compared to T0 (23.83 ± 0.97 mmol/L). In the Se group (Figure 6b),
sham rats displayed no significant difference of pH and
[HCO₃⁻]ₚ with T0. Furthermore, no significant pH differ-
ence was observed in septic rats while [HCO₃⁻]ₚ was re-
duced in septic rats at T2 (19.3 ± 0.85 mmol/L) compared to
T0 (25.25 ± 0.75 mmol/L). For the Spi groups (Figure 6c)
pH was not different in sham and septic condition. At T2,
in septic rats, [HCO₃⁻]ₚ was lower (19.71 ± 1.47 mmol/L)
than the T0 value (25.13 ± 0.76 mmol/L). Finally, in SeSP
septic and sham rats (Figure 6d), no significant difference
in pH and [HCO₃⁻]ₚ was observed. Finally, pH in the Se
septic group (7.32 ± 0.02) was higher compared to the
SeSP septic group (7.25 ± 0.02) at T1h. At T2, pH was

higher in the Se septic group (7.29 ± 0.02) when compared
to the D septic group (7.21 ± 0.02).

No difference of plasmatic lactate concentrations was observed
in the sham groups (whatever the time and the condition, see
Figure 6e). In the D group, 2 h after sepsis induction, plasma lac-
tate concentration (2.16 ± 0.73 mmol/L) was increased compared
to T0 (0.76 ± 0.06 mmol/L), T1 sepsis (0.98 ± 0.25 mmol/L) and
T2 sham (0.92 ± 0.28 mmol/L). In the Se group, no significant dif-
ference in lactate concentration with time in both sham and septic
conditions was observed. Both the Spi and SeSP groups seem to
exhibit a similar profile. In fact, lactate concentrations increased
in septic groups at T1 (1.26 ± 0.36 and 1.52 ± 0.58 mmol/L, re-
spectively) and T2 (2.4 ± 0.62 and 2.76 ± 1.03 mmol/L) com-
pared to T0 (0.33 ± 0.04 and 0.41 ± 0.05 mmol/L, respectively),
and T1 sham. Moreover, the Spi and SeSP groups showed a rise
in lactate concentration compared to their own T1 sepsis and
T2 sham. Finally, at T2h, the sepsis Se group showed lower lac-
tate concentration (1.06 ± 0.23 mmol/L) than the T2 sepsis D
(2.16 ± 0.73 mmol/L), Spi (2.4 ± 0.62 mmol/L) and SeSP groups
(2.76 ± 1.03 mmol/L).

<figure><img src="image_12.png" /><figcaption>FIGURE 3 Plasma selenium concentration after 8 weeks of selenium-deficient diet and 4 weeks of sodium selenite, spirulina, or selenium-enriched supplementation. Data are expressed as mean ± SEM (n = 16 for each group). D, deficient, non-supplemented group; Se, selenium-supplemented group; Spi, spirulina-supplemented group; SeSP, selenium-enriched spirulina-supplemented group. Different letters indicate significant difference between groups (*p* < 0.05)</figcaption></figure>

3.6 | Hemoglobin, Hematocrit, and P<sub>02</sub>

No significant difference appeared for any of these param-
eters in any condition (data not shown). Mean hemoglobin
concentration, hematocrit, and P₀₂ were 13.4 ± 0.10 g/dL,
40.06 ± 0.31%, and 59.05 ± 0.84 mmHg, respectively.

3.7 | Plasma IL-6, TNF-α, and IL-10 concentrations

No significant difference in plasma IL-6 concentrations was
observed between sham groups (Figure 7). In the D group
sepsis, a rise was observed at T2 (2206.0 ± 2696.5 pg/ml)

<figure><img src="image_13.png" /><figcaption>FIGURE 4 Survival duration rate after induction of sepsis or Sham operations. Data are expressed as percentage of T0 animal number (n = 8 for all group and condition except D sham n = 6 and D sepsis n = 7). D, deficient, non-supplemented group; Se, selenium-supplemented group; Spi, spirulina-supplemented group; SeSP, selenium-enriched spirulina-supplemented group. *indicates a significant difference with Se Sepsis group (p < 0.05)</figcaption></figure>
sham rats displayed no significant difference of pH and
[HCO₃⁻]ₚ with T0. Furthermore, no significant pH differ-
ence was observed in septic rats while [HCO₃⁻]ₚ was re-
duced in septic rats at T2 (19.3 ± 0.85 mmol/L) compared to
T0 (25.25 ± 0.75 mmol/L). For the Spi groups (Figure 6c)
pH was not different in sham and septic condition. At T2,
in septic rats, [HCO₃⁻]ₚ was lower (19.71 ± 1.47 mmol/L)
than the T0 value (25.13 ± 0.76 mmol/L). Finally, in SeSP
septic and sham rats (Figure 6d), no significant difference
in pH and [HCO₃⁻]ₚ was observed. Finally, pH in the Se
septic group (7.32 ± 0.02) was higher compared to the
SeSP septic group (7.25 ± 0.02) at T1h. At T2, pH was

higher in the Se septic group (7.29 ± 0.02) when compared
to the D septic group (7.21 ± 0.02).

No difference of plasmatic lactate concentrations was observed in the sham groups (whatever the time and the condition, see Figure 6e). In the D group, 2 h after sepsis induction, plasma lactate concentration (2.16 ± 0.73 mmol/L) was increased compared to T0 (0.76 ± 0.06 mmol/L), T1 sepsis (0.98 ± 0.25 mmol/L) and T2 sham (0.92 ± 0.28 mmol/L). In the Se group, no significant difference in lactate concentration with time in both sham and septic conditions was observed. Both the Spi and SeSP groups seem to exhibit a similar profile. In fact, lactate concentrations increased in septic groups at T1 (1.26 ± 0.36 and 1.52 ± 0.58 mmol/L, respectively) and T2 (2.4 ± 0.62 and 2.76 ± 1.03 mmol/L) compared to T0 (0.33 ± 0.04 and 0.41 ± 0.05 mmol/L, respectively), and T1 sham. Moreover, the Spi and SeSP groups showed a rise in lactate concentration compared to their own T1 sepsis and T2 sham. Finally, at T2h, the sepsis Se group showed lower lactate concentration (1.06 ± 0.23 mmol/L) than the T2 sepsis D (2.16 ± 0.73 mmol/L), Spi (2.4 ± 0.62 mmol/L) and SeSP groups (2.76 ± 1.03 mmol/L).

<figure><img src="image_14.png" /><figcaption>FIGURE 3 Plasma selenium concentration after 8 weeks of selenium-deficient diet and 4 weeks of sodium selenite, spirulina, or selenium-enriched supplementation. Data are expressed as mean ± SEM (n = 16 for each group). D, deficient, non-supplemented group; Se, selenium-supplemented group; Spi, spirulina-supplemented group; SeSP, selenium-enriched spirulina-supplemented group. Different letters indicate significant difference between groups (*p* < 0.05)</figcaption></figure>

3.6 | Hemoglobin, Hematocrit, and P<sub>02</sub>

No significant difference appeared for any of these param-
eters in any condition (data not shown). Mean hemoglobin
concentration, hematocrit, and P<sub>02</sub> were 13.4 ± 0.10 g/dL,
40.06 ± 0.31%, and 59.05 ± 0.84 mmHg, respectively.

3.7 | Plasma IL-6, TNF-α, and IL-10 concentrations

No significant difference in plasma IL-6 concentrations was observed between sham groups (Figure 7). In the D group sepsis, a rise was observed at T2 (2206.0 ± 2696.5 pg/ml)

<figure><img src="image_15.png" /><figcaption>FIGURE 4 Survival duration rate after induction of sepsis or Sham operations. Data are expressed as percentage of T0 animal number (n = 8 for all group and condition except D sham n = 6 and D sepsis n = 7). D, deficient, non-supplemented group; Se, selenium-supplemented group; Spi, spirulina-supplemented group; SeSP, selenium-enriched spirulina-supplemented group.
\*indicates a significant difference with Se Sepsis group (*p* < 0.05)</figcaption></figure>
<figure><img src="image_14.png" /></figure>

FIGURE 5 Plasma Gpx1 mRNA (a) and plasma Gpx3 mRNA (b) levels in D, Se, Spi, and SeSP rats. Data are expressed as mean ± SEM (n = 16 for each group). D, deficient, non-supplemented group; Se, selenium-supplemented group; Spi, spirulina-supplemented group; SeSP, selenium-enriched spirulina-supplemented group. Different letters indicate significant difference between groups (*p* < 0.05)

<figure><img src="image_15.png" /></figure>

FIGURE 6 Davenport diagrams showing pH, PCO2, and bicarbonate (HCO3-) concentrations of sham and septic rats in the D (a), Se (b), Spi (c), and SeSP (d) groups. Lactate concentrations in the D, Se, Spi, and SeSP groups at different time (e). Data are expressed as mean ± SEM. D, deficient, non-supplemented group; Se, selenium-supplemented group; Spi, spirulina-supplemented group; SeSP, selenium-enriched spirulina-supplemented group. “*” indicates a significant difference with T0 in same group (*p* < 0.05). “#” indicates a significant difference with sham at the same time in the same group (*p* < 0.05). “$” indicates a significant difference with T1 septic in same group. “&” indicates a significant difference (*p* < 0.05)
<figure><img src="image_16.png" /></figure>

FIGURE 5 Plasma Gpx1 mRNA (a) and plasma Gpx3 mRNA (b) levels in D, Se, Spi, and SeSP rats. Data are expressed as mean ± SEM (n = 16 for each group). D, deficient, non-supplemented group; Se, selenium-supplemented group; Spi, spirulina-supplemented group; SeSP, selenium-enriched spirulina-supplemented group. Different letters indicate significant difference between groups (*p* < 0.05)

<figure><img src="image_17.png" /></figure>

FIGURE 6 Davenport diagrams showing pH, PCO2, and bicarbonate (HCO3-) concentrations of sham and septic rats in the D (a), Se (b), Spi (c), and SeSP (d) groups. Lactate concentrations in the D, Se, Spi, and SeSP groups at different time (e). Data are expressed as mean ± SEM. D, deficient, non-supplemented group; Se, selenium-supplemented group; Spi, spirulina-supplemented group; SeSP, selenium-enriched spirulina-supplemented group. “*” indicates a significant difference with T0 in same group (*p* < 0.05). “#” indicates a significant difference with sham at the same time in the same group (*p* < 0.05). “$” indicates a significant difference with T1 septic in same group. “&” indicates a significant difference (*p* < 0.05)
<figure><img src="image_16.png" /></figure>

<figure><img src="image_17.png" /></figure>

FIGURE 7 IL-6 (pg/ml) plasma concentration (a), TNF-α (pg/ml) plasma concentration (b), and IL-10 (pg/ml) plasma concentration (c) in sham and septic rats at T0, T1h, and T2h. Data are expressed as median ± IQR (n = depending on survival rate). “*” indicates a significant difference with T0 in same group (*p* < 0.05). D, deficient, non-supplemented group; Se, selenium-supplemented group; Spi, spirulina-supplemented group; SeSP, selenium-enriched spirulina-supplemented group; IQR, interquartile range. “#” indicates a significant difference with sham at same time in same group (*p* < 0.05). “$” indicates a significant difference with T1 septic in same group. “&” indicates a significant difference (*p* < 0.05).

<figure><img src="image_18.png" /></figure>

<figure><img src="image_19.png" /></figure>

<figure><img src="image_20.png" /></figure>

compared to T0 (0.0 ± 32.25 pg/ml). A similar difference (between T0 and T2 sepsis) was observed in all other groups.

Concerning plasma TNF-α concentration, no significant difference between the sham groups was observed.

In the D groups, no significant difference was observed between treatments. In the Se groups, plasma TNF-α concentration was increased in T2 sepsis (106.2 ± 905.5 pg/ml) compared to T0 (0 ± 31.3 pg/ml) and T1 sepsis
<figure><img src="image_18.png" /></figure>

<figure><img src="image_19.png" /></figure>

FIGURE 7 IL-6 (pg/ml) plasma concentration (a), TNF-α (pg/ml) plasma concentration (b), and IL-10 (pg/ml) plasma concentration (c) in sham and septic rats at T0, T1h, and T2h. Data are expressed as median ± IQR (n = depending on survival rate). “*” indicates a significant difference with T0 in same group (*p* < 0.05). D, deficient, non-supplemented group; Se, selenium-supplemented group; Spi, spirulina-supplemented group; SeSP, selenium-enriched spirulina-supplemented group; IQR, interquartile range. “#” indicates a significant difference with sham at same time in same group (*p* < 0.05). “$” indicates a significant difference with T1 septic in same group. “&” indicates a significant difference (*p* < 0.05).

<figure><img src="image_20.png" /></figure>

<figure><img src="image_21.png" /></figure>

<figure><img src="image_22.png" /></figure>

compared to T0 (0.0 ± 32.25 pg/ml). A similar difference (between T0 and T2 sepsis) was observed in all other groups.

Concerning plasma TNF-α concentration, no significant difference between the sham groups was observed.

In the D groups, no significant difference was observed between treatments. In the Se groups, plasma TNF-α concentration was increased in T2 sepsis (106.2 ± 905.5 pg/ml) compared to T0 (0 ± 31.3 pg/ml) and T1 sepsis
<figure><img src="image_21.png" /></figure>

(0 ± 94.1 pg/ml). The same results were observed for Spi and SeSP rats.

No difference in plasmatic IL-10 concentrations between the groups was observed, except for the SeSP septic group at 2 h that was significantly increased when compared to the corresponding T0.

## 3.8 | DISCUSSION

Selenium deficiency is associated with an increase of mortality in intensive care units and spirulina is known to reduce inflammation in many models. Hence, the association of spirulina with a selenium supplementation as a selenium-enriched spirulina, may combine antioxidant and anti-inflammatory properties. In this context, the aim of our study was (on the basis of a Se deficiency) to investigate the effects of selenium-enriched spirulina supplementation on sepsis outcomes.

In this study, weight gain and food intake were not affected by the supplementations. These results are consistent with those of Cases et al., (2001) that showed no difference in food intake after 6 weeks of selenium deficiency and 8 weeks of selenium supplementation. However, if spirulina beneficial effects on weight gain had previously been described (Khalil et al., 2018), no difference appears in our study. spirulina supplementation (Spi and SeSP groups), increases water intake during the first week of supplementation. As spirulina is cultivated in a saline environment, it could amplify the thirst sensation.

Plasmatic selenium concentrations were measured in order to validate our experimental procedures. According to Tanguy et al., (2012), in rat a plasma selenium concentration lower than 300 ppb corresponds to a selenium deficiency. This was the case in the non-supplemented D group and in the Spi-group. It can consequently be concluded that the protocol used has been efficient to induce a selenium deficiency. On this basis, a 4 weeks supplementation with sodium selenite (Se)- or selenium-enriched spirulina made it possible to reach normal plasmatic selenium concentrations. Nevertheless, in SeSP-supplemented rats, plasma selenium concentration was slightly lower than Se-supplemented rats. Interestingly, several studies obtained similar results (Cases et al., 2001; Suzuki et al., 2006; Takahashi et al., 2017), while others did not evidence any difference between sodium selenite- and spirulina-enriched supplementation (Falk et al., 2020; Zhang et al., 2020). When observed, the reduction of plasma selenium concentration in the SeSp group could be explained by the selenium form. In fact, during spirulina enrichment, 85% of selenite is present as organic selenium (Selenomethionine, SeMet) (Li et al., 2003) and SeMet when absorbed, has to be metabolized (either by trans-sulfuration or conversion

into methylselenol, Okuno et al., 2001) before its incorporation into selenoproteins.

The survival analysis after sepsis induction was performed with the sham and sepsis subgroups. Interestingly, in the sham groups, survival at 6h ranged from 88% in the Se group to 50% and 40% in the SeSp and Spi groups, respectively. The high survival in the non-deficient sham rats (Se group) provide evidence for the validity of the surgical and anesthesia procedure but the survival seems to be strongly reduced when animals are supplemented with spirulina (p = 0.06). Similarly, the comparison of septic subgroups showed that Se rats displayed a better survival than SeSP rats. This point clearly need further investigation but if it is confirmed that spirulina reduces the positive outcome of surgical procedures and sepsis, it stresses a potentially strong negative effect of this nutraceutic product.

The effect of a selenium repletion after a selenium deficiency on the oxidative stress-associated selenoproteins has been evaluated through plasma GPx1 and GPx3 mRNA levels. GPx1 but not GPx3 mRNA levels were increased in Se- and SeSP-supplemented group compared to the groups without selenium supplementation (Spi and D groups). Moreover, the SeSP-supplemented group shows a GPx1 mRNA content 2.5 times higher than the Se-supplemented group. This results is consistent with those presented by Xia et al., (2005) who demonstrated that the highest human GPx activity was achieved with a 37 µg SeMet supplemental dose while the same levels of GPx activity required 66 µg of sodium selenite supplementation. Most of all, it highlights the selenoproteins expression hierarchy already described during selenium-deficiency (Burk & Hill, 2015). In our model, GPx3 transcription seems to have priority on GPx1. However, our results also suggest that organic selenium absence downregulates GPx1 transcription (when compared to sodium selenite supplementation). Organic selenium could be better to enhance protein expression but does not appear to be sufficient to improve plasma selenium concentration and survival duration after sepsis induction compared to sodium selenite. It seems obvious that a better understanding of selenium form involvement in selenium repletion after a deficiency period is needed.

Acid-base disorders is a major issue in septic patients and in intensive care units, severe septic and septic shock patients exhibit elevated blood lactate concentration (Casserly et al., 2015; Levy, 2006). In fact, lactate concentration is considered as a good marker of sepsis severity and low lactate is associated with a reduction of mortality and a faster weaning of mechanical ventilation (Jansen et al., 2010; Nichol et al., 2011; Shapiro et al., 2005). In this experiment, a metabolic acidosis is observed 2 h after sepsis induction in the case of selenium deficiency. This metabolic acidosis was not observed in Se-, Spi-, and SeSp-supplemented groups. In case of Se and Spi supplementation, the absence of acidosis can be explained by a respiratory compensation (since pH is maintained while $pCO_2$ and/or $[HCO_3^-]$ is decreased).
<figure><img src="image_23.png" /></figure>

(0 ± 94.1 pg/ml). The same results were observed for Spi and SeSP rats.

No difference in plasmatic IL-10 concentrations between the groups was observed, except for the SeSP septic group at 2 h that was significantly increased when compared to the corresponding T0.

**3.8 | DISCUSSION**

Selenium deficiency is associated with an increase of mortal-
ity in intensive care units and spirulina is known to reduce
inflammation in many models. Hence, the association of
spirulina with a selenium supplementation as a selenium-
enriched spirulina, may combine antioxidant and anti-
inflammatory properties. In this context, the aim of our study
was (on the basis of a Se deficiency) to investigate the effects
of selenium-enriched spirulina supplementation on sepsis
outcomes.

In this study, weight gain and food intake were not af-
fected by the supplementations. These results are consistent
with those of Cases et al., (2001) that showed no difference
in food intake after 6 weeks of selenium deficiency and
8 weeks of selenium supplementation. However, if spiru-
lina beneficial effects on weight gain had previously been
described (Khalil et al., 2018), no difference appears in our
study. spirulina supplementation (Spi and SeSP groups),
increases water intake during the first week of supplemen-
tation. As spirulina is cultivated in a saline environment, it
could amplify the thirst sensation.

Plasmatic selenium concentrations were measured in
order to validate our experimental procedures. According
to Tanguy et al., (2012), in rat a plasma selenium con-
centration lower than 300 ppb corresponds to a selenium
deficiency. This was the case in the non-supplemented D
group and in the Spi-group. It can consequently be con-
cluded that the protocol used has been efficient to induce
a selenium deficiency. On this basis, a 4 weeks supple-
mentation with sodium selenite (Se)- or selenium-enriched
spirulina made it possible to reach normal plasmatic sele-
nium concentrations. Nevertheless, in SeSP-supplemented
rats, plasma selenium concentration was slightly lower
than Se-supplemented rats. Interestingly, several studies
obtained similar results (Cases et al., 2001; Suzuki et al.,
2006; Takahashi et al., 2017), while others did not evidence
any difference between sodium selenite- and spirulina-
enriched supplementation (Falk et al., 2020; Zhang et al.,
2020). When observed, the reduction of plasma selenium
concentration in the SeSp group could be explained by the
selenium form. In fact, during spirulina enrichment, 85% of
selenite is present as organic selenium (Selenomethionine,
SeMet) (Li et al., 2003) and SeMet when absorbed, has to
be metabolized (either by trans-sulfuration or conversion

into methylselenol, Okuno et al., 2001) before its incorpora-
tion into selenoproteins.

The survival analysis after sepsis induction was per-
formed with the sham and sepsis subgroups. Interestingly,
in the sham groups, survival at 6h ranged from 88% in
the Se group to 50% and 40% in the SeSp and Spi groups,
respectively. The high survival in the non-deficient sham
rats (Se group) provide evidence for the validity of the sur-
gical and anesthesia procedure but the survival seems to be
strongly reduced when animals are supplemented with spi-
rulina (p = 0.06). Similarly, the comparison of septic sub-
groups showed that Se rats displayed a better survival than
SeSP rats. This point clearly need further investigation but
if it is confirmed that spirulina reduces the positive out-
come of surgical procedures and sepsis, it stresses a po-
tentially strong negative effect of this nutraceutic product.

The effect of a selenium repletion after a selenium deficiency
on the oxidative stress-associated selenoproteins has been eval-
uated through plasma GPx1 and GPx3 mRNA levels. GPx1
but not GPx3 mRNA levels were increased in Se- and SeSP-
supplemented group compared to the groups without selenium
supplementation (Spi and D groups). Moreover, the SeSP-
supplemented group shows a GPx1 mRNA content 2.5 times
higher than the Se-supplemented group. This results is consis-
tent with those presented by Xia et al., (2005) who demonstrated
that the highest human GPx activity was achieved with a 37 µg
SeMet supplemental dose while the same levels of GPx activ-
ity required 66 µg of sodium selenite supplementation. Most of
all, it highlights the selenoproteins expression hierarchy already
described during selenium-deficiency (Burk & Hill, 2015). In
our model, GPx3 transcription seems to have priority on GPx1.
However, our results also suggest that organic selenium absence
downregulates GPx1 transcription (when compared to sodium
selenite supplementation). Organic selenium could be better to
enhance protein expression but does not appear to be sufficient
to improve plasma selenium concentration and survival duration
after sepsis induction compared to sodium selenite. It seems ob-
vious that a better understanding of selenium form involvement
in selenium repletion after a deficiency period is needed.

Acid-base disorders is a major issue in septic patients and
in intensive care units, severe septic and septic shock patients
exhibit elevated blood lactate concentration (Casserly et al.,
2015; Levy, 2006). In fact, lactate concentration is consid-
ered as a good marker of sepsis severity and low lactate is
associated with a reduction of mortality and a faster wean-
ing of mechanical ventilation (Jansen et al., 2010; Nichol
et al., 2011; Shapiro et al., 2005). In this experiment, a met-
abolic acidosis is observed 2 h after sepsis induction in the
case of selenium deficiency. This metabolic acidosis was
not observed in Se-, Spi-, and SeSp-supplemented groups.
In case of Se and Spi supplementation, the absence of aci-
dosis can be explained by a respiratory compensation (since
pH is maintained while pCO₂ and/or [HCO₃⁻] is decreased).
Lactate concentration increased 1 h after sepsis induction in spirulina- and SeSP-supplemented rats, but 2 h after sepsis induction in the D group. Spirulina appears consequently to be responsible for an early increase of lactate. This effect raises again the aforementioned concern of a potentially negative effect of spirulina in a surgical context. Interestingly, a sodium selenite supplementation totally prevents the increase in plasmatic lactate concentration, but the restoration of this disorder does not appear to be sufficient to significantly increase the survival of septic rats. During sepsis, hypovolemia leads to tissue hypoxia which in turn leads to the switch from aerobic to anaerobic metabolism. Thus, the pyruvate generated by glycolysis cannot be metabolized by mitochondria and is converted to lactate by lactate dehydrogenase (LDH). In our study, no changes in PO₂, PCO₂, and hemoglobin were observed over time (data not shown). Therefore, the sepsis-associated hyperlactatemia occurring after 2 h of sepsis appears to have a metabolic origin and the hypothesis of a respiratory distress can be ruled out. Interestingly Se supplementation reduced lactate production but not SeSp despite their normal plasmatic selenium concentration: the selenium form appears to be important to face the metabolic disorders associated with sepsis. Since tissue hypoxia in septic patients appears to be insufficient to explain all the associated metabolic disturbances (Astiz et al., 1988; Hayes et al., 1994; Mouncey et al., 2015; Ronco et al., 1993), an evaluation of mitochondria activity, and of glycolytic enzyme involved in anaerobic ATP production could be pertinent to clarify the role of sodium selenite in lactate production.

In this study, pro-inflammatory and anti-inflammatory cytokines were measured. Indeed, as soon as a pathogen is detected by the immune system, pro-inflammatory cytokines will be released (Faix, 2013), and, an early release of TNF-α by macrophages (through TLR4 pathway) may induce disseminated intravascular coagulation, hypotension and multiple organ failure (Lv et al., 2014). IL-6, also released by macrophages, is directly correlated with sepsis severity, inflammatory response and associated complications (Gouel-Chéron et al., 2012). In our study, IL-6 and TNF-α expression displayed the same profile: 2 h after sepsis induction, a sharp increase was detected, with nevertheless one exception: for TNF-α, in non-supplemented rats where this increase was not significant (*p* = 0.058). These results are in accordance with clinical trials that demonstrate that the pro-inflammatory cytokines release peak is reached 2 h after sepsis induction (Copeland et al., 2005). Nevertheless, beneficial effects of spirulina (Abdel-Daim et al., 2015) and selenium (Duntas & Benvenga, 2015; Huang et al., 2013; Tan et al., 2013) supplementations had already been described but are not evidenced in our experiment. The control of the inflammation pathways is ensured by the release of anti-inflammatory cytokines by the immune system and, in particular of IL-10 (Chousterman et al., 2017). In our study, only SeSP rats reveal an increase of plasmatic

IL-10 level 2 h after sepsis induction. The others supplementations display extremely low IL-10 level even 2 h after sepsis induction. SeSP supplementation could precociously activate the anti-inflammatory pathway. Nevertheless, all together, these results do not suggest any clear beneficial effects of the different supplementations on inflammatory status during sepsis.

## 4 | CONCLUSION

Sepsis is a complex pathology leading—via inflammation and oxidative stress—to potentially fatal multiple organ failures. Moreover, a selenium deficiency could play a role in sepsis evolution. In this context, and on a basis of a selenium deficiency, sodium selenite supplementation appears to improve metabolic disorders and acido-basic equilibrium without reducing mortality. However, selenium-enriched spirulina does not seem to be indicated in sepsis and raises questions about the selenium form involvement and its bioassimilation.

## ACKNOWLEDGMENTS

The authors thank Nathalie GUEGUENIAT¹ and the Pôle de Spectrométrie Océan (PSO) (IUEM/Ifremer, Brest, France) for their technical assistance. The authors also thank Brittany council.

## CONFLICT OF INTEREST

The authors declare no conflict of interest.

## AUTHOR'S CONTRIBUTIONS

Castel T, Théron M, Pichavant-Rafini K, and Léon K conceived and designed the experiments and contributed to the writing and revising of the article manuscript. Joublin-Delavat A, Guernec A, and Gueguen B contributed to the acquisition, the analyses of the data and the revision of manuscript. All authors have seen and approved the final manuscript.

## DATA AVAILABILITY STATEMENT

The authors declare that data and material are available upon reasonable request.

## ORCID

Thomas Castel https://orcid.org/0000-0003-4339-8859

## REFERENCES

Abdel-Daim, M. M., Abushouk, A. I., Alkhalf, M. I., Toraih, E. A., Fawzy, M. S., Ijaz, H., Aleya, L., & Bungau, S. G. (2018). Antagonistic effects of Spirulina platensis on diazinon-induced hemato-biochemical alterations and oxidative stress in rats. *Environmental Science and Pollution Research International, 25*, 27463-27470. https://doi.org/10.1007/s11356-018-2761-0.
Lactate concentration increased 1 h after sepsis induction in
spirulina- and SeSP-supplemented rats, but 2 h after sepsis
induction in the D group. Spirulina appears consequently
to be responsible for an early increase of lactate. This effect
raises again the aforementioned concern of a potentially neg-
ative effect of spirulina in a surgical context. Interestingly, a
sodium selenite supplementation totally prevents the increase
in plasmatic lactate concentration, but the restoration of this
disorder does not appear to be sufficient to significantly in-
crease the survival of septic rats. During sepsis, hypovolemia
leads to tissue hypoxia which in turn leads to the switch from
aerobic to anaerobic metabolism. Thus, the pyruvate gener-
ated by glycolysis cannot be metabolized by mitochondria
and is converted to lactate by lactate dehydrogenase (LDH).
In our study, no changes in PO2, PCO2, and hemoglobin were
observed over time (data not shown). Therefore, the sepsis-
associated hyperlactatemia occurring after 2 h of sepsis
appears to have a metabolic origin and the hypothesis of a
respiratory distress can be ruled out. Interestingly Se supple-
mentation reduced lactate production but not SeSp despite
their normal plasmatic selenium concentration: the selenium
form appears to be important to face the metabolic disorders
associated with sepsis. Since tissue hypoxia in septic patients
appears to be insufficient to explain all the associated met-
abolic disturbances (Astiz et al., 1988; Hayes et al., 1994;
Mouncey et al., 2015; Ronco et al., 1993), an evaluation of
mitochondria activity, and of glycolytic enzyme involved in
anaerobic ATP production could be pertinent to clarify the
role of sodium selenite in lactate production.

In this study, pro-inflammatory and anti-inflammatory cytokines were measured. Indeed, as soon as a pathogen is detected by the immune system, pro-inflammatory cytokines will be released (Faix, 2013), and, an early release of TNF-α by macrophages (through TLR4 pathway) may induce disseminated intravascular coagulation, hypotension and multiple organ failure (Lv et al., 2014). IL-6, also released by macrophages, is directly correlated with sepsis severity, inflammatory response and associated complications (Gouel-Chéron et al., 2012). In our study, IL-6 and TNF-α expression displayed the same profile: 2 h after sepsis induction, a sharp increase was detected, with nevertheless one exception: for TNF-α, in non-supplemented rats where this increase was not significant (p = 0.058). These results are in accordance with clinical trials that demonstrate that the pro-inflammatory cytokines release peak is reached 2 h after sepsis induction (Copeland et al., 2005). Nevertheless, beneficial effects of spirulina (Abdel-Daim et al., 2015) and selenium (Duntas & Benvenga, 2015; Huang et al., 2013; Tan et al., 2013) supplementations had already been described but are not evidenced in our experiment. The control of the inflammation pathways is ensured by the release of anti-inflammatory cytokines by the immune system and, in particular of IL-10 (Chousterman et al., 2017). In our study, only SeSP rats reveal an increase of plasmatic

IL-10 level 2 h after sepsis induction. The others supplementa-
tions display extremely low IL-10 level even 2 h after sepsis in-
duction. SeSP supplementation could precociously activate the
anti-inflammatory pathway. Nevertheless, all together, these
results do not suggest any clear beneficial effects of the dif-
ferent supplementations on inflammatory status during sepsis.

4 | CONCLUSION

Sepsis is a complex pathology leading—via inflammation and
oxidative stress—to potentially fatal multiple organ failures.
Moreover, a selenium deficiency could play a role in sepsis
evolution. In this context, and on a basis of a selenium defi-
ciency, sodium selenite supplementation appears to improve
metabolic disorders and acido-basic equilibrium without re-
ducing mortality. However, selenium-enriched spirulina does
not seem to be indicated in sepsis and raises questions about
the selenium form involvement and its bioassimilation.

ACKNOWLEDGMENTS

The authors thank Nathalie GUEGUENIAT¹ and the Pôle de Spectrométrie Océan (PSO) (IUEM/Ifremer, Brest, France) for their technical assistance. The authors also thank Brittany council.

CONFLICT OF INTEREST

The authors declare no conflict of interest.

AUTHOR'S CONTRIBUTIONS

Castel T, Théron M, Pichavant-Rafini K, and Léon K con-
ceived and designed the experiments and contributed to the
writing and revising of the article manuscript. Joublin-Delavat
A, Guernec A, and Gueguen B contributed to the acquisition,
the analyses of the data and the revision of manuscript. All
authors have seen and approved the final manuscript.

DATA AVAILABILITY STATEMENT

The authors declare that data and material are available upon
reasonable request.

ORCID

Thomas Castel https://orcid.org/0000-0003-4339-8859

REFERENCES

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<figure><img src="image_22.png" /></figure>

<figure><img src="image_23.png" /></figure>

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Cirino Ruocco, M. A., Pacheco Cechinatti, E. D., Barbosa, F., & Navarro, A. M. (2018). Zinc and selenium status in critically ill patients according to severity stratification. *Nutrition*, **45**, 85–89. https://doi.org/10.1016/j.nut.2017.07.009.

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Falk, M., Bernhoft, A., Reinoso-Maset, E., Salbu, B., Lebed, P., Framstad, T., Fuhrmann, H., & Oropeza-Moe, M. (2020). Beneficial antioxidant status of piglets from sows fed selenomethionine compared with piglets from sows fed sodium selenite. *Journal of Trace Elements in Medicine and Biology*, **58**, 126439. https://doi.org/10.1016/j.jtemb.2019.126439.

Forceville, X., Laviolle, B., Annane, D., Vitoux, D., Bleichner, G., Korach, J.-M., Cantais, E., Georges, H., Soubirou, J.-L., Combes, A., & Bellissant, E. (2007). Effects of high doses of selenium, as sodium selenite, in septic shock: a placebo-controlled, randomized,
<figure><img src="image_24.png" /></figure>

<figure><img src="image_25.png" /></figure>

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Chen, T., Zheng, W., Wong, Y.-S., Yang, F., & Bai, Y. (2006). Accumulation of selenium in mixotrophic culture of Spirulina platensis on glucose. *Bioresource Technology*, **97**, 2260–2265. https://doi.org/10.1016/j.biortech.2005.10.038.

Chousterman, B. G., Swirski, F. K., & Weber, G. F. (2017). Cytokine storm and sepsis disease pathogenesis. *Seminars in Immunopathology*, **39**, 517–528.

Cirino Ruocco, M. A., Pacheco Cechinatti, E. D., Barbosa, F., & Navarro, A. M. (2018). Zinc and selenium status in critically ill patients according to severity stratification. *Nutrition*, **45**, 85–89. https://doi.org/10.1016/j.nut.2017.07.009.

Copeland, S., Warren, H. S., Lowry, S. F., Calvano, S. E., Remick, D., & Inflammation and the Host Response to Injury Investigators. (2005). Acute inflammatory response to endotoxin in mice and humans. *Clinical and Diagnostic Laboratory Immunology*, **12**, 60–67. https://doi.org/10.1128/CDLI.12.1.60-67.2005.

Duntas, L. H., & Benvenga, S. (2015). Selenium: an element for life. *Endocrine*, **48**, 756–775. https://doi.org/10.1007/s1202 0-014-0477-6.

Faix, J. D. (2013). Biomarkers of sepsis. *Critical Reviews in Clinical Laboratory Sciences*, **50**, 23–36. https://doi.org/10.3109/10408 363.2013.764490.

Falk, M., Bernhoft, A., Reinoso-Maset, E., Salbu, B., Lebed, P., Framstad, T., Fuhrmann, H., & Oropeza-Moe, M. (2020). Beneficial antioxidant status of piglets from sows fed selenomethionine compared with piglets from sows fed sodium selenite. *Journal of Trace Elements in Medicine and Biology*, **58**, 126439. https://doi.org/10.1016/j.jtemb.2019.126439.

Forceville, X., Laviolle, B., Annane, D., Vitoux, D., Bleichner, G., Korach, J.-M., Cantais, E., Georges, H., Soubirou, J.-L., Combes, A., & Bellissant, E. (2007). Effects of high doses of selenium, as sodium selenite, in septic shock: a placebo-controlled, randomized,
double-blind, phase II study. *Critical Care, 11*, R73. https://doi.org/10.1186/cc5960.

Forceville, X., Vitoux, D., Gauzit, R., Combes, A., Lahilaire, P., & Chappuis, P. (1998). Selenium, systemic immune response syndrome, sepsis, and outcome in critically ill patients. *Critical Care Medicine, 26*, 1536–1544. https://doi.org/10.1097/00003246-199809000-00021.

Gargouri, M., Soussi, A., Akrouti, A., Magné, C., & El Feki, A. (2018). Ameliorative effects of Spirulina platensis against lead-induced nephrotoxicity in newborn rats: Modulation of oxidative stress and histopathological changes. *EXCL J, 17*, 215–232.

Gattinoni, L., Vasques, F., Camporota, L., Meessen, J., Romitti, F., Pasticci, I., Duscio, E., Vassalli, F., Forni, L. G., Payen, D., Cressoni, M., Zanella, A., Latini, R., Quintel, M., & Marini, J. J. (2019). Understanding lactatemia in human sepsis: potential impact for early management. *American Journal of Respiratory and Critical Care Medicine, 200*, 582–589. https://doi.org/10.1164/rccm.201812-2342OC.

Gouel-Chéron, A., Allaouchiche, B., Guignant, C., Davin, F., Floccard, B., & Monneret, G., & AzuRea Group (2012). Early interleukin-6 and slope of monocyte human leukocyte antigen-DR: a powerful association to predict the development of sepsis after major trauma. *PLoS One, 7*, e33095. https://doi.org/10.1371/journal.pone.0033095.

Habib, M. A. B. (2008). Review on culture, production and use of Spirulina as food for humans and feeds for domestic animals and fish. Food and Agriculture Organization of the United Nations. Available at [Accessed July 1, 2020].

Hallisey, S. D., & Greenwood, J. C. (2019). Beyond mean arterial pressure and lactate: perfusion end points for managing the shocked patient. *Emergency Medicine Clinics of North America, 37*, 395–408. https://doi.org/10.1016/j.emc.2019.03.005.

Hayes, M. A., Timmins, A. C., Yau, E. H., Palazzo, M., Hinds, C. J., & Watson, D. (1994). Elevation of systemic oxygen delivery in the treatment of critically ill patients. *New England Journal of Medicine, 330*, 1717–1722. https://doi.org/10.1056/NEJM199406163302404.

Hosnedlova, B., Kepinska, M., Skalickova, S., Fernandez, C., Ruttkay-Nedecky, B., Malevu, T. D., Sochor, J., Baron, M., Melcova, M., Zidkova, J., & Kizek, R. (2017). A summary of new findings on the biological effects of selenium in selected animal species—a critical review. *International Journal of Molecular Sciences, 18*(10), 2209. https://doi.org/10.3390/ijms18102209.

Huang, T.-S., Shyu, Y.-C., Chen, H.-Y., Lin, L.-M., Lo, C.-Y., Yuan, S.-S., & Chen, P.-J. (2013). Effect of parenteral selenium supplementation in critically ill patients: a systematic review and meta-analysis. *PLoS One, 8*, e54431.

Jansen, T. C., van Bommel, J., Schoonderbeek, F. J., Sleeswijk Visser, S. J., van der Klooster, J. M., Lima, A. P., Willemse, S. P., Bakker, J., & LACTATE study group. (2010). Early lactate-guided therapy in intensive care unit patients: a multicenter, open-label, randomized controlled trial. *American Journal of Respiratory and Critical Care Medicine, 182*, 752–761. https://doi.org/10.1164/rccm.200912-1918OC.

Khalil, S. R., Khalifa, H. A., Abdel-Motal, S. M., Mohammed, H. H., Elewa, Y. H. A., & Mahmoud, H. A. (2018). Spirulina platensis attenuates the associated neurobehavioral and inflammatory response impairments in rats exposed to lead acetate. *Ecotoxicology and Environmental Safety, 157*, 255–265. https://doi.org/10.1016/j.ecoenv.2018.03.068.

Köhrle, J. (2015). Selenium and the thyroid. *Current Opinion in Endocrinology, Diabetes, and Obesity, 22*, 392–401. https://doi.org/10.1097/MED.0000000000000190.

Kong, Z., Wang, F., Ji, S., Deng, X., & Xia, Z. (2013). Selenium supplementation for sepsis: a meta-analysis of randomized controlled trials. *American Journal of Emergency Medicine, 31*, 1170–1175. https://doi.org/10.1016/j.ajem.2013.04.020.

Lamela, T., & Rocha, F. J. M. (2000). Phycocyanin production in seawater culture of Arthrospira maxima. *Ciencias Marinas, 26*(4), 607–619. https://doi.org/10.7773/cm.v26i4.619.

Landucci, F., Mancinelli, P., De Gaudio, A. R., & Virgili, G. (2014). Selenium supplementation in critically ill patients: a systematic review and meta-analysis. *Journal of Critical Care, 29*, 150–156. https://doi.org/10.1016/j.jcrc.2013.08.017.

Leema, J. T. M., Kirubagaran, R., Vinithkumar, N. V., Dheenan, P. S., & Karthikayulu, S. (2010). High value pigment production from Arthrospira (Spirulina) platensis cultured in seawater. *Biosource Technology, 101*, 9221–9227. https://doi.org/10.1016/j.biortech.2010.06.120.

Léon, K., Pichavant-Rafini, K., Quéméner, E., Sébert, P., Egreteau, P.-Y., Ollivier, H., Carré, J.-L., & L'Her, E. (2012). Oxygen blood transport during experimental sepsis: effect of hypothermia*. *Critical Care Medicine, 40*, 912–918. https://doi.org/10.1097/CCM.0b013e3182373134.

Levraut, J., Ciebiera, J. P., Chave, S., Rabary, O., Jambou, P., Carles, M., & Grimaud, D. (1998). Mild hyperlactatemia in stable septic patients is due to impaired lactate clearance rather than overproduction. *American Journal of Respiratory and Critical Care Medicine, 157*, 1021–1026. https://doi.org/10.1164/ajrccm.157.4.9705037.

Levy, B. (2006). Lactate and shock state: the metabolic view. *Current Opinion in Critical Care, 12*, 315–321. https://doi.org/10.1097/01.CCCX.0000235208.77450.15.

Li, S., Tang, T., Guo, P., Zou, Q., Ao, X., Hu, L., & Tan, L. (2019). A meta-analysis of randomized controlled trials: Efficacy of selenium treatment for sepsis. *Medicine (Baltimore), 98*, e14733. https://doi.org/10.1097/MD.00000000000014733.

Li, Z.-Y., Guo, S.-Y., & Li, L. (2003). Bioeffects of selenite on the growth of Spirulina platensis and its biotransformation. *Biosource Technology, 89*, 171–176.

Lv, S., Han, M., Yi, R., Kwon, S., Dai, C., & Wang, R. (2014). Anti-TNF-α therapy for patients with sepsis: a systematic meta-analysis. *International Journal of Clinical Practice, 68*, 520–528. https://doi.org/10.1111/ijcp.12382.

Mantzarlis, K., Tsolaki, V., & Zakynthinos, E. (2017). Role of oxidative stress and mitochondrial dysfunction in sepsis and potential therapies. *Oxidative Medicine and Cellular Longevity, 2017*, 5985209. https://doi.org/10.1155/2017/5985209.

Manzanares, W., Lemieux, M., Elke, G., Langlois, P. L., Bloos, F., & Heyland, D. K. (2016). High-dose intravenous selenium does not improve clinical outcomes in the critically ill: a systematic review and meta-analysis. *Critical Care, 20*, 356. https://doi.org/10.1186/s13054-016-1529-5.

Mertens, K., Lowes, D. A., Webster, N. R., Talib, J., Hall, L., Davies, M. J., Beattie, J. H., & Galley, H. F. (2015). Low zinc and selenium concentrations in sepsis are associated with oxidative damage and inflammation. *British Journal of Anaesthesia, 114*, 990–999. https://doi.org/10.1093/bja/aev073.

Morsy, M. A., Gupta, S., Nair, A. B., Venugopala, K. N., Greish, K., & El-Daly, M. (2019). Protective Effect of Spirulina platensis extract against dextran-sulfate-sodium-induced ulcerative colitis in rats. *Nutrients, 11*(10), 2309. https://doi.org/10.3390/nu11102309.

Mouncey, P. R., Osborn, T. M., Power, G. S., Harrison, D. A., Sadique, M. Z., Grieve, R. D., Jahan, R., Harvey, S. E., Bell, D., Bion, J. F.,
double-blind, phase II study. *Critical Care, 11*, R73. https://doi.org/10.1186/cc5960.

Forceville, X., Vitoux, D., Gauzit, R., Combes, A., Lahilaire, P., & Chappuis, P. (1998). Selenium, systemic immune response syndrome, sepsis, and outcome in critically ill patients. *Critical Care Medicine, 26*, 1536–1544. https://doi.org/10.1097/00003246-199809000-00021.

Gargouri, M., Soussi, A., Akrouti, A., Magné, C., & El Feki, A. (2018). Ameliorative effects of Spirulina platensis against lead-induced nephrotoxicity in newborn rats: Modulation of oxidative stress and histopathological changes. *EXCL J, 17*, 215–232.

Gattinoni, L., Vasques, F., Camporota, L., Meessen, J., Romitti, F., Pasticci, I., Duscio, E., Vassalli, F., Forni, L. G., Payen, D., Cressoni, M., Zanella, A., Latini, R., Quintel, M., & Marini, J. J. (2019). Understanding lactatemia in human sepsis: potential impact for early management. *American Journal of Respiratory and Critical Care Medicine, 200*, 582–589. https://doi.org/10.1164/rccm.201812-2342OC.

Gouel-Chéron, A., Allaouchiche, B., Guignant, C., Davin, F., Floccard, B., & Monneret, G., & AzuRea Group (2012). Early interleukin-6 and slope of monocyte human leukocyte antigen-DR: a powerful association to predict the development of sepsis after major trauma. *PLoS One, 7*, e33095. https://doi.org/10.1371/journal.pone.0033095.

Habib, M. A. B. (2008). Review on culture, production and use of Spirulina as food for humans and feeds for domestic animals and fish. Food and Agriculture Organization of the United Nations. Available at [Accessed July 1, 2020].

Hallisey, S. D., & Greenwood, J. C. (2019). Beyond mean arterial pressure and lactate: perfusion end points for managing the shocked patient. *Emergency Medicine Clinics of North America, 37*, 395–408. https://doi.org/10.1016/j.emc.2019.03.005.

Hayes, M. A., Timmins, A. C., Yau, E. H., Palazzo, M., Hinds, C. J., & Watson, D. (1994). Elevation of systemic oxygen delivery in the treatment of critically ill patients. *New England Journal of Medicine, 330*, 1717–1722. https://doi.org/10.1056/NEJM199406163302404.

Hosnedlova, B., Kepinska, M., Skalickova, S., Fernandez, C., Ruttkay-Nedecky, B., Malevu, T. D., Sochor, J., Baron, M., Melcova, M., Zidkova, J., & Kizek, R. (2017). A summary of new findings on the biological effects of selenium in selected animal species—a critical review. *International Journal of Molecular Sciences, 18*(10), 2209. https://doi.org/10.3390/ijms18102209.

Huang, T.-S., Shyu, Y.-C., Chen, H.-Y., Lin, L.-M., Lo, C.-Y., Yuan, S.-S., & Chen, P.-J. (2013). Effect of parenteral selenium supplementation in critically ill patients: a systematic review and meta-analysis. *PLoS One, 8*, e54431.

Jansen, T. C., van Bommel, J., Schoonderbeek, F. J., Sleeswijk Visser, S. J., van der Klooster, J. M., Lima, A. P., Willemse, S. P., Bakker, J., & LACTATE study group. (2010). Early lactate-guided therapy in intensive care unit patients: a multicenter, open-label, randomized controlled trial. *American Journal of Respiratory and Critical Care Medicine, 182*, 752–761. https://doi.org/10.1164/rccm.200912-1918OC.

Khalil, S. R., Khalifa, H. A., Abdel-Motal, S. M., Mohammed, H. H., Elewa, Y. H. A., & Mahmoud, H. A. (2018). Spirulina platensis attenuates the associated neurobehavioral and inflammatory response impairments in rats exposed to lead acetate. *Ecotoxicology and Environmental Safety, 157*, 255–265. https://doi.org/10.1016/j.ecoenv.2018.03.068.

Köhrle, J. (2015). Selenium and the thyroid. *Current Opinion in Endocrinology, Diabetes, and Obesity, 22*, 392–401. https://doi.org/10.1097/MED.0000000000000190.

Kong, Z., Wang, F., Ji, S., Deng, X., & Xia, Z. (2013). Selenium supplementation for sepsis: a meta-analysis of randomized controlled trials. *American Journal of Emergency Medicine, 31*, 1170–1175. https://doi.org/10.1016/j.ajem.2013.04.020.

Lamela, T., & Rocha, F. J. M. (2000). Phycocyanin production in seawater culture of Arthrospira maxima. *Ciencias Marinas, 26*(4), 607–619. https://doi.org/10.7773/cm.v26i4.619.

Landucci, F., Mancinelli, P., De Gaudio, A. R., & Virgili, G. (2014). Selenium supplementation in critically ill patients: a systematic review and meta-analysis. *Journal of Critical Care, 29*, 150–156. https://doi.org/10.1016/j.jcrc.2013.08.017.

Leema, J. T. M., Kirubagaran, R., Vinithkumar, N. V., Dheenan, P. S., & Karthikayulu, S. (2010). High value pigment production from Arthrospira (Spirulina) platensis cultured in seawater. *Biosource Technology, 101*, 9221–9227. https://doi.org/10.1016/j.biortech.2010.06.120.

Léon, K., Pichavant-Rafini, K., Quéméner, E., Sébert, P., Egreteau, P.-Y., Ollivier, H., Carré, J.-L., & L’Her, E. (2012). Oxygen blood transport during experimental sepsis: effect of hypothermia*. *Critical Care Medicine, 40*, 912–918. https://doi.org/10.1097/CCM.0b013e3182373134.

Levraut, J., Ciebiera, J. P., Chave, S., Rabary, O., Jambou, P., Carles, M., & Grimaud, D. (1998). Mild hyperlactatemia in stable septic patients is due to impaired lactate clearance rather than overproduction. *American Journal of Respiratory and Critical Care Medicine, 157*, 1021–1026. https://doi.org/10.1164/ajrccm.157.4.9705037.

Levy, B. (2006). Lactate and shock state: the metabolic view. *Current Opinion in Critical Care, 12*, 315–321. https://doi.org/10.1097/01.CCCX.0000235208.77450.15.

Li, S., Tang, T., Guo, P., Zou, Q., Ao, X., Hu, L., & Tan, L. (2019). A meta-analysis of randomized controlled trials: Efficacy of selenium treatment for sepsis. *Medicine (Baltimore), 98*, e14733. https://doi.org/10.1097/MD.00000000000014733.

Li, Z.-Y., Guo, S.-Y., & Li, L. (2003). Bioeffects of selenite on the growth of Spirulina platensis and its biotransformation. *Biosource Technology, 89*, 171–176.

Lv, S., Han, M., Yi, R., Kwon, S., Dai, C., & Wang, R. (2014). Anti-TNF-α therapy for patients with sepsis: a systematic meta-analysis. *International Journal of Clinical Practice, 68*, 520–528. https://doi.org/10.1111/ijcp.12382.

Mantzarlis, K., Tsolaki, V., & Zakynthinos, E. (2017). Role of oxidative stress and mitochondrial dysfunction in sepsis and potential therapies. *Oxidative Medicine and Cellular Longevity, 2017*, 5985209. https://doi.org/10.1155/2017/5985209.

Manzanares, W., Lemieux, M., Elke, G., Langlois, P. L., Bloos, F., & Heyland, D. K. (2016). High-dose intravenous selenium does not improve clinical outcomes in the critically ill: a systematic review and meta-analysis. *Critical Care, 20*, 356. https://doi.org/10.1186/s13054-016-1529-5.

Mertens, K., Lowes, D. A., Webster, N. R., Talib, J., Hall, L., Davies, M. J., Beattie, J. H., & Galley, H. F. (2015). Low zinc and selenium concentrations in sepsis are associated with oxidative damage and inflammation. *British Journal of Anaesthesia, 114*, 990–999. https://doi.org/10.1093/bja/aev073.

Morsy, M. A., Gupta, S., Nair, A. B., Venugopala, K. N., Greish, K., & El-Daly, M. (2019). Protective Effect of Spirulina platensis extract against dextran-sulfate-sodium-induced ulcerative colitis in rats. *Nutrients, 11*(10), 2309. https://doi.org/10.3390/nu11102309.

Mouncey, P. R., Osborn, T. M., Power, G. S., Harrison, D. A., Sadique, M. Z., Grieve, R. D., Jahan, R., Harvey, S. E., Bell, D., Bion, J. F.,
<figure><img src="image_24.png" /></figure>

<figure><img src="image_25.png" /></figure>

Coats, T. J., Singer, M., Young, J. D., & Rowan, K. M. (2015). Trial of early, goal-directed resuscitation for septic shock. *New England Journal of Medicine*, **372**, 1301–1311. https://doi.org/10.1056/NEJMoa1500896.

Nasirian, F., Dadkhah, M., Moradi-kor, N., & Obeidavi, Z. (2018). Effects of *Spirulina platensis* microalgae on antioxidant and anti-inflammatory factors in diabetic rats. *Diabetes, Metabolic Syndrome and Obesity: Targets and Therapy*, **11**, 375–380.

Nichol, A., Bailey, M., Egi, M., Pettila, V., French, C., Stachowski, E., Reade, M. C., Cooper, D. J., & Bellomo, R. (2011). Dynamic lactate indices as predictors of outcome in critically ill patients. *Critical Care*, **15**, R242. https://doi.org/10.1186/cc10497.

Okuno, T., Kubota, T., Kuroda, T., Ueno, H., & Nakamuro, K. (2001). Contribution of enzymic alpha, gamma-elimination reaction in detoxification pathway of selenomethionine in mouse liver. *Toxicology and Applied Pharmacology*, **176**, 18–23.

Pan, J., Peng, M., Liao, C., Hu, X., Wang, A., & Li, X. (2019). Relative efficacy and safety of early lactate clearance-guided therapy resuscitation in patients with sepsis: A meta-analysis. *Medicine (Baltimore)*, **98**, e14453. https://doi.org/10.1097/MD.00000000000014453.

Pereira, M. I. B., Chagas, B. M. E., Sassi, R., Medeiros, G. F., Aguiar, E. M., Borba, L. H. F., Silva, E. P. E., Neto, J. C. A., & Rangel, A. H. N. (2019). Mixotrophic cultivation of *Spirulina platensis* in dairy wastewater: Effects on the production of biomass, biochemical composition and antioxidant capacity. *PLoS One*, **14**, e0224294. https://doi.org/10.1371/journal.pone.0224294.

Pfaffl, M. W. (2001). A new mathematical model for relative quantification in real-time RT-PCR. *Nucleic Acids Research*, **29**, e45. https://doi.org/10.1093/nar/29.9.e45.

Pham, T. X., Lee, Y., Bae, M., Hu, S., Kang, H., Kim, M.-B., Park, Y.-K., & Lee, J.-Y. (2019). *Spirulina* supplementation in a mouse model of diet-induced liver fibrosis reduced the pro-inflammatory response of splenocytes. *British Journal of Nutrition*, **121**, 748–755. https://doi.org/10.1017/S0007114519000126.

Pieczyńska, J., & Grajeta, H. (2015). The role of selenium in human conception and pregnancy. *Journal of Trace Elements in Medicine and Biology*, **29**, 31–38. https://doi.org/10.1016/j.jtemb.2014.07.003.

Razazi, K., Boissier, F., Surenaud, M., Bedet, A., Seemann, A., Carteaux, G., de Prost, N., Brun-Buisson, C., Hue, S., & Mekontso Dessap, A. (2019). A multiplex analysis of sepsis mediators during human septic shock: a preliminary study on myocardial depression and organ failures. *Annals of Intensive Care*, **9**, 64. https://doi.org/10.1186/s13613-019-0538-3.

Ronco, J. J., Fenwick, J. C., Tweeddale, M. G., Wiggs, B. R., Phang, P. T., Cooper, D. J., Cunningham, K. F., Russell, J. A., & Walley, K. R. (1993). Identification of the critical oxygen delivery for anaerobic metabolism in critically ill septic and nonseptic humans. *JAMA*, **270**, 1724–1730.

Rudd, K. E., Johnson, S. C., Agesa, K. M., Shackelford, K. A., Tsoi, D., Kievlan, D. R., Colombara, D. V., Ikuta, K. S., Kissoon, N., Finfer, S., Fleischmann-Struzek, C., Machado, F. R., Reinhart, K. K., Rowan, K., Seymour, C. W., Watson, R. S., West, T. E., Marinho, F., Hay, S. I., ... Naghavi, M. (2020). Global, regional, and national sepsis incidence and mortality, 1990–2017: analysis for the Global Burden of Disease Study. *The Lancet*, **395**, 200–211. https://doi.org/10.1016/S0140-6736(19)32989-7.

Sakr, Y., Reinhart, K., Bloos, F., Marx, G., Russwurm, S., Bauer, M., & Brunkhorst, F. (2007). Time course and relationship between plasma selenium concentrations, systemic inflammatory response,

sepsis, and multiorgan failure. *British Journal of Anaesthesia*, **98**, 775–784. https://doi.org/10.1093/bja/aem091.

Shapiro, N. I., Howell, M. D., Talmor, D., Nathanson, L. A., Lisbon, A., Wolfe, R. E., & Weiss, J. W. (2005). Serum lactate as a predictor of mortality in emergency department patients with infection. *Annals of Emergency Medicine*, **45**, 524–528. https://doi.org/10.1016/j.annemergmed.2004.12.006.

Singer, M., Deutschman, C. S., Seymour, C. W., Shankar-Hari, M., Annane, D., Bauer, M., Bellomo, R., Bernard, G. R., Chiche, J.-D., Coopersmith, C. M., Hotchkiss, R. S., Levy, M. M., Marshall, J. C., Martin, G. S., Opal, S. M., Rubenfeld, G. D., van der Poll, T., Vincent, J.-L., & Angus, D. C. (2016). The Third International Consensus Definitions for Sepsis and Septic Shock (Sepsis-3). *JAMA*, **315**, 801–810. https://doi.org/10.1001/jama.2016.0287.

Suzuki, K. T., Doi, C., & Suzuki, N. (2006). Metabolism of 76Se-methylselenocysteine compared with that of 77Se-selenomethionine and 82Se-selenite. *Toxicology and Applied Pharmacology*, **217**, 185–195.

Takahashi, K., Suzuki, N., & Ogra, Y. (2017). Bioavailability comparison of nine bioselenocompounds in vitro and in vivo. *International Journal of Molecular Sciences*, **18**(3), 506. https://doi.org/10.3390/ijms18030506.

Tan, L., Sang, Z. N., Shen, J., Wu, Y. T., Yao, Z. X., Zhang, J. X., Zhao, N., & Zhang, W. Q. (2013). Selenium supplementation alleviates autoimmune thyroiditis by regulating expression of TH1/TH2 cytokines. *Biomedical and Environmental Sciences*, **26**, 920–925.

Tanguy, S., Grauzam, S., de Leiris, J., & Boucher, F. (2012). Impact of dietary selenium intake on cardiac health: experimental approaches and human studies. *Molecular Nutrition & Food Research*, **56**, 1106–1121.

Weber, S. U., Lehmann, L. E., Schewe, J.-C., Thiele, J. T., Schröder, S., Book, M., Hoeft, A., & Stüber, F. (2008). Low serum alpha-tocopherol and selenium are associated with accelerated apoptosis in severe sepsis. *BioFactors*, **33**, 107–119.

Xia, Y., Hill, K. E., Byrne, D. W., Xu, J., & Burk, R. F. (2005). Effectiveness of selenium supplements in a low-selenium area of China. *American Journal of Clinical Nutrition*, **81**, 829–834. https://doi.org/10.1093/ajcn/81.4.829.

Zahran, W. E., & Emam, M. A. (2018). Renoprotective effect of *Spirulina platensis* extract against nicotine-induced oxidative stress-mediated inflammation in rats. *Phytomedicine*, **49**, 106–110. https://doi.org/10.1016/j.phymed.2018.06.042.

Zhang, K., Zhao, Q., Zhan, T., Han, Y., Tang, C., & Zhang, J. (2020). Effect of different selenium sources on growth performance, tissue selenium content, meat quality, and selenoprotein gene expression in finishing pigs. *Biological Trace Element Research*, **196**, 463–471. https://doi.org/10.1007/s12011-019-01949-3.

**How to cite this article:** Castel, T., Theron, M., Pichavant-Rafini, K., Guernec, A., Joublin-Delavat, A., Gueguen, B., & Leon, K. (2021). Can selenium-enriched spirulina supplementation ameliorate sepsis outcomes in selenium-deficient animals? *Physiological Reports*, **9**, e14933. https://doi.org/10.14814/phy2.14933
<figure><img src="image_26.png" /></figure>

<figure><img src="image_27.png" /></figure>

Coats, T. J., Singer, M., Young, J. D., & Rowan, K. M. (2015). Trial of early, goal-directed resuscitation for septic shock. *New England Journal of Medicine*, **372**, 1301–1311. https://doi.org/10.1056/NEJMoa1500896.

Nasirian, F., Dadkhah, M., Moradi-kor, N., & Obeidavi, Z. (2018). Effects of *Spirulina platensis* microalgae on antioxidant and anti-inflammatory factors in diabetic rats. *Diabetes, Metabolic Syndrome and Obesity: Targets and Therapy*, **11**, 375–380.

Nichol, A., Bailey, M., Egi, M., Pettila, V., French, C., Stachowski, E., Reade, M. C., Cooper, D. J., & Bellomo, R. (2011). Dynamic lactate indices as predictors of outcome in critically ill patients. *Critical Care*, **15**, R242. https://doi.org/10.1186/cc10497.

Okuno, T., Kubota, T., Kuroda, T., Ueno, H., & Nakamuro, K. (2001). Contribution of enzymic alpha, gamma-elimination reaction in detoxification pathway of selenomethionine in mouse liver. *Toxicology and Applied Pharmacology*, **176**, 18–23.

Pan, J., Peng, M., Liao, C., Hu, X., Wang, A., & Li, X. (2019). Relative efficacy and safety of early lactate clearance-guided therapy resuscitation in patients with sepsis: A meta-analysis. *Medicine (Baltimore)*, **98**, e14453. https://doi.org/10.1097/MD.00000000000014453.

Pereira, M. I. B., Chagas, B. M. E., Sassi, R., Medeiros, G. F., Aguiar, E. M., Borba, L. H. F., Silva, E. P. E., Neto, J. C. A., & Rangel, A. H. N. (2019). Mixotrophic cultivation of *Spirulina platensis* in dairy wastewater: Effects on the production of biomass, biochemical composition and antioxidant capacity. *PLoS One*, **14**, e0224294. https://doi.org/10.1371/journal.pone.0224294.

Pfaffl, M. W. (2001). A new mathematical model for relative quantification in real-time RT-PCR. *Nucleic Acids Research*, **29**, e45. https://doi.org/10.1093/nar/29.9.e45.

Pham, T. X., Lee, Y., Bae, M., Hu, S., Kang, H., Kim, M.-B., Park, Y.-K., & Lee, J.-Y. (2019). *Spirulina* supplementation in a mouse model of diet-induced liver fibrosis reduced the pro-inflammatory response of splenocytes. *British Journal of Nutrition*, **121**, 748–755. https://doi.org/10.1017/S0007114519000126.

Pieczyńska, J., & Grajeta, H. (2015). The role of selenium in human conception and pregnancy. *Journal of Trace Elements in Medicine and Biology*, **29**, 31–38. https://doi.org/10.1016/j.jtemb.2014.07.003.

Razazi, K., Boissier, F., Surenaud, M., Bedet, A., Seemann, A., Carteaux, G., de Prost, N., Brun-Buisson, C., Hue, S., & Mekontso Dessap, A. (2019). A multiplex analysis of sepsis mediators during human septic shock: a preliminary study on myocardial depression and organ failures. *Annals of Intensive Care*, **9**, 64. https://doi.org/10.1186/s13613-019-0538-3.

Ronco, J. J., Fenwick, J. C., Tweeddale, M. G., Wiggs, B. R., Phang, P. T., Cooper, D. J., Cunningham, K. F., Russell, J. A., & Walley, K. R. (1993). Identification of the critical oxygen delivery for anaerobic metabolism in critically ill septic and nonseptic humans. *JAMA*, **270**, 1724–1730.

Rudd, K. E., Johnson, S. C., Agesa, K. M., Shackelford, K. A., Tsoi, D., Kievlan, D. R., Colombara, D. V., Ikuta, K. S., Kissoon, N., Finfer, S., Fleischmann-Struzek, C., Machado, F. R., Reinhart, K. K., Rowan, K., Seymour, C. W., Watson, R. S., West, T. E., Marinho, F., Hay, S. I., ... Naghavi, M. (2020). Global, regional, and national sepsis incidence and mortality, 1990–2017: analysis for the Global Burden of Disease Study. *The Lancet*, **395**, 200–211. https://doi.org/10.1016/S0140-6736(19)32989-7.

Sakr, Y., Reinhart, K., Bloos, F., Marx, G., Russwurm, S., Bauer, M., & Brunkhorst, F. (2007). Time course and relationship between plasma selenium concentrations, systemic inflammatory response,

sepsis, and multiorgan failure. *British Journal of Anaesthesia*, **98**, 775–784. https://doi.org/10.1093/bja/aem091.

Shapiro, N. I., Howell, M. D., Talmor, D., Nathanson, L. A., Lisbon, A., Wolfe, R. E., & Weiss, J. W. (2005). Serum lactate as a predictor of mortality in emergency department patients with infection. *Annals of Emergency Medicine*, **45**, 524–528. https://doi.org/10.1016/j.annemergmed.2004.12.006.

Singer, M., Deutschman, C. S., Seymour, C. W., Shankar-Hari, M., Annane, D., Bauer, M., Bellomo, R., Bernard, G. R., Chiche, J.-D., Coopersmith, C. M., Hotchkiss, R. S., Levy, M. M., Marshall, J. C., Martin, G. S., Opal, S. M., Rubenfeld, G. D., van der Poll, T., Vincent, J.-L., & Angus, D. C. (2016). The Third International Consensus Definitions for Sepsis and Septic Shock (Sepsis-3). *JAMA*, **315**, 801–810. https://doi.org/10.1001/jama.2016.0287.

Suzuki, K. T., Doi, C., & Suzuki, N. (2006). Metabolism of 76Se-methylselenocysteine compared with that of 77Se-selenomethionine and 82Se-selenite. *Toxicology and Applied Pharmacology*, **217**, 185–195.

Takahashi, K., Suzuki, N., & Ogra, Y. (2017). Bioavailability comparison of nine bioselenocompounds in vitro and in vivo. *International Journal of Molecular Sciences*, **18**(3), 506. https://doi.org/10.3390/ijms18030506.

Tan, L., Sang, Z. N., Shen, J., Wu, Y. T., Yao, Z. X., Zhang, J. X., Zhao, N., & Zhang, W. Q. (2013). Selenium supplementation alleviates autoimmune thyroiditis by regulating expression of TH1/TH2 cytokines. *Biomedical and Environmental Sciences*, **26**, 920–925.

Tanguy, S., Grauzam, S., de Leiris, J., & Boucher, F. (2012). Impact of dietary selenium intake on cardiac health: experimental approaches and human studies. *Molecular Nutrition & Food Research*, **56**, 1106–1121.

Weber, S. U., Lehmann, L. E., Schewe, J.-C., Thiele, J. T., Schröder, S., Book, M., Hoeft, A., & Stüber, F. (2008). Low serum alpha-tocopherol and selenium are associated with accelerated apoptosis in severe sepsis. *BioFactors*, **33**, 107–119.

Xia, Y., Hill, K. E., Byrne, D. W., Xu, J., & Burk, R. F. (2005). Effectiveness of selenium supplements in a low-selenium area of China. *American Journal of Clinical Nutrition*, **81**, 829–834. https://doi.org/10.1093/ajcn/81.4.829.

Zahran, W. E., & Emam, M. A. (2018). Renoprotective effect of *Spirulina platensis* extract against nicotine-induced oxidative stress-mediated inflammation in rats. *Phytomedicine*, **49**, 106–110. https://doi.org/10.1016/j.phymed.2018.06.042.

Zhang, K., Zhao, Q., Zhan, T., Han, Y., Tang, C., & Zhang, J. (2020). Effect of different selenium sources on growth performance, tissue selenium content, meat quality, and selenoprotein gene expression in finishing pigs. *Biological Trace Element Research*, **196**, 463–471. https://doi.org/10.1007/s12011-019-01949-3.

**How to cite this article:** Castel, T., Theron, M., Pichavant-Rafini, K., Guernec, A., Joublin-Delavat, A., Gueguen, B., & Leon, K. (2021). Can selenium-enriched spirulina supplementation ameliorate sepsis outcomes in selenium-deficient animals? *Physiological Reports*, **9**, e14933. https://doi.org/10.14814/phy2.14933