Novel Oral Edaravone Attenuates Diastolic Dysfunction Of Diabetic Cardiomyopathy In Rats By Activating The Nrf2 Signaling Pathway

Objective:Diabetic cardiomyopathy is the principal reason of heart failure in diabetes mellitus,which has been proved to be independent of hypertension,coronary atherosclerotic heart disease,and valvular heart disease.Diabetic cardiomyopathy is characterized by diastolic dysfunction,which is more common and earlier than systolic dysfunction.The main pathological mechanisms of diabetic myocardial dysfunction are cardiomyocyte hypertrophy,apoptosis,collagen accumulation and interstitial fibrosis.Myocardial remodeling induced by these pathophysiological changes is the primary cause of diabetic myocardial diastolic dysfunction.Oxidative stress injury induced by hyperglycemia and hyperlipidemia toxicity is the main pathogenic factor of diabetic cardiomyocyte injury and dysfunction.Oxidative injury causes imbalances in the physiological regulation of cardiomyocyte and collagen synthesis,leading to cardiac hypertrophy,apoptosis,collagen accumulation and interstitial fibrosis.Increased attention has been paid to the exploration of effective antioxidant drugs and their potential mechanisms.Novel oral edaravone(OED)has potent non-specific free radical scavenging effects that can alleviate oxidative stress,which is effective for treating target organ damage caused by oxidative stress and convenient for maintaining effective and sufficient courses of treatment for patients with chronic disease like diabetic cardiomyopathy.In the present study,we hypothesized that OED could ameliorate the diabetic cardiomyopathy pathophysiological changes by alleviating oxidative stress injury,finally attenuate diastolic dysfunction associated with diabetic cardiomyopathy.We further explored the potential mechanism.Methods:1.Diabetic cardiomyopathy model was established by high-glucose and high-fat diet with intraperitoneal injection of streptozotocin to induced type 2 diabetic rats.The T2DM+OED group was orally administered OED for 4 w.Transthoracic echocardiography was performed to evaluate the cardiac function of rats at the termination of experiments,measuring heart rates(HR),early diastolic peak velocity(E velocity),late diastolic peak velocity(A velocity),left ventricular internal dimension(LVIDd),left ventricular internal dimension in systole(LVIDs),interventricular septal thickness in diastole(IVSd),interventricular septal thickness in systole(IVSs),left ventricular posterior wall thickness at end diastole(LVPWd),left ventricular posterior wall thickness at end systole(LVPWs)and left atrial diameter(LAD)in two-dimensional and M-mode imaging.In addition,left ventricular ejection fraction(LVEF),left ventricular fractional shortening(LVFS),ratio of E and A velocity(E/A ratio),left ventricular mass(LVW),and ratio of left ventricular mass to body weight(LVW/BW)were calculated for analysis.Superoxide dismutase(SOD)and malondialdehyde(MDA)contents in tissue were determined with ELISA kits.Cardiomyocyte cross-sectional areas,collagen-positive areas,and collagen Ⅰ/Ⅲaccumulation were assessed by HE staining,Masson staining,and immunohistochemistry.Cell apoptosis was detected by TUNEL staining and protein expression levels of Caspase3,Bax and Bcl-2 were detected using Western blotting.mRNA expression of β-MHC,Nrf2,NQO1,HO-1 was detected using RT-qPCR and protein expression levels of Nrf2,NQO1,HO-1 were detected using Western blotting.2.In vitro,glucolipotoxicity injured H9c2 cardiomyocyte was established and siRNA was used to silence Nrf2.Cell viability was determined by CCK-8 assay.Intracellular ROS content was detected by DCFH-DA assay.Cytoskeleton was stained by Actin-Stain 555 Phalloidin to measure cell surface area.Apoptotic cells were detected by hoechst33342 and TUNEL staining.mRNA expression of β-MHC,Nrf2,NQO1,HO-1 was detected using RT-qPCR.Protein expression levels of Nrf2,NQO1,HO-1 were detected using Western blotting.Results:1.OED significantly improved E/A ratio in diabetic rats and reduced IVSd,LVPWd,LAD,LVW and LVW/BW.There were no significant changes in the percentages of LVEF and LVFS among groups.2.OED decreased tissue content of MDA and increased tissue content of SOD in the diabetic myocardium.The cross-sectional area of cardiomyocytes was significantly increased and mRNA relative expression of β-MHC was elevated in the T2DM group.Oral administration of OED for 4 w prevented these changes.OED significantly reduced the proportion of apoptotic cells and reversed changes in Caspase3,Bax and Bcl-2 expression.OED significantly reduced collagen volume fractions and protein expression levels of collagen Ⅰ/Ⅲ increased.3.In vitro,OED improved H9c2 cells injury induced by glucolipotoxicity.The protective effect of OED against HG/HF-induced H9c2 on cell viability gradually increased as its concentrations increased.OED significantly decreased ROS levels,cell surface areas,and ratio of apoptotic cells induced by high glucose and high fat in H9c2 cardiomyocytes.4.In vivo and in vitro,OED significantly increased the protein expression level of Nrf2 and its nuclear translocation,followed by activated downstream antioxidant gene transcription including upregulated mRNA and protein expression levels of NQO1 and HO-1.5.The expression of Nrf2 decreased significantly in H9c2 cells with Nrf2 gene silence.The improvement of oxidative damage in H9c2 cells elicited by OED was abolished by silencing of Nrf2.Conclusion:OED improved hypertrophy and reduced cardiomyocyte apoptosis,collagen accumulation and interstitial fibrosis,eventually relieving diabetic myocardial diastolic dysfunction.This effect was associated with alleviation of oxidative stress injury by activation of the Nrf2 signaling pathway.Our study identifies novel OED maybe as a potential therapeutic agent for future clinical trials to treat chronic diabetic cardiomyopathy.