Effect Of Danshiliuhao Granule On Hepatic Fibrosis In Rats Induced By Carbon Tetrachloride And Its Mechanism

Objective:The contents of three characteristic components in Danshiliuhao Granule?DSLHG?were determined for quality control.Based on the anti-hepatic fibrosis effect of DSLHG,the mechanisms of DSLHG anti-liver fibrosis were predicted and clarified by network pharmacology,and the main signaling pathway was explored.Methods:1.HPLC method was used to determine the contents of geniposide,polydatin and naringin in DSLHG.2.The animal model with liver fibrosis was induced by Carbon tetrachloride?CCl₄?,and the index levels of liver function and liver fibrosis in rats'serum were detected.Hematoxylin and eosin?H&E?staining and Masson trichromic staining were used to observe the inflammatory reaction and fibrosis of liver tissue.3.The active ingredients of DSLHG were screened from public databases,and the potential targets of these compounds were predicted through the PharmMapper server database.Furthermore,a compound-target network was constructed,and GO analyzed enrichment and relevant pathway enrichment were performed for the core targets to predict the possible signaling pathways.4.Western blot?WB?was used to test the expression levels of PI3K,p-PI3K,Akt,p-Akt,Caspase-3,cleaved Caspase-3,Bax,Bcl-2 protein,and immunohistochemical?IHC?was used for examining the expression levels of TGF-?1 and?-SMA in rat liver.Results:1.Methodological investigation showed that the established method of content determination was specific,simple and reliable.The contents of gardenoside,polydatin and naringin were 3.75 mg/g,2.66 mg/g,3.07 mg/g.2.DSLHG could significantly reduce the serum levels of ALT,AST,ALP,HA,LN,PIIINP,and Col IV,increase ALB level?P<0.01?,and significantly improve hepatic fibrosis.3.According to databases,108 main active ingredients were identified in the DSLHG formula,which were involved in reputation of 192 potential targets,86 of which were related to liver fibrosis.The results of network prediction and analysis showed that the anti-fibrosis effect of DSLHG mainly involves multiple biological processes such as cell proliferation,signaling transduction,and protein phosphorylation.Mechanistically,the anti-liver fibrosis effect of DSLHG was exerted by interfering with 47 signaling pathways,such as Rap1 signaling pathway,PI3K/Akt signaling pathway and FoxO signaling pathway,and the second-ranked PI3K/Akt signaling pathway may be a key pathway for DSLHG to play an anti-fibrotic role in the liver.4.Effects of DSLHG on PI3K/Akt signaling pathway and the expression of apoptosis-related proteins:?1?The results of WB showed that compared with normal control group,the levels of p-PI3K,p-Akt,cleaved Caspase-3/Caspase-3 and Bax/Bcl-2 in liver tissue of model group were significantly increased?P<0.01,P<0.05?.Compared with the model group,the positive control group?Silibinin?and DSLHG groups significantly reduced the levels of p-PI3K,p-Akt,cleaved Caspase-3/Caspase-3 and Bax/Bcl-2 in rat liver?P<0.01,P<0.05?.?2?IHC results showed that,the expression levels of TGF-?1 and?-SMA in model group significantly increased compared with normal control group?P<0.05,P<0.01?.DSLHG could effectively reduce the expression levels of TGF-?1 and?-SMA?P<0.05?.Conclusion:1.The established detection method was simple,reliable,and good repeatability for the determination of contents.It could realize the quality control of Gardeniae Fructus,Polygoni Cuspidati Rhizoma Et Radix,and Aurantii Fructus,which could be used as one of DSLHG quality control methods.2.DSLHG had the effect on anti-liver fibrosis in rats of model,which could improve significantly liver function and alleviate the liver fibrosis.3.Network pharmacology predicted that DSLHG could exert anti-liver fibrosis effect through regulating multiple targets and multiple pathways,and the PI3K/Akt signaling pathway may be the key pathway.4.DSLHG could alleviate liver fibrosis in rats induced by CCl₄,and the mechanism may be related to TGF-?1/PI3K-Akt/Caspase-3 dependent apoptosis pathway.