The Protective Effect Of Dimethyl Fumarate On Diabetic Cardiomyopathy And The Underlying Mechanisms

Background:Diabetes Mellitus(DM)has become a worldwide threat,and diabetic complications,especially cardiovascular complications and diabetic cardiomyopathy(DCM)lead the mortality rate among all diabetic complications.Diabetic patients undergo a series of pathophysiological disorders,such as glucose and fatty acid energy metabolic disorder,insulin resistance,neurohormonal activation(e.g.the activation of Renin-angiotensin-aldosterone system),oxidative stress,altered Calcium handling etc.These then lead to the dysfunction,hypertrophy,fibrosis,arrhythmia,apoptosis/necrosis and microvascular abnormalities of the diabetic heart.Thus efficient prevention and treatment methods for DCM are urgently needed.The pathophysiological mechanisms of DCM are not yet fully understood.However,most studies agree that oxidative stress plays a key role in the development of DCM.Reactive oxygen species and reactive nitrogen species can directly damage cell protein and DNA,while enhance inflammatory reactions,induce cell apoptosis and contribute to DCM exacerbation.Previous investigations have extensively shown that antioxidant treatment such as with sulforaphane or resveratrol can partially or completely prevent DCM deterioration.Nuclear factor erythroid-2 related factor 2/antioxidant reactive element(Nrf2/ARE)promotes the body's defense against endogenous and exogenous oxidative stress.They upregulate multiple antioxidative genes and is considered the central transforming regulator of cell oxidative status.There are several studies that shows the upregulation of Nrf2 is beneficial to treating DCM.Furthermore,in vitro experiments found that silencing of Nrf2 gene leads to the lose of cardiac protection.As we better understand the mechanism of the Nrf2/ARE antioxidant pathway and the pathophysiology of DCM,treatment targeting activation of Nrf2 has come into sight.Though there has been evidence in animal models that activation of Nrf2 can attenuate tissue damage caused by ROS,inflammation and fibrogenesis,clinically safe and efficient Nrf2 activator remains under development.Dimethyl fumarate(DMF),also known as BG-12,is an FDA approved oral chemical for treatment of multiple sclerosis(MS).By experiments on human and animals,DMF sets a good safety record and reveals its ability to activate Nrf2 in both clinical patients and experimental animal models.By releasing Nrf2 from its inhibitor Keap1,DMF can help increase the downstream antioxidant genes' expression to fight against pathological factors.Due to the fact that oxidative stress is a central pathological impact on the diabetic heart,it is likely that anti-oxidant,such as DMF treatment is a potentially promising treatment strategy for DCM.This study also provides supporting evidence for repurpose of DMF in clinical application.Aim:In this study,we aim to establish DCM mouse models(type 1 diabetes)via multiple low-dose STZ administration,and analyze the therapeutic effect of DMF on pathophysiological mechanisms of myocardial injury in DCM.We also try to clarify the molecular mechanism of DCM myocardial protection.This study will help provide theoretical data and application reference for clinical prevention and treatment of DCM.Methods:Eight-week-old male C57BL/6J mice was injected via intraperitoneal with multiple low-dose(50mg/kg)of streptozotocin(STZ)to induce type 1 diabetes model,and as diabetes progress,cardiac damage will develop.At the same time,age-matched male C57BL/6J mice were given sodium citrate(p H4.5,0.1mol/L)buffer as control group.Then all mice were randomly divided into the following 4 groups:(1)control group(Ctrl): non-diabetic mice were injected with saline as treatment;(2)Ctrl+DMF group: non-diabetic mice given DMF treatment;(3)T1D group: type 1 diabetic mice with saline as treatment;(4)T1D+DMF group: type 1 diabetic mice given DMF treatment.DMF was dissolved in saline solution and administered intraperitoneally at 10mg/kg daily for 12 weeks.During the 12 weeks of administration,we weighed/measured and recorded the body weight and blood glucose of all the mice monthly;the mice cardiac structure and function were evaluated by non-invasive transthoracic echocardiography.At the end of 12-weeks of treatment,all mice were anesthetized and euthanized.We collected the mouse hearts and observed the morphological changes and anatomy of the cardiac tissue.Extraction of protein and tissue section biopsy observation were to detect myocardial fibrosis(e.g.,Sirius red staining,CTGF,TGF-?1).By ELISA,Western blot methods and real-time quantitative PCR,we assayed the myocardial tissue inflammation factors(TNF-?,PAI-1,IL-1?),adhesion molecules(ICAM-1,VCAM-1),oxidative stress level(SOD activity,CML adduct,NADPH oxidase activity,3-NT,4 HNE)and a variety of signaling transduction pathways.Furthermore,the transcription and translation levels of Nrf2 and its downstream antioxidant enzymes(SOD2,HO-1,catalase)were analysed in the anti-oxidative stress pathway.Conclusions are drawn by comparing and analysis of the experimental data.Results:(1)DMF treatment did not affect the blood glucose level or body weight of T1 D mice and Ctrl mice.The blood glucose(BG)level in Type 1 diabetic mice were significantly elevated after STZ injection(3-h fasting blood glucose ?250 mg/dl)compared to the Ctrl mice.Over the course of 12 weeks of DMF or vehicle treatment,neither T1 D nor Ctrl mice was affected on BG levels.Compared to the Ctrl mice,the T1 D mice heart weight to tibia length ratio(HW/TL)showed a slight drop(but no significant difference).Meanwhile DMF and vehicle treatment did not affect HW/TL within T1 D or Ctrl mice.(2)DMF treatment attenuated DCM cardiac dysfunction increasing ejection fraction and inhibited myocardial dilatation.In T1 D mice,echocardiography measurements showed significantly increased left ventricular internal diameter(LVID)(both systolic and diastolic phases),decreased ejection fraction(EF)percentage and fractional shortening(FS)and elevated LV volume(both systolic and diastolic phases).These indicated enlarged LV chamber and cardiac systolic dysfunction in T1 D mice,compared to the Ctrl mice.T1 D mice with DMF treatment showed significantly augmented EF and reduced systolic LV volume compared to T1 D mice.Other parameters of T1D+DMF mice(diastolic LVID,systolic LVID,FS and diastolic LV Vol)were moderateyl ameliorated,respectively,compared to the T1 D mice.(3)DMF treatment alleviated myocardial fibrosis by reducing TGF-?1 and other pro-fibrosis factors in DCM.Sirius Red staining on myocardial tissue showed a noticeable increase of collagen deposition in T1 D mice,which was significantly alleviated by DMF treatment.Real-time PCR assessment showed significantly increased pro-fibrotic markers TGF-?1,CTGF and ECM components.Fibronectin and collagen-1 in the heart of T1 D mice was blunted by DMF treatment.Western blot analysis found markedly increased protein levels of TGF-?1 and CTGF in T1 D mice,which was also repressed by DMF treatment,but Ctrl mice were not affected by DMF treatment.These results demonstrated that DMF treatment efficiently reduced diabetes-induced cardiac fibrosis.(4)DMF reduced the inflammatory response and inflammatory infiltration of myocardial tissue in DCM by eliminating pro-inflammatory markers,adhesion molecules,etc.Using real-time PCR,we found that T1 D mice cardiac tissue exhibited significant elevations of pro-inflammatory cytokines and adhesion molecules such as TNF-?,IL-1?,ICAM-1 and VCAM-1,and these cytokines and molecules were suppressed upon DMF treatment.By Western blot analysis,inflammatory markers TNF-?,ICAM-1,PAI-1 were also found to be elevated at the protein level in T1 D mice,and DMF significantly decreased these markers in the cardiac tissue.(5)DMF significantly inhibited the level of oxidative stress in the DCM cardiac tissue by reducing the source of free radicals and enhancing the expression of antioxidant enzymes and proteins.Oxidative stress levels were significantly elevated in T1 D mice,indicated by increased the activity of macrophage-associated NADPH-oxidase,a key source for O2-,lipid protein oxidation product 4HNE,nitrate stress marker 3-NT and protein oxidation product-CML adduct.But treatment with DMF efficaciously reduced these activities.Also,in T1 D mice,the anti-oxidative stress system was damaged,indicated by markedly reduced SOD activity and diminished GSH levels,corroborated with increased accumulation of lipid peroxides.Treatment with DMF sufficiently maintained the SOD and GSH production in T1 D mice while the Ctrl mice were not affected.(6)The protective effect of DMF on DCM was correlated with the up-regulation of Nrf2 and its downstream anti-oxidative enzyme expression in the myocardial tissue of T1 D mice.Myocardial tissues were subjected to Western blots and real-time PCR determinations for Nrf2 activation and its downstream effectors.Western blot analysis showed increased total Nrf2(t Nrf2)protein in T1 D mice(P < 0.01 vs.Ctrl)and DMF treatment further enhanced this increase(P < 0.01 vs.T1D).Only T1D+DMF mice had increased nuclear Nrf2(n Nrf2)levels,compared to all other groups.In addition to Nrf2 activation,we also found repressed or unaltered transcription levels of HO-1,SOD1 and catalase in T1 D mice,which were all prominently elevated by DMF treatment.Western blot analysis confirmed the m RNA findings and showed significantly decreased HO-1,catalase and increased SOD2 proteins in T1 D mice(P < 0.05 vs.Ctrl),and in T1 D + DMF mice HO-1,SOD2 and catalase were all augmented(P < 0.05 vs.T1D).Conclusions:(1)DMF can attenuate diabetes-induced cardiac structural damage and dysfunction.(2)The protective effect of DMF on DCM is anti-oxidative and highly correlated with the activation of Nrf2 and its downstream antioxidant enzymes.(3)DMF has a high potential in applying as prevention or treatment of DCM.Highlights:(1)The current study found for the first time the protective effect of DMF on DCM.(2)The mechanism of protective effect of DMF on DCM achieved by upregulation of Nrf2 and its downstream anti-oxidative gene expressions was first elucidated.