Study On The Mechanisms Of Ginsenoside CK For Reducing Oxygen-glucose Deprivation/Reoxygenation Injury In Neuronal Cells Based On Mul1/Mfn2 Pathway

Objective:This study took ginsenosides(GS)as the starting point,combined with energy metabolism analyzer,fluorescence staining,immunofluorescence and other efficacy evaluation systems,and took Mul1’s involvement in the regulation of mitochondrial quality control as the starting point,selected the anti-neural I/R saponin monomer in GS and identified its mechanism,providing ideas and reliable experimental basis for the elucidation of the core efficacy of ginseng.Methods:1.The oxygen-glucose deprivation/reoxygenation(OGD/R)injury model of PC12 cells was constructed by the method of sugar-free serum-free culture medium and hypoxia culture.CCK-8 assay was used to screen the protective components of ginsenosides against GOG/R injury in PC12 nerve cells.The content of ATP was determined by ATP fluorescence quantitative kit.Mitochondrial function and source of ATP were evaluated by mitochondrial energy metabolizer.The protein expression and enzyme activity of mitochondrial respiratory chain complex were detected by Western Blot and kit respectively.To clarify the mechanism of ginsenoside CK against OGD/R damage induced neurobiogenic energy imbalance based on Mul1/Mfn2 pathway.2 Through the Mito-tracker fluorescence staining observation PC12 cells by OGD/R and ginseng saponin CK morphological changes after treatment.The key proteins of autophagy were verified by Western Blot.The number of autophagolysosomes was evaluated by m Cherry-e GFP-LC3 B tandem fluorescent probe immunofluorescence staining.Mfn2 knockout PC12 cells were traced by Mito-tracker,immunofluorescence,fluorescence probe and other methods.It was determined that ginsenoside CK was based on Mul1/Mfn2 pathway mediated mitochondrial autophagy against OGD/R damage.3 Using Western Blot respectively for cell plasma total protein and total protein in mitochondria Mul1,P-Drp1(S637),Drp1,Fis1,OPA1,Mfn1,Mfn2 protein expression evaluation.It was determined by co-IP that Mul1 played a protective role against OGD/R-induced PC12 cell damage by interacting with Mfn2 protein.This effect was reversed with the addition of the small molecule inhibitor Mdivi-1.The production of ROS in mitochondria was studied by Mito-SOX fluorescent probe.Results:1 Ginsenosides and their ginsenoside monomers ginsenoside Rb2 and ginsenoside CK have obvious protective effects on OGD/R induced PC12 nerve cell damage,and significantly improve the cell activity of PC12 cells after the model.Ginsenoside CK can significantly restore the decrease of ATP content in PC12 cell injury model induced by OGD/R,and ATP mainly comes from mitochondria.After ginsenoside CK pretreatment,the basic oxygen consumption,maximum oxygen consumption,respiratory potential,and ATP of PC12 cells were significantly increased in a dose-dependent manner.The effect of ginsenoside CK on mitochondrial function of PC12 cells is mediated by regulating OCR level.The protein expression of mitochondrial complex did not change significantly,but significantly promoted the enzyme activity of complex Ⅰ and Ⅲ,and enhanced the function of mitochondrial electron transport chain.2 Ginsenoside CK after pretreatment,OGD/R caused mitochondrial divide significantly reduced;After Mul1 was knocked out,the above phenomenon was significantly inhibited,suggesting that mitochondrial division was related to Mul1 expression.The expressions of autophagy related key proteins LC3-Ⅰ and LC3-Ⅱ were significantly decreased,while the expression of positive regulatory protein p62 was significantly increased.After ginsenoside CK pretreatment,the Parkin-mitochondrial co-localization fluorescence intensity of PC12 cells was significantly reduced,and the above phenomenon was significantly inhibited after Mul1 knockout.After Mfn2 knockout,the effect of ginsenoside CK pretreatment on significantly reducing mitochondrial and lysosome colocalization induced by OGD/R injury was offset.3 Ginsenoside CK regulates Mul1 protein expression,reduces Mfn2 ubiquitination and degradation,promotes Mfn2 expression,and inhibits mitochondrial division,mitochondrial autophagy and mitochondrial apoptosis induced by OGD/R induced nerve injury.Conclusion:1 Ginsenoside CK changes the mitochondrial dysfunction of PC12 cells induced by OGD/R by increasing intracellular ATP production,enhancing mitochondrial respiratory capacity and electron transport chain activity.2 Ginsenoside CK by adjusting the ginsenosides Mul1 pathway,affect mitochondrial dynamics and mitochondrial autophagy,relieve the PC12 cells induced by OGD/R injury.3 Ginsenoside CK ginsenosides regulate Mul1 protein expression,reduce Mfn2 ubiquitin and degradation,and thus promote the expression of Mfn2,suppress the nerve injury induced by OGD/R caused by split mitochondrial mitochondria,mitochondrial autophagy and apoptosis.