Analysis And Experimental Verification Of Panax Notoginseng Saponins Improving RPE Damage Based On Network Pharmacology Combined With GEO Gene Chip

Objective The potential molecular mechanism of PNS against age-related macular degeneration(AMD)was explored by using online pharmacology and GEO database.The anti-apoptosis ability of Panax Notoginseng saponins on retinal pigment epithelium(RPE)cells damaged by sodium iodate(SI)and its protective effect on retinal injury were verified by biological experiments.Method 1、Bioinformatics analysis: Through searching three drug target databases,such as TCMSP and Swiss Target Prediction,we can obtain the information of the main active ingredients and the target of action of PNS.AMD disease targets are obtained from four GEO gene chips combined with Gene Cards and OMIM disease databases.Venny2.1.0 was used to obtain the intersection Wayne diagram and common target of PNS and AMD,and STRING(11.0)was used to construct protein-protein interaction network.Filter core targets with the help of network topology.Use the R language related installation package to conduct GO and KEGG enrichment analysis,and establish a "drug component target disease pathway" network.The binding of the main compounds to the target gene protein was verified by molecular docking.2.Experimental verification: In vitro experiments,human retinal pigment epithelial(RPE)cells were selected as oxidative stress models induced by SI,and the experiments were set as control(Control)group,model(SI)group,and treatment(PNS+SI)group.Cell viability was detected by MTT assay.DCFH-DA measures the level of reactive oxygen species(ROS).Changes in the membrane potential of cell mitochondria were detected by JC-1.Hoechst 33342 staining was used to detect cell apoptosis.Western blot was used to detect the expression changes of Bcl-2,Bax,cleaved caspase-3 and related proteins in PI3K/AKt-P53 pathway in RPE cells.For in vivo experiments,15 male C57BL/6J mice were randomly divided into three groups: control(Control)group,model(SI)group,and treatment(PNS+SI)group.The treatment group was treated with 25 mg/ml PNS,and the SI group was treated with the same dose of 0.1% DMSO normal saline.One week later,both the SI group and the treatment group were injected with SI in the tail of the rats to establish the model,and the administration was continued according to the original plan,4 One week later,retinal thickness was measured by OCT.After the mice were sacrificed,retinal tissue sections were taken and stained with hematoxylin-eosin(HE)to observe the structural changes of each layer of the retina.Result 1、Five active ingredients of panax notoginseng saponins were obtained.According to the screening criteria of ADME,it is speculated that Ginsenoside Rd may be the main component that plays a role in the efficacy of panax notoginseng saponins during oral administration;There are 85 cross targets of drug PNS and disease AMD,among which AKT1,CASP3,INS,ALB,TP53,etc.may be potential therapeutic targets;GO and KEGG analysis showed that PNS may play a role in antagonizing AMD by regulating biological processes such as oxidative stress response,drug response,active oxygen metabolism process,apoptosis pathway,involving PI3K-AKT signal pathway and apoptosis signal pathway;Network relationship analysis shows that Ginsenoside Rb1 and Ginsenoside Re may play a leading role in the biomolecular mechanism of PNS to improve AMD;Molecular docking showed that the binding activity of the active component ligand and target protein receptor was strong.2、 MTT assay showed that when PNS concentration was 50-100 u M,it was non-toxic to ARPE-19 cells and had protective effect on SI-induced oxidative damage to ARPE-19 cells;DCFH-DA probe detected that PNS reduced the production of reactive oxygen species(ROS)in ARPE-19,thus inhibiting the oxidative stress induced by sodium iodate;JC-1 staining results showed that PNS could effectively improve ROS-induced mitochondrial membrane potential damage in RPE cells,and had a certain repair effect on mitochondrial function;Hoechst 33342 staining showed that PNS significantly reduced the apoptosis of ARPE-19 cells induced by SI;Western blot showed that PNS regulated the phosphorylation expression of Akt and the expression of downstream key apoptotic molecules,including reducing the ratio of Bax/BCL-2 and the expression of P53 and Cleared-Case-3;OCT and HE staining showed that PNS protected the retina by slowing down the progression of retinal degeneration and inhibiting the formation of lipid deposits on the RPE layer induced by SI.Conclusion Panax notoginseng saponins may partially inhibit sodium iodate-induced RPE cell apoptosis by regulating the PI3K/Akt-P53 apoptosis pathway,and at the same time,it can effectively protect the retina from damage.