Effects And Mechanisms Of PP2A/Nrf2 On Experimental Diabetic Cardiomyopathy

With the improvement of people's living standard,changes in dietary pattern and lifestyle have led to a rapid increase in diseases related to glycolipid metabolic disorders,and diabetes and diabetes complications have become a global health threat.Diabetic cardiomyopathy(DCM)is a kind of specific cardiomyopathy which occurs in diabetic patients with abnormal cardiac structure and functions.It is independent of heart disease such as coronary heart disease,hypertension,and heart valve disease and so on.It is one of the main causes of death in diabetic patients.The exact pathogenesis of DCM is unclear.It involves a variety of factors,including hyperglycemia,hyperinsulinemia,abnormal fatty acid metabolism,oxidative stress,cardiac autonomic neuropathy,and overactivation of local renin-angiotensin-aldosterone system.Hyperglycemia is generally considered to be the key to activate and abnormally regulate several metabolic pathways to initiate diabetic myocardial damage.Hyperglycemia in diabetes can cause abnormal metabolism of myocardium and increase the factors such as glucose autoxidation,free fatty acid and leptin,cause the increase of reactive oxygen species(ROS)generation,resulting in oxidative stress,and eventually induce autophagy and apoptosis of cardiac myocytes.Keapl/Nrf2 signaling pathway is important to regulate oxidative stress in cells.It can produce antioxidant activities by regulating antioxidant enzymes and phase II detoxification enzymes.It is also an important protective mechanism for cell resistance to foreign substances and tumor formation.Protein phosphatase 2A(PP2A)is a serine/threonine protein phosphatase that dephosphorylates the proteins activated by reversible phosphorylation,which is negatively regulated in signal transduction.Studies have shown that PP2A can participate in insulin activation and improve insulin resistance in metabolism.The results also showed that the increase of PP2A activity could improve the apoptosis of myocardial cells induced by high glucose,and the inhibition of PP2A activity would lead to the continuous phosphorylation of IKK ?/IKK ?,and the activation of NF-?B signaling pathway could not be terminated.However,how PP2A/Nrf2 regulates cardiomyocytes through autophagy and apoptosis in high glucose environment is not clear.In this study,the roles of PP2A/Nrf2 in experimental diabetic cardiomyopathy were studied in vivo and in vitro,and the mechanism of its action was discussed in order to lay the foundation for the development of the drugs for the prevention and treatment of diabetic cardiomyopathy.The study of autophagy and apoptosis pathway will be helpful to explore the process and mechanism of diabetic cardiomyopathy,and to explore new targets for the development of new drugs for the prevention and treatment of diabetic cardiomyopathy,heart failure and myocardial infarction.In vitro:MTT assay showed that glucose inhibited the viability of cardiomyocytes with the increase of concentration and time.Cardiomyocytes were induced by high glucose,and cell surface area was measured by Image Pro Plus software.The results showed that 50 mmol/L glucose could induce the increase of myocardial surface area.The above results showed that high glucose could induce experimental diabetic cardiomyopathy.The effects of 3-MA combined with high glucose on myocardial autophagy were investigated by AO staining and Western blotting.The results showed that 3-MA pretreatment could inhibit the increase of autophagy induced by high-glucose.In the experiment,NAC was used to inhibit the ROS produced in the cells,and DCFH-DA staining was used to detect the ROS generation.The results showed that the ROS level in the high glucose group was significantly higher than that in the control group.AO and LTG staining showed that NAC could inhibit the increase of autophagy level in high glucose group.The results of Western blotting showed that high-glucose group could increase the expression of HIF-1 a and Nrf2,increase the ratio of autophagy related LC3B2/LC3B1,and down-regulate the expression of P62.PI3K/Akt/mTOR pathway was down-regulated and the expression of ERK was up-regulated.These results suggested that high-glucose may induce the increase of ROS generation and then activate autophagy by regulating PI3K/Akt/mTOR and ERK signaling pathways.In order to study the effect and mechanism of PP2A on high glucose induced cardiomyopathy.PP2A inhibitor(OA)and agonist(FTY720)and adenovirus-PP2A were used.AO staining and LTG staining showed that high expression and low expression of PP2A group could reduce the level of autophagy induced by high glucose.Western blotting results showed that low expression of PP2A+NAC group and high expression PP2A group could reduce the expression of Nrf2 protein,and reduce the ratio of autophagy related protein LC3B2/LC3B1,and increase P62 expression.The results showed that PP2A could affect the autophagy induced by high glucose by inhibiting the production of ROS;Hoechst 33342 staining and Annexin V/PI double staining showed that the apoptosis level of high glucose group was not obvious,and the level of apoptosis was increased after inhibition of ROS.Apoptosis was inhibited in low expression group PP2A,but when combined with NAC,the level of apoptosis increased significantly.The expression of high expression of PP2A was contrary to the group OA.When the expression level of PP2A was increased,the expression of Bcl-2 was inhibited and the level of apoptosis was significantly increased,and apoptosis was inhibited after the combined use of NAC.The results showed that PP2A might affect myocardial apoptosis by inhibiting ROS production.it was found that the expression of adenovirus-PP2A was the most obvious effect on the expression of Caspase-3.The expression of Caspase-3 in the low expression PP2A group was completely suppressed and the level of apoptosis was decreased.In vivo:Kunming mice were fed with high-glucose and high-fat diet for 4 weeks,then intraperitoneal injection of low dose STZ was used to establish diabetic cardiomyopathy model,and continuous intraperitoneal injection for 2 weeks.The results showed that compared with the blank control group,the ratio of heart/body weight in the model group was increased.The results of HE staining showed that the cardiomyocytes of the model group were hypertrophy,the color of the cells became pale,the nucleus increased,and the part of the cell contact appeared to be fused.Compared with the model group,the ratio of heart/weight of OA group decreased obviously,the ratio of heart/weight in FTY720 group increased.The result of HE staining showed that group FTY720 could significantly reduce the degree of myocardial lesion,while the OA group was significantly worse.Compared with the blank group,the contents of MDA,CAT,NOS and other enzymes in the serum of model group increased and SOD content decreased.Compared with the model group,the content of MDA,CAT,NOS and other enzymes in the FTY720 group increased,and the Western blotting results showed that the expression of Nrf2 protein in the model group increased,the autophagy increased and the apoptosis inhibited.Compared with the model group,the expression of Nrf2 protein in OA group decreased,autophagy decreased,apoptosis inhibited,and the expression of Nrf2 protein in FTY720 group increased,and the level of autophagy was not changed,and apoptosis was increasedConclusion:we studied the roles of PP2A/Nrf2 in diabetic cardiomyopathy and explored its mechanism for the first time.The results found that the expression of PP2A increased and the protective autophagy and apoptosis also increased in diabetic cardiomyopathy.This protective increase in autophagy and apoptosis might be achieved by initiating the antioxidant and reductive pathway and regulating the expression of Nrf2.However,how PP2A regulates Nrf2 and further regulates autophagy and apoptosis to protect cardiomyocytes are needed further research.