| Liver injury is a common result of pathological changes in varieties of liver diseaseses.If Liver injury was long-term persistence that would lead to liver fibrosis , cirrhosis and even liver cancer. There was the classic model of liver injury induced by the Carbon tetrachloride. In carbon tetrachloride induced liver injury, Oxidative stress played a leading role in liver cells degeneration, necrosis and inflammation.During liver damage and damage repair process, hepatic stellate cells of in the stationary state were activated into myofibroblasts (MFB). MFB cuold to generate a lot of the ECM as its characteristics. when the damaging stimulus was not removed timly, that most would lead to liver fibrosis. Therefore, prevention and treatment measures of liver injury was included to remove the cause, to reduce liver damage, and decrease the excessive activation of HSC ,and so on. Norcantharidin( NCTD) was derivatives of the cantharidin that retained not only to promote anti-tumor activity and white blood cells count, but also significantly reduced side effects. NCTD has been reported to protect the liver function, but its mechanisms have not been detailed report. And it also has the effect of anti-hepatitis B virus.Studies have shown that NCTD could induce apoptosis of tumor cells, so we speculated that NCTD could induce apoptosis of HSC.Through the establishment of carbon tetrachloride liver injury model, this study was to explore the norcantharidin on protecting of liver function and its anti- oxidative stress mechanism, as well as to explore the effect of norcantharidin on activated hepatic stellate cells apoptosis.Objective: To explore the protection on rat acute liver injury induced by carbon tetrachlorie and the effection on activated hepatic stellate cell apoptosis of norcantharidin Methods:①The experimental rats were randomly divided into normal control group,liver injury model group(model group),bicyclol group(positive control group)and two norcantharidin(NCTD)-treated groups (10mg/kg, 20mg/kg). Liver injury of rats was induced by carbon tetrachloride in model group, bicyclol group and two cantharidin treatment groups (10mg/kg, 20mg/kg).The norcantharidin treatment groups were simultaneously fed with NCTD (10mg/kg/d, 20mg/kg/d) for two weeks, bicyclol group were fed with bicyclol (120mg/kg/d) for two weeks, instead of that the normal control group and liver injury model group were fed with the same dose distilled water for the same time.The rats were sacrificed to get samples at two weeks. Histopathological changes were evaluated by hematoxylin and eosin(HE) staining and by Masson's trichrome methods. The software of pathological image analysis was used to detect Masson staining of collagen surface density. Alanine aminotransferase(ALT)and aspartate aminotransferase (AST)were evaluated in the serum of samples.Maleic dialdehyde(MDA) and Superoxide dismutase (SOD) were evaluated in samples of Liver tissue. The distribution ofα-SMA in the livers was assessed by immunohitochemistry. Apoptosis of hepatic stellate cells was tested by tunel and a-SMA dual staining.②The experiments mainly through norcantharidin intervention rat HSCs in vivo were investigated the effect on stimutaniously changes in mitochondrial membrane potential, caspase3 activity and activated HSCs apoptosis.Results:①Compared with the normal group,The serum levels of ALT, AST in model group were significantly higher than those in the normal control group, and the liver of the model was obvious swelling and the appearance was dark yellow or yellowish brown. HE and Masson's trichrome staining on model proved liver injury induced by carbon tetrachloride in rats is successful. Paraffin sections of liver tissue was showed histopathologic changes inclouding fatty degeneration, infiltration of inflammatory cells and fibrous tissue proliferation and so on. Bicyclol treated and NCTD treated could all attenuate the histopathologic changes above of liver injury model.②The effects of NCTD treatment on liver injury: The serum levels of ALT and AST in the model group were significantly increased with a 559.78%and a 212.29% higher than those in the normal control group(P<0.01).The level of ALT bicyclol group and NCTD treatment group (20 mg/kg) decreased with a 35.19% and with a 27.19%, AST decreased with a 42.45% and with a 39.18%(P<0.05). The results of ALT and AST assay showed that no significant difference was found between the bicyclol group and the NCTD treatment group(P>0.05)③Area density of collagen by semi-quantitative analysis showed that the model group than in the control group had significantly higher collagen content 317.3%(P <0.01).Compared with model group , Bicyclol treated and NCTD treated attenuated the collagen deposition in liver injury by analyzing masson-stained area (P<0.05). The results showed that the three treatment groups were not statistically different(P>0.05).④The effects of NCTD treatment on oxidative stress in liver injury model: Compared with normal group,liver tissue SOD content of model group decreased to 75.5% and MDA content increased to 405.64%, the results were statistical significantlly difference (P <0.01);After the treatment, liver tissue SOD and MDA levels of bicyclol treated group and NCTD treated group content was different than those in model group(P <0.05).Although bicyclol and NCTD(20mg/kg) the same treatment ,no significant difference was found between the bicyclol group and the NCTD treatment group(P>0.05).⑤Compared with normal group,The expression ofα-SMA of liver injury model increased to 648.8% by immunohistochemistry analysis(P<0.01). the positive cells ofα-SMA in NCTD (10mg/kg, 20mg/kg) treatment group than in model group decreased 17.6% and 39.25%.the difference was statistically significant(P<0.05) NCTD-treated decreased the positive cells ofα-SMA in a dose-dependent manner.⑥Both the number and the rate of apoptosis in treatment group were higher than model group by tunel and a-SMA dual staining (P<0.05). In particular, NCTD (20mg/kg) increased significantly actived HSC apoptosis.⑦The effect of 5.0μg/ml norcantharidin on HSCs mitochondrial membrane potential:The membrane potential were detected by flow cytometry analysis method with JC-1 kit.Compared with the negative control group (1.872±0.019),The membrane potential decreased after 5.0μg/ml norcantharidin group treatment for 6h, 12h, 24h(0.891±0.057, 0.591±0.010, 0.714±0.010)and the difference were significant (P<0.05).⑧Caspase3 activity analysis showed that Caspase3 activity overall mean was statistically significant compared with the control group(0.325±0.015)after 5.0μg/ml norcantharidin treatment for 6h, 12h, 24h, 36h(0.657±0.033, 0.785±0.037, 1.771±0.177,4.806±0.15)(P<0.05), the difference of caspase3 activity overall mean was statistically significant between the control group and norcan- tharidin group(P<0.05),we found that Caspase3 activity of incubated 36h group was one of the highest in the five groups.However,the casepase3 activity was decrease after Ac-EDVE-CHO(2.763±0.152)and norcantharidin treatment with for 36h compared with norcantharidin group (P<0.05). It was showed that norcantharidin could increase the activity of the HSCs in a time dependent manner and the effection could be inhibited by Ac-EDVE-CHO.Conclusion: NCTD can alleviate the degree of liver injury induced by carbon tetrachloride in rat by its antioxidation activity. NCTD can induce activated HSCs apoptosis. |
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