Protective Effect And Mechanism Of Canagliflozin Pretreatment On Hyperglycemic Hypoxia Reoxygenation Injury Of H9c2 Cardiomyocytes

Background and objectiveOne of the main chronic diseases,diabetes is becoming more and more prevalent worldwide.The incidence of cardiovascular disease in individuals with diabetes is 2 to 3times higher than in non-diabetic patients,the heart is more vulnerable to ischemiareperfusion damage,and diabetic patients have a higher risk of developing coronary artery occlusion than non-diabetic patients do.The lipidemic drug sodium-glucose cotransporter 2 inhibitor(SGLT2i)has greater cardiovascular advantages over other hypoglycemic drugs.The CANVAS study further supported the cardioprotective effects of CANA,which decreased major adverse cardiac events by 14% in patients with T2 DM.The CVD-REAL study evaluated the cardiovascular protective effects of SGLT2 i in the real world,with canagliflozin(CANA)at 53%,higher than other SGLT2 i.Similar to this,in animal models of acute myocardial infarction,CANA has been found to have cardioprotective effects by enhancing cardiac function during ischemia,decreasing infarct size,and delaying the development of heart failure.In vitro studies have shown that CANA can preserve the myocardium by lowering endothelial dysfunction,decreasing inflammatory response,inhibiting apoptosis,and other factors.In contrast to apoptosis,which results in a slow,reversible breakdown of the membrane,pyroptosis is an inflammatory form of programmed cell death.The PI3K/Akt/m TOR signaling pathway is a key negative regulatory route for autophagy,which is a lysosome-dependent process that exploits the double-membrane structure as a type of intracellular transport to breakdown damaged,denatured,or senescent organelles and proteins in cells.In this research,we generated an in vitro cardiomyocyte HG+ H/R damage model by treating H9c2 cardiomyocytes with high glucose(HG)+hypoxia/reoxygenation(H/R).As a result,we hypothesize that CANA may have a protective impact on the myocardium through regulating autophagy and anti-pyroptosis.As a result,the effect of CANA on H/R damage caused by cardiomyocytes in an HG environment was discussed,and the cardioprotective mechanism of CANA was clarified,which gave rise to a new concept for the clinical treatment strategy for patients with T2 DM who have suffered myocardial ischemia-reperfusion injury.MethodsAfter subculture H9c2 cardiomyocytes for 48 h,they were randomly divided into 5groups: CON group(blank control group),HG+H/R group(high glucose + hypoxic reoxygenation group),HG+H/R+Ly294002 group(Ly294002 inhibitor control group),HG+H/R+CANA group(canagliflozin pretreatment group),HG+H/R+CANA+Ly294002 group(canagliflozin + Ly294002 inhibitor group).Cardiomyocytes in the CON group were cultured normally with low-glucose DMEM without any treatment;The cells of HG+H/R group were cultured in 33.30 m M highglucose medium for 48 h,followed by 12 h hypoxia and 4 h reoxygenation treatment to construct the HG+H/R damage model of the cells.Cells in HG+H/R+Ly294002 and HG+H/R+CANA groups were incubated with 33.30 m M high-glucose medium containing 10μmol/L Ly294002 or CANA 12 h before H/R injury,and the rest of the treatment was the same as HG+H/R group.Twelve hours before to H/R damage,the cells of the HG+H/R+CANA+Ly294002 group were cultured with 10 mol/L of LCANA and 10 mol/L of Ly294002 complete culture.The rest of the treatment was the same as it was for the HG+H/R group.Following modeling of each group,the intracellular ROS level was determined by flow cytometry,p-Akt/Akt,p-m TOR/m TOR,LC3II/I,Beclin 1,pro-caspase-1,and expression of proteins like NLRP3,GSDMD.Cell viability was determined using the CCK-8 method,LDH activity in cardiomyocytes was determined using a kit,and the content of MDA in cardiomyocytes was determined using a kit.Results1.Effects of CANA pretreatment on H/R-induced cardiomyocyte damage in HG environment: Compared with the CON group,cell viability in HG+H/R group was significantly reduced(P<0.05),LDH activity,MDA content and ROS level were significantly increased(P<0.05),and intracellular mitochondrial membrane potential level decreased(P<0.05).Under the condition of HG+H/R damage,CANA pretreatment can significantly improve cell survival rate(P<0.05)and inhibit the increase of LDH activity,MDA and ROS in cells(P<0.05).At the same time,CANA improved H/R damage-induced mitochondrial dysfunction(P<0.05).2.Effect of CANA pretreatment on H/R-induced autophagy in cardiomyocytes in HG environment: Compared with the CON group,the level of autophagy in cells under HG+H/R damage conditions was significantly increased.Cardiomyocytes pretreated with CANA expressed significantly lower Beclin 1 and LC3 II/I proteins under HG+H/R damage(P<0.05).3.Effect of CANA pretreatment on H/R-induced pyroptosis of cardiomyocytes in HG environment: Compared with the CON group,the staining rate of cardiomyocyte pyrozosis under HG+H/R injury conditions was increased.After pretreatment with CANA,a decrease in the staining rate of pyroptosis and a significant decrease in the expression levels of pro-caspase-1,ASC,GSDMD,and NLRP3 proteins(P<0.05)were observed.4.Effect of CANA on the expression of proteins related to PI3K/Akt signaling pathway: Compared with the CON group,a significant decrease in the expression of p-Akt/Akt in cardiomyocytes was observed in the HG+H/R group,and the expression of p-Akt/Akt in cardiomyocytes was increased after adding CANA pretreatment(P<0.05).After adding Ly294002,the expression level of p-Akt/Akt in cardiomyocytes was significantly reduced compared with the HG+H/R+CANA group(P<0.05).5.Effect of CANA on H/R damage of cardiomyocytes in HG environment through PI3K/Akt/m TOR signaling pathway: Compared with HG+H/R+CANA group,cell survival rate decreased in HG+H/R+CANA+Ly294002 group,intracellular LDH activity,MDA content and ROS level increased significantly(P<0.05),while mitochondrial membrane potential decreased(P <0.05),Ly294002 reversed the cardioprotective effects of CANA.6.Effect of CANA on autophagy through PI3K/Akt/m TOR signaling pathway:Compared with the HG+H/R group,the expression levels of autophagy proteins LC3II/I and Beclin 1 were significantly reduced(P<0.05)and the proportion of pm TOR/m TOR increased significantly(P<0.05)after CANA pretreatment,indicating that CANA can inhibit the expression of autophagy protein and promote m TOR protein phosphorylation.Ly294002 reverses the inhibitory effect of CANA on autophagy protein expression(P<0.05).7.Effect of CANA on cell pyroptosis through PI3K/Akt/m TOR signaling pathway:Compared with the HG+H/R group,the staining rate of cardiomyocytes pyroptosis decreased after CANA pretreatment,the protein expression levels of pro-caspase-1,ASC,GSDMD,and NLRP3 decreased(P<0.05),and the expression level of pm TOR/m TOR protein increased(P<0.05).The addition of Ly294002 reversed the inhibitory effect of CANA on cell pyrozoosis staining and protein expression(P<0.05).Conclusions1.Under HG+H/R damage circumstances,autophagy and cell pyrosis alter in H9c2 cardiomyocytes.2.Reduced autophagy,oxidative stress,improved mitochondrial dysfunction,decreased cell pyrosis,and other protective effects of CANA on H9c2 cardiomyocyte damage in HG environments.3.CANA can regulate the PI3K/Akt/m TOR pathway to inhibit the excessive autophagy of cardiomyocytes and reduce pyroptosis of cardiomyocytes,and the addition of PI3 K inhibitor Ly294002 can eliminate the protective effect of CANA on cardiomyocytes,which indicates that CANA’s inhibition of autophagy and reduction of cell pyroptosis are related to the PI3K/Akt/m TOR pathway.We draw the conclusion that CANA may protect against cardiomyocyte ischemiareperfusion injury in high glucose environments by reducing cell damage,oxidative stress,mitochondrial dysfunction,inhibiting cell hyperphagy,and reducing cell pyroptosis,which may be mediated by the PI3K/Akt/m TOR signaling pathway.NLRP3 inflammasome activation can be decreased by CANA by preventing excessive autophagy,which is a key factor in cardiomyocyte damage.