Study Of Longhu Rendan Effect On The Treatment Of Liver Fibrosis And Its Mechanism

Aim:Hepatic fibrosis is a pathological change caused by chronic liver injury due to various factors.The occurrence of liver fibrosis affects the normal function of liver to some extent,and it may further develop into cirrhosis or liver cancer if uncontrolled.Hepatic fibrosis is still a reversible process and can be cured with effective means or drugs.At present,there are no specific drugs for the prevention and treatment of liver fibrosis.Therefore,the search for specific anti-liver fibrosis drug not only has become a research focus of scholars both at home and abroad,but also an urgent need for the prevention and treatment of liver disease in China.Longhu Rendan,a Chinese patent drug,which has been used for anti-emesis drugs or sunstroke deprevention for decades.However,other aspects of the function has not been reported.In this study,Longhu Rendan was applied to the study of liver fibrosis in order to find specific anti-fibrotic drugs.Methods:1.Experimental animals,groups and models:The model of liver fibrosis was induced by CCl₄.The SPF male C57BL/6 mice were randomly divided into three groups:the solvent control group?Oil?,the model group?CCl₄?and the LHRD group?LHRD?.CCl₄ and LHRD groups were injected with CCl₄ for 6 weeks.The LHRD group were treated with LHRD by intragastric administration every day until the end of the experiment.The control group was injected with corn oil daily.The model of bile duct ligation?BDL?:kunming mice were randomly divided into three groups:Sham operation group?Sham?,model group?BDL?,and LHRD group?LHRD?.Both BDL and LHRD groups were treated with bilateral bile duct ligation for 15 days.The LHRD group was treated with the drug in advance for 2 weeks before BDL,and after BDL,the drug treatment continued to be performed on the next day until the end of the experiment.The Sham group only exposed bile duct,not ligatured.2.Materials and sample processing:24 h after the establishment of animal model experiment,blood samples were collected from the eye socket in mice and the serum was collected by centrifugation for follow-up experiments.The liver tissue was harvested and divided into two parts,one of which was used for morphological analysis after dehydration and embedding,another which was cryopreserved for use on extract protein,RNA and other experiments.3.To determine the therapeutic effect of LHRD on liver fibrosis,the serum ALT and AST activity were measured,and HE,Sirius red staining,immunohistochemical,and western blot were performed to observe the protein expression of liver fibrosis index.The level of RNA were also detected by fluorescence quantitative PCR.4.Explore the potential molecular mechanism of anti-liver fibrosis of LHRD by detecting the changes of NF?B pathway,NOX4,ROS etc.Result:1.The results of serological indicators showed that ALT and AST in the CCl₄ group or the BDL group were significantly increased,indicating that both models of liver damage had been formed.Compared with the model group,ALT and AST in LHRD group of the two models were significantly reduced,indicating that the Longhu Rendan could effectively resist liver damage caused by two different models.2.HE and Sirius Red results showed:Compared with the model group,the necrosis of liver cells and inflammatory cells infiltration in LHRD group decreased significantly.In the model group,the collagen fiber around the central vein form false flocculus,while false flocculus in LHRD group has almost disappeared,collagen fiber bridging between the manifold obviously improved.The activation of hepatic stellate cell is considered to be the central link of liver fibrosis by immunohistochemistry and western blot detection the expression of stellate cell activation markers of?-SMA,in order to further illustrate the Longhu Rendan effect on the treatment of liver fibrosis,the results show that the expression of?-SMA LHRD group was obviously lower than model group,by fluorescence quantitative PCR detection and stellate cell activation related molecular RNA level changes,also found that Longhu Rendan can obviously reduce the activation of stellate cells,improve liver fibrosis.3.The expression of CD68 in the liver macrophages was detected by immunohistochemistry,and in the LHRD group it was significantly reduced compared with that in the model group,which suggested that LHRD anti-hepatic fibrosis may be associated with anti-inflammation.Then we examined the classical inflammatory pathway named NF?B by observing the phosphorylation of p-65.The results showed that Longhu Rendan significantly inhibits the phosphorylation of NF and B p-65 in hepatic fibrosis mice,and then reduces the development of inflammation in mouse and inhibiting liver fibrosis.The changes of oxidative stress were observed by ROS staining.Compared with the model group,the amount of ROS in LHRD group was significantly reduced.Then the changes of NOX4 and 4-HNE in upstream are detected and the results are consistent with the above results,which indicated that LHRD can improve liver fibrosis by reducing oxidative stress.Conclusion:LHRD alleviates liver fibrosis significantly,and its mechanism may be related to reducing inflammation and oxidative stress in liver fibrosis.