The Effect Of Carabin Overexpression On AC16 Cells Apoptosis Induced By Oxygen Glucose Deprivation

Background and ObjectiveIschemic heart disease is caused by insufficient or interrupted blood supply to the coronary arteries,resulting in insufficient energy of the heart,which leads to cardiomyocyte apoptosis or necrosis.In severe cases,it will develop into cardiac insufficiency and heart failure.Cardiomyocytes are terminally differentiated cells and have almost no regenerative capacity.So we hope to inhibit cardiomyocyte apoptosis in myocardial ischemia early,which will protect the cardiomyocytes.Carabin is a protein of the TBC1 family discovered in recent years.It prevents myocardial hypertrophy through the CaN and Ras signaling pathways.However,whether Carabin plays an important role is still inconclusive on the study of myocardial ischemia.Our previous results showed that Carabin protein was significantly down-regulated in human ventricular myocytes AC16 induced by oxygen glucose deprivation(OGD)24h.Is Carabin associated with the myocardial ischemia? Our study aimed to establish a model of apoptosis induced by oxygen glucose deprivation in AC16 cells.Overexpression of Carabin was used to observe the effect of Carabin on AC16 apoptosis induced by oxygen glucose deprivation,in order to provide a scientific theoretical basis for the prevention and treatment of ischemic heart disease.MethodsPart Ⅰ.Changes of Carabin induced by oxygen glucose deprivation(OGD)in AC16 cells and transfect Carabin into AC16 cells1.Detected the Carabin mRNA by qRT-PCR and Carabin protein by Western Blot in AC16 cells induced by oxygen glucose deprivation(OGD).2.Construction of Carabin overexpression lentivirus.3.Transfected Carabin into AC16 cells.Part Ⅱ.Carabin overexpression reduces apoptosis of AC16 cells induced by oxygen glucose deprivation1.Observed the cell morphology of AC16 cells using the microscope.2.Detected the apoptosis of AC16 cells by flow cytometry.3.Detected the apoptosis of AC16 cells by TUNEL.4.Detected the Caspase-3 activity of AC16 cells using the Caspase-3 activity assay kit.5.Detected the lactate dehydrogenase(LDH)activity of AC16 cells using the lactate dehydrogenase activity assay kit.6.Detected the Caspase-3 protein in AC16 cells by Western Blot.Part Ⅲ.Carabin overexpression enhances the mitochondrial metabolism in AC16 cells1.Detected the mitochondrial metabolism of AC16 cells under normoxic conditions by mitochondrial pressure test.2.Detected the COX IV protein in AC16 cells by Western Blot.3.Detected the COX IV activity of AC16 cells using the COX IV activity assay kit.ResultsPart Ⅰ.The Carabin mRNA and protein change significantly in AC16 induced by oxygen glucose deprivation.1.The results of qRT-PCR and Western Blot showed that the Carabin mRNA and protein levels decreased in AC16 cells induced by oxygen glucose deprivation(p<0.05).2.Successfully transfected Carabin into AC16 cells.The results of qRT-PCR and Western Blot showed that the lentivirus-transfected AC16 cells had Carabin overexpression continuously(p<0.05).Part Ⅱ.Carabin overexpression reduces apoptosis of AC16 cells induced by oxygen glucose deprivation1.The morphology of AC16 cells was observed under the inverted microscope.The cells of the no-load-normoxic groups and the Carabin-overexpression-normoxic groups grew well: cells spread the culture dish,the cells were intact,cell adherence was firm,cell boundary was clear,cell size was uniform,and the cell shape was fusiform.Compared with the no-load-normoxic groups and the Carabin-overexpression-normoxic groups,the cells of the no-load OGD 24 h groups had poorer cell growth,only a small number of cells were adherent,and most of cells were circular and floating.Compared with the no-load-normoxic groups and the Carabin-overexpression-normoxic groups,the Carabin-overexpression OGD 24 h groups had poorer cell growth,low cell density and uneven cell size.However,compared with the no-load OGD 24 h groups,the Carabin-overexpression OGD 24 h groups had the better cell growth,the cell density was higher,and only a few cells were floating.2.Flow cytometry showed that the apoptosis rates of the no-load OGD 24 h groups and the Carabin-overexpression OGD 24 h groups were higher than that of the no-load-normoxic groups(p<0.01),the survival rates were decreased(p<0.01).However,the apoptotic rate of the Carabin-overexpression OGD 24 h groups was significantly lower than that of the no-load OGD 24 h groups(p<0.01),and the survival rate was significantly increased(p<0.01).3.The number of apoptosis in the no-load OGD 24 h groups and the Carabin-overexpression OGD 24 h groups increased,compared with the no-load-normoxic groups by TUNEL.The apoptosis number of the Carabin-overexpression OGD 24 h groups was reduced,compared the no-load OGD 24 h groups(p< 0.05).4.The activity of Caspase-3 was significantly higher in the no-load OGD 24 h groups than the no-load normoxic groups(p<0.01).The Caspase-3 activity of the Carabin-overexpression OGD 24 h groups was significantly reduced,compared with the no-load-OGD 24 h groups(p< 0.05).5.The LDH of the no-load OGD 24 h groups was significantly increased,compared with the no-load normoxic groups(p<0.0001),while the LDH of the Carabin-overexpression OGD 24 h groups was significantly lower than that of the no-load-OGD 24 h groups(p< 0.001).6.The Caspase-3 protein of the no-load OGD 24 h groups was significantly higher than that of the no-load normoxic groups(p<0.05).The Caspase-3 protein of the Carabin-overexpression OGD 24 h groups was significantly lower than of the no-load OGD 24 h groups(p<0.05).Part Ⅲ.Carabin overexpression enhances the mitochondrial metabolism in AC16 cells1.The mitochondrial pressure test results showed that under normoxic culture conditions,the Carabin-overexpression-normoxic groups were compared with the no-load-normoxic groups,the mitochondrial basal oxygen consumption rate increased(p<0.05),the maximum oxygen consumption rate increased(p<0.05),the oxygen spare capacity(p<0.05)and the ATP production increased significantly(p<0.05).2.The expression of COX IV protein in AC16 cells was detected by Western Blot.Under normal oxygen culture conditions,there was no significant difference between the COX IV protein in the Carabin-overexpression-normoxic groups and the no-load-normoxic groups(p>0.05).After OGD for 24 h,there was no significant difference COX IV protein between in the Carabin-overexpression OGD 24 h groups and the no-load OGD 24 h groups(p>0.05).However,the COX IV protein of the no-load groups after OGD for 24 h was significantly lower than that under normoxic conditions(p<0.05).The COX IV protein of the Carabin-overexpression groups after OGD for 24 h was also significantly lower than that under normoxic conditions(p<0.05).3.The COX IV activity in AC16 cells showed that the COX IV activity of the Carabin-overexpression-normoxic groups was significantly higher than that of the no-load-normoxic groups,under normal oxygen culture conditions(p<0.05).After OGD for 24 h,the COX IV activity of the Carabin-overexpression OGD 24 h groups was significantly higher than that of the no-load OGD 24 h groups(p<0.05).The COX IV activity in the no-load AC16 cells induced by OGD 24 h was significantly lower than that in the AC16 cells under normal oxygen culture conditions(p<0.05).And the COX IV activity in the Carabin-overexpression AC16 cells was also lower than that in the AC16 cells under normal oxygen culture conditions(p<0.05).ConclusionOverexpression of Carabin increases the mitochondrial metabolism in AC16 cells,and reduces the AC16 cells apoptosis induced by oxygen glucose deprivation.