Study On Effects And Mechanisms Of Clearing Liver Prescription On Liver Injury And Regeneration In Acute Liver Failure Induced By D-GalN In Rats

ObjectiveTo further discuss mechanisms of Clearing Liver Prescription (CLP) against D-GalN-induced acute liver failure(ALF) rats with heat toxin and blood stasis syndrome based on effects of CLP on liver injury and regeneration.Methods125healthy clean grade SD rats, female, weighing160-200g, were randomly divided into four groups, that was normal group(each subgroup had10), model group, SNMC group and CLP group(each subgroup had20and15, respectively). One subgroup was used to collect blood and liver tissue samples and the other was applied to observe the survival rate of rats at hour96. All groups were fasted12hour before the establishment of the model. Normal group was injected with0.9%saline, whi le other groups were injected intraper i toneal ly with D-Ga1N1.4g/kg to induce ALF rat model with heat toxin and blood stasis syndrome. From the3rd day before modeling to the end of the study, normal group and model group were given saline gavage according to10ml/kg while SNMC group and CLP group were respectively treated with15.6mg/kg/d and29.7g/kg/d. All rats had free diet. At36hours, the first subgroup was utilized to collect6ml blood through the femoral artery and two liver tissue samples. The behavioral changes and survival rate of rats at hour96were mainly observed; serum ALT, AST, TBil, ALB and CHE levels were measured via automatic biochemical analysis; plasma PT was measured via automatic coagulation analysis; PCNA in the liver tissue was detected via SABC immunohistochemical method; expression levels of TLR4、NF-κB、HMGB1and caspase-3mRNA in the liver tissue were detected via RT-PCR fluorescence method.Results 1. Survival rate after96-hour treatment:①there are33.3%who survived in model group,46.7%in SNMC group and66.7%in CLP group, There was no significant difference (P>0.05) in comparing the survival and death rate among the three groups.②At96hours, mean survival times of the three groups were64.6,71.9and83.3hours respectively; cumulative survival rate of rats in CLP group was higher than that of rats in model group (P<0.05). However, there was no significant difference between SNMC group and model group or CLP group (P>0.05).2. Liver function and coagulation function:the levels of ALT, AST, TBiL, ALB, CHE, PT and liver tissue pathologic score at hour36in CLP group were superior to those in model group with statistical significance (P<0.05). It was in the levels of ALT, AST and TBiL that there were significant difference between SNMC group and model group (P<0.05).3. Liver tissue pathologic score:pathological damage score in model group was significantly higher than that in normal group. Meanwhile, both CLP group and SNMC group were lower than model group, especially the former. There were significant differences (P<0.05)4. Index detected by RT-RCR:expression level of liver tissue TLR4, NF-kB, HMGB1, caspase-3mRNA was usually low in normal group, but those in model group was obviously high with statistical significance (P<0.05). Both CLP and SNMC could decrease the express level of TLR4, NF-κB, HMGB1and caspase-3, especially the former. There were significant differences (P<0.05)5. PCNA expression of the liver tissue:after the establishment of the model, PCNA expression would increase in the liver tissue. However, gavage with CLP or SNMC would further improve the PCNA expression, especially CLP.Conclusion1. Clearing Liver Prescription can obviously relieve liver cell’s damage, promote liver cell’s regeneration, improve liver function and liver pathology, extend survival time, reduce mortality in D-Ga1N-induced acute liver failure rats with heat toxin and blood stasis syndrome.2. The possible mechanisms of Clearing Liver Prescription against acute liver failure induced by D-Ga1N in rats:①CLP might decline the.expression of TLR4not only to reduce expression of proinflammatory cytokines via blocking TLR4/NF-κB signal passage activated by related pathogen molecules, LPS and so on, but also to adjust organism immune system through affecting dendritic cells’maturity.②CLP might low NF-κB expression in liver tissue to reduce excessive activation of the signals, such as TNF-a, IL-1, IL-6, thereby interrupting the inflammatory cascade reaction.③CLP might decrease the expression level of HMGB1in the liver tissue to block HMGB1/TLR4/NF-κB signal pathway.④CLP might reduce expression level of caspase-3not only to inhibit liver cell apoptosis, but also to decrease secondary liver cell necrosis caused by the invasion of neutrophils.⑤CLP might also increase PCNA expression in the liver tissue, regulate the regeneration of residual liver cells, promote the differentiation of liver stem cells, aiming to further improve the prognosis of ALF rats.