Study On The Therapeutic Effect And Related Mechanism Of Qi-Tonifying Decoction In Rats With Hepatic Fibrosis Induced By CCL4

Background:Traditional Chinese medicine has great research value in anti-fibrosis.In our previous research,we found that Astragalus and its compound preparations have good effects.The main ingredients of the Qi-Tonifying Decoction used in this experiment are Astragalus and Atractylodes.Studies have shown that astragalus can effectively inhibit CCL4-induced hepatic fibrosis in rats.It also has a good effect on the liver’s microcirculation system and helps the regeneration of liver cells.Atractylodes macrocephala can maintain the integrity of liver tissue structure and improve liver antioxidant function.Astragalus and Atractylodes macrocephala are common representative drugs,which are mainly used to benefiting qi in clinical practice.They are often used in combination in traditional Chinese medicine.Although Astragalus and Atractylodes macrocephala can effectively inhibit the formation of liver fibrosis,the specific mechanism of action is not very clear.In this paper,some key targets were selected from the in vivo level to observe the anti-fibrosis effect and mechanism of Qi-Tonifying Decoction,so as to provide a basis for the treatment of hepatic fibrosis with Qi-Tonifying Decoction.Part 1 Study on the effect of Qi-Tonifying Decoction on inhibiting CCL4-induced hepatic fibrosis via MAPK pathway in Rats.Objective:Establish a rat liver fibrosis model caused by CCL4,and then use the Qi-Tonifying Decoction(Astragalus,Atractylodes macrocephala)for treatment.By detecting the expression of α-SMA,p-ERK1/2,p-p38,MMP-2,MMP-9,and TIMP-1 in rat liver tissues.We observed the therapeutic effect of Qi-Tonifying Decoction on liver fibrosis,and then determine its possible relationship.Methods:Purchase seventy6-week-old rats and feed in the laboratory for one week.Randomly choose fifty rats intraperitoneally injected with CCl4 corn oil solution(40%)and feed with the diet of high-fat and low-protein.Ten rats in group A were fed normally.During the model-building period,according to statistics,a total of 19 died,and 31 were left.Randomly opted three of them to verify the success of the model.The remaining model rats were grouped and numbered.10 rats in the liver fibrosis model group were group B,9 rats in the Qi-Tonifying Decoction group were group C,and 9 rats in the colchicine group were group D.They were given daily for 84 consecutive days.Volume gavage treatment.After 12 weeks,all were decapitated and put to death and fresh liver tissues were taken.Hepatic histopathology was observed by HE and Masson staining,through the method of the western blot to determined the variety of α-SMA,p-ERK1/2,and p-p38 protein,and the transformation of MMP-2 and MMP-9 and TIMP-1 was analysed by IHC.Results:From the results of HE staining,we found that the liver cells in the model group showed swelling and necrosis to varying degrees,and the hepatic sinusoids disappeared with extensive inflammatory cell infiltration;The infiltration of inflammatory cells in the groups of treatment was obvious decrease.The experiment of masson showed that there were abundant of collagen fibers aggregated in the liver tissue and divided into relatively obvious pseudolobules in model group,the degree of liver fibrosis in each drug intervention group is all effectively improved compared with the model group,.The liver tissue protein was detected by western blot,and it was found that the protein of α-SMA and p-ERK1/2 were higher in the model group than in the normal group,and the content of α-SMA and p-p38 in group C and group D less obvious contrast to group B,the content of p-ERK1/2protein in group D was decreased,indicating that the Qi-Tonifying Decoction can inhibit α-SMA,P-ERK1/2 and p-p38 expression alleviate the process of liver fibrosis.Through the IHC,we fond that the content of the MMP-2 increased obviously.The content of MMP-2 in the Qi-Tonifying Decoction group and the colchicine group was up-regulated.MMP-9 was clearly observed in the group B.The content of MMP-9 in the Qi-Tonifying Decoction group was up-regulated contrasted to the group B,The content of TIPM-1 was fewer than group A,but augment obviously in the group B.The synthesis of TIPM-1 in the Qi-Tonifying Decoction group and the colchicine group was lower than that in the model group.Conclusion: The liver fibrosis degree of the liver fibrosis model rats was effectively improved and the occurrence and development of liver fibrosis were effectively inhibited.The expressions of α-SMA,p-ERK1/2,p-p38,MMP-2,MMP-9 and TIMP-1 were changed to different degrees,and colcoline had the best effect,followed by the group of Qi-Tonifying Decoction.In this study,by inhibiting the activation of ERK1/2 and P38 signaling pathways,Qi-Tonifying Decoction blocked the signal transmission to phosphorylated P-ERK1/2 and P-P38 in the nucleus,so as to suppress TIMP-1 production and have a boost in MMP-2 and MMP-9synthesis,increasing the rate of ECM decomposition,reducing deposition,and thus effectively promoting liver fibrosis.Part 2 Study on the effect of Qi-Tonifying Decoction on inhibiting CCL4 induced liver fibrosis in rats by regulating MCP-1a.Objectives: To observe the effect of Qi-Tonifying Decoction on the expression of monocyte chemokine MCP-1a in rat liver fibrosis model group induced by CCL4,and to investigate the anti-fibrosis effect and mechanism of Qi-Tonifying Decoction.Methods: The expression of MCP-1a gene in liver tissues was detected by Quantitative real-time PCR.Results: From the point of view of QPCR experiment results,the RNA content of MCP-1 in group B was obviously height,the content of group C and group D were significant decline contrasted to the model group(P<0.05),the difference is statistically significant.Conclusion: Qi-Tonifying Decoction may alleviate the progression of liver fibrosis by down-regulating the expression of MCP-1a.,suggesting that this cytokine is one of the targets of anti-fibrosis effect of qi-supplementing agent.