Protective Effect Of Mu-Xiang-You-Fang On PC12 Cell Injury Induced By Oxygen-glucose Deprivation And Reperfusion Based On AMPK/mTOR Signaling Pathway

Objective:We applied the platform of integrative pharmacology to reveal the Chemical Constituents and targets of Mu-Xiang-You-Fang?MXYF?in the treatment of stroke,and to evaluate the mechanisms of Mu-Xiang-You-Fang protect PC12 cells induced by oxygen-glucose deprivation/reperfusion injury.Methods:1.Based on an internet-based computation platform for integrated pharmacology Chinese Medicine?TCM?component database and disease/symptoms target database),search for chemical composition information contained in Mu-Xiang-You-Fang,as well as targets for stroke diseases,constructed a visual network of"Chinese herbal medicines-chemical components-core targets-critical pathways"and analyzed them.2.?1?PC12 cells were divided into control group,Mu-Xiang-You-Fang group?final concentration was 32,16,8,4,2,1?g/mL?.CCK-8 was used to detect the cytotoxicity of Mu-Xiang-You-Fang on PC12 cells.?2?PC12 cells were used to establish OGD/R injury models and the PC12 cells were divided into Control group,OGD/R group,OGD/R+Nimo group?5?g/ml?,OGD/R+MXYF group?1,2,4?g/ml?,CCK-8 was used to test the survival rate,LDH viability assay was used to detect LDH release rate,chemical fluorescence staining was used to detect changes of Ca²⁺levels and Flow cytomertry was used to detect changes of mitochondrial membrane potential.3.?1?PC12 cells were divided into control group,OGD/R group,OGD/R+MXYF group,OGD/R+3-MA group,OGD/R+3-MA+MXYF group?4?g/ml?immunofluorescence and western blot was used to test the expression of related protein of autophagy LC3,Beclin 1 and p62.MDC staining was used to detect PC12cell autophagy rate in each group.?2?PC12 cells were divided into control group,OGD/R group,OGD/R+MXYF group,OGD/R+Compound C group,OGD/R+Compound C+MXYF group?4?g/ml?.Western Blot was used to detect the expression of AMPK,p-AMPK^Thr172,p-mTOR^Ser2448,ULK1,p70S6K,p-p70S6K^Thr389,Beclin1 and p62 protein.Results:1.Material basis and mechanism of Mu-Xiang-You-Fang for the Treatment of Type Stroke Based on Integrated Pharmacology PlatformA total of 189 chemical ingredients were searched from Aucklandia,Pepper and Asarum.Its pharmacological effect was relevant to 99 core targets.Among them,targets directly related to stroke include targets for reducing brain injury?SLC1A2,AR,NMDA receptors?,targets for improving neurological recovery after brain injury?PGR?,targets for the development and prognosis of cerebral infarction?CAT?,inhibitory neurotransmitter?-aminobutyric acid rate-limiting enzyme GAD2,and two subunits of PSENEN,which can reduce infarct area,and AMPK.GO and KEGG enrichment analysis showed that these key targets may involve neurotrophic factor signaling pathways,nervous system,endocrine system,cell growth,death and cell metabolism.2.Protective effects of Mu-Xiang-You-Fang on PC12 cells induced by oxygen-glucose deprivation/reperfusion?1?The results of CCK-8 assay showed that PC12 cells treated with different concentrations of Mu-Xiang-You-Fang,with the increase of the concentration of Mu-Xiang-You-Fang,the effect of inhibiting the survival of PC12 cells was enhanced.When the dosage of Mu-Xiang-You-Fang was 4,2,1?g/mL,the toxicity to PC12 cells was relatively low,so we choose the dosage of Mu-Xiang-You-Fang was 4,2,1?g/mL?p<0.01?.?2?The results of CCK-8 assay showed that compared with the control group,the cell survival rate of OGD/R group was significantly lower;compared with OGD/R group,Mu-Xiang-You-Fang?1,2,4?g/ml?group and the nimodipine group wereincreased?p<0.01?,and the effect of the Mu-Xiang-You-Fang?4?g/ml?was comparable to that of the nimodipine group.?3?The LDH viability test showed that compared with the control group,the leakage rate of LDH in OGD/R group was significantly higher;compared with OGD/R group,the leakage rate of LDH in Mu-Xiang-You-Fang?1,2,4?g/ml?group and the nimodipine group were decreased?p<0.01?,and the effect of Mu-Xiang-You-Fang?4?g/ml?was comparable to that of the nimodipine group.?4?The results of the mitochondrial membrane potential?MMP?detected by JC-1staining showed that compared with the control group,the mitochondrial membrane potential of OGD/R group decreased significantly;compared with OGD/R group,Mu-Xiang-You-Fang?1,2,4?g/ml?group and the nimodipine group enhance the mitochondrial membrane potential?p<0.01?,and the Mu-Xiang-You-Fang?4?g/ml?was equivalent to the nimodipine group.?5?The change of Ca²⁺concentrate on PC12 cells showed that compared with the Control group,the green fluorescence of OGD/R group was higher.Compared with the OGD/R group,the green fluorescence of the Mu-Xiang-You-Fang?1,2,4?g/ml?group was weakened?p<0.01?,and the Mu-Xiang-You-Fang?4?g/ml?was equivalent to that of the nimodipine group.3.Effect of Mu-Xiang-You-Fang on autophagy-related factors after PC12 cells injury induced by oxygen-glucose deprivation/reperfusion?1?MDC staining results showed that compared with the control group,the green fluorescence of the OGD/R group was enhanced;compared with the OGD/R group,the Mu-Xiang-You-Fang and the 3-MA group could reduce the green fluorescence intensity;Compared with the group,the combination of Mu-Xiang-You-Fang and3-MA could significantly reduce the green fluorescence intensity?p<0.05?.?2?Immunofluorescence results showed that compared with the control group,the expression of LC3 and Beclin1were up-regulated and the expression of p62 protein was down-regulated in the OGD/R group.Compared with the OGD/R group,the expression of LC3 and Beclin1 were down-regulated and the expression of p62 was up-regulated in Mu-Xiang-You-Fang and the 3-MA group.Compared withMu-Xiang-You-Fang group,the combination of Mu-Xiang-You-Fang and 3-MA could significantly down-regulate the expression of LC3 and Beclin1 and up-regulate the expression of p62 protein?p<0.05?.?3?The results of immunoblotting showed that compared with the control group,the expression of LC3 and Beclin1 was up-regulated and the expression of p62 protein was down-regulated in the OGD/R group.Compared with the OGD/R group,the expression of LC3 and Beclin1 down-regulated the expression of p62 protein was up-regulated.Compared with the Mu-Xiang-You-Fang group,the combination of Mu-Xiang-You-Fang and 3-MA group could significantly down-regulate the expression of LC3 and Beclin1 and up-regulate the expression of p62 protein?p<0.05?.?4?The results of immunoblotting results showed that compared with the control group,the expression of p-AMPK^Thr172,ULK1,Beclin1 was up-regulated and the expression of p-mTOR^Ser2448,p-p70S6K^Thr389 and p62 protein was down-regulated in OGD/R group;compared with OGD/R group,the expression of p-AMPK^Thr172,ULK1and Beclin1 was down-regulated and the expression of p-mTOR^Ser2448,p-p70S6K^Thr389and p62 protein was up-regulated in Mu-Xiang-You-Fang and the compound C group;Compared with the Mu-Xiang-You-Fang,the Mu-Xiang-You-Fang and Compound C significantly down-regulated p-AMPK^Thr172,ULK1,and Beclin1 protein expression,and up-regulated p-mTOR^Ser2448,p-p70S6K^Thr389,and p62 protein expression.?p<0.05?.Conclusion:1.With the help of integrated pharmacological platform,the chemical components,key targets AMPK and related pathways of Mu-Xiang-You-Fang in the treatment of stroke were revealed,which provided scientific basis for the study of the mechanism of Mu-Xiang-You-Fang in the treatment of stroke.2.Mu-Xiang-You-Fang has protective effect on PC12 cell injury induced by oxygen-glucose deprivation and reperfusion,and its mechanism is related to the inhibition of autophagy mediated by AMPK/mTOR signaling pathway.