A Role Of SDHA In Protective Effect Of Hypoxic Postconditioning In Adult Rat Cardiomyocytes In High Glucose State

Objective: 1.To observe the role of succinate dehydrogenase flavoprotein(SDHA)in relieving hypoxia reoxygenation injury of adult rat cardiomyocytes after hypoxic postconditioning;2.To observe the effect of high glucose concentration environment on hypoxic postconditioning in adult rat cardiomyocytes;3.To observe the hypoxic postconditioning attenuates little about hypoxia reoxygenation in adult rat cardiomyocytes under high glucose condition,and it's relationship with SDHA.Methods: Adult rat cardiomyocytes were isolated by in vitro cardiac perfusion device(MPA).Myocardial cells were cultured for 48 h after normal glucose concentration and high glucose concentration medium,and were randomly divided into normal group(N group),hypoxia reoxygenation group(HR group),hypoxic postconditioning group(HPO group),siRNA silencing SDHA treatment + hypoxia/reoxygenation group(HR + siRNA SDHA group),siRNA silencing SDHA treatment + hypoxia postconditioning group(HPO+ siRNA SDHA group)and the corresponding blank adenovirus were infected into the control group.The treatment was carried out according to the following experimental conditions: N group: continuous incubation for 105 min in a 37 °C incubator;HR group:reoxygenation for 60 min after 45 min of hypoxia;HPO group: on the basis of HR,after 45 min of hypoxia,reoxygenation for 5 min,hypoxia for 5 min,a total of 3 cycles,reoxygenation for 30 min;HR + siRNA SDHA group: adenovirus successfully infected with silencing SDHA protein expression and then given corresponding HR treatment;HPO+ siRNA SDHA group: adenovirus was infected successfully to silence the expression of SDHA protein,the corresponding HPO treatment was given,the corresponding blank adenovirus was infected into the control group: after the blank adenovirus was infected successfully,the corresponding HR and HPO were treated.At the end of reoxygenation,the indicators of each group of cardiomyocytes were compared and observed(1)The change of viability of cardiomyocytes in each group was detected by multi-function microplate reader;(2)the changes of mitochondrial membrane potential(MMP)in each group were detected by laser confocal microscopy and multi-function microplate reader;(3)The changes of reactive oxygen species(ROS)in cardiomyocytes of each group weredetected by inverted phase contrast fluorescence microscopy and Image Pro Plus;(4)ATP kit was used to detect ATP content in cardiomyocytes of each group;(5)Western-Blot method to detect the expression level of SDHA protein in each group.Results:1.Changes of myocardial cell viability: The changes of myocardial cell viability under normal conditions: the viability of myocardial cells in HR group was lower than that in N group and HPO group,and the activity of cardiomyocytes in HPO group was lower than that in N group(P<0.05).The changes of cardiomyocyte viability in each group after infection of siRNA SDHA with adenovirus: The changes of cardiomyocyte viability in the HR group and the HR + siRNA SDHA NC group were lower than those in the HR +siRNA SDHA group and the HPO group,the HR + siRNA SDHA group had lower cardiomyocyte viability than the HPO group(P<0.05),there was no significant difference in myocardial cell viability between HR group and HR + siRNA SDHA NC group(P>0.05).Changes in cardiomyocyte viability at high glucose: GHR,GHR + siRNA SDHA NC,GHPO,and GHPO + siRNA SDHA NC group had lower cardiomyocyte viability than GN,GHR + siRNA SDHA,and GHPO + siRNA SDHA group.The myocardial cell viability of GHR + siRNA SDHA group was lower than that of GHPO + siRNA SDHA group(P<0.05).There was no significant difference in cardiomyocyte viability between GHR,GHR + siRNA SDHA NC,GHPO and GHPO + siRNA SDHA NC group(P>0.05).2.Changes of MMP: MMP changes under normal conditions: MMP in myocardial cells of HR group was lower than that of N group and HPO group(P<0.05).MMP changes in cardiomyocytes of each group after infection of siRNA SDHA with adenovirus: MMP in HR group and HR + siRNA SDHA NC group were lower than HR + siRNA SDHA group and HPO group,and MMP in HR + siRNA SDHA group was lower than HPO group(P<0.05),there was no significant difference in MMP between HR group and HR + siRNA SDHA NC group(P>0.05).MMP changes in cardiomyocytes in high glucose state: The MMP of cardiomyocytes in GHR,GHR + siRNA SDHA NC,GHPO and GHPO + siRNA SDHA NC group were lower than GN,GHR + siRNA SDHA and GHPO + siRNA SDHA group.The MMP of GHR + siRNA SDHA group was lower than that of GHPO + siRNA SDHA group(P<0.05).There was no significant difference in MMP of cardiomyocytes between GHR,GHR + siRNA SDHA NC,GHPO and GHPO + siRNA SDHA NC group(P>0.05).3.Changes in ROS: Changes of ROS content in cardiomyocytes under normal conditions: ROS content in myocardial cells of HR group was higher than that in N group and HPO group(P<0.05).Changes content of ROS in cardiomyocytes of each group after infection of siRNA SDHA with adenovirus: The changes of ROS content in cardiomyocytes of HR group and HR +siRNA SDHA NC group were higher than those in HR + siRNA SDHA group and HPO group,and ROS content in HR +siRNA SDHA group was higher than HPO group(P<0.05).There was no significant difference in ROS content between HR group and HR +siRNA SDHA NC group(P>0.05).Changes of ROS content in myocardial cells under high glucose: The ROS content of cardiomyocytes in GHR,GHR+ siRNA SDHA NC,GHPO and GHPO + siRNA SDHA NC group was higher than GN,GHR + siRNA SDHA and GHPO + siRNA SDHA group.The ROS content in cardiomyocytes of GHR + siRNA SDHA group was higher than that in GHPO + siRNA SDHA group(P<0.05).There was no significant difference in ROS content between GHR,GHR + siRNA SDHA NC,GHPO and GHPO + siRNA SDHA NC group(P>0.05).4.Changes of ATP content: The changes of ATP content in cardiomyocytes under normal conditions: the ATP content of myocardial cells in HR group was less than that in N group and HPO group(P<0.05).The ATP content of cardiomyocytes in each group after infection of siRNA SDHA with adenovirus: The content of ATP in cardiomyocytes of HR group and HR + siRNA SDHA NC group was lower than that of HR + siRNA SDHA group and HPO group,and the content of ATP in cardiomyocytes of HR + siRNA SDHA group was lower than that of HPO group(P<0.05).There was no significant difference in ATP content between HR group and HR + siRNA SDHA NC group(P>0.05).Changes in ATP content in cardiomyocytes in high glucose state: ATP content in cardiomyocytes of GHR,GHR + siRNA SDHA NC,GHPO and GHPO + siRNA SDHA NC group was lower than that of GN,GHR + siRNA SDHA and GHPO + siRNA SDHA group.The ATP content of cardiomyocytes in GHR + siRNA SDHA group was lower than that in GHPO +siRNA SDHA group(P<0.05).There was no significant difference in ATP content between GHR,GHR + siRNA SDHA NC,GHPO and GHPO + siRNA SDHA NC(P>0.05).5.Changes of SDHA protein expression level: the change of SDHA protein expression in cardiomyocytes under normal conditions: the expression of SDHA protein in HR group was higher than that of N group and HPO group,and the expression of SDHA protein in HPO group was higher than that in N group(P<0.05).The expression of SDHA protein ofcardiomyocytes in each group after infection of siRNA SDHA with adenovirus: the expression of SDHA protein in HR group and HR + siRNA SDHA NC group was higher than that in HR + siRNA SDHA group and HPO group,the expression level of SDHA protein in HR + siRNA SDHA group was lower than that in HPO group(P<0.05).There was no significant difference in the expression of SDHA protein between HR group and HR + siRNA SDHA NC group(P>0.05).The expression of SDHA protein in cardiomyocytes under high glucose status: The expression levels of SDHA in G15 N group,G15 HR group and G15 HPO group were higher than those in normal culture group(P<0.05).The expression of SDHA protein in cardiomyocytes of GHR,GHR + siRNA SDHA NC,GHPO and GHPO + siRNA SDHA NC group was higher than that of GN,GHR + siRNA SDHA and GHPO + siRNA SDHA group,the expression of SDHA protein in cardiomyocytes of GHR + siRNA SDHA group and GHPO + siRNA SDHA group was lower than that of GN group(P<0.05).There was no significant difference in the expression of SDHA protein between GHR + siRNA SDHA group and GHPO + siRNA SDHA group.There was no significant difference in SDHA protein expression between GHR,GHR + siRNA SDHA NC,GHPO and GHPO + siRNA SDHA NC group(P>0.05).Conclusion: Hypoxia postconditioning has protective effects on hypoxia reoxygenation injury in normal adult rat cardiomyocytes,and its mechanism is related to inhibition of SDHA protein expression.The protective effect of hypoxia postconditioning on adult rat cardiomyocytes in high glucose state disappears.Inhibition of SDHA protein expression can restore the protective effect of HPO on cardiomyocytes in high glucose state,which may be related to reducing ROS production to protect membrane integrity,thereby protecting MMP and promoting mitochondrial ATP synthesis.