Bioinformatics And Experimental Studies Of Kinado In The Treatment Of NAION Through The The VEGFA-PI3K/AKT Signaling Pathway

Objective: To study the potential mechanism of Ginkgo biloba in the treatment of non-arteritic ischemic optic neuropathy(NAION)based on network pharmacology and molecular docking.The in vitro experimental study was carriedout by using the prediction target of Bioinformation to explore the functional regulation mechanism of the Ginkgo biloba injection on RGC-5 cells under the condition of ischemia or hypoxia.Method:(1)Bioinformatics part : First,used the traditional Chinese medicine network database to collect the main chemical components and potential protein targets of Ginkgo biloba.Then used databases such as OMIM to search for NAION disease-related targets.Import the above targets into Venn software to obtain the intersection targets of the two.And imported the String database for protein interaction analysis and use the data results to import the Cytoscape-v3.8 database to construct a network diagram of medicinal materials-compounds-targets-pathways.And continue to use Cytoscape-v3.8.2 software and its plug-ins to screen out core targets.Used David database for bio-enrichment and signal pathway transduction analysis,and use R language to visualize it.Finally,using molecular docking software to perform molecular docking simulation to verify the degree of connection between the drug and the target molecule.(2)Experimental validation parts:(1)A cell ischemia and hypoxia model was established and the optimal concentration of Ginkgo biloba injection was screened:cobalt chloride hexahydrate medium containing 2% serum was used to induce ischemia and hypoxia in RGC-5 cells,and the CCK8 method was used to screen the half lethal concentration(IC50)and time of RGC-5 cells,and the optimal concentration of RGCs cells in the case of alleviation of ischemia and hypoxia by Ginkgo biloba injection.Therefore,the cells were divided into control group(group A),ischemic hypoxia group(group B)and ginkgo biloba injection treatment group(group C),and the mitochondrial membrane potential JC-1 fluorescence method was used in 6-well plates to observe the status of cells under ischemic and hypoxia conditions.PCR and Western blot were used to verify the expression of hypoxia-related gene hif-1α under hypoxic conditions;2)According to the first part of the network pharmacology part,the eight main targets of Ginkgo biloba treatment NAION VEGFA,JUN,EGF,RELA,APP,ESR1,FOS,MYC were screened out by PCR experiments,and the expression of these eight genes in RGC-5 cells under three group conditions was screened out by PCR experiments,and the genes with obvious differential expression were screened;3)The above screened genes were selected using the results of previous bioinformation enrichment analysis to select the corresponding signal pathways of the genes for verification by PCR and Western blot methods.Results:(1)Bioinformatics part : Screening showed that there are 20 main active ingredients of Ginkgo biloba for treatment,which are mainly quercetin,kaempferol,digital flavonoids,etc.and the corresponding 521 protein targets,and NAION has 756 targets.There are 26 targets in the intersection of the two,and the number of targets in the Hub set,12 in the MCC set,and 24 in the MCODE setare obtained through the Cytoscape-v3.8.2 plug-in.The target points of the 3 datasets are selected from the intersection and a total of 8 are selected.The core targets are mainly VEGFA,JUN,EGF,RELA,APP,ESR1,FOS,MYS.It mainlyinvolves cancer pathways,T cell receptor signaling pathways,PI3K-Akt signaling pathways,MAPK signaling pathways and other pathways,which play a therapeutic role in anti-inflammatory,antioxidant,immune regulation,and neuro-protection.(2)Experimental validation parts:1)After screening,RGC-5 cells containing 2% serum concentration of 300umol/L were incubated with RGC-5 cells for 24 hours(cell survival rate was 57.18%).100ug/ml of Ginkgo biloba injection was the optimal concentration for alleviating cell ischemia and hypoxia(cell survival rate was 79.32%,which was closest to the cell survival rate of the control group).And this experiment found that the injection with too high concentration not only could not alleviate the situation of cell ischemia and hypoxia,but also increased the apoptosis rate of cells.Mitochondrial membrane potential JC-1 fluorescence observation cells showed that the ratio of green light cells / red light cells containing green light cells(group B)treated with 2% serum cobalt chloride hexahydrate ischemia and hypoxia increased significantly,while the ratio could be reduced in the 100ug/ml Jinnado injection treatment group(group C).PCR verification showed that the expression rate of hypoxia factor hif-1 in group B was higher than that in group A and group,and the expression of hif-1 protein in group B was higher than that in group A and group C.2)After PCR verification,it was found that among the 8 core genes screened out by Biologics,only rela,vegf,and jun genes were expressed differently in group B and group C.The expression of rela gene in group B and C showed a gradual decline trend compared with the control group(group A).The expression of jun gene in group B showed a downward trend and an upward trend in group C(compared to the control group).The expression of vegfa gene in group B showed an increasing trend,and the expression in group C showed a trend similar to that of the control group.3)The selection of vegfa gene and its enriched pathway pi3k/akt pathway was verified by PCR and WB methods,and it was found that the expression of m RNA genes and proteins of pi3k/akt pathway in both groups B and C was similar to that of vegfa gene: expression increased in group B and decreased expression in group C.Conclusion: Through the use of network pharmacology and comprehensive analysis methods,a new idea is proposed for the mechanism of action of using the active ingredients of Ginkgo biloba in the treatment of NAION,which provides a bioinformatics basis for the experimental verification in the later stage.In this study,Ginkgo biloba extract injection can alleviate the damage caused by ischemia and hypoxia to RGC-5 cells,and the mechanism of action is related to reducing the activation and expression of pi3k/akt pathway protein under the action of VEGFA by reducing the expression of VEGFA gene.This lays a foundation for exploring the mechanism of NAION disease itself and exploring targeted treatment for NAION disease.