Study On The Mechanism Of Yiqi Huoxue Recipe Inhibiting Liver Fibrosis By Regulating Hippo-YAP1 Pathway

Objective:Liver fibrosis is a common pathological change of chronic hepatitis that could be caused by viral infection,drugs,alcohol and other causes,which is mainly manifested as abnormal proliferation and excessive deposition of hepatic extracellular matrix,and the key is the activation of hepatic stellate cells.Liver fibrosis is progressive and will progress to cirrhosis or even liver failure.Currently,there is still a lack of effective anti-hepatic fibrosis drugs in clinical practice.Our previous animal studies on liver fibrosis have found that the self-designed prescription for Yiqi Huoxue recipe(YQHX)has a clear therapeutic effect on liver fibrosis,but the mechanism remains to be further explored.Recently,a number of studies have shown that dysfunction of Hippo-YAP1 pathway is directly related to liver fibrosis.Therefore,our study explored the anti-fibrosis effect of key factors regulating Hippo-YAP1 pathway by yiqi huoxue recipe through animal experiments,in order to lay a theoretical foundation for the development of new anti-fibrosis drugs.Methods:Healthy male Wistar rats were selected and randomly divided into 3 groups: 1)control group(n=6),given normal diet and the same method of the same amount of cosolvent(intraperitoneal injection of olive oil instead of 30% CCl4,gastric saline instead of yiqi huoxue granule);2)the model group(n=6)was given a normal diet and intraperitoneal injection of 30% carbon tetrachloride(CCl4)olive oil solution with a dosage of 0.2ml/100g;and the model group was given saline solution twice a week;3)yiqi huoxue group(n=6),normal diet,with 30% CCl4 olive oil solution intraperitoneal injection,two times a week,and yiqi huoxue granule(3.4g/10ml/Kg/d)gavage,feeding 8 weeks after the death of the animals.Hematoxylin-eosin(HE)staining was used to observe the level of inflammation in liver tissue.The degree of liver fibrosis was observed by Masson trichrome staining.Immunohistochemistry was used to detect the downstream transcription factor YAP1(YAP1)and its target gene connective tissue growth factor(CTGF).Western blot was used to detect the expression of liver tissue-smooth muscle actin(α-SMA)and key factors of Hippo pathway,and real-time quantitative PCR was used to detect the expression of key target genes of Hippo pathway.One-way anova was used for statistical analysis,and LSD test was used for comparison between groups.P<0.05 meant that the difference was statistically significant.Results:1.Liver histopathological observation and the change of liverfibrosis-related indicators:The liver histopathological observation of each group showed that the hepatic lobule structure of the control group was normal,and the central venules of the CCl4 hepatic fibrosis model group were interconnected with fibrous septum,and the liver structure was deformed.The degree of hepatic fibrosis was obviously reduced in the intervention group.The protein expression of α-sma in CCl4 liver fibrosis model group was significantly increased,and the protein expression of α-sma in YQHX group was down-regulated.2.Changes in protein and mRNA expression levels of key genes inHippo-YAP1 pathway.2.1 Protein expression levels of key factors in the Hippo pathway in livertissues:Compared with the control group and the qi-invigorating and blood-activating group,the expression levels of YAP1 protein in the liver of the model group were significantly increased(P < 0.05),while the expression levels of phosphorylated YAP1 and the upstream key factor MST2 protein were significantly decreased(P < 0.05),both of which were statistically significant.There was no significant difference in the expression level of MST1 protein among the three groups.2.2 The mRNA expression levels of key factors in Hippo pathway in liver tissues:Compared with the control group,the liver YAP and TAZ mRNA expression level in the model group was significantly decreased(P < 0.001).YAP1 mRNA expression decreased in the model group compared with the intervention group(P < 0.001).However,there was no significant difference in the expression level of TAZ mRNA between the model group and the intervention group.The expression levels of CTGF and AREG mRNA in the liver of the model group were significantly higher than those of the control group and the intervention group(P < 0.001),while the expression levels of MST1 and LATS1 and LATS2 mRNA,the key upstream factors of YAP1/TAZ in the Hippo-YAP1 pathway were significantly lower than those of the control group and the intervention group(P < 0.05).There was no significant difference in liver MST2 mRNA expression among the three groups.3.Immunohistochemical analysis: the expression of YAP1 in the liver tissues of the model group was significantly higher than that of the normal group and the YQHX group(P < 0.05).In the model group,the upstream factors of YAP1,MST1 and LATS1,were significantly lower than those of the YQHX group(P < 0.05).The expression of YAP1 target gene CTGF in the model group was significantly higher than that in the control group and YQHX group(P < 0.05).Conclusions:1.Intraperitoneal injection of CCL4 can successfully establish rat liver fibrosis model.2.YQHX recipe can significantly improve the degree of liver fibrosis in rats,and at the same time down-regulate the expression of α-SMA3.YQHX recipe can affect the expression of MST1/MST2,up-regulate the expression of LATS1/LATS2,increase the phosphorylated YAP1 and down-regulate the activated YAP1,and ultimately inhibit the expression of downstream target genes of YAP1/TAZ,inhibit the Hippo-YAP1 pathway,and prevent the progression of fibrosis.Among them,the regulation of YAP1 protein may be regulated by a post-transcriptional mechanism and the mechanism of action of MST1 and MST2 remains to be studied.