Study On The Function And Mechanism Of Antihypoxia Effective Parts Of Gentiana Stramines

Objective:to screen the effective anti-hypoxia parts of Gentiana straminea and study the mechanism of the effective anti-hypoxia parts of Gentiana straminea.Methods:1.Using Hydrogen peroxide?H₂O₂?,sodium bisulfite?Na₂S₂O₄?and physical anoxia?three-gas incubator?to establish PC12 cell anoxia models,and the best modeling conditions of the three methods were found.2.Treatting PC12 cells at different extraction sites for 24h.using MTT assay to detect the cell activity and to screen the safe concentration range of drugs at different sites.3.PC12 cells were pretreated with safe concentration of drugs for 24h and then hypoxia was performed in H₂O₂,Na₂S₂O₄ and three-gas incubator respectively.After hypoxia treatment,cell viability was detected by MTT method,and the effective anti-hypoxia sites were screened.4.Mice were given the drug by gavage for 15 days,low-pressure oxygen chamber was used to simulate the hypoxia environment at an altitude of 10000m,and the survival rate of mice was calculated within 1h to screen the effective anti-hypoxia parts of Gentiana straminea.5.The physical hypoxia PC12 cell model was established,and Hoechst33258 staining was used to detect cell apoptosis after the intervention of effective parts of gentiana straminea,and the expression levels of hypoxia-related genes and proteins were detected by SOD,MDA and other indicators of oxidative stress,as well as by qPCR and Westren Blot with the kit.6.Network pharmacology was used to predict the antihypoxia material basis and mechanism of ethyl acetate of Gentiana straminea.Results and conclusions:1.The results of cytotoxicity studies in different sites showed that the safe concentration of drugs in ethyl acetate sites ranged from 0-600?g/mL,n-butanol sites from 0-200?g/mL,and aqueous sites from 0-400?g/mL.Therefore,the drug concentration of 50,100 and 200?g/mL was selected for the follow-up study.2.Research results of different hypoxia models showed that the hypoxia model of H₂O₂ was established under the following conditions:concentration of 40?mol/L,action of 4h;The hypoxia model of Na₂S₂O₄ was established under the following conditions:concentration of 2.5mmol/L,action for 24h;The hypoxia model was established in the three-gas incubator under the conditions of 1%O₂ concentration and 48h of action.3.The screening results of three hypoxia damage models for the effective parts of antihypoxia showed that all parts of Gentiana straminea had anti-hypoxia effects,among which ethyl acetate had better effects than n-butanol and aqueous phase parts.4.Through the acute hypoxic endurance experiment at an altitude of 10000m,the fatality rate of mice within 1h was calculated.The results showed that all polar parts of Gentiana straminea extract had certain anti-hypoxic effect compared with the model group,and the ethyl acetate extract had better effect than n-butanol and aqueous phase extraction.5.Hoechst33258 staining results showed that the ethyl acetate of gentiana macrophylla could significantly reduce the apoptosis of hypoxic PC12 cells and have a certain protective effect on hypoxic PC12 cells.The resulte of qPCR showde that Hif-1??NF-?BmRNA expression level decreased obviously?P<0.01,P<0.05?,and the ratio of Bcl-2/Bax was increased.Results of Westren Blot showed that the levels of Bcl-2 and Bcl-2/Bax in Ethyl Acetate group were significantly higher than those in Model Group?P<0.05?,and the levels of Bax,Hif-1 and NF-?B were significantly lower than those in model group?P<0.01,P<0.05?.6.Network pharmacology was used to systematically analyze the network correlation between 11 active components at the extraction site of ethyl acetate and hypoxia-related targets.Among them,the results of c-t network topology analysis showed that all 11 compounds had good anti-hypoxia effects,among which naphthol,luteolin and ursolic acid were the main active components,and PTGS2 and MMP9 were the main targets.The results of PPI network topology analysis showed that TP53,STAT3 and AKT1 were the main anti-hypoxia target proteins at the extraction site of ethyl acetate.KEGG pathway analysis showed that PI3k-akt signaling pathway,Hif-1signaling pathway and TNF signaling pathway were the main antihypoxia signaling pathways at the extract site of ethyl acetate.Conclusion:the ethyl acetate site of Gentiana straminea may be the active site of its antihypoxia,and its mechanism of action may be achieved by improving antioxidant capacity and affecting Hif-1a/NF-?B pathway.