MiR-126-5p Mediates Hepatocyte Apoptosis In ACLF And The Intervention Mechanism Of Jiedu Huayu Ⅱ Decoction

Objective:1.To observe the expression of miR-126-5p in serum of normal people,patients with compensated cirrhosis and Acute-on-chronic liver failure(ACLF),and the effect of Jiedu Huayu II recipe on the expression of miR-126-5p in ACLF patients.2.To observe the expression of miR-126-5p and its related downstream factors Bcl-2,Bax and Caspase-3 in ACLF rats and the regulatory effect of Jiedu Huayu II recipe on the apoptosis pathway of miR-126-5p.Methods:1.Clinical study:(1)Ten normal people,10 patients diagnosed with compensated cirrhosis,and 36 patients diagnosed with ACLF were enrolled and divided into normal group,cirrhosis group,and ACLF group.The expression levels of miR-126-5p in each group were detected by real-time fluorescent quantitative PCR.(2)Thirty-six patients diagnosed with ACLF were randomly divided into control group and traditional Chinese medicine group by SPSS statistical software.Both groups were treated with basic comprehensive treatment of western medicine,and the traditional Chinese medicine group was treated with Jiedu Huayu II decoction granules orally on the basis of the control group for 8 weeks.The expression levels of miR-126-5p in the two groups were observed before and after treatment,and the liver function(total bilirubin,transaminase,albumin),coagulation function(PTA),TCM symptom score,and clinical comprehensive efficacy rate were recorded and analyzed before and after treatment.2.Experimental study: The liver cirrhosis model was established by carbon tetrachloride(CCL4).After the liver cirrhosis model was completed,the ACLF rat model was established by D-galactosamine(D-Gal N)combined with Lipopolysaccharide(LPS).The experiment was divided into liver cirrhosis group,model group and Jieduhuayu II decoction group.The degree of liver fibrosis and necrosis were observed by Masson staining and HE staining.The expressions of miR-126-5p,Bcl-2 m RNA,and Bax m RNA in liver tissue were detected by real-time fluorescence quantitative PCR.The expressions of Bcl-2,Bax,and Caspase-3 in liver tissue were detected by ELISA.The apoptosis rate of liver cells was detected by TUNEL fluorescence.The content of TBi L in serum was detected by automatic biochemical analyzer.Results:1.Clinical study results(1)Compared with the normal group,the expression of miR-126-5p in liver cirrhosis group and ACLF group was increased,and the difference was statistically significant(P<0.05).Compared with the cirrhosis group,the expression of miR-126-5p in the ACLF group was increased,and the difference was statistically significant(P<0.05).(2)After 8 weeks of treatment,the expression of miR-126-5p in the control group and the traditional Chinese medicine group was significantly decreased(P<0.05).The expression of miR-126-5p in the traditional Chinese medicine group was lower than that in the control group,and the difference was statistically significant(P<0.05).(3)After 8 weeks of treatment,ALT,AST and TBi L in the control group and the TCM group were significantly decreased,and PTA was significantly increased,and the differences were statistically significant(P<0.05).After treatment,the decrease degree of AST,ALT and TBi L and the increase degree of PTA in the traditional Chinese medicine group were better than those in the control group,and the difference was statistically significant(P<0.05).2.Experimental research results(1)In the liver cirrhosis group,hepatocytes were divided into limited cell clusters of different sizes by proliferating fibrous tissue,normal hepatic lobules were divided,obvious pseudolobules were formed,hepatocytes in the pseudolobules were arranged disorderly,chronic inflammatory cell infiltration was observed,and hepatocyte apoptosis,necrosis and regeneration in different periods were observed.Model group and Jiedu Huayu II Decoction group: On the basis of the pathological results of liver cirrhosis group,different degrees of hepatocyte necrosis were observed in both groups,with large areas,cell swelling or atrophy,nuclei could become large,binuclear,multinuclear and other forms,and hepatic sinusoid dilatation and congestion.However,compared with the model group,the area of liver necrosis and inflammatory infiltration in the Jiedu Huayu II decoction group were reduced.(2)Compared with the liver cirrhosis group,the expression levels of miR-126-5p and Bax m RNA in the model group and the Jieduhuayu II Decoction group were increased,the expression levels of Bcl-2 m RNA were decreased,the expression levels of Bax and Caspase-3 were increased,and Bcl-2 mrna was decreased,the differences were statistically significant(P<0.05).Compared with the model group,the expression of miR-126-5p and Bax m RNA in the Jieduhuayu II decoction group decreased,the expression of Bcl-2 m RNA increased,the expression of Bax and Caspase-3 decreased,and the expression of Bcl-2 increased,with statistically significant differences(P<0.05).(3)The effect of Jieduhuayu II Decoction on hepatocyte apoptosis in ACLF rats: In the detection of hepatocyte apoptosis,fluorescence staining results showed that compared with the liver cirrhosis group,the model group and Jiedu Huayu II decoction group had significantly increased apoptotic cells and apoptosis rate in liver tissue(P<0.05);Compared with the model group,the Jieduhuayu II decoction group had a significant reduction in the number of apoptotic cells in the liver tissue and a significant reduction in the apoptosis rate(P<0.05).Conclusions:1.The expression of miR-126-5p in ACLF patients is increased,which is related to TBil and PTA.miR-126-5p can promote the progression of ACLF.Jieduhuayu Ⅱ decoction can reduce the serum miR-126-5p level in ACLF patients,improve the liver function and coagulation function of patients,and improve the clinical efficacy.2.The expression of miR-126-5p in the liver tissue of ACLF rats is increased,which is positively correlated with the pro-apoptotic proteins Bax and Caspase-3,which can promote the apoptosis of hepatocytes in ACLF rats and participate in the disease process of ACLF.Jieduhuayu Ⅱ decoction can down-regulate miR-126-5p expression,reduce hepatocyte apoptosis in ACLF rats,and antagonize ACLF.