Matrine attenuates endotoxin-induced acute liver injury after hepatic ...

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3 Department of General Surgery, Hepatosplenic Surgery Center, the First Affiliated Hospital of Harbin Medical University, Harbin 150001,. China. Abstract.
Surg Today (2011) 41:1075–1084 DOI 10.1007/s00595-010-4423-9

Original Article Matrine Attenuates Endotoxin-Induced Acute Liver Injury After Hepatic Ischemia/Reperfusion in Rats FENG ZHANG1, XUN WANG2, LIQUAN TONG1, HAIQUAN QIAO3, XINLEI LI1, LIGUANG YOU1, HONGCHI JIANG3, and XUEYING SUN3 1

Department of General Surgery, the Fifth Affiliated Hospital of Harbin Medical University, Daqing, China Department of General Surgery, Daqing Oilfield General Hospital, Daqing, China 3 Department of General Surgery, Hepatosplenic Surgery Center, the First Affiliated Hospital of Harbin Medical University, Harbin 150001, China 2

Abstract Purpose. Hepatic ischemia/reperfusion (HIR) injury is an unavoidable consequence of major liver surgery, during which endotoxemia often takes place. This study aimed to investigate whether matrine has a protective effect against HIR-induced liver injury aggravated by endotoxin. Methods. Wistar rats were subjected to 30 min of HIR followed by lipopolysaccharide (LPS) (0.5 mg/kg) administration. At the indicated time points, six rats from each group (24 rats) were randomly euthanized to collect blood samples and livers. Results. Preadministration of matrine protected livers from injury induced by HIR+LPS as the histological score, myeloperoxidase activity and malondialdehyde contents, expression of macrophage-inflammatory protein-2, DNA-binding activity of nuclear factor κB, and serum levels of alanine aminotransferase, aspartate aminotransferase, lactate dehydrogenase, alkaline phosphatase, tumor necrosis factor-α, soluble intercellular adhesion molecule-1, and nitric oxide were significantly reduced, and serum levels of interleukin-6 were further increased. HIR+LPS markedly induced cell apoptosis and necrosis, and upregulated the expression of cleaved caspase-3, Fas, and FasL. Matrine significantly reduced cell necrosis, but had a nonsignificant inhibitory effect on cell apoptosis and expression of cleaved caspase-3 and FasL Conclusions. Matrine attenuates endotoxin-induced acute liver injury after HIR mainly by its antiinflammatory and antioxidative activities, and has little inhibitory effect on cell apoptosis.

Reprint requests to: X. Sun Received: December 27, 2009 / Accepted: August 16, 2010 F.Z. and X.W. contributed equally to this work.

Key words Matrine · Endotoxemia · Hepatic ischemia/ reperfusion · Inflammatory cytokine · Oxidative stress · Apoptosis

Introduction Hepatic ischemia/reperfusion (HIR) injury is an unavoidable consequence of major liver surgery including liver transplantation, resulting in the release of proinflammatory cytokines and reactive oxygen species (ROS), which can cause liver injury and dysfunction, and even liver failure.1 During the ischemic phase, occlusion of the portal vein and hepatic artery interrupts the flow of mesenteric blood, leading to intestinal ischemia, stagnation, and damage to the intestinal barriers, thus endotoxin produced in the gut can be released into the liver via the portal vein in the reperfusion phase.2,3 In addition, postoperative infections, which often occur after major liver surgery, may also cause endotoxemia. It has been demonstrated that endotoxin can aggravate HIR-induced liver injury because it induces oxidative stress,4 free radical formation,5 and the release of inflammatory mediators.6–8 The role of endotoxin in HIR injury is supported by the fact that endotoxin tolerance protects organs against HIR injury in rats.9 Despite improvements in surgical techniques and perioperative management, liver failure remains one of the major complications after major liver surgery. Therefore, it is worthwhile investigating novel agents that can protect against HIR-induced liver injury aggravated by endotoxin. Matrine (with a molecular formula of C15H24N2O),10 one of the main components extracted from a traditional Chinese herb, Sophora flavescens Ait, has become an attractive candidate for attenuating HIR-induced liver injury. Matrine has exhibited multiple pharmaceutical benefits including anti-inflammatory, antiviral, and

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antifibrosis effects in treating viral hepatitis and liver cirrhosis patients.11,12 Matrine inhibited the activity of peritoneal macrophages to produce fibrogenic cytokines induced by lipopolysaccharide (LPS),13 and also reduced LPS-induced damage to rat intestinal microvascular endothelial cells by reducing the production of interleukin (IL)-6, IL-8, and soluble intercellular adhesion molecule-1 (sICAM-1).14 Matrine also exhibited a potential protective effect against 17α-ethinyl estradiolinduced acute liver cholestasis in rats,15 and attenuated acetaminophen-induced hepatotoxicity and reduced the number and area of γ-glutamyltranspeptidase-positive foci by its anti-inflammatory effect, when combined with glycyrrhizin.16 It also decreased the total bilirubin and improved survival in liver transplantation patients when combined with Kushen, a traditional Chinese medicinal herb.17 Furthermore, matrine has shown neuroprotective effects against focal cerebral ischemia as a result of its antiapoptotic activity through inhibition of caspase-3 activity.18 It also attenuated the injury of sinusoidal endothelial cells induced by cold HIR, and decreased the plasma level of hyaluronic acid and the expression of ICAM-1 in sinusoidal endothelial cells in a rat model of orthotopic liver transplantation.19 These studies suggest that matrine may have protective effects on HIR and endotoxin-induced liver injury. We therefore tested this hypothesis in a rat model of HIRinduced liver injury aggravated by LPS as described in our previous report.20

Materials and Methods Animal Experiments Male Wistar rats (200–250 g) were supplied by the Animal Research Center at the First Affiliated Hospital of Harbin Medical University, Harbin, China. The animals were maintained under standard conditions and fed rodent chow and water. All surgical procedures and care administered to the animals had been approved by the institutional ethics committee. Seventy-two rats were randomly assigned to three groups (each group had 24 rats): sham, control, and matrine treatment. The rats in the control and matrine groups received daily intraperitoneal injections of 1 ml of physiological saline or the same volume of matrine (Shaanxi Huike Botanical Development, Xi’an, China) at a dose of 100 mg/kg body weight, respectively, for 7 days. Then the rats underwent the same procedure of HIR and LPS administration as described previously.20–22 In brief, hepatic ischemia was induced by occluding the portal vein and hepatic artery with a vascular clamp. Reperfusion was initiated by removal of the clamp 30 min later, and the abdominal wall was sutured by layers. Immediately after

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the procedure, LPS (Sigma-Aldrich, Shanghai, China) was injected intravenously (0.5 mg/kg body weight). The rats in the sham group underwent laparotomy and closure of the abdominal cavity without HIR or LPS administration. At the indicated time points (1, 3, 6, and 9 h after the operation), 6 rats from each group were randomly euthanized, blood was collected via cardiac puncture, and the liver harvested. Blood samples were centrifuged at 3000× g for 10 min to collect sera, which were stored at −80°C. Each liver was divided into two halves: one half was fixed with 10% buffered formalin and the other half was kept at −80°C. Histological Examination Liver specimens were fixed in 10% buffered formalin, embedded in paraffin, stained with hematoxylin–eosin (H&E), and examined by light microscopy. The liver histopathological scoring analysis was performed in a blinded fashion according to previously described methods.20,22,23 The assessment was expressed as the sum of the individual score grades from 0 (no findings), 1 (mild), 2 (moderate), to 3 (severe) for each of the following six parameters: cytoplasmic color fading, vacuolization, nuclear condensation, nuclear fragmentation, nuclear fading, and erythrocyte stasis. Measurement of Parameters in Sera The serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), lactate dehydrogenase (LDH) and alkaline phosphatase (ALP) were measured with an autobiochemical analyzer (Toshiba, Tokyo, Japan) as described previously.20,22,24 The serum levels of tumor necrosis factor (TNF)-α, IL-6, and sICAM-1 were measured with enzyme-linked immunosorbent assay kits (Wuhan Boster Biological Technology, Wuhan, China) according to the manufacturer’s instructions. The serum level of nitric oxide (NO) was measured by determining the nitrate reductase activity with an NO detection kit (Nanjing Science and Technology, Nanjing, China) according to the manufacturer’s manual. Myeloperoxidase (MPO) Activity in Liver Tissue The methodology for measuring the activity of MPO in liver tissue specimens has been described in our previous report.22 Malondialdehyde (MDA) in Liver Tissues The contents of MDA in liver tissues were measured to assess lipid peroxidation. Samples of liver tissues were homogenized with ice-cold 150 mM potassium chloride,

F. Zhang et al.: Matrine Protects the Liver from HIR/LPS

and the MDA levels were measured spectrophotometrically. The results were expressed as nmol of MDA per gram tissue.25

Cell Death Assays Serial liver sections of 5 μm thickness were stained with terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) (Roche, Shanghai, China) according to the manufacturer’s instructions, and were examined by fluorescence microscopy to detect apoptotic cells. Adjacent sections were counterstained with H&E and the apoptosis index was calculated as the percentage of stained cells. The liver sections were also stained with propidium iodide (PI) (BD Biosciences, San Jose, CA, USA) to detect necrotic cells. The number of PI-stained cells was counted in ten blindly chosen random fields at ×200 magnification.

Western Blot Analysis The methodology for the Western blot analysis has been described previously.20–23,26 In brief, tissues were homogenized in protein lysate buffer. The homogenates were resolved on polyacrylamide–sodium dodecyl sulfate gels, and electrophoretically transferred to polyvinylidene difluoride membranes. The membranes were blocked with 3% bovine serum albumin, incubated with antibodies against cleaved caspase-3, Fas, Fas ligand (FasL), and macrophage-inflammatory protein-2 (MIP-2) (Santa Cruz Biotechnology, Santa Cruz, CA, USA), and subsequently with alkaline phosphataseconjugated secondary antibodies. They were developed by 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium (Tiangen Biotech, Beijing, China). Blots were stained with an anti-β-actin antibody, and the levels of proteins were normalized with respect to β-actin band density.

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Statistical Methods The results were expressed as the mean values ± standard deviation (SD). An analysis of variance test followed by a least significant difference test was used to evaluate the statistical significance. A P value of less than 0.05 was considered to be significant.

Results Matrine Attenuates Liver Injury As shown in Fig. 1, the serum levels of ALT (Fig. 1A), AST (Fig. 1B), LDH (Fig. 1C), and ALP (Fig. 1D) were significantly higher in the control group than in the sham group at all of the indicated time points (all P < 0.001), in accordance with our previous report.20 However, pretreatment with matrine significantly attenuated the increase of ALT (6 and 9 h), AST (9 h), LDH (1, 6 and 9 h), and ALP (6 and 9 h) induced by HIR+LPS. These results suggest that matrine can attenuate the liver dysfunction that occurs as a result of hepatic damage by HIR+LPS. The serological changes were further confirmed by histological analysis. The liver histological scores in the control group were significantly greater than those in the sham group at all of the indicated time points (all P < 0.001), and pretreatment with matrine significantly decreased the histological scores compared with controls, 1, 6, and 9 h after the operation (all P < 0.05) (Fig. 2A). Although the histological score in the matrine group was lower than that in the control group at 3 h after the operation, the difference did not reach significance. Representative illustrations of H&Estained liver sections revealed that livers from the sham group exhibited normal morphology (Fig. 2B). Histological alteration of the livers from control rats was characterized as inflammatory cell infiltration, hemorrhagic changes, and focal necrosis in the midzone and periportal regions of the liver 6 h after the operation (Fig. 2C). In contrast, pretreatment with matrine attenuated the pathological changes (Fig. 2D).

Electrophoretic Mobility Shift Assay (EMSA) The methodology for EMSA has been described previously.27 In brief, nuclear extracts (10 μg) were incubated with 1 μg of poly(deoxyinosinic-deoxycytidylic acid) in binding buffer for 30 min at 4°C. The DNA-binding activity was confirmed with a biotin-labeled oligonucleotide bio-nuclear factor (NF)-κB probe (5-AGT TGA GGG GAC TTT CCC AGG C-3′) using an EMSA kit according to the manufacturer’s instructions (Viagene, Beijing, China). The probe was resolved on a 4% polyacrylamide gel containing 0.25× TBE (Tris/borate/ethylenediamine tetraacetic acid) buffer, and visualized with a CoolImager imaging system (IMGR002).

MPO Activity and MDA Contents in Liver Tissues The liver MPO activity in the control group was dramatically increased compared with the sham group at all of the indicated time points (all P < 0.001) (Fig. 3A). These increases were significantly diminished by pretreatment with matrine at the time points 3 and 6 h after the operation (both P < 0.05). Although the level of MPO was slightly reduced in matrine-treated rats compared with control rats at 1 and 9 h after the operation, the difference did not reach significance (Fig. 3A). Similarly, the level of MDA in liver tissues was significantly (P < 0.001) increased in control rats 6 h after the

Fig. 1A–D. Matrine attenuates liver dysfunction induced by hepatic ischemia/reperfusion plus lipopolysaccharide (HIR+LPS). Blood samples were taken at the indicated time points from sham-operated rats or from the rats pretreated with saline (control) or matrine, then subjected to HIR and LPS. The serum levels of alanine aminotransferase (ALT; A),

aspartate aminotransferase (AST; B), lactate dehydrogenase (LDH; C), and alkaline phosphatase (ALP; D) were measured. The results are expressed as the mean ± SD (n = 6). *P < 0.01, significant increase from sham-operated rats and ‡P < 0.05, significant reduction from control rats at the respective time points

Fig. 2. A Matrine attenuates the histological alterations of livers induced by HIR+LPS. Six rats were randomly euthanized from the sham, control, or matrine groups, and livers were harvested and examined for histological scoring of hepatic injury. The results are expressed as the mean ± SD (n = 6). *P < 0.01, significant increase from sham-operated rats and ‡P < 0.05, significant reduction from control rats at the respective time points. B–D Representative photographs (×400) of hematoxylin–eosin-stained sections of livers, which were taken 6 h after the operation from sham-operated rats (B), or from the rats pretreated with saline (C) or matrine (D) and then subjected to HIR and LPS

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of MIP-2 and the increase of NF-κB activity (Fig. 3C and D).

Parameters in Sera As shown in Table 1, the serum levels of TNF-α, sICAM1, IL-6, and NO were dramatically increased by HIR+LPS at all of the indicated time points. The increase in TNF-α at 3, 6, and 9 h after the operation was significantly diminished by preadministration of matrine (all P < 0.05). The elevated levels of sICAM-1 were attenuated by matrine at 3 and 6 h after the operation (both P < 0.05). In addition, the increase in NO was significantly diminished (P < 0.01) by matrine at the time point 3 h after the operation. However, preadministration further increased the level of IL-6 that was already elevated by HIR+LPS, resulting in a significant (P < 0.05) increase 1 h after operation.

Cell Death in Situ

Fig. 3. Myeloperoxidase (MPO) activity, malondialdehyde (MDA) content, expression of macrophage-inflammatory protein-2 (MIP-2), and nuclear factor kappa-B (NF-κB) activity in livers. Liver tissues in Fig. 2 were used to measure the levels of MPO (A), MDA (B), the expression of MIP-2 (C), and NF-κB activity (D) as described in Materials and Methods. The results are expressed as the mean ± SD (n = 6). *P < 0.01, significant increase from sham-operated rats and ‡P < 0.05, significant reduction from control rats at the respective time points

operation as compared with sham-operated rats, but preadministration of matrine significantly (P < 0.05) decreased the level of MDA (Fig. 3B). Expression of MIP-2 and NF-κB Activity We further demonstrated that the hepatic expression of MIP-2 was upregulated and the DNA-binding activity of NF-κB increased, in the control group as compared to the sham group, at 6 h after the operation. The preadministration of matrine attenuated the upregulation

The liver sections were stained with TUNEL to examine cell apoptosis. There were very few apoptotic cells sparsely distributed in the liver sections taken from sham-operated rats (Fig. 4A), whereas large numbers of apoptotic cells were observed in liver sections from the control (Fig. 4B) and matrine (Fig. 4C) groups at 6 h after the operation. The apoptosis indexes in the livers from the control and matrine groups were significantly (both P < 0.001) greater than in the sham group (Fig. 4D). Although the apoptosis index in the matrine group was slightly lower than that in the control group, the difference did not reach significance (Fig. 4D). We further detected cell necrosis by staining the liver sections with PI, and counted necrotic cells under microscopy. As shown in Fig. 4E, the number of necrotic cells in livers from the control or matrine group was significantly (both P < 0.001) greater than that in the sham group, but the matrine group had a significantly (P < 0.05) smaller number of necrotic cells than the control group. We previously demonstrated that HIR+LPS increased the activation of caspase-3 and the expression of Fas and FasL in the liver tissues.20 In accordance with this result, in the present study the Western blotting analysis of liver homogenates revealed that the expression of cleaved caspase-3, Fas, and FasL was upregulated in liver tissues from both the control and matrine groups compared with the sham group (Fig. 4F). Compared to controls, pretreatment with matrine slightly reduced the expression of cleaved caspase-3 and FasL, but the difference did not reach significance, and matrine had no effect on the expression of Fas.

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Table 1. Parameters in sera collected from rats at different time points

TNF-α (pg/ml)

sICAM-1 (pg/ml)

IL-6 (pg/ml)

NO (μmol/l)

Time point (h)

Sham

Control

Matrine

1 3 6 9 1 3 6 9 1 3 6 9 1 3 6 9

14.6 ± 4.3 10.5 ± 5.9 16.2 ± 4.7 12.5 ± 3.6 46.3 ± 11.9 75.5 ± 12.6 79.6 ± 9.7 64.3 ± 15.5 38.3 ± 8.4 36.2 ± 11.0 30.7 ± 9.6 26.3 ± 5.7 4.2 ± 1.7 3.3 ± 2.6 5.5 ± 1.3 4.7 ± 2.4

53.6 ± 19.3* 129.8 ± 25.6* 205.1 ± 37.4* 156.5 ± 22.7* 172.0 ± 29.3* 246.7 ± 58.4* 353.8 ± 47.6* 279.5 ± 75.4* 75.8 ± 16.0* 338.0 ± 41.5* 235.4 ± 26.7* 179.4 ± 41.3* 20.1 ± 6.8* 37.3 ± 5.7* 22.4 ± 4.3* 21.9 ± 5.6*

44.10 ± 8.3* 82.7 ± 23.0*,‡ 154.1 ± 30.7*,‡ 93.1 ± 11.5*,‡ 157.6 ± 40.5* 183.6 ± 25.3*,‡ 212.7 ± 42.4*,‡ 255.8 ± 37.5* 127.8 ± 14.5*,‡ 353.1 ± 35.4* 241.1 ± 26.8* 206.4 ± 10.8* 18.6 ± 5.9* 23.5 ± 6.1*,‡ 18.7 ± 5.8* 16.5 ± 4.3*

Data are expressed as the mean ± SD, and an analysis of variance test followed by a least significant difference test was used to evaluate statistical significance. P < 0.05 was considered to be significant TNF-α, tumor necrosis factor-α; sICAM-1, soluble intercellular adhesion molecule-1; IL-6, interleukin-6; NO, nitric oxide * Significant increase compared to sham-operated rats ‡ Significant reduction compared to control rats

Discussion We have previously reported that LPS aggravated liver injury induced by HIR.20 We herein again demonstrated that subjection of the liver to HIR+LPS led to enhanced liver injury as the serum levels of AST, ALT, LDH, and ALP, and histological scores of livers were dramatically increased compared to the sham group. It has been shown that the cytotoxic sensitivity to endotoxin increased after major liver surgery, such as hepatectomy,28 and the increased sensitivity was partly associated with the reduced phagocytic function of the reticuloendothelial system.29 Several studies have demonstrated that the HIR-induced liver injury was increased by LPS.9,30,31 Hepatic ischemia/reperfusion not only caused systemic and portal endotoxemia9 but also activated LPS signaling pathways, initiating a cascade of complications of septic shock and multiple organ failure.32 Therefore, endotoxemia represents one of the main causes of death after massive liver surgery. The present study has demonstrated that daily administration of matrine for 1 week attenuated the endotoxinaggravated liver injury induced by HIR, evidenced by the reduction of serum levels of AST, ALT, LDH, and ALP, attenuation of histological alterations, and inhibition of cell necrosis. Matrine has displayed anti-inflammation properties that protect livers.11,12 Accordingly, the present study has demonstrated that matrine reduced the production of MPO in livers and several inflammatory factors in the sera. During endotoxemia, inflammatory cytokines

and neutrophil infiltration play important roles in liver injury.33 Myeloperoxidase, an enzyme present in neutrophils, is a widely used marker of neutrophil infiltration.34 By producing oxidative stress, neutrophils activate Kupffer cells, and contribute to microvascular dysfunction and edema.35 Hepatic ischemia/reperfusion could increase the MPO activity in livers,22 and the combination of HIR and LPS further increased MPO activity in accordance with our previous report.20 The present results showing that matrine decreased MPO activity may indicate that matrine ameliorates inflammation-induced liver injury by inhibiting neutrophil infiltration. Cytokines play an important role in the responses after surgical stress.36 We have previously reported that HIR increased the release of TNF-α,22 and later reported that LPS induced acute liver injury after partial hepatectomy by promoting the translocation of NF-κB into the nuclei and release of TNF-α.23 The present study has shown that HIR+LPS markedly increased the serum levels of TNF-α and the DNA-binding activity of NF-κB, and upregulated expression of MIP-2. TNF-α, a key mediator of the cytokine cascade and tissue injury in sepsis, is involved in the pathogenesis of LPS-induced liver injury.33 Lipopolysaccharide triggers sepsis syndrome by activating monocytes to produce proinflammatory cytokines, which potently stimulate the activation of neutrophils. Lipopolysaccharide binds to LPS-binding protein, and interacts with CD14 to form a ternary complex, which in turn leads to activation of toll-like receptor 4 and subsequent activation of NF-κB.37 Once activated, NF-κB is translocated into the nucleus, where

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Fig. 4A–F. Cell death. Representative photographs (×400 magnification) were from transferase-mediated dUTP nick end-labeling (TUNEL)-stained sections of livers (in Fig. 2), which were taken 6 h after the operation from sham-operated rats (A) or from the rats pretreated with saline (control, B) or matrine (C), and then subjected to HIR and LPS. D TUNELpositive cells were counted to record the apoptosis index. E The liver sections above were stained with propidium iodide (PI) to detect cell necrosis, and PI-stained cells were counted

under microscopy. F A Western blot analysis of cleaved caspase-3, Fas, and FasL in homogenates of livers, which were taken 6 h after the operation from the sham-operated rats, or from the rats pretreated with saline (control) or matrine, and then subjected to HIR and LPS. The density of each band was measured and compared to that of the internal control, β-actin. *P < 0.001, significant difference from sham-operated rats; ‡ P < 0.05, significant difference from control rats

it induces transcriptional upregulation of various proinflammatory mediators such as TNF-α38 and MIP-2.39 The present study showed that matrine reduced the serum level of TNF-α, activity of NF-κB, and expression of MIP-2 in liver tissues elevated by HIR+LPS.

Intercellular adhesion molecule-1 plays an important role in the rolling, adherence, and migration of leukocytes. Intercellular adhesion molecule-1 was overexpressed in the liver tissues in a rat model of orthotopic liver transplantation,19 and the protein participated in

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HIR-induced liver injury.40 During HIR, microcirculatory disturbance, activation of Kupffer cells, and endotoxin upregulate the expression of ICAM-1, which enters the circulation from the activated endothelial cell surface.41 Anti-ICAM-1 blockade was shown to attenuate the injury to transplanted livers by reducing leukocyte adherence following cold HIR.42 In agreement with this finding, we have demonstrated that the serum level of sICAM-1 was dramatically increased by HIR+LPS, and matrine attenuated this increase, which may contribute to its anti-inflammatory effects to attenuate liver injury. Interleukin-6 has shown a protective function in experimental liver disease models, as it plays a role in the induction of the acute phase response.43 The serum level of IL-6 is elevated in patients with acute and chronic liver diseases.44 Interleukin-6 binds directly to hepatocytes by interacting with the gp80/gp130 complex.45 In experimental models of liver injury, mice deficient for the gp130 receptor showed an abolished acute-phase response and an increased susceptibility to LPS-induced liver failure.46 In a model of concanavalin-A-induced hepatitis, pretreatment with IL-6 protected mice from liver injury.47 The present study has shown that subjection of livers to HIR+LPS dramatically increased the serum levels of IL-6, and pretreatment of matrine resulted in an even higher level of IL-6, thus implying that the elevated IL-6 level may be induced by a self-defense mechanism of the host, and matrine may exhibit its anti-inflammatory activity partly by enhancing IL-6 production. During the reperfusion phase of HIR, reoxygenation of the ischemic liver leads to generation of free radicals, which are important factors responsible for HIRinduced injury.48 The liver tissue MDA content has been identified as an indicator for such oxidative damage.49 We have herein shown that the content of MDA in liver tissues was dramatically increased by HIR+LPS, while pretreatment with matrine significantly attenuated this increase, indicating that matrine may inhibit the HIR+LPS-induced liver injury through its antioxidative activity. Nitric oxide, a short-lived reactive nitrogen species from l-arginine, plays a role in physiological processes in essentially every organ and tissue.50 The process of ischemia and reperfusion is known to cause the induction and activation of inducible nitric oxide synthase (iNOS), but the role of NO in HIR has not been finalized.51 Both beneficial and harmful effects of NO have been reported, and the NO molecule has been described as having a “Janus face.”52 General nitric oxide synthase inhibitors have been found to exert a protective effect during HIR in rats,53 although both endothelial nitric oxide synthase (eNOS) and iNOS deficiency have been shown to increase liver injury.54 In the present study the

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serum level of NO was significantly increased by HIR+LPS, and matrine attenuated this increase, indicating that matrine may exert its antioxidative effect by reducing NO production. Apoptosis has been regarded as a central mechanism responsible for the injury induced by HIR.55 We have previously reported that the apoptosis of hepatocytes was increased in damaged livers by HIR or HIR+LPS.20,22,34 In both death receptor and mitochondrial apoptotic pathways, caspase-3, the final “effector” protease, plays a key role in cell apoptosis.56 The present study has demonstrated that HIR+LPS increased the activation of caspase-3 and expression of Fas/FasL in liver tissues, in accordance with our previous report.20 However, matrine did not significantly attenuate the increased apoptosis or inhibit the upregulation of apoptosisrelated proteins, but could decrease the cell necrosis elevated by HIR+LPS. These results indicate that the protective effect of matrine against HIR+LPS-induced liver injury mainly relies on protecting cells from necrosis. In conclusion, the present study has, for the first time, demonstrated the protective effect of matrine on acute liver injury induced by HIR+LPS. Its protective mechanisms may be attributable to its anti-inflammatory activity and inhibition of NF-κB, its release of proinflammatory mediators and neutrophil accumulation, its increase in the production of anti-inflammatory cytokines, and its antioxidative activity by reducing the production of lipid peroxidation. Our results therefore support the potential utilization of matrine for protecting livers against endotoxin-induced injury, especially after HIR. Acknowledgments. This work was supported by the National Natural Scientific Foundation of China (30872987, 30973474), and Heilongjiang Provincial Natural Scientific Foundation (D2007-34, 41400298-9-09007). The experiments were carried out in the Key Laboratory of Hepatosplenic Surgery, the First Affiliated Hospital of Harbin Medical University, China.

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