1

Scientific Reports, in press.

2
3

Levosimendan pretreatment improves survival of septic rats after partial

4

hepatectomy and suppresses iNOS induction in cytokine-stimulated hepatocytes

5
6

Tatsuma Sakaguchi1*, Yuki Hashimoto1, Hideyuki Matsushima1, Hidehiko Hishikawa1,

7

Mikio Nishizawa2, Tadayoshi Okumura1, 3 and Masaki Kaibori1

8
91

Department of Surgery, Kansai Medical University, Hirakata, Osaka, Japan

10

2

Department of Biomedical Sciences, and 3Research Organization of Science and Technology,

11

College of Life Sciences, Ritsumeikan University, Kusatsu, Shiga, Japan

12
13

Email addresses: sakaguct@hirakata.kmu.ac.jp

(TS)

14

hashimoy@hirakata.kmu.ac.jp (YH)

15

matsushh@hirakata.kmu.ac.jp (HM)16

auss0211@gmail.com

(HH)

17

nishizaw@sk.ritsumei.ac.jp

(MN)

18

okumura@hirakata.kmu.ac.jp

(TO)

19

kaibori@hirakata.kmu.ac.jp

(MK)

20
21

*Correspondence: Tatsuma Sakaguchi, MD, Department of Surgery, Kansai Medical University,22

2-5-1 Shinmachi, Hirakata, Osaka, 573-1010, Japan

23

1

Abstract2

We evaluated the survival effects and biochemical profiles of levosimendan in septic rats

3

after partial hepatectomy and investigated its effects in cultured hepatocytes. Thirty-two

4

rats underwent 70% hepatectomy and were randomised equally into four groups, followed

5

by lipopolysaccharide (LPS) injection (250 µg/kg, i.v.) after 48 h. Levosimendan was given

6(i.p.) 1 h before LPS injection [group (A) levosimendan 2 mg/kg; (B) 1; (C) 0.5; (D)7vehicle]. Survival at 7 days was increased significantly in group A compared with that in

8group D [A: 63%; B: 38%; C: 13%; D: 0%]. In serum, levosimendan decreased the level of

9tumour necrosis factor-α, interleukin (IL)-1β, IL-6 and nitric oxide (NO). In remnant livers,

10

levosimendan inhibited inducible nitric oxide synthase (iNOS) gene expression. In primary11

cultured rat hepatocytes stimulated by IL-1β, levosimendan suppressed NO production by

12

inhibiting iNOS promoter activity and stability of its mRNA.

13
14

Keywords: levosimendan, sepsis, partial hepatectomy, primary cultured rat hepatocyte,

15

inducible nitric oxide synthase

16

2

1

Levosimendan is a calcium sensitiser licensed in numerous countries to treat2

decompensated heart failure1. It acts by: (i) increasing the sensitivity of troponin C to

3

calcium in myocardial cells, leading to inotropy; (ii) opening mitochondrial adenosine

4

triphosphate (ATP)-sensitive potassium channels in smooth muscle cells, resulting in

5vasodilation2. Moreover, it is known that levosimendan treatment leads to reduction in

6

proinflammatory cytokines and apoptosis signaling pathways in patients with heart failure3.

7

Furthermore, in experimental sepsis model induced by cecal ligation and puncture (CLP),

8

levosimendan showed cardioprotective effects through preventing cardiac inflammation4.

9The composite actions of levosimendan as an inotrope and anti-inflammatory support raise10

the theoretical possibility that levosimendan may have a value as a treatment of sepsis5. In

11animal studies, accumulating evidences suggest that levosimendan may mitigate multiple

12organ injuries besides the heart in conditions of septic shock or ischemia–reperfusion2, 5, 6,

13

including lung injury in CLP model7-9, renal failure in LPS-induced endotoxemia10 and

14

liver injury in hepatic ischemia–reperfusion11. In terms of underlying mechanisms,

15

levosimendan exerted anti-inflammatory effects through probably decreasing nitric oxide

16(NO) release in sepsis2, 12. However, the inhibitory effect of levosimendan on

17

proinflammatory cytokine production cannot be solely attributed to alterations in the18

nuclear factor (NF)-κB pathway12. In a few limited case series and trials have shown a19

beneficial potential of levosimendan on cardiac13, renal14, pulmonary15 and hepatic1620

function in patients with sepsis. However, the survival and organ protective benefits of21

levosimendan in patients with septic shock were not demonstrated in one randomised

22

controlled clinical trial6. The optimal indications and protocols of levosimendan for sepsis

31

treatment have not been established.

2
3

Sepsis after major hepatectomy is a major issue. There is general agreement that 70%

4

hepatectomy alone is not fatal in rodents17, but intravenous injection of a sub-lethal dose of5

lipopolysaccharide (LPS) 48 h after partial hepatectomy (‘PH/LPS’-model) is associated6with high mortality18–21. Reduced phagocytic function of the reticuloendothelial system

7

after hepatectomy is considered to enhance endotoxin sensitivity22. LPS has a direct effect8

on macrophages (or Kupffer cells) to activate NF-κB, which induces expression of

9

proinflammatory cytokines and inducible nitric oxide synthase (iNOS). The latter produces

10an excess of NO, which has been implicated in tissue injury and assumed to be one of the11

triggers leading to septic shock and multiple-organ failure23.

1213

Previously, we reported that fibronectin18, pirfenidone19, edaravone20 and sivelestat21

14

improved survival and prevented liver injury in PH/LPS-model rats. These agents

15

commonly exerted survival benefits if they were administered before LPS injection and had

16inhibitory effects on iNOS induction in hepatocytes24–26. In primary cultured rat

17

hepatocytes, interleukin (IL)-1β stimulates production of iNOS and NO markedly in the18absence of other cytokines27, and prevention of expression of those proinflammatory

19

mediators is a reliable indicator of liver protection28.

20
21

We hypothesised that levosimendan pretreatment improves the survival of PH/LPS-model

22

rats by preventing the endotoxin-induced systemic inflammatory response and liver injury.

4

1

In addition to experiments using the PH/LPS-model, we conducted analyses of2

IL-1β-stimulated primary cultured rat hepatocytes, as a simple in vitro model of liver injury,

3in the presence or absence of levosimendan for better understanding of the intracellular4mechanisms involved.

5
6
7

Results

8

Effect of levosimendan pretreatment on survival of PH/LPS-model rats

9

A scheme of the experimental protocol of PH/LPS is shown in Fig. 1. Although we have10

less than 10% failure rate of PH/LPS model during the induction of anaesthesia and11

laparotomy, there was no rat to be lost during the interval between randomisation after

12

laparotomy and LPS injection. Thirty-two operated rats were randomised equally into four

13groups and evaluated the survival during 7 days after LPS injection (Fig. 2). Rats

14

administered vehicle (group D) began to die at 6 h and all rats died within 1 day after LPS

15

injection. Survival of groups A, B and C at 7 days was 63%, 38% and 13%, respectively.

16

The significant difference among four groups was confirmed (P < 0.01). According to post17

hoc analysis, survival of group A was significantly improved compared with group D (P <180.01). A dose of 2 mg/kg was used in subsequent in vivo experiments.

19
20

Effect of levosimendan on expression of cytokines, NO and transaminase in serum51

The levels of tumour necrosis factor (TNF)-α, IL-1β, IL-6, NO, aspartate transferase (AST)

2

and alanine transaminase (ALT) in serum were inhibited significantly (P <0.01, 0.01, 0.02,

3

0.02, 0.04 and 0.02, respectively) by levosimendan at 4 h (Fig. 3a–f).

4
5

Effect of levosimendan on expression of NF-κB, iNOS and cytokines in remnant livers

6

The level of NF-κB in remnant liver was activated by LPS injection at 1 h, and then7

attenuated at 4 h, after LPS injection was examined by electrophoretic mobility shift assays

8(EMSAs). Activation of NF-κB tended to be decreased by levosimendan at 4 h after LPS

9

injection, without significant differences (P = 0.056) (Fig. 4a). iNOS expression in remnant

10

livers was inhibited significantly (P = 0.01) by levosimendan at 4 h (Fig. 4b).

11

Levosimendan inhibited expression of the mRNA of TNF-α, IL-1β and IL-6 significantly

12(P = 0.03, 0.03 and 0.03, respectively) at 1 h, but insignificantly (P = 0.8, 0.09 and 0.15,

13respectively) at 4 h (Fig. 4c–f). iNOS mRNA was inhibited significantly (P = 0.03) at 4 h,

14

but insignificantly (P = 0.1) at 1 h. Expression of cytokine-induced neutrophil

15

chemoattractant (CINC)-1 mRNA tended to be inhibited at both 1 h and 4 h, without

16

significant differences (P = 0.1 and 0.2) (Fig. 4g). Expression of IL-10 mRNA tended to be17

increased at both 1 h and 4 h, without significant differences (P = 0.6 and 0.4) (Fig. 4h).

18
19

Effect of levosimendan on histopathological changes

20

Histopathology revealed the change in regeneration of rat livers 48 h after 70%

21

hepatectomy: ballooning hepatocytes and spreading of lipid droplets (Fig. 5a). After 4 h of

22

LPS injection, focal necrotic hepatocytes were prominent at the centrilobular zone and6

1

midzone in both groups of PH/LPS with vehicle and levosimendan (Fig. 5b, c). Few

2myeloperoxidase (MPO)-positive cells were infiltrated in livers 48 h after 70%

3

hepatectomy: (0.3 cells/mm2, Fig. 5d). Severe infiltration of MPO-positive cells was

4

recognized in specimens of rat livers after 4 h of LPS injection with vehicle (Fig. 5e).

5Levosimendan pretreatment did not inhibit the infiltration of MPO-positive cells

6significantly in remnant livers (P = 0.7) (Fig. 5f, g). Apoptotic bodies were evaluated by

7

terminal deoxynucleotidyl transferase-mediated dUTP-digoxigenin nick-end labelling

8(TUNEL) staining, and few positive nuclei were detected in rats 48 h after 70%

9

hepatectomy (4 per 1,000 nuclei, Fig. 5h). The difference in the percentage of

10

TUNEL-positive cells in all nuclei was not significant in the absence (Fig. 5i) or presence11of levosimendan pretreatment (P = 0.9) (Fig. 5j, k).

12
13

Effect of levosimendan on induction of expression of NO, iNOS protein and iNOS

14mRNA in IL-1β-stimulated cultured hepatocytes

15

In the culture medium, simultaneous administration of levosimendan (20 µM) with IL-1β

16(1 nM) reduced the level of nitrite (NO metabolite) time-dependently, which was increased17

by single administration of IL-1β (Fig. 6a). Levosimendan reduced the production of NO

18

and iNOS protein dose-dependently, and decreased production to a near-basal level at a19

concentration of 20 µM (Fig. 6b, upper and middle). The level of lactate dehydrogenase

20(LDH) in the culture medium was not increased by ≤20 µM of levosimendan (Fig. 6b,

21lower). A dose of 20 µM was used in subsequent in vitro experiments. Reverse

7

1transcription-polymerase chain reaction (RT-PCR) revealed that levosimendan reduced

2

expression of iNOS mRNA in each hour (Fig. 6c).

34Effect of levosimendan on the activity of iNOS promoters, iNOS antisense

5transcription and intranuclear level of NF-κB in IL-1β-stimulated hepatocytes

6

The scheme of the constructs containing firefly luciferase controlled by the iNOS promoter

7(pRiNOS-Luc-SVpA and pRiNOS-Luc-3′UTR) is shown in Fig. 7a. Levosimendan

8

inhibited relative luciferase activities on both constructs, which were increased by IL-1β9single administration (Fig. 7b). RT-PCR revealed that levosimendan inhibited expression of10

the iNOS antisense transcript at 3 h and 6 h (Fig. 7c). EMSAs with nuclear extracts did not

11show an inhibitory effect of levosimendan on NF-κB activation (Fig. 7d). Further, we could

12

not detect significant influences of levosimendan on NF-κB nuclear translocation, IκB

13

degradation and phosphorylation of NF-κB p65 (Ser536) (Supplementary information file;

14

M, K, and L).

15
16

Effect of levosimendan on the mRNA expression of pro-inflammatory cytokines in

17

IL-1β-stimulated hepatocytes

18

RT-PCR revealed that levosimendan suppressed expression of the mRNA of TNF-α,

19

CINC-1 and the type-I IL-1 receptor (IL-1RI) at certain times (Fig. 8).

20
21
22

Discussion8

1

Two experimental models of sepsis with acute liver injury can be employed: (i)2simultaneous administration of D-galactosamine/LPS 29–31; (ii) PH/LPS. The lethal activity

3of endotoxins is enhanced considerably under both models, but the PH/LPS-model exhibits

4

more severe and refractory symptoms32, and is closer to a specific clinical situation.

5
6

A pilot study revealed that a 50% lethal dose of LPS for this model was ≈100 µg/kg, and7

that >90% of rats died at a LPS dose of 250 µg/kg (i.v.) (T. O., unpublished observation).

8

We chose the doses of levosimendan by reference to a similar study of ischemia–

9reperfusion injury in rat mesenteries33. In our preliminary study, administration of

10levosimendan (2 mg/kg) 1 h after LPS injection showed no effect on survival (data not

11

shown). In contrast, pretreatment of levosimendan increased the survival of PH/LPS-model

12

rats in a dose-dependent fashion, though a significant difference was only found between

13

group A (doses of 2 mg/kg) and group D (vehicle) by post hoc analysis. Levosimendan

14

pretreatment prevented an increase in expression of proinflammatory cytokines in serum15

and their mRNAs in remnant livers. Expression of iNOS in remnant livers and NO in serum16(which are proinflammatory mediators) was also inhibited by levosimendan pretreatment.

17

Those effects would probably involve inhibition of NF-κB activation, because NF-κB has

18

an important role as a transcriptional factor of iNOS gene28. However, levosimendan did

19

not inhibit NF-κB activation significantly shown in EMSA experiments in remnant livers.

20

We should mention of the limited number of experimental animals we used and there

21

probably existed the influence of other transcriptional factors such as hypoxia-inducible

22

factor-1α34 or nuclear respiratory factor 235. According to a reported study of septic mice,

9

1

Wang et al. concluded that levosimendan did not inhibit the LPS-induced activation of2

NF-κB significantly, which is a similar result to our study8. Levosimendan demonstrated a3

hepatoprotective effect in that levels of transaminases in serum decreased significantly in4

the levosimendan group 4 h after LPS injection. However, histopathology revealed that5levosimendan did not inhibit both the infiltration of MPO-positive cells (i.e., necrotic

6

change) and TUNEL-positive cells (i.e., apoptotic change). The results of histopathology

7will cause controversy whether a hepatoprotective effect of levosimendan determined the8survival benefit in our study. We assume that D-galactosamine/LPS-model would be

9essential for examining a heaptoprotective effect of levosimendan against LPS-induced

10

acute liver injury36, but this model would not surely represent for septic shock37. As a11limitation, we could not adopt a blinded maneuver of each group for a practical reason

12

when we injected LPS and/or levosimendan. However, two researchers (T. S. and T. O.)

13

assured the quality of experiments of PH/LPS.

14
15

In IL-1β-stimulated primary cultured hepatocytes, levosimendan suppressed NO production

16

in a time- and dose-dependent fashion through inhibition of iNOS gene expression. We set

17

the concentration of levosimendan at 20 µM in the experiments, because the levels of LDH

18

in culture medium were slightly elevated at the concentration of 100 µM of levosimendan

19(data not shown), which implied cytotoxicity caused by the overdose of levosimendan, but

20

levosimendan had no such effects at 1-20 µM. The experiments with iNOS promoter

21

constructs demonstrated that levosimendan inhibited iNOS expression during the synthesis

22

and stabilisation of mRNA. iNOS promoter activity measured with the constructs

10

1

represented the intensity of NF-κB-dependent transcription because both constructs have2

two NF-κB binding sites (κB) in each promoter area. However, EMSAs revealed that the3binding activity of nuclear extracts to the NF-κB consensus oligonucleotide was not4

inhibited by levosimendan. We conducted the additional experiments to investigate the5

NF-κB nuclear translocation, IκB degradation and phosphorylation of NF-κB p65 (Ser536),

6

which are the important signalling steps to stimulate NF-κB activation. However, we could

7

not detect significant influences of levosimendan on these steps (Supplementary file). From8

the results above, we concluded that levosimendan did not inhibit the activating steps of

9

NF-κB in cultured hepatocytes. This result suggests that levosimendan might affect the10synthesis of iNOS mRNA through signalling pathways and transcription factors other than

11

NF-κB. We found that the iNOS antisense-transcript had a key role in stabilising iNOS

12mRNA by interacting with the 3′-ultratranslated region (UTR) and adenylate-uridylate-rich

13

sequence elements-binding proteins37. Levosimendan demonstrated an inhibitory effect on

14

expression of iNOS antisense transcripts. An anti-inflammatory profile of levosimendan

15

was also shown in hepatocytes because of inhibition of the mRNA expression of TNF-α,

16

CINC-1 and IL-1RI. Note that our in vitro study was not a complete reproduction of

17

PH/LPS-model in two points that we did not use the direct cultured hepatocytes from all18groups in PH/LPS-model, and we used a single cytokine (IL-1β) to stimulate the19

hepatocytes. The results from our in vitro study should be considered as reference to20

understand the anti-inflammatory mechanism of levosimendan.

21

11

1

Some in vitro studies have shown that levosimendan can down-regulate iNOS induction

2and NO production in response to inflammatory stimuli in macrophages12, cardiac

3

fibroblasts39 and hepatocytes40. Differences in the signalling events leading to activation of

4iNOS transcription between cell types might exist. Sareila et al. reported that levosimendan5

did not affect the activation, nuclear translocation or DNA binding of NF-κB in J774

6

macrophages, but inhibited NF-κB-dependent transcription in L929 fibroblasts12. Okada et7

al. reported that levosimendan inhibited IL-1β-induced apoptosis via activation of the8phosphatidylinositol-4, 5-bisphosphate 3-kinase/Akt pathway in the cardiac fibroblasts of9

adult rats39. The data from those studies are similar to our results.

10
11

As an in vivo model of sepsis, CLP-model7, 8 has previously been used to show a survival

12

benefit of levosimendan. Authors selected continuous infusion via a catheter in the jugular13

vein7 or an intraperitoneal osmotic pump8 of levosimendan, whereas we used

14

intraperitoneal bolus administration. One may argue that intraperitoneal bolus

15

administration of levosimendan does not represent the clinical situation accurately.

16

However, the sepsis model caused by LPS injection does not fully represent human sepsis

17

because LPS causes a much earlier peak of expression of pro-inflammatory cytokines

18

compared with that seen in human sepsis. A survival curve of PH/LPS model is more

19

precipitous that the majority of positive control rats died at 6 h after LPS injection

20

compared with CLP-model that two-thirds of controlled rats survived at 9 h after operation7.

21

Levosimendan has a half-life of ≈1 h but its active metabolite, OR-1896, has a half-life of12

1

80 h41, which could cover the duration of effect of a LPS bolus administration. Continuous

2

infusion of levosimendan in the PH/LPS-model may merit further study.

3
4

As in vivo model of liver injury, Grossini et al. reported that levosimendan protected5against ischemia–reperfusion injury through mechanisms related to NO production and6

mitochondrial ATP-dependent potassium-channel function11. Taken together, levosimendan

7

would have a beneficial effect in liver surgery/transplantation. The results of our study lead8

us to recommend levosimendan pretreatment for sepsis management after acute liver injury.

9
10
11

Methods

12

Animals13

All animal experiments were undertaken in accordance with the Guidelines for the care and14

use of laboratory animals (National Institutes of Health, Bethesda, MD, USA). The study

15

protocol was approved by the Animal Care Committee of Kansai Medical University

16(Permission numbers: 17-023(01) and 18-027(01)).

17

Rats (specific pathogen-free) were purchased from Charles River Laboratories Japan

18(Yokohama, Japan) and maintained in a room at 22°C under a 12-h light–dark cycle with a19diet of γ-irradiated CRF-1 (Oriental Bioservice, Kyoto, Japan) and water ad libitum.

20
21

Drugs

13

1

Levosimendan was purchased from Wako Pure Chemical Industries (Osaka, Japan).

2

Levosimendan was resolved in dimethyl sulfoxide (DMSO) and stored at −80°C. For

3

PH/LPS experiments, resolved levosimendan was diluted by 1 ml of normal saline for each4

rat so that the DMSO concentration was 2%. Isoflurane, pentobarbital sodium, collagenase,5

Transaminase CII-test kit, LDH-Cytotoxicity Assay kit and PicaGene Luminescence kit

6

were from Wako Pure Chemical Industries. LPS (Escherichia coli O111:B4) and mouse7

anti-β-tubulin were from Sigma–Aldrich Japan (Tokyo, Japan).

89Recombinant human IL-1β (2 × 107 U/mg protein) was purchased from MyBioSource (San10

Diego, CA, USA). Enzyme-linked immunosorbent assay (ELISA) kits for TNF-α, IL-1β

11

and IL-6 were from Life Technologies Japan (Tokyo, Japan). Rabbit anti-iNOS, TRIzol™

12

Reagent, and UltraPure™ DNase/RNase-Free Distilled Water were from Thermo Scientific13(Waltham, MA, USA). ECL western blotting detection reagents were from GE Healthcare

14

Japan (Tokyo, Japan). Luminate Forte Western Horseradish Peroxidase (HRP) was from

15

Merck Japan (Tokyo, Japan). Oligo (dT) Primer (25 ng), 5×RT Buffer, dNTPs Mixture,

16

RNase Inhibitor and Rever Tra Ace® were from Toyobo (Osaka, Japan). Magnet-assisted

17transfection (MATra) Reagent was from IBA (Gottingen, Germany). Beta-Glo kits, mouse18

immunoglobulin κ light chain was from Promega (Fitchburg, WI, USA). An In situ

19

Apoptosis Detection kit (MK500) was from Takara Bio (Shiga, Japan). An

20

Anti-myeloperoxidase rabbit polyclonal antibody (A0398) was from DAKO (Glostrup,21

Denmark).

22

14

1

Creation of the PH/LPS model2

The procedure for 70% partial hepatectomy is based on experiments described elsewhere42.

3

Briefly, male Sprague–Dawley rats (8 weeks; 310 ± 10 g) were anaesthetised using

4

pentobarbital sodium (40 mg/kg, i.p.) and isoflurane (0–2%). A laparotomy was done with5

a midline incision (≈3 cm). The left lateral and left median lobe of the liver were removed

6

after ligation, followed by wound closure. Operated rats were randomised immediately and7equally into four groups: (A) levosimendan 2 mg/kg; (B) levosimendan 1 mg/kg; (C)8levosimendan 0.5 mg/kg; (D) vehicle (normal saline). Forty-eight hours after surgery, 250

9µg/kg body weight of LPS in saline was injected into the penile vein. Levosimendan (i.p.)

10

was given 1 h before LPS injection. Survival was evaluated during 7 days after LPS

11

injection, and then rats were killed by isoflurane. As an exploratory experiment, samples of

12

blood and remnant liver were taken from Sprague–Dawley rats 0 h, 1 h and 4 h after LPS

13

injection with or without levosimendan pretreatment (n = 3–5 in each group). A scheme of14

the experimental protocol is shown in Fig. 1.

15
16

Isolation and culture of primary hepatocytes

17

The isolation and culture of rat hepatocytes is based on experiments described elsewhere43,

18

44

19

perfusion via the portal vein, followed by centrifugation (50×g, 70 sec, 4℃; four times).

20

Isolated hepatocytes were suspended at 6×105 cells/mL in Williams’ E (WE) culture

21

medium, supplemented with 10% newborn calf serum, Hepes (5 mM), penicillin (10022

U/mL), streptomycin (100 µg/mL), fungisone (0.25µg/mL), aprotinin (0.1µg/mL),. Hepatocytes were isolated from livers of Wister rats (200-220 g) by collagenase15

1

dexamethasone (10 nM) and insulin (10 nM). The cells were seeded into 35- or 100-mm

2

plastic dishes (2 or 10 mL/dish; Falcon Plastic, Oxnard, CA, USA) and cultured at 37℃ in

3

a CO2 incubator under a humidified atmosphere of 5% CO2 in air for 2 h. The medium (1.54mL/35-mm dish) was replaced with fresh serum-free and hormone-containing WE medium

5(first medium change), then with fresh serum- and hormone-free WE medium at 5 h

6(second medium change), and the cells were cultured overnight. As cells were cultured two

7days or more before use in experiments, fresh serum-free and hormone-containing WE

8medium was used in the second medium change, with this medium subsequently changed

9

every day. Then, cells were treated with recombinant human IL-1β (1 nM) in the presence10

or absence of levosimendan.

11
12

Biochemical analyses

13

Serum levels of TNF-α, IL-1β and IL-6 were measured using commercial ELISA kits. The

14

sum of nitrite and nitrate (stable metabolites of NO) in the serum, or nitrite in the culture

15

medium, was measured using the Griess reagent method45. Serum levels of AST and ALT

16

were determined using commercial kits. LDH activity in the culture medium was measured

17

using a commercial kit according to manufacturer instructions.

18
19

Western blotting

20

Protein extracts of liver sections and hepatocytes were prepared for western blotting, as

21

described previously29. They were subjected to a 7.5% gel, and electroblotted.

22

Immunostaining was done using primary antibodies against iNOS and β-tubulin (internal

16

1

control), followed by visualisation with ECL Western Blotting Detection Reagents for

2

iNOS and Luminate Forte Western HRP for β-tubulin. The bands corresponding to each3

protein were quantified by densitometry using ImageJ (San Diego, CA, USA)46.

4
5

RT-PCR

6

Total RNAs of liver sections and hepatocytes were extracted in TRIzol Reagent using the7

guanidinium–phenol–chloroform method47. cDNA was synthesised from 1 µg of total RNA

8from each sample with Oligo(dT)20 Primer (25 ng), 5×RT Buffer (5 µl), 10 mM of dNTPs

9

Mixture (2.5 µl), RNase Inhibitor (0.5 µl), Rever Tra Ace (100 U) and UltraPure™

10

DNase/RNase-Free Distilled Water. The conditions of thermal cycling using iCycler

11(Bio-Rad Laboratories, Hercules, CA, USA) were 42°C for 60 min and 95°C for 5 min.

12

Real-time PCR was done using SYBR Green and primers for each gene. Primer sequences13

were synthesised by Eurofins Genomics (Tokyo, Japan) (Table 1). The conditions of

14

thermal cycling using Rotor-Gene Q (Qiagen, Stanford, VA, USA) were 95°C for 5 min

15followed by 40 cycles of 95°C for 5 s and 60°C for 10 s. Collection and analyses of data

16

were done using the software included with the system. mRNA levels of each gene were17

measured as CT threshold levels and normalised to those of eukaryotic elongation

18

factor-1α.

1920Transfection and luciferase assay

21

Transfection of cultured hepatocytes was undertaken as described previously38. Briefly, on

22

day-0, hepatocytes were cultured for 7 h before being subjected to MATra. Reporter17

1

plasmid pRiNOS-Luc-SVpA or pRiNOS-Luc-3′UTR (1 µg) and the cytomegalovirus

2

promoter-driven β-galactosidase plasmid pCMV-LacZ (1 ng) as an internal control were3

mixed with 1.5 µg of MATra-A reagent in 200 µL of Williams’ E medium. After4

incubation for 15 min on a magnetic plate, the medium was replaced and cultured overnight,

5

and then treated with IL-1β in the presence or absence of levosimendan. Activities of6

luciferase and β-galactosidase in cell extracts were measured using PicaGene and Beta-Glo

7

kits, respectively. Luciferase activity was normalised by β-galactosidase activity. Fold8

activation was calculated by dividing luciferase activity by control activity (without IL-1β

9and levosimendan).

10
11

EMSA

12

EMSA was carried out as described previously48, 49 with a minor modification, as described13

elsewhere20, 50. Nuclear extracts were prepared from frozen liver at −80ºC or cultured

14

hepatocytes. Binding reactions were undertaken by incubating the nuclear extracts in

15

reaction buffer (20 mM of HEPES-KOH, pH 7.9; containing 1 mM of EDTA, 60 mM of

16

KCl, 10% glycerol, and 1 µg of poly[dI-dC]) with a probe (40,000 dpm) for 20 min at room

17

temperature. Products were electrophoresed on a 4.8% polyacrylamide gel in

18high-ionic-strength buffer, and dried gels were analysed by autoradiography. An NF-κB

19

consensus oligonucleotide (5′-AGTTGAG GGGA-CTTTCCCAGGC) from the mouse20

immunoglobulin κ light chain was purchased and labelled with [γ-32P]-ATP and T4

21polynucleotide kinase. Protein was measured using the Bradford method51. Bands

22

corresponding to NF-κB were quantified by densitometry using ImageJ46.

18

1
2

Histopathology

3

Specimens of remnant liver were fixed in 10% formalin solution and embedded in paraffin.

4

Sections (3–5 µm) were cut and stained with haematoxylin and eosin. Neutrophil

5

infiltration

6

Anti-myeloperoxidase rabbit polyclonal antibody（A0398）per 20 HPFs under light7

microscopy. Apoptotic bodies were evaluated by TUNEL staining using the In Situ

8

Apoptosis Detection kit (MK500) per 20 HPFs under light microscopy. The Labeling Index

9of TUNEL-positive cells per 1,000 hepatocyte nuclei was counted in duplicate.

wasevaluatedbythecountsof

MPO-positive

cells

using

the

10
11

Statistical analyses

12

Comparison of rats’ survival among four groups was analysed statistically by one-way

13

ANOVA, followed by Tukey-Kramer method (JMP® 14, SAS Institute Inc., Cary, NC,

14USA). The results of in vitro studies in the figures are representative of at least three15

independent experiments that yielded similar findings. Data are the mean ± standard error

16(SE). Differences were analysed using the Student’s t-test and P < 0.05 was considered17significant.

18
19

Authors’ contributions

20

TS carried out the experiments, acquired and analysed the data, and wrote the major part of21

the manuscript. YH helped create the PH/LPS-model under the supervision of MK. HM

22

and HH assisted in the isolation and culture of primary hepatocytes. MN helped in the19

1experiments on antisense transcripts and provided reporter plasmids. TO was a mentor of2

this study and attended all experiments. All authors approved the final version of the3manuscript for submission.

4
5Competing interests

6

Authors have no competing interest to declare.

78

Funding

9This study was supported by the research fund of the Department of Surgery of Kansai

10

Medical University.

11
12

Acknowledgements

13

Authors thank Dr. Mitsuaki Ishida (Department of Pathology and Laboratory Medicine,

14

Kansai Medical University) for analysing liver specimens by histopathology; Kyodo Byori.

15(Kobe, Japan) for staining the liver specimens; and Arshad Makhdum, PhD, from Edanz

16

Group (www.edanzediting.com/ac), for editing a draft of this manuscript. Prof. A-Hon

17

Kwon and his colleagues had established the experimental model of PH/LPS.

18
19

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19

248–254, https://doi.org/10.1016/0003-2697(76)90527-3 (1976).

20

27

1

Table 1. Primer sets for RT-PCR

2

Nucleotide sequences of primers

3
4
5
6
7
8
9
10
11
12

Gene

RT Primer

PCR Forward Primer

PCR Reverse Primer

EF-1αoligo (dT)20

5'-TCTGGTTGGAATGGTGACAACATGC-3'

5'-CCAGGAAGAGCTTCACTCAAAGCTT-3'

Amplification(bp)
332

iNOS

oligo (dT)205'-CCAACCTGCAGGTCTTCGATG-3'

5'-GTCGATGCACAACTGGGTGAAC-3'

257

TNF-α

oligo (dT)20

5'-TCCCAACAAGGAGGAGAAGTTCC-3'5'-GGCAGCCTTGTCCCTTGAAGAGA-3'

275

IL-1β

oligo (dT)20

5'-TCTTTGAAGAAGAGCCCGTCCTC-3'5'-GGATCCACACTCTCCAGCTGCA-3'

321

IL-6

oligo (dT)205'-GAGAAAAGAGTTGTGCAATGGCA-3'

5'-TGAGTCTTTTATCTCTTGTTTGAAG-3'

286

CINC-1oligo (dT)205'-GCCAAGCCACAGGGGCGCCCGT-3'5'-ACTTGGGGACACCCTTTAGCATC-3'

231

IL-10

oligo (dT)205'-GCAGGACTTTAAGGGTTACTTGG-3'5'-CCTTTGTCTTGGAGCTTATTAAA-3'

245

IL-1RI

oligo (dT)20

5’-CGAAGACTATCAGTTTTTGGAAC-3’5’-GTCTTTCCATCTGAAGCTTTTGG-3’

327

iNOS AST

5’-TGCCCCTCCCCCACATTCTCT-3’5'-ACCAGGAGGCGCCATCCCGCTGC-3'5'-CTTGATCAAACACTCATTTTATTAAA-3'

185

13
14

EF-1α, elongation factor-1-alpha; iNOS, inducible nitric oxide synthase; TNF-α, tumour

15

necrosis

16

cytokine-induced neutrophil chemoattractant-1; IL-10, interleukin-10; IL-1R1, type-I IL-1

17

receptor; iNOS AST, iNOS-antisense transcript.

factor-alpha;IL-1β,

interleukin-1beta;

28

IL-6,

interleukin-6;

CINC-1,Experimental protocol of PH/LPS-1 h 0 h-48 h
Levo (i.p)

LPS (i.v)

70% hepatectomy
and randomization

Randomized Group
A
B
C
D

4h

1h

7 daysSample analyses(20 rats)

Survival evaluation(32 rats)

Condition
Levo 2 mg/kg (n = 8)
Levo 1 mg/kg (n = 8)
Levo 0.5 mg/kg (n = 8)
Vehicle (saline contg. 2% DMSO, n = 8)

Figure 1: Experimental protocol of PH/LPS.
Rats were treated with lipopolysaccharide (LPS, 250 µg/kg, i.v.) 48 h after 70%
hepatectomy (PH/LPS). Levosimendan (Levo) or vehicle [saline containing 2%
dimethyl sulfoxide (DMSO)] was administered (i.p.) 1 h before LPS injection.
Survival of 32 rats was evaluated during 7 days. Samples from 20 rats were obtained at
0 h, 1 h or 4 h after LPS administration and analysed for an exploratory experiment.

1

Cumulative survival

0.8

* (A)

0.6

0.4

(B)

0.2

(C)

(D)
0
0

12

24

36

48

60

72

84

96

108 120 132 144 156 168

Time after LPS injection (h)Figure 2. Effects of levosimendan on rat survival.
Kaplan–Meier curves of PH/LPS are shown. (A) Levosimendan, 2 mg/kg, square; (B)1 mg/kg, triangle; (C) 0.5 mg/kg, open circle; (D) vehicle, dot (8 rats per group). Eachmark represents the death of rat in the indicated time. *: P < 0.05

30

8

6

4

*

2

ー

Time (h)

0

ー

＋

ー

＋

4

1

(d)

15

n.s.

10

*

400

Time (h)

0

ー

＋

ー

1

＋

0

4

ー

Time (h)

0

(e)

400

*

ー

＋

ー

1

＋

4

(f)

*

800

n.s.

600
400

300

200

n.s.

200

＋

1

n.s.

Levo

0

ー

4

100

n.s.
ー

6

1000

600

200

8

0

ー

1200

*

2

Levo

ALT (IU/L)

NO (nM)

800

Time (h)

20

10

(c)

12

0

0

Levo

*

5

Levo

0

14

25

IL-1β (ng/ml)

TNF-α (ng/ml)

n.s

(b)

IL-6 (ng/ml)(a)

AST (IU/L)

10

ー

＋

4

0

Levo

ー

Time (h)

0

ー

＋

1

ー

＋

4

Levo

ー

Time (h)

0

ー

＋

1

ー

＋

4

Figure 3. Effects of levosimendan on expression of cytokines, NO andtransaminases in serum.
Biochemical analyses of serum samples for (a) TNF-α, (b) IL-1β, (c) IL-6, (d) NO, (e)ALT and (f) AST are shown. Each graph consists of 5 bars representing 0 h (48 h after
70% hepatectomy without LPS or levosimendan treatment) as well as 1 h and 4 h after
LPS treatment with levosimendan or vehicle. * and n.s. stand for P < 0.05 and not
significant, respectively, between the shown pair.

Figure 4 (a, b). Effects of levosimendan on expression of NF-κB, iNOS andcytokines in livers.
(a): The result of EMSA for remnant-liver samples is shown (upper), which consists ofrepresentatives of PH/LPS with vehicle at 0 h (3 rats), PH/LPS with vehicle at 1 h (3rats), PH/LPS with levosimendan (Levo) at 1 h (4 rats), PH/LPS with vehicle at 4 h (4rats) and PH/LPS with Levo at 4 h (5 rats). Full-length gels and full sample data areshown in a Supplementary information file. The density of blots in each group wasquantified by densitometry (lower). (b): Results of western blotting for remnant-liver
samples using primary antibodies against iNOS (upper) and β-tubulin (lower) at 1 h(representative 2 samples) and 4 h (5 samples in each group) after LPS treatment withlevosimendan or vehicle are shown (Full-length gels are shown in a Supplementary
Information file). * and n.s. stand for P < 0.05 and not significant, respectively,
between the shown pair.

Figure 4 (c-h). Effects of levosimendan on expression of NF-κB, iNOS andcytokines in livers.
RT-PCR results for (c): TNF-α, (d): IL-1β, (e): IL-6, (f): iNOS, (g): CINC-1 and (h):IL-10. Each graph consists of five bars representing 0 h (48 h after 70% hepatectomy
without LPS or levosimendan treatment), 1 h and 4 h after LPS treatment withlevosimendan or vehicle. * and n.s. stand for P < 0.05 and not significant, respectively,
between the shown pair.

After 48 h of PHa

After 4 h of LPS
with levosimendan

After 4 h of LPS
with vehicle
b

c

HE

g

MPO positive cells

500

e

f

n.s.

Positive cells/
mm2

d

400
300

MPO

200
100
0

0
Levo ーTime (h)

k
h

i

j

2.0

4

4

ー

＋

TUNEL L.I.
n.s.

TUNEL

%

1.5
1.0
0.5
0.0

Time (h) 0
Levo ー

44

ー＋

Figure 5. Effects of levosimendan on liver histopathology.
Histopathology of remnant liver specimens of H&E staining ((a)–(c)), MPO staining((d)–(f)) and TUNEL staining ((h)–(j)) are shown. Figure (a), (d) and (h) are
specimens of rats after 48 h of PH; figure (b), (e) and (i) are rats after 4 h of LPS
injection with vehicle; figure (c), (f) and (j) are rats after 4 h of LPS injection withlevosimendan. Graph (g) shows the result of MPO-positive cell counts (per mm2) ineach group. Graph (h) shows the result of TUNEL-positive cell counts (per mm2) ineach group. Each figure is a representative of each condition, and data of each graphrepresent the mean ± SE (n = 3–5 specimens/group). N.s. stands for not significant.
Bar = 100 microns.

Figure 6. Effects of levosimendan on NO and iNOS induction in IL-1β-stimulated
primary cultured hepatocytes.
(a) Effects of levosimendan (Levo, 20 µM) on NO production for the indicated times(IL-1β only, open circles ○; IL-1β and Levo, closed circles ●; Levo only, closed
triangles ▲; control, open triangles △). (b) Effects of Levo (1–20 µM) for 8 h on NO
production (upper), iNOS and β-tubulin levels (middle, full-length gels are shown in a
Supplementary Information file), and LDH activity (lower). (c) Effects of Levo (20µM) on expression of iNOS mRNA for the indicated times. *P < 0.05 vs. IL-1β alone.
n = 3 dishes/point or indication.

Figure 7. Effects of levosimendan on transactivation of iNOS promoters, iNOSAST expression, and binding of nuclear extracts to NF-κB consensus
oligonucleotide.
(a) Promoter region of iNOS (schematic). Two reporter constructs consisting of the rat
iNOS promoter (1.0 kb), a luciferase gene, and the SV40 poly(A) region (pRiNOSLuc-SVpA) or iNOS 3′-UTR (pRiNOS-Luc-3′UTR). “An” indicates the presence of apoly(A) tail. The iNOS 3′-UTR contains AREs (AUUU(U)A × 6), which contribute tomRNA stabilisation. (b) Relative luciferase activity of pRiNOS-Luc-SVpA andpRiNOS-Luc-3′UTR. *P < 0.05 vs. IL-1β alone (n = 6 dishes/indication). (c)Expression of iNOS AST for the indicated times. *P < 0.05 vs. IL-1β alone (n = 3
dishes/indication). (d) Nuclear extracts were analysed by EMSA (full-length gel isshown in a Supplementary Information file).

(a)

(b)
TNF-α mRNA expression

800
600
400

*

200
0

150
Fold change vs. control

Fold change vs. control

1,000

CINC-1 mRNA expression

100

*

50
0

*

Levo (20 µM)

ー

＋

—

＋

—

＋

Levo (20 µM)

—

＋

—

＋

—

＋

IL-1β (1nM)

＋

＋

＋

＋

＋

＋

IL-1β (1nM)

＋

＋

＋

＋

＋

＋

1

Time (h)(c)

Time (h)

6

1

3

6

IL-1RI mRNA expression

20

Fold change vs. control

3

15
10

*

5
0

Levo (20 µM)

—

＋

—

＋

—

＋

IL-1β (1nM)

＋

＋

＋

＋

＋

＋

Time (h)136Figure 8. Effects of levosimendan on the mRNA expression of pro-inflammatory
cytokines in IL-1β-stimulated hepatocytes.
(a) TNF-α mRNA, (b) CINC-1 mRNA, and (c) IL-1RI mRNA. *P < 0.05 vs. IL-1βalone. n = 3 dishes/indication.

Supplementary Information File
Supplementary Figures: (A) – (I)
(A)

(C)

(G)

(B)

(D)

(H)

(I)
(E)

(F)

Supplementary Figure legends(A): A full-length gel of Figure 4a is shown.
(B): The overall result of EMSA for remnant-liver samples is shown.
(C-F): Full-length gels of Figure 4b are shown; (C) using anti-iNOS antibodies at 0 h and 1
h after LPS treatment with levosimendan or vehicle; (D): using anti-β-tubulin antibodies at
0 h and 1 h; (E) using anti-iNOS antibodies at 4 h; (F) using anti-β-tubulin antibodies at 4 h.
(G, H): Full-length gels of Figure 6b are shown; (G) using anti- iNOS antibodies; (H) using
anti-β-tubulin antibodies.
(I): A full-length gel of Figure 7d is shown.

Supplementary Information File
Supplementary Figures: (J) – (M)(J)

Nuclear translocation of NF-κB p65(L) Phosphorylation of NF-κB p65 (Ser536) in cell lysates

(M)
(K)

NF-κB p65 in cell lysatesIκB degradation in cell lysates

Supplementary Figure legends(J): Nuclear translocation of NF-kB subunit p65 at 30 and 60 minutes after treatment with
IL-1β (1 nM) in the presence or absence of levosimendan (20 nM).
(K-M): Cell lysates (20 µg of protein) were subjected to SDS-PAGE in a gel, followed byimmunoblotting (K): using an anti-IκBα antibody (arrow: 36kDa), (L): using an phosphoNF-κB p65 (Ser536), (M): using an NF-kB p65 antibody.

Supplementary Information File
Methods: (J) – (M)Western Blot Analysis for intranuclear NF-κB p65
Nuclear extracts were immunoprecipitated with an anti-p65 antibody (H286; Santa Cruz
Biotechnology). The bands were analyzed by western blotting using an antibody against
human NF-κB p65 (BD Transduction Laboratories, Lexington, KY, USA).

Western Blot Analysis for IκB and NF-kappa B p65 phosphorylated at serine-536
The bands were analyzed by western blotting using using an antibody against human IκBα(Santa Cruz Biotechnology, Santa Cruz, CA, USA) and phospho-NF-κB p65 (Ser536) (Cell
Signaling Technology, Tokyo, Japan).