Proteasome Inhibitor Bortezomib Attenuates Hepatic Injury In The Common Bile Ducts-ligatured Rats

Abstract:Objective:Obstruction Jaundice is a clinically common surgical disease. The circulation of bile acids will be interfered and finally lead to the damage of liver cell.Ggradually, the liver fibrosis could be induced, which result in cirrhosis, even the liver function exhaustion and serious complications could be induced and finally lead to the death of the patient. So far, there is not particularly effective treatment to control the liver fibrosis in clinic. The reason, which may cause the specific mechanism of the liver damage and fibrosis, is unknown. Recent research indicates that obstruction jaundice will activate the nuclear factor (NF-κB), then the activation of NF-κB may play an important role in liver cell damage and fibrosis. Therefore, how to curb obstruction jaundice pathological process of NF-κB activation will be an important treatment to attenuate the liver damage. The purpose of this experiment is to research bortezomib protection liver cell through establishing the model of the rat obstruction jaundice.Methods:Healthy male Spague-Dawlay (SD) rats were 48 and the weight of each was about 200-250g. They were randomly divided into three groups according to principles. The sham operation group (Ⅰ/SO+NS)was 12, the common bile ducts group by ligatured (Ⅱ/BDL+NS)was 12, the common bile ducts group by ligatured and Bortezomib (Ⅲ/BDL+Bor) was 12. And specially set up other group by observed survival rate (Ⅳ/ survival rate group)and the number was 12. Preoperative 24 hours, the groupsⅠ,ⅡandⅣinjected saline water 2ml, the groupⅢinjected bortezomib 0.2mg/kg and saline water to dilute 2ml into abdominal cavity. Preoperative fasted 10 hours, could drink water freely. Ketamine (150 mg/kg) were injected into abdominal cavity before operation and opened abdomen in the axenic conditions. The groupⅠjust separated the common bile ducts from rats and didn’t ligature, and exposureed the same operation time with groupsⅡ,Ⅲ,Ⅳ,then closed abdominal cavity. The groupsⅡ,ⅢandⅣnext to the liver from 0.5cm ligatured the common bile ducts by4-0#thread, then closed abdominal cavity. Postoperation each rat injected 0.9% saline water 2ml in the neck. Postoperation 4 days the groupsⅠ,ⅡandⅣinjected saline water 2ml, the groupⅢinjected bortezomib 0.2mg/kg and saline water 2ml into the abdominal cavity. Postoperation 7 days repeated the same action. Postoperation 15 days the groupsⅠ,ⅡandⅢrats used ketamine anesthesia, then opened, photoed the liver and expansion of the common bile ducts and gathered blood 5ml. Part of serum used automatic biochemical analyzer and measured the rats serum ALT、AST、TBIL and TBA, the other serum used Enzyme linked immunosorbent assay and measured the rats serum IL-6 and TGF-β.Before blood samples were collected and went on collecting specimens of the liver organization. The specimen of the liver organization used 4% of the neutral formaldehyde and fixed. The paraffin was sliced and watched the liver pathological changes with HE staining, and observed the collagen fiber of liver with Masson staining and detected the level of NF-κB with Immunohistochemistry in the liver. All data were tested with SPSS 16.0 statistical software. Count information used Chi-square test. The results were measured by the mean and standard deviation (x±S). To use T inspection, several groups used party’s analysis and the single factor used H inspection. The difference of p<0.05 has statistical significance. Results:Postoperation 15 days all the rats were alive. (1) HE staining, the liver organization of GroupⅠrats weren’t exception. The liver organization of GroupⅡrats had expansion of the liver bile, liver cells swelling, hydropic degeneration, vacuolar degeneration and a small amount of point-like or spotty necrosis; the groupⅢrats were changed in the liver which significantly reduced comparison with the groupⅡ, showed a small amount of fibrous tissue, new bile duct hyperplasia, but still showed a small amount of liver cell swelling and focal necrosis. (2) Serology showed TBIL (umol/L),TBAL (umol/L),ALT (U/L) and ALT, levels of groupⅠwere 12.66±3.56,6.99±2.51,30.92±4.21; 26.99±9.79; the groupⅡwere 142.95±8.18,200.75±17.80,128.61±16.88, 811.28±43.49; the groupⅢwere 88.70±5.37,132.86±14.03 44.80±7.73,223.59±42.94, (p<0.05). (3) ELISA detected serum IL-6 and TGF-(3 levels. levels of groupⅠwere 73.59±3.59,78.32±3.84; the groupⅡwere 117.43±3.26,147.10±5.18; the groupⅢwere 90.11±3.51, 116.69±4.54 (p<0.05). (4) Immunohistochemistry of experimental data used integral optical density (IOD) for semi-quantitative analysis, NF-κB/p65 protein expression of the groupⅢrats were significantly lower than the groupⅡ(p<0.05). (5) Masson staining, the groupⅠrats liver samples had hardly expressed collagen protein, the liver of the groupⅡrats had a large number of expression of collagen protein,the liver samples of the groupⅢrats showed a few collagen protein expression markedly lower than the groupⅡ(p< 0.05).(6)Observation survival rate, postoperation 15 days the groupⅣrats were alive. Postoperation 28 days the first rat died, then rats intermittently appeared to die, Postoperation 132 days all the rats died. Conclusion:1) NF-κB/p65 expression could up-regulate inflammatory factors IL-6 and lead to liver damage and up-regulate TGF-(3 expression in the liver and make liver fibrosis.2) Bortezomib could protect liver cell. Bortezomib could inhibit NF-κB activation and attenuate the liver damage and the liver fibrosis which had been caused by obstructive jaundice.