Protective Effects And Mechanisms Of Costunolide On Injury Of Mouse Brain Slices And C12 Cells Induced By Oxygen-glucose Deprivation/reperfusion

Objective:To study the protective effects and mechanisms of costunolide on injury induced by oxygen-glucose deprivation/reperfusion in mouse brain slices and PC12 cells.Methods:（1）OGD/R injury model was established by isolated brain slices.The experiment was divided into six groups:Control group,OGD/R group,OGD/R+Nim 10μM group,OGD/R+CT 1μM group,OGD/R+CT 5μM group and OGD/R+CT 10μM group.The activity of the brain slices and the degree of injury were detected by the lactatedehydrogenase release test and 2,3,5-trichlorophenyltetrazolium chloride staining test.The expression of the mitochondrial pathway related proteins Bcl-2,Bax,Cyto-c,Caspase-9,Caspase-7 and Caspase-3 expression levels were detected by Western blot.（2）PC12 cells were used to establish OGD/R injury models,and the experiments were divided into Control group,OGD/R group,OGD/R+Nim5μM group,OGD/R+CT 2.5μM group,OGD/R+CT 5μM group and OGD/R+CT 10μM group.Light microscope was used to observe the morphological changes of PC12 cells;CCK-8 was used to test the cell viability;LDH viability assay was used to detect the activity of LDH in cell culture medium;Annexin V-FITC/Propidium Iodide dual staining flow cytometry was used to detect the mortality rate of PC12 cells;JC-1 staining was used to detect changes in mitochondrial membrane potential of PC12 cells;Fluo-3/AM fluorescence staining was used to detect calcium levels in PC12cells,and Western blot was used to further detect Bcl-2,Bax,Cyto-c,Apaf-1,Caspase-9,Caspase-7,and Caspase-3 proteins expression.Finally,the expression levels of Bcl-2,Bax,Cyto-c and Caspase-3 proteins were detcted by cell immunofluorescence.Results:（1）In the brain slice experiment,the TTC staining results of brain slices showed that compared with OGD/R group,the OD₄₉₀ values at the TTC staining on A₄₉₀nanometer in brain slices at 1μM,5μM,and 10μM were significantly higher（p<0.05）;The results of LDH release test showed that compared with the OGD/R group,the LDH release of CT1μM,5μM,10μM three dose group were significantly reduced（p<0.05）;Western blot showed that compared with OGD/R group,the expression of Bax,Cyto-c,Caspase-9,Caspase-7,Caspase-3 in CT1μM,5μM,10μM groups were significantly reduced（p<0.05）,while the Bcl-2 was distinctly increased（p<0.05）.（2）In the PC12 cell experiment,the effect of CT on the morphology of PC12 cells showed that compared with the OGD/R group,the cell bodies of CT2.5μM,5μM,10μM groups were plump,and the cell membranes were relatively smooth and intact.Most of the cells were interwoven into a network structure;The results of CCK-8 assay showed that compared with the OGD/R group,the survival rate of the cells in CT2.5μM group and CT5μM group was significantly higher（p<0.05）,while CT10μM group was no significantly different compared with OGD/R group;The LDH viability test showed that compared with the OGD/R group,the activity of LDH in the culture medium of PC12 cells was significantly reduced in the three dose groups of CT2.5μM,5μM and 10μM（p<0.05）;The apoptosis rate of PC12 was detcted by flow cytometry.The result showed that compared with OGD/R group,the apoptosis rates of CT2.5μM,5uM and 10u M groups were all reduced.But the apoptotic rate of CT10uM group was no significant;the results of the mitochondrial membrane potential detected by JC-1 staining showed compared with the OGD/R group,the red fluorescence of the CT5μM group was significantly increased（p<0.05）,while the red fluorescence of theCT2.5μM,CT10μM group was not significantly enhanced;The results of the change of Ca²⁺concentration in PC12 cells showed that green fluorescence was weakened in CT2.5μM,5μM,10μM three dose groups;Western blot showed that CT could increase the expression of anti-apoptotic protein Bcl-2 and decreased the expression of promote apoptosis protein Bax,the expression of Cyto-c,Apaf-1,Caspase-9,Caspase-7 and Caspase-3 protein;The results of immunofluorescence staining showed that CT can increased the expression of Bcl-2,and decreased the expression of Bax,Cyto-c and Caspase-3.Conclusion:The costunolide has certain protective effects on OGD/R injury in mouse brain slices and PC12 cells.Its protective mechanism may be related to the regulation of the mitochondria-Cyto-c pathway,which increases the expression of Bcl-2 protein,making Bax The expression levels of Cyto-c,Apaf-1,Caspase-9,Caspase-7 and Caspase-3 proteins were related to the decrease.