Establishment Of Danhongqing Compound Formula And Pharmacodynamic Mechanism Of Anti-hepatic Fibrosis

Objective:Under the guidance of TCM theory,the function of liver fibrosis in link clear several herbal ingredients composition of compound Chinese medicine,to make it both has the traditional Chinese medicine compound ingredients targets more play to the characteristics of the efficacy,and make up for the traditional Chinese medicine compound is difficult to conduct mechanism in vitro studies,to research and can clarify mechanism have quality assurance of traditional Chinese medicine compound as a useful exploration.Methods:1.Screening the best compatible dose of four components by uniform design:Salvianolic acid B,salidroside,artesunate and resveratrol were used as the research objects and CCl₄-induced liver fibrosis model in mice was established by intraperitoneal injection for 6 weeks and administration from the second week.Then"four factors and eight levels"grouping design was adopted by uniform design method.Hyp content in liver tissue of mice was used as screening index,and the optimal effective dose was obtained by NOMEN?Non-Linear Mixed-Effect Modeling?analysis with DAS 3.0software.2.Screening the best compatible dose of three components by uniform design:Salvianolic acid B,salidroside,and artesunate were studied and CCl₄-induced liver fibrosis model in mice was established by intraperitoneal injection for 6 weeks and administration from the second week.Then"three factors and eight levels"grouping design was adopted by uniform design method.Hyp content in liver tissue of mice was used as main screening index.AST,ALT and collagen area were the assistant screening indexes.The optimal effective dose was obtained by NOMEN?Non-Linear Mixed-Effect Modeling?analysis with DAS 3.0 software.3.The validation of compound Chinese herbal medicinal ingredients on CCl4-induced hepatic fibrosis in mice:Basedon the results of three components screened,with Fuzheng Huayu extract as the control,different components and dosages were added or subtracted on the basis of the best compatible components.The groups were designed to observe the changes of serum hepatic function and Hyp content of liver tissue,liver histopathology as well as collagen fibers in CCl₄ mice,and the optimum formula were verified to determine the final optimum compatibility,called Danhongqing formula.4.The validation of compound Chinese herbal medicinal ingredients on BDL-induced hepatic fibrosis in rats:Based on the results of the third part,Danhongqing formula was used as the object of observation and Fuzheng Huayu Extract as the control.The changes of serum liver function,Hyp content of liver tissue,liver histopathology and collagen fibers in BDL rats were observe and the anti-hepatic fibrosis effect of Danhongqing formula was verified.5.Inhibition of HSCs activation,migration and contraction by Danhongqing formula:?1?HSCs were treated with Danhongqing formula?Salvianolic acid B 10?M,salidroside 10?M,artesunate 50?M?for 24 hours,incubated for 12 hours,and treated with TGF-?1?10ng/ml?for 12 hours.The effects of Danhongqing formula on the activation and proliferation of HSCs were observed.Western blot was used for protein detection and RT-PCR was used for RNA detection.?2?LX-2 were treated by the scratch assay,then incubated with Danhongqing formula for 8 hours,meanwhile treated with platelet-derived growth factor-BB?10ng/ml?,using Image J to analyze the migration area to observe the effect of LX-2 migration.?3?JS 1 were inoculated in the collagen gel for24h,and incubated with Danhongqing formula for 30 minutes,then induced by 10 n M ET-1 for 12h,using Image J to analyze the contraction area ratio to study the effect of Danhongqing formula on inhibiting contraction of JS 1.6.Transcriptome sequencing experiment:The library was prerared by KAPA RNA Hyper Prep Kit with Ribo Erase?HMR?for Illumina?to detect the gene changes of Danhongqing formula on anti-CCl₄ mice.The results were mapped to the latest UCSC transcripts by Bowite 2.1.0.and the gene expression level was estimated by RSEM software.Finally,the main differential genes were analyzed by GO function enrichment and KEGG enrichment.Results:1.?1?The combination of salvianolic acid B,salidroside,artesunate and resveratrol at different doses could reduce the Hyp content,the activity of ALT,AST and collagen deposition area in liver fibrosis model mice to different degrees and play an anti-fibrosis role?P?0.05?.?2?The results of digital model analysis showed that the optimal formula was salidroside+artesunate+resveratrol,and the compatible dosage was salidroside 474 mg·kg^-1,artesunate 62 mg·kg^-1 and resveratrol 319 mg·kg^-1,but there was no interaction between drugs.2.The optimal formula was salidroside+artesunate according to the analysis of the digital model of the changes in the degree of liver fibrosis.Considering the actual effect of salvianolic acid B on hepatic fibrosis,three components of salvianolic acid B,salidroside and artesunate were finally determined.3.The validation experiment shows that each ingredient in compound prescriptions have remarkable curative effect,among which the three ingredients in group B have relatively prominent anti-hepatic fibrosis effect,and are not inferior to Fuzheng Huayu Formula,which is named Danhongqing Formula.4.To evaluate the pharmacodynamics of Danhongqing Formula in treating hepatic fibrosis in BDL rats comprehensively,the pharmacodynamics of Danhongqing compound is relatively prominent?P<0.05?,which is not inferior to Fuzheng Huayu Formula and any other ingredients.5.Danhongqing Formula inhibits HSC activation and proliferation induced by TGF?1?P<0.05?,inhibits HSC migration and activation induced by PDGF?P<0.05?,and inhibits contraction of JS1 induced by ET-1?P<0.05?.6.Danhongqing Formula can significantly change the gene differences in liver tissues of mice with liver fibrosis,which are mainly concentrated in four signal pathways related to tissue metabolism.Conclusions:1.Danhongqing Formula of specific proportion dose of salvianolic acid B,salidroside and artesunate was obtained by uniform design of animal experiment and pharmacodynamic index evaluation.2.Danhongqing Formula was veritied to have the effect of anti-liver fibrosis by two animal models,CCl₄ and BDL.Comprehensive evaluation of its efficacy is relatively prominent than its components,and is similar to fuzheng huayu formula.3.Danhongqing Formula can retain the biological function of each component of the formula,which inhibits HSC activation induced by TGF-?1,proliferation,migration induced by PDGF and contraction induced by ET-1.4.Danhongqing Formula can significantly change the gene differences in liver tissues of mice with liver fibrosis,which are mainly concentrated in four signal pathways related to tissue metabolism.