| Objective:To investigate the protective effect of dehydrocostus lactone（DHL）on PC12 cell injury induced by oxygen-glucose deprivation-reperfusion（OGD/R）,and to explore its mechanism from the view of autophagy and apoptosis.Methods:（1）the mechanism of anti-apoptosis and autophagy of dehydroandrographolide was predicted by docking with autophagy and apoptosis-related proteins.（2）PC12 cells were divided into the control group and DHL group（32,16,8,4,2,1μmol/L）.The cytotoxicity of DHL on PC12 cell was detected by the CCK-8 method.（3）The model of OGD/R injury in PC12 cells were established（hypoxia and glucose deprivation for 4 h,and reperfusion for 24 h）.Cells were divided into eight groups,including Control,OGD/R,OGD/R+DHL（0.5,1,2,4,8μmol/L）,and Nimodipine（5μg/m L）group.CCK-8 method was used to detect the cell survival rate.（4）PC12 cells were divided into control group,OGD/R group,nimodipine group,OGD/R+DHL（1,2,4,μmol/L）group.Observing the changes in cell morphology,The level of lactate dehydrogenase（LDH）was detected by colorimetry,and the changes of reactive oxygen species（ROS）and intracellular Ca2+concentration were detected by chemical fluorescence staining.（5）PC12 cells were divided into control group,OGD/R group and OGD/R+DHL（2μmol/L）group.The formation of autophagosomes was detected by the transmission electron microscope.（6）The following groups were set up,including control,OGD/R,OGD/R+DHL（1,2,4μmol/L）and OGD/R+3-MA group,the number of apoptotic cells was detected by flow cytometry,and the autophagy rate of PC12 cells in each group was detected by MDC staining.Immunofluorescence and western blots were used to detect LC3-Ⅱ/LC3-Ⅰ,Beclin1,P62,Cyto-c,Cleaved-Caspase-3,Cleaved-caspase-9,Cleaved-Caspase-7,Bcl-2,Bax,P-PI3K,P-AKT,P-m TOR expression changes.Results:1.Results of molecular docking of dehydroandrographolideDehydroandrographolide has good binding potential with LC3,Beclin1,Caspase-3,Caspase-9,Caspase-7,Bcl-2,Bax,PI3K,AKT and m TOR.The score of Bax is the highest.2.Protective effect of DHL on PC12 cell injury induced by OGD/R.（1）The cytotoxicity of DHL on PC12 cells showed that DHL at different concentrations could inhibit the survival of PC12 cells.The inhibitory effect on the survival of PC12 cells was enhanced with the increase of DHL concentration.The IC50 of DHL was 8μmol/L.（2）The effect of DHL on the viability of PC12 cells:the cell survival rate of OGD/R group was lower than that of the control group,while DHL（1,2,4,μmol/L）group was higher than that of OGD/R group,There was no significant difference between the DHL（0.5,8μmol/L）group and the OGD/R group（P<0.05）.So the dose of DHL was 1,2,4,μmol/L.（3）The morphological changes of PC12 cells treated with DHL:the results showed that most of the cells in the OGD/R group became round or had short-axis mutation.The cell condition was significantly improved in DHL and nimodipine group.（4）The effect of DHL on the leakage rate of LDH in PC12 cells:the leakage rate of LDH in the culture medium of the OGD/R group was higher than the control group.After treatment with DHL（1,2,4μmol/L）,the leakage rate of LDH in cell culture medium was significantly lower than the OGD/R group,and the effect of DHL（2μmol/L）was the most obvious（P<0.05）.（5）Effect of DHL on ROS changes in PC12 cells:the fluorescence intensity of PC12cells treated with OGD/R was significantly higher than the control group.The accumulation of ROS and fluorescence intensity was significantly decreased in the DHL（1,2,4μmol/L）group（P<0.05）.（6）The effect of DHL on the changes of intracellular[Ca2+]in PC12 cells:the green fluorescence intensity of PC12 cells treated with OGD/R was significantly higher than that control group.The accumulation of[Ca2+]and fluorescence intensity decreased substantially in the DHL group（P<0.05）.3.Effects of DHL on autophagy and apoptosis of PC12 cells after OGD/R injury（1）MDC staining showed that the green fluorescence intensity of PC12 cells in the OGD/R group was significantly higher than the control group.The green fluorescence intensity was significantly decreased in the 3-MA（10μmol/L）group and DHL（1,2,4μmol/L）group（P<0.05）.（2）The transmission electron microscope results showed that compared with the control group,the number of autophagosomes in the OGD/R group was significantly increased,accompanied by mitochondrial swelling,vacuolation,and crest rupture.DHL（2μmol/L）could dramatically reduce the number of autophagosomes and improve cell damage.（3）The results of immunofluorescence showed that compared with the OGD/R group,3-MA（10μmol/L）and DHL（1,2,4μmol/L）could significantly down-regulate the expression of LC3-ΙΙ/LC3-Ιand Beclin1.（4）The results of western blot showed that compared with OGD/R group,3-MA（10μmol/L）and DHL（1,2,4μmol/L）could significantly down-regulate the expression of LC3-ΙΙ/LC3-Ιand Beclin1 while up-regulating the expression of p62.（5）The effect of DHL on the apoptosis rate of PC12 cells:Compared with the control group,the apoptosis rate of the OGD/R group was significantly increased,while the apoptosis rate was significantly decreased in DHL（1,2,4μmol/L）and autophagy inhibitor3-MA（10μmol/L）group（P<0.05）.（6）Western blot was used to detect apoptotic proteins Cyto-c,Caspase-3,Caspase-9,Caspase-7.The results showed that compared with the OGD/R group,DHL（1,2,4μmol/L）and 3-MA（10μmol/L）significantly decreased Bax expression and increased Bcl-2expression.The expression of Cyto-c,Cleaved-Caspase-3,Cleaved-caspase-9 and Cleaved-Caspase-7 decreased significantly（P<0.05）.4.Effect of DHL on PI3K/Akt/m TOR pathway in PC12 cells after OGD/R induced injuryThe expression of P-PI3K,P-AKT,P-m TOR were detected by western blot.The results showed that 3-MA（10μmol/L）and DHL（1,2,4μmol/L）could significantly down-regulate the expression of P-PI3K,P-AKT and P-m TOR（P<0.05）.Conclusion:1.Dehydroandrographolide can protect PC12 cells from OGD/R-induced injury by inhibiting autophagy and apoptosis.2.Dehydroandrographolide can protect PC12 cells from OGD/R-induced injury,the mechanism of inhibition of apoptosis and autophagy may be related to PI3K/Akt/m TOR pathway activation. |
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