Sini San Ameliorates Liver Fibrosis By Inhibiting AKT-mediated Hepatocyte Apoptosis

Objective:This study aimed to evaluate the ameliorative effect of SNS on carbon tetrachloride（CCl₄）-induced liver fibrosis in mice and the underlying mechanisms.Methods:Part onePharmacodynamic study and preliminary mechanism of Sini San on liver fibrosis induced by CCl₄in miceThe mice were injected intraperitoneally with CCl₄（0.5 m L/kg）thrice weekly for 6weeks to establish a liver fibrosis model.Starting from the 3rd week,the mice were gavaged with different doses of SNS（6.2,3.1,and 1.5 g/kg）daily.The experiment was divided into a normal control group,model group and SNS（6.2,3.1,and 1.5 g/kg）groups.Observe the general conditions of mice,measure the weight of mice once a week,and set up mice in the 6th week to collect serum and liver tissue.Detecting the serum biochemical indicators of rats AST,ALT and T-BIL.The pathological characteristics of the liver were observed using hematoxylin and eosin（HE）and Masson’s staining.Hepatocyte apoptosis was assessed using terminal deoxynucleotidyl transferase d UTP nick end labeling（TUNEL）staining.The m RNA levels of Col1α1,Col1α2,Acta2,Timp1 and Timp2 were detected by RT-qPCR;Hepatocyte apoptosis was assessed using terminal deoxynucleotidyl transferase d UTP nick end labeling（TUNEL）staining;The proteins expression of p-PI3K,PI3K,p-AKT,AKT,FXR,Caspase-8,Bax and Bcl-2 were detected by Western blot;FXR m RNA levels were detected by quantitative real-time reverse transcription-polymerase chain reaction（RT-qPCR）and immunofluorescence.Part twoComponent screening and mechanism verification of SNS to improve apoptosis by inhibiting AKTThrough network pharmacology and bioinformatics,the active components,targets and disease targets of SNS were found,and the material basis of SNS to regulate liver fibrosis was found by constructing component-target-disease network interaction maps,and on this basis,the degree of binding between the active components of SNS and the core targets of further inflammation was carried out through molecular docking.Hep G2cell lines were cultured in vitro,the CCK-8 method screened the optimal effective time and concentration of stimulation factor TNF-αintervention in Hep G2 cells,and AKT inhibitor MK-2206 was added,which was divided into Control group,TNF-αgroup,Control+MK-2206 group,TNF-α+MK-2206 group,apoptosis rate was detected by flow cytometry,and Western blot was used to detect p-PI3K,PI3K,p-AKT,AKT,protein expression levels of FXR,Caspase-8,Bcl-2 and Bax;RT-qPCR and immunofluorescence detected FXR m RNA levels,and the optimal administration concentrations of quercetin,kaempferol and isorhamnetin were screened by CCK-8.Control group,TNF-αgroup,TNF-α+quercetin group,TNF-α+kaempferol group,TNF-α+isorhamnetin group were randomly established.Control group,TNF-αgroup,TNF-α+5%drug-containing serum group,TNF-α+10%drug-containing serum group,TNF-α+15%drug-containing serum group,the protein expression of AKT,P-AKT and FXR was detected by Western blot,and the best active ingredient and drug-containing serum concentration were screened.Control group,TNF-αgroup,TNF-α+15%drug-containing serum group,TNF-α+isorhamnetin group,TNF-α+15%drug-containing serum group+MK-2206 group,TNF-α+isorhamnetin group+MK-2206 group were randomly set up,and the protein expression levels of AKT,p-AKT,FXR,Caspase-8,Bax and Bcl-2 were detected by Western blot.Results:Part onePharmacodynamic study and preliminary mechanism of Sini San on liver fibrosis induced by CCl₄in mice1.The CCl₄induced mouse liver fibrosis model showed a significant decrease in body weight compared to the normal group（P<0.05）.After receiving SNS treatment,the body weight significantly increased compared to the model group（P<0.05）2.The serum biochemical levels of mice were detected by a fully automated biochemical analyzer,and it was found that the serum AST,ALT,and T-BIL levels in the model group were significantly increased（P<0.01）.Compared with the model group,the serum AST,ALT,and T-BIL levels in the SNS intervention groups were significantly reduced,and the AST,ALT,and T-BIL levels in the SNS（6.2/3.1g/kg）dose group were also significantly reduced（P<0.01）.3.According to the results of liver pathology,HE staining showed that the model group had a large number of inflammatory cells infiltrate,the liver tissue structure was destroyed,the liver cell necrosis,pseudolobular formation,SNS treatment can reduce the inflammatory cell infiltration,restore the liver tissue and hepatocytes structure,and improve the formation of pseudolobules.Masson’s results also showed that SNS reduced the expression of CCl₄-induced collagen fibers and improved the formation of pseudolobules and fiber septas.4.Western blot results showed that TNF-αThe expression of p-PI3K/PI3K,p-AKT/AKT ratio,and Caspase-8 was significantly increased in the group,while the expression of Bcl-2/Bax ratio and FXR was significantly reduced（P<0.05）.After the addition of AKT inhibitor MK-2206,the expression of p-PI3K/PI3K,p-AKT/AKT ratio,and Caspase-8 was significantly decreased,while the expression of Bcl-2/Bax ratio and FXR was significantly increased（P<0.05）.5.TUNEL staining results showed that a large number of TUNEL-positive cells appeared in the model group,and the apoptosis index increased,in addition,we found that TUNEL-positive cells decreased and hepatocyte apoptosis decreased after SNS administration.6.Western blot results showed that in the model group,p-PI3K/PI3K,p-AKT/AKT ratio,and Caspase-8 expression were significantly increased,while Bcl-2/Bax ratio and FXR expression were significantly reduced（P<0.05）.After SNS administration intervention,p-PI3K/PI3K,p-AKT/AKT ratio,and Caspase-8 expression were significantly decreased,while Bcl-2/Bax ratio and FXR expression were significantly increased.The difference in SNS（6.2g/kg）dose group was statistically significant（P<0.05）.7.The RT-qPCR results showed a significant decrease in FXR m RNA levels in the model group（P<0.05）,and a significant increase in FXR m RNA levels in each SNS treatment group.Among them,the SNS（6.2 g/kg）dose group had the most significant increase in FXR m RNA levels,with a statistically significant difference（P<0.05）.8.The immunofluorescence results showed that the expression of FXR in the model group was significantly reduced,while the expression of FXR in the SNS administration group was significantly increased compared to the model group.Part twoComponent screening and mechanism verification of SNS to improve apoptosis by inhibiting AKT1.Through network pharmacology screening,36 intersection genes between SNS and liver fibrosis were identified,and it was found that AKT1 and FXR targets may play an important role in the anti liver fibrosis process of SNS;Among all active ingredients,ranked by Degree value,the top three are quercetin,kaempferol,and isorhamnetin,with Degree values of 103,103,102,which may be the main active ingredients for SNS in preventing and treating liver fibrosis.The molecular docking of quercetin,kaempferol and isorhamnetin with AKT1 target showed that the binding energy was-7.8 kcal/mol,-8.3 kcal/mol,and-7.5 kcal/mol,respectively,with good binding.2.Using the CCK-8 method,actinomycin D（0.2μM）/TNF-α,The most suitable intervention time and concentration is to induce liver cell apoptosis for 12 hours（20ng/ml）;When the administration concentration of quercetin,kaempferol,and isorhamnetin is 10μM,the cell vitality reaches its optimal state.3.Flow cytometry results showed that apoptosis increased after TNF-αintervention（P<0.01）,and the apoptosis rate decreased significantly after the addition of AKT inhibitor MK-2206（P<0.01）。4.Western blot results showed that the expression of p-PI3K/PI3K,p-AKT/AKT and Caspase-8 in TNF-αgroup was significantly increased,the Bcl-2/Bax ratio was reduced and the expression of FXR was significantly reduced（P<0.05）,and after the intervention of AKT inhibitor MK-2206,the expression of p-PI3K/PI3K,p-AKT/AKT and Caspase-8 decreased significantly,the Bcl-2/Bax ratio and the expression of FXR increased significantly（P<0.05）.5.RT-qPCR results showed that the m RNA level of FXR in the TNF-αgroup decreased significantly（P<0.05）,while the m RNA level of FXR increased significantly after AKT inhibition（P<0.05）.6.Immunofluorescence results showed that the protein expression level of FXR in the TNF-αgroup was significantly reduced,while the protein expression of FXR was significantly increased compared with the model group after the addition of AKT inhibitors.7.By detecting the protein levels of AKT,p-AKT and FXR by Western blot,we found that the p-AKT/AKT ratio in the TNF-αgroup was significantly increased,and the expression of FXR was significantly reduced（P<0.05）,and by comparing the effects of quercetin,kaempferol and isorhamnetin on p-AKT and FXR,it was found that isorhamnetin acted on p-AKT and FXR the most significant,and the difference was statistically significant（P<0.05）;Comparing the three different concentrations of drug-containing serum at 5%,10%and 15%,we found that 15%drug-containing serum had the most significant inhibitory effect on apoptosis（P<0.05）.8.The expression of p-PI3K/PI3K,p-AKT/AKT and Caspase-8 in TNF-αgroup was significantly increased,the Bcl-2/Bax ratio was reduced and the expression of FXR was significantly reduced（P<0.05）,and p-PI3K/PI3K,p-AKT/AKT ratio and Caspase-8expression decreased significantly,Bcl-2/Bax ratio decreased,and FXR expression level increased significantly（P<0.05）.Conclusion:1.SNS can improve CCl₄-induced liver fibrosis in mice,and may improve hepatocyte apoptosis through the PI3K/AKT signaling pathway.2.SNS may be inhibiting the expression of AKT targets through its active ingredient isorhamnetin,increasing FXR expression,inhibiting apoptosis of hepatocyte,and preventing liver fibrosis.