Study On The Anti Renal Fibrosis Mechanism Of Yishen Huoxue Recipe Regulating PI3K/AKT/HIF-1α Signal Pathway Based On Network Pharmacology

Objective1.To study the effective components and possible targets of Yishen Huoxue Recipe in the treatment of renal fibrosis(RIF)and to explore the mechanism of Yishen Huoxue Recipe in the treatment of RIF.2.Objective to investigate the effect of Yiqi Huoxue Method on the expression of PI3 K,Akt,HIF-1α,VEGF protein and the concentration of inflammatory factors IL-6 and TNF-α in rats with renal fibrosis.Methods1.Use Tcmsp to establish the compound library of seven traditional Chinese medicines(Astragalus membranaceus,Angelica sinensis,Salvia miltiorrhiza,rhubarb,safflower,Panax notoginseng and Achyranthes bidentata);Genecards database and OMIM database were used to predict the target of RIF;the interaction network of compound target of Yishen Huoxue Recipe and RIF disease target was constructed,and the go analysis and KEGG enrichment analysis of the target network were carried out through David website.Finally,the analysis results were visualized by Cytoscape software.2.Animal experiment: thirty six male SD rats(weighing 260 ± 20g)were randomly divided into six groups: sham operation group,model group,losartan group,low-dose Yishen Huoxue Recipe group,medium dose Yishen Huoxue Recipe group and high-dose Yishen Huoxue Recipe group,with 6rats in each group.After 3 days of adaptive feeding,rats in sham operation group were treated with unilateral ureteral ligation,rats in other five groups were treated with unilateral ureteral ligation,rats in different dosage groups of Yishen Huoxue Recipe and losartan group were treated with corresponding drugs by gavage,rats in sham operation group and model group were treated with normal saline by gavage for 14 days.The protein expression of VEGF,PI3 K,Akt and HIF-1 α in renal tissue was detected by Western blot,and the concentrations of serum inflammatory factors IL-6 and TNF-α were detected by ELISA.Results1 Network pharmacologyIn this study,105 compounds were screened by tcmsp compound analysis platform;188 target sites of Yishen Huoxue Formula compound RIF disease were found by GENECARD,OMIM and other target prediction platforms for protein interaction analysis.It can be seen that protein kinase related receptors(AKT1,MAPK1,MAPK 8,JUN,etc.)and inflammation related receptors(IL-6,IL-1B,TNF,etc.),growth factor related receptors(VEGFA,EGFR,etc.).According to go analysis,these core targets are mainly involved in steroid hormone receptor activity,transcription factor binding catalytic function,protein kinase activity,cytokine activity and other molecular functions,as well as regulation of inflammatory chemokines,production of interleukin-6,activation of epidermal growth factor and activation of MAPK activity by adrenergic receptor signaling pathway It may be through inhibiting the growth and differentiation of fibroblasts,inhibiting inflammatory chemotaxis,inhibiting inflammatory response,inhibiting apoptosis,anti oxidative stress and other multiple targets to exert the therapeutic effect on RIF.Cell signal pathways such as P13 K Akt,TNF,MAPK,HIF-1,VEGF,TLR and NF-κ B signal transduction pathway suggest that Yishen Huoxue recipe is an important pathway involved in renal fiber.Taking the core targets of IL-6,VEGF,MAPK,AKT1,EGFR and PTGS2 as the research vectors,the chemical components of Yishen Huoxue Formula were studied by molecular docking.The results showed that the chemical components of Yishen Huoxue Formula had good binding activity with the key targets.2.1 HE stainingCompared with the sham operation group,the renal parenchyma of the model group was severely atrophied and thinned,the number of glomeruli was reduced and atrophied,some renal tubules were cystic expansion,and interstitial fibrous tissue hyperplasia.Compared with the model group,the renal parenchyma of losartan group and Yishen Huoxue Decoction groups showed mild to moderate atrophy,the number of glomeruli atrophied and decreased,and some renal tubules showed cystic dilatation.The degree of fibrosis was reduced.2.2 Masson stainingCompared with the sham operation group,the blue staining of collagen fibers increased,the formation of interstitial collagen fibers was obvious,and the tubular basement membrane and the fibrous tissue around the tubule proliferated significantly in the model group.Compared with the model group,the blue staining of collagen fibers in the obstructed kidney tissue of losartan group and Yishen Huoxue Decoction groups decreased,and the tubule basement membrane and surrounding fibrous tissue showed moderate hyperplasia.2.3 PI3 K,Akt,HIF-1 α,VEGF protein expressionOn the 14 th day,the protein expressions of PI3 K,Akt,HIF-1 α and VEGF in the model group were higher than those in the sham operation group(P < 0.05).The protein expressions of PI3 K,Akt,HIF-1 α and VEGF in Yishen Huoxue Decoction groups and losartan group were lower than those in model group,and the comparison between groups was statistically significant(P < 0.05).The protein expressions of PI3 K,Akt,HIF-1 α and VEGF in high-dose Yishen Huoxue Decoction group and losartan group were similar,but there was no significant difference between the two groups(P > 0.05).2.4 Concentration of IL-6 and TNF-αOn the 14 th day,the serum concentrations of IL-6 and TNF-α in the model group were higher than those in the sham operation group(P < 0.05).Compared with the model group,the levels of IL-6 and TNF-α in serum of all treatment groups decreased in varying degrees(P<0.05).The levels of IL-6and TNF-α in serum of high-dose Yishen Huoxue Recipe group and losartan group were significantly lower than those of low-dose and medium dose Yishen Huoxue Recipe group(P<0.05).There was no significant difference in the levels of IL-6 and TNF-α between high-dose Yishen Huoxue Recipe group and losartan group(P >0.05).Conclusions1.Network pharmacology conclusion shows that Yishen Huoxue recipe may delay the development of RIF by participating in the biological processes such as inflammatory reaction,oxidative stress,growth and differentiation of vascular endothelial factor,immune response,regulating P13K/Akt,HIF-1α,VEGF,MAPK,NF-κ B and other signaling pathways.2.Experimental studies have proved that Yishen Huoxue recipe can inhibit the expression of PI3 K,Akt,HIF-1 α and VEGF protein in rats with renal fibrosis,reduce the concentration of serum inflammatory factors IL-6and TNF-α,and its mechanism may be that Yishen Huoxue recipe can inhibit the P13K/Akt/HIF-1α signal transduction pathway,inhibit the inflammatory response,inhibit the disordered proliferation of microvessels under the condition of ischemia and hypoxia,so as to play an anti RIF role.