Study On The Protective Effect And Mechanism Of Notopterol On The Injury Of H9c2 Cells Induced By Isoproterenol

ObjectiveThe isoproterenol(ISO)of this disease processes large mouse H9c2 cells and establishes the H9c2 cells damage mode and studies the protective action and possible mechanism of this notopterol induced H9c2 cells injury of this disease.Method1.First,we construct an H9c2 cells damage model through ISO and examine the effect of notopterol on the survival rate of H9c2 cells and the effect of the survival rate of ISO damaged H9c2 cells;using colorimetric detection kit to detect the amount of lactate dehydrogenase(LDH)released in the cells.2.The experimental groups were as follows: blank control group(control group),model group(ISO group),different concentrations of notopterol group(10,20,40 μ m).Using active oxygen detection kit,xanthine oxidase method and thiobarbituric acid method to detect reactive oxygen species(ROS)levels,superoxide dismutase(SOD)activity and intracellular malondialdehyde(MDA)content;to investigate the effect of notopterol on the level of oxidative stress in H9c2 cells with ISO damage.3.The effects of notopterol on mitochondrial apoptosis induced by ISO induced H9c2 were studied by Hoechst 33342 staining,Annexin V-FITC/PI apoptosis test kit,mitochondrial membrane potential detection kit(JC-1)and Western blotting method.Finally,the effects of Akt/GSK3 β mediated mitochondrial apoptosis signal pathway were simply verified.Result1.ISO could affect the survival rate of H9c2 cells.With the increase of ISO concentration,the survival rate of H9c2 cells decreased gradually.At ISO concentration of 30 μm,the cell survival rate decreased about 60% at 48 h.The appropriate concentration can determine the model for subsequent tests.2.Different concentrations of notopterol had obvious proliferative effect on H9c2 cells.Compared with ISO group,different concentrations of notopterol group can significantly improve the survival rate of H9c2 cells injury cells induced by ISO,and reduce the release of LDH,with statistical significance.It is suggested that notopterol has a certain protective effect on ISO induced H9c2 cells injury.3.Compared with ISO group,different concentrations of notopterol gradually reduced the intracellular green fluorescence intensity,decreased the ROS content,increased the SOD activity and decreased the MDA content in H9c2 cells injury induced by ISO in a concentration dependent manner.These results suggest that notopterol has antioxidant effect on ISO induced H9c2 cells injury.4.Compared with ISO group,different concentrations of notopterol attenuated the blue fluorescence in H9c2 cells injury cells induced by ISO,significantly reduced the apoptotic cells and the proportion of apoptotic cells,and increased the mitochondrial membrane potential in a concentration dependent manner.At the same time,the expression of anti apoptotic protein Bcl-2 was up-regulated,and the expression of Pro apoptotic protein Bax,caspase-3 and cyto-C was down regulated.These results suggest that notopterol may play a protective role in ISO induced H9c2 cells injury through mitochondrial apoptosis pathway.In addition,we found that Akt and GSK3βphosphorylation protein expression can be up-regulated,suggesting that Akt /GSK3β signaling pathway may participate in the protective effect.ConclusionNotopterol can protect H9c2 cells from ISO induced injury to some extent,and may reduce the injury by enhancing the resistance of cells to oxidative stress and Akt / GSK3β-mediated mitochondrial apoptosis pathway.