Author Affiliations: Department of Infectious Diseases, Second Clinical Hospital, Harbin Medical University, Harbin 150086, China (Yan BZ, Chen LY, Kang L, Wang XR, Bi MR, Wang W and Yang BS)

 

Corresponding Author: Bao-Shan Yang, Professor, Department of Infectious Diseases, Second Clinical Hospital, Harbin Medical University, Harbin 150086, China (Tel: 86-451-86297420; Fax: 86-451-86605330; Email: baoshanyang@126.com)

 

© 2013, Hepatobiliary Pancreat Dis Int. All rights reserved.

doi: 10.1016/S1499-3872(13)60010-7

 

Contributors: YBZ and YBS designed the study. YBZ wrote the manuscript. YBZ, CLY, KL, WXR, BMR and WW performed most of the experiments. All authors contributed to the interpretation of the study and to further drafts. YBS is the guarantor.

Funding: None.

Ethical approval: The study was approved by the Ethics Committee of Harbin Medical University.

Competing interest: No benefits in any form have been received or will be received from a commercial party related directly or indirectly to the subject of this article.

 

 

BACKGROUND: Increasing evidence suggests that the inactiva-tion of cathepsin B attenuates hepatocyte apoptosis and liver damage. This study aimed to investigate the protective effects of a cathepsin B inhibitor (CA-074me) on lipopolysaccharide (LPS)/D-galactosamine (D-GalN)-induced acute hepatic failure (AHF) in mice.

 

METHODS: Mice were intraperitoneally injected with a combination of LPS/D-GalN to induce AHF with or without CA-074me pretreatment. The cumulative survival rates were calculated 48 hours after the induction of AHF. As well as changes in biochemical indicators and liver histology, hepatocyte apoptosis was assessed using a TUNEL method. Serum tumor necrosis factor-α (TNF-α) production, caspase-3, caspase-8, and caspase-9 activity was evaluated. Cytosolic cytochrome c and Bcl-2 expression were measured by Western blotting.

 

RESULTS: The marked elevation in serum aminotransferase activity and prothrombin time found in LPS/D-GalN-treated mice was significantly improved by pretreatment with CA-074me. The efficacy of CA-074me was also confirmed by histological analysis and TUNEL assay. The survival rate significantly improved in LPS/D-GalN-induced mice given CA-074me compared with untreated mice. LPS/D-GalN-induced caspase-3 and caspase-9 activation was remarkably suppressed by CA-074me. However, the increased levels of serum TNF-α and elevated caspase-8 activity in AHF mice were not significantly reduced by CA-074me. Moreover, CA-074me sharply reduced the increased expression of cytosolic cytochrome c and markedly augmented Bcl-2 expression.

 

CONCLUSION:These results suggest that CA-074me has a protective effect in acute hepatic failure induced by LPS/D-GalN.

 

(Hepatobiliary Pancreat Dis Int 2013;12:80-86)

 

KEY WORDS: acute hepatic failure; cathepsin B inhibitor; hepatocyte apoptosis; caspases; cytochrome c

The clinical syndrome of acute hepatic failure (AHF) is life-threatening and complex, including hepatic encephalopathy, severe coagulopathy, jaundice, and hydroperitoneum^{[1, 2]} The mortality rate of AHF is extremely high without liver transplantation, and there is still a lack of satisfactory therapeutic approaches.^{[3, 4]} Although recent advances in the treatment of AHF have been widely reported,^[5] the precise mechanisms involved in recovery remain to be fully elucidated. Therefore, prevention and management of AHF is still a major clinical challenge in the field of liver disease.

 

Lipopolysaccharide (LPS) in combination with D-galactosamine (D-GalN)-induced liver injury is a well-known experimental model used to develop AHF as a result of massive hepatocyte death.^[6] It is generally accepted that the model mimics clinical liver dysfunction and is useful for evaluating the efficiency of treatment.^[7] A previous study^[8] showed that the progression of acute liver injury is cloesely associated with the overproduction of proinflammatory cytokines such as tumor necrosis (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6).

 

Cathepsin B, a lysosomal cysteine protease, is thought to play a pivotal role in apoptosis.^[9] Another study^[10] reported that cathepsin B is released from lysosomes to the cytosol in response to TNF-α and appears to be due to the activation of caspase-8. Furthermore, another research group reported that cathepsin B is involved in TNF-α-mediated hepatocyte apoptosis by promoting the release of cytochrome c from mitochondria.^{[11, 12]} CA-074me (N-[L-trans-propylcarbamoyloxirane-2-carbonyl]- L-isoleucyl-L-prolinemethylester), which is a highly specific cathepsin B inhibitor,^[13] is considered to have a protective effect on acute pancreatitis and reduce its severity. ^[14]

 

However, no research has been performed on the protective capacity of this cathepsin B inhibitor in hepatic failure. In this study, we focused on exploring the possible mechanisms responsible for the protection by CA-074me against AHF.

 

 

Male Kunming mice weighing 20-22 g obtained from the Laboratory Animal Center of Harbin Medical University were used. All experiments were carried out according to the guidelines of Harbin Medical University for the care and use of laboratory animals. The mice were maintained under controlled conditions (22±1?��, 55% humidity and 12 hours day/night rhythm). All animals were allowed for standard laboratory food and water.

 

The following reagents and kits were used: LPS, D-GalN and CA-074me (Sigma, USA); TUNEL reagent kit (Zhongshan Biotechnical Ltd., Beijing, China); DAB kits (Wuhan Boster Biological Technology Co., Ltd., Wuhan, China); alanine aminotransferase (ALT), aspartate aminotransferase (AST) and prothrombin time (PT) detection kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China); TNF-α ELISA kit (BD Biosciences, San Diego, CA., USA); cytosol fractionation kit (Biovision, CA., USA); caspase-3, caspase-8 and caspase-9 colorimetric assay kits (Bio Vision, CA., USA); and goat anti-mouse cytochrome c mAb and Bcl-2 (Santa Cruz Biotechnology Inc., Santa Cruz, USA).

 

For induction of AHF, the mice were intraperitoneally injected with LPS (100 µg/kg) and D-GalN (800 mg/kg). All animals were randomly divided into three groups with 15 mice/group: control group: mice were given physiological saline; LPS/D-GalN group: mice were given only LPS/D-GalN; LPS/D-GalN+CA-074me group: mice were intraperitoneally administered CA-074me (10 mg/kg) 30 minutes before LPS/D-GalN. CA-074me alone did not cause liver damage (data not shown). The survival rate was monitored for 48 hours after LPS/D-GalN administration. Another 75 mice were also grouped as described above. Some of them were sacrificed at 1.5 or 6 hours after LPS/D-GalN and serum was collected for measurement of TNF-α. The remaining mice were sacrificed by decapitation at 6 hours after LPS/D-GalN administration, and biochemical and histological analyses of blood and liver samples were performed.

 

Blood samples were collected at 6 hours after LPS/D-GalN injection. After centrifugation for 10 minutes at 3000×g, all serum samples were hemolysis-free and kept at -70?�� for determination of biochemical parameters. The serum levels of ALT, AST and PT were measured with an automatic analyzer (AU2700, Olympus, Japan) and a Sysmex 1500 automatic coagulometer.

 

For light microscopy, liver specimens were processed by standard histological techniques (fixed in 10% formalin, embedded in paraffin and stained with hematoxylin and eosin). The degree of histological changes including necrosis, hemorrhage and inflammation were classified on a severity scale of - to +++ (-: no change; ±: slight change; +: mild change; ++: moderate change; +++: strong change), according to previous descriptions.^[15]

 

The extent of apoptosis was determined by terminal deoxynucleotidy1-transferase (TdT)-mediated deoxyuridine triphosphate-digoxigenin (dUTP) nick-end labeling (TUNEL), according to the manufacturer's instructions. Hepatocyte apoptosis was quantified by counting the number of TUNEL-positive cells (number of apoptotic nuclei/total number of nuclei ×100). The positive cells were identified in randomly-selected high-power fields (×400).

 

Serum TNF-α levels were measured using a com-mercially available enzyme-linked immunosorbent assay (ELISA) kit, following the manufacturer's instructions.

 

Caspase protease activity was measured using in vitro fluorogenic peptide substrates, referred to previous studies.^[16] Briefly, a liver sample was homogenized in lysis buffer. After centrifugation for 15 minutes at 40?000×g, the supernatant was incubated with substrate peptide (DEVD-AFC for caspase-3, IETD-AFC for caspase-8 and LEHD-AFC for caspase-9). The change in fluorescence (excitation at 400 nm and emission at 490 nm) was monitored after 120-minute incubation.

 

The cytosolic proteins of liver samples were prepared using cytoplasmic extraction reagents according to the manufacturer's instructions. Briefly, equivalent aliquots of protein extract were separated on 12% SDS-PAGE gel. After electrophoretic separation, proteins were transferred to polyvinylidene fluoride (PVDF) membranes, followed by incubation with primary antibodies against cytochrome c, Bcl-2 and β-actin and treatment with secondary antibodies. The image density of specific bands was quantified and scanned using a bio-imaging analyzer.

 

Data were expressed as mean±standard deviation, and differences between the groups were calculated by Student's t test or by ANOVA where appropriate. Survival curves were drawn using the Kaplan-Meier method and analyzed by the log-rank test. P values ≤0.05 were considered statistically significant.

 

 

All mice in the control group survived. After the induction of AHF, severe hepatic failure rapidly developed and the mice began to die at 8 hours. The mortality of mice not given CA-074me was 53.3% (8/15) at 24 hours and reached 100% (15/15) at 48 hours. However, mice treated with CA-074me had markedly better survival, the mortality being 13.3% (2/15) at 24 hours and 33.3% (5/15) at 48 hours (the Kaplan-Meier method, P<0.01) (Fig. 1).

 

To identify the acute hepatic injury induced by LPS/D-GalN, the serum levels of ALT and AST were measured. The results showed that they were significantly elevated at 6 hours after LPS/D-GalN treatment. In the mice pretreated with CA-074me, the ALT and AST activities were remarkably decreased by 59.23% and 49.27%, respectively, compared to the LPS/D-GalN group (P<0.05, Fig. 2A). A marked change in blood coagulation was found as indicated by prolonged PT in LPS/D-GalN mice. Administration of CA-074me shortened PT compared with LPS/D-GalN mice (P<0.05, Fig. 2B).

 

The severity of the pathological changes induced by LPS/D-GalN exposure were scored (Table 1). The histological analysis of the liver in the control group revealed normal lobular architecture and cell structure. However, administration of LPS/D-GalN resulted in extensive hepatocyte damage, including inflammatory cell infiltration, massive necrosis, cellular vacuolar degeneration and hemorrhage. The mice given CA-074me showed clear improvement indicated by a significant reduction in the amount of necrosis (Fig. 3).

 

Hepatocyte apoptosis plays a central role in the hepatic injury induced by LPS/D-GalN, which was confirmed by the TUNEL assay (Fig. 4). A large number of apoptotic hepatocytes were observed in the liver after administration of LPS/D-GalN. However, only a few apoptotic hepatocytes were found in the liver of mice pretreated with CA-074me compared with the LPS/D-GalN group (P<0.01).

 

As TNF-α is a critical mediator of the hepatic injury caused by LPS/D-GalN, the effect of CA-074me on TNF-α was assayed by ELISA. Our result showed that the serum levels of TNF-α peaked at 1.5 hours after LPS/D-GalN administration in comparison with the control group (P<0.01). But administration of CA-074me did not notably decrease this elevation compared with the LPS/D-GalN group (Table 2). A similar observation was made at 6 hours after LPS/D-GalN treatment (415.4±31.2 vs 398.2±29.4 pg/mL, P>0.05).

 

The activity of caspases 3, 8 and 9 in the cytosol fraction at 6 hours after LPS/D-GalN administration was rapidly increased in comparison with the control group (P<0.01) (Table 2). The elevated activity of caspases 3 and 9 was sharply attenuated by pretreatment with CA-074me (P<0.01). However, CA-074me had no depressive effect on the augmentation of caspase-8 activity induced by LPS/D-GalN injection (P>0.05).

 

After injection of LPS/D-GalN, the cytosolic level of cytochrome c protein increased remarkably compared with the control group. Administration of CA-074me sharply reduced its expression compared with the LPS/D-GalN group. While Bcl-2 protein showed little expression in the LPS/D-GalN group, pretreatment with CA-074me augmented it (Fig. 5).

 

 

Cathepsin B has been shown to act as the main executor of apoptosis^[17] or necrosis.^[18] The inactivation of cathepsin B attenuates hepatocyte apoptosis and improves liver damage in several liver disease models.^{[19, 20]} Our earlier study^[21] showed that cathepsin B plays an essential role in the pathogenesis of AHF, and that LPS/D-GalN-induced elevation of cathepsin B is significantly suppressed by pretreatment with CA-074me.

 

Exposure to LPS and D-GalN was done to characterize the development of AHF, which is similar to clinical AHF.^[22] In this experiment, the death of mice treated with LPS/D-GalN began at 8 hours, and reached 100% by 48 hours. The survival rate of mice was clearly improved by pretreatment with CA-074me. It is generally accepted that serum ALT and AST activity and PT are important indicators, which reflect the extent of liver damage in AHF^{[23, 24]} Our study showed that pretreatment with CA-074me ameliorated liver function by lowering the serum aminotransferases and reversing PT.

 

Massive hepatocyte death by apoptosis and necrosis is a key feature of the development of AHF.^{[25, 26]} There is growing evidence that anti-apoptosis treatment is likely to be therapeutic in AHF.^{[27, 28]} In the present study, the classical histological injuries of inflammation, necrosis and apoptosis of hepatocytes induced by LPS/D-GalN were massive. However, the histological grading revealed that the aggravation of hepatic injury was ameliorated by CA-074me. According to the TUNEL assay, CA-074me greatly reduced the hepatocyte apoptosis induced by LPS/D-GalN.

 

It is well documented that TNF-α is an important pathological factor in mediating hepatic injury, and is involved in the development of AHF induced by administration of LPS/D-GalN.^[29] Therefore, the regulation of TNF-α production may be a target for the prevention and treatment of AHF. To explore this possibility, we assessed the effects of CA-074me on the production of TNF-α. As expected, LPS significantly promoted the release of TNF-α in GalN-sensitized mice. However, the up-regulation of the TNF-α level was not significantly reduced by CA-074me pretreatment. Therefore, CA-074me-mediated protection against LPS/D- GalN-treated liver injury is independent of the inhibition of TNF-α production.

 

A family of cysteine-aspartyl proteases called caspases plays an essential part in the induction and execution of apoptosis.^[30] There are two major apoptotic pathways: the extrinsic pathway triggered by the activation of caspase-8, and the intrinsic pathway triggered by the activation of caspase-9.^[31] In addition, caspase-3 is a predominant downstream effector activated by caspases 8 and 9.^[32] Massive activation of caspases is a crucial process in the induction of apoptosis involved in the pathogenesis of acute hepatic failure.^[33] To further confirm the specific anti-apoptotic mechanisms of CA-074me, caspase activity (caspases 3, 8 and 9) was measured. The activity of all three caspases significantly increased after administration of LPS/D-GalN. Pretreatment with CA-074me sharply reduced the elevated activity of caspases 3 and 9, but not caspase 8.

 

In the process of apoptosis, the caspase cascade is mainly associated with the release of cytochrome c from mitochondria, and caspases 3 and 9.^[31] It is established that the Bcl-2 family, including pro-apoptotic and anti-apoptotic proteins, takes part in the control of apoptosis, and the overexpression of Bcl-2 is anti-apoptotic.^[34] So, the levels of cytosolic cytochrome c and Bcl-2 were determined by Western blotting analysis in this study. Our results showed that the levels of the anti-apoptotic Bcl-2 protein markedly decreased, while the levels of cytosolic cytochrome c strikingly increased after administration of LPS/D-GalN. Pretreatment with CA-074me inhibited cytochrome c release and augmented Bcl-2 expression. The possible mechanisms underlying the protective effect CA-074me are correlated with the inhibition of apoptosis.

 

In conclusion, the administration of cathepsin B inhibitor (CA-074me) can increase survival and attenuate hepatic injury. Our findings show that the amelioration is mainly attributable to the inhibition of apoptosis, which is mediated by its blockade of the LPS/D-GalN-induced mitochondrial apoptosis pathway. Therefore, CA-074me may offer an alternative therapy for the prevention of AHF.

 

 

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