Exploring The Mechanism Of Astragalus Propinquus Schischkin And Panax Notoginseng (A&P) In The Treatment Of Renal Fibrosis In Chronic Kidney Disease Based On Network Pharmacology And Experimental Verification

Objective:To explore the potential molecular mechanism of Astragalus propinquus Schischkin and Panax notoginseng(A&P)on chronic kidney disease based on network pharmacology.Astragalus propinquus Schischkin and Panax notoginseng is a compound preparation made under the guidance of traditional Chinese medicine theory.It is composed of Astragalus,Panax notoginseng,Achyranthes bidentata,Laminaria and other drugs.It has been widely used in clinical practice for many years to treat chronic kidney disease and renal fibrosis and has remarkable curative effect.However,the specific mechanism of improving CKD is unclear.Therefore,it is necessary to explore its effective pharmacodynamic components and action mechanism through advanced technical means.Methods:1.Network pharmacology and molecular docking:1)Using the TCMSP database,the active ingredients of Astragalus notoginseng mixture were screened according to oral bioavailability(OB)and drug-like properties(DL)(conditional parameters:OB≥30&DL≥0.18).Predict the target of each active ingredient on the Swiss Target Prediction database to obtain drug-related targets.Through the mining of CKD targets,the two target data sets were analyzed to find the intersection targets,and differential gene analysis was performed to obtain the core genes.Corresponding components were used as active ingredients and the control network of traditional Chinese medicine compound prescriptions was constructed using Cytoscape 3.6 software.Find important active ingredients and targets by analyzing the active ingredient-target network Degree parameter.GO/KEGG enrichment analysis was performed to find out the potential molecules of Astragalus Sanqi mixture in the treatment of chronic kidney disease.2)Molecular docking technology:Use molecular docking technology to dock a target with a high degree value and an active ingredient,predict and visualize the binding performance of the target and the active ingredient.2.Animal experiment verification:The unilateral ureteral ligation(UUO)mouse animal model was constructed as a CKD disease model,and divided into sham operation group,UUO group,and A&P low group(1972 mg/kg/day,added to the experimental diet)and A&P high-dose group(3943 mg/kg/day,added to the experimental diet).The renal pathological damage and fibrosis were observed by HE and Masson staining,the expression of renal α-SMA was detected by immunohistochemistry,and the protein expressions of heat shock transcription factor 1(HSF1)and RELA proto-oncogene(RELA)were detected by western blotting.The mRNA expressions of α-SMA,heat shock transcription factor 1(HSF1)and RELA proto-oncogene(RELA)were detected by real-time quantitative PCR.Results:1.Network pharmacology and molecular docking technology:1)The results of network pharmacology showed that a total of 37 active ingredients were found,including 15 Astragalus,11 Achyranthes,6 kelp,4 Notoginseng and 1 Angelica.In the Swiss Target Prediction database,target predictions were made for each active ingredient,and 582 targets of Astragalus Sanqi mixture were finally obtained.The NCBI GEO database was searched with "CKD" as the keyword(conditional parameter:gene expression profile microarray data,the number of samples in a single group was greater than 20),and a total of 2 sets of chips were obtained;the differential gene annotation was performed by the R language "Limma" package to obtain the difference Genes,and their co-expression profiles were obtained,and then the detected redundant and messy disease-related target information was eliminated,and finally 1546 disease-related targets were obtained.Take the target of Astragalus Notoginseng mixture and the common target of the two chips as an intersection to build a drug-target-disease molecular network,and obtain 98 common targets;further,by strengthening the screening criteria for targets(conditional parameters:|logFC|>1&p-vaule<0.05),24 differential genes were obtained,namely HSF1,HSP90AB1,HDAC5,CA12,CD81,MYLK,BCL2,BCAT2,PRKCD,ICMT,CDK4,PTPRF,FAAH,HSD11B2,CAPN1,CSNK2A2,GRK6,AKR1B1,PREP,CYP1B1,IGF1R,ERCC5,RELA,MAP2K1,among which HSF1 and RELA were most differentially expressed.Considering the central roles of heat shock transcription factor 1(HSF1)and RELA proto-oncogene(RELA)in gene regulatory networks,they are considered as hub genes.GO enrichment analysis confirmed that the biological processes mainly include the response of cells to oxidative stress,the response to reactive oxygen species,the regulation of reactive oxygen species metabolism,and the response to toxic substances;cellular components include transcription factor complexes,histone deacetylases Complexes,nuclear chromatin;molecular functions involve protein kinase activity,transcription factor activity,chromatin DNA binding protein phosphatase binding,etc.The results of KEGG analysis showed that the signaling pathway of Huangqi Sanqi mixture in the treatment of renal fibrosis(RF)and CKD involved PI3K-Akt,endocrine resistance,and EGFR tyrosine kinase inhibitor resistance.2)Molecular docking:In order to further explain the effect of active ingredients in A&P on HSF1 and RELA,we carried out molecular docking analysis,and the results showed that the minimum binding energies of the core active ingredients and key target genes HSF1 and RELA were mostly lower than-5kcal/mol,indicating that they have strong binding activity and can build a stable binding conformation.2.Animal experiment verification:The mechanism of action of key genes was further verified by establishing unilateral ureteral ligation(UUO)to induce RF in a rodent model.By observing the results of HE staining of tissue sections,it was found that A&P could improve some renal pathological damage caused by UUO;Masson staining and immunohistochemical results showed that the protein and mRNA levels of fibrosis-related index a-SMA were significantly down-regulated after A&P intervention,indicating that It significantly reduces fiber deposition caused by UUO.In addition,the expressions of heat shock transcription factor 1(HSF1)and RELA proto-oncogene(RELA)were significantly up-regulated at both mRNA and protein levels in the UUO model,but their expressions were significantly reversed after A&P intervention.Conclusion:This paper partially reveals the potential molecular mechanism of Astragalus propinquus Schischkin and Panax notoginseng in the treatment of CKD through the combination of traditional Chinese medicine compound network pharmacology and molecular docking methods combined with experimental verification.Astragalus propinquus Schischkin and Panax notoginseng may act on HSF1,RELA and PI3K-Akt signaling pathways through active components such as berberine,coptisine,wogonin,ginsenoside rh2,biphenyl ester,formononetin,calyxine,and kaempferol.Regulates the biological processes such as fibroblast aggregation,renal tubular epithelial cell apoptosis and immune cell infiltration caused by renal fibrosis in chronic kidney disease,and Astragalus propinquus Schischkin and Panax notoginseng in the treatment of chronic kidney disease has the characteristics of multi-target,multi-path and multi-pathway.This provides a basis for further understanding of the mechanism of Astragalus propinquus Schischkin and Panax notoginseng in the treatment of renal fibrosis and chronic kidney disease.