Sulforaphane Prevents Angiotensin Ⅱ-induced Cardiomyopathy By Activiation Of Nrf2 Through Epigenetic Modification

Background and objective Angiotensin Ⅱ(Ang Ⅱ)is involved in the development and progression of various cardiac diseases.Oxidative stress is the main cause of Ang Ⅱ-induced cardiac damage.The failure of exogenous ROS scavengers in large clinical trials suggested that improving the endogenous antioxidant defenses will be the most effective and safe strategy for cardiovascular patients.Nuclear factor erythroid 2-related factor(Nrf2)is a redox-sensitive transcription factor that up-regulates multiple antioxidant and detoxification genes against oxidative stress.Sulforaphane(SFN),a strong activator of Nrf2,is a safe and non-toxic compound that extracted from cruciferous vegetables.SFN can effectively protect oxidative stress-related cardiomyopathy associated with upregulation of Nrf2.SFN can activate Nrf2 by directly modifying the cysteine residue of Keap1 or indirectly inducing the activity of several kinases in cells.In our previous study of diabetic cardiomyopathy,SFN up-regulated the expression of Nrf2 in the myocardium as a long-term effect.The reports indicated that SFN exerted the chemopreventive effects of cancer by epigenetic modification of Nrf2.Therefore,our study aims to investigate the potential of SFN to activate Nrf2 by epigenetic modification,which provides a theoretical and experimental basis for clinical prevention and treatment of Ang Ⅱrelated cardiomyopathy.Methods Eight-week-old male C57/BL mice were injected subcutaneously with Ang Ⅱ(0.5 mg/kg)every other day for 2 months,with or without SFN(0.5 mg/kg)treatment for 3 months(3M),and observed for 6 months(6M);the control group was given the same dose of SFN formulation solvent.Animal experiments were divided into four groups(n=5): control group,SFN group,Ang Ⅱ group and Ang Ⅱ/SFN.At the 6th month after the initial modeling,echo analysis was used to detect the structure and function of the heart,and then the mice were sacrificed,cardiac tissues were collected for Masson and HE staining,and protein and RNA were extracted for detection of related indicators,including oxidative damage indicators(3-NT,4-HNE);inflammatory factors(IL-1β,ICAM-1);fibrosis-related factor of CTGF;the expression of Nrf2 and its downstream antioxidant genes(Nrf2,HO-1,CAT)and HDACs and DNMTs.The kit detected the enzymatic activity of HDACs and DNMTs.Methylation sequencing assay was performed to detect the methylation status of the first 15 Cp G islands and Chromatin immunoprecipitation(Ch IP)assay for histone H3 acetylation status in Nrf2 promoter region.Results Low-dose Ang Ⅱ could cause cardiac hypertrophy,ventricular dilatation,and decreased cardiac function in mice;SFN intervention effectively improved cardiac structural abnormality and disfunction.Ang Ⅱ induced the expression of cardiac inflammatory factors of IL-1β and ICAM-1,the increase of oxidative damage(3-NT)and nitriding damage(4-HNE),the expression of myocardial fibrosis molecular CTGF and accumulation of collagen(reflected by Masson staining),whereas SFN treatment inhibited Ang Ⅱ-induced cardiac inflammation,oxidative stress and fibrosis.SFN significantly increased the expression of Nrf2 and its downstream genes CAT and HO-1.Ang Ⅱ promoted the hypermethylation of the first 15 Cp G islands and decreased the acetylation of histone H3 in the Nrf2 promoter region,while SFN treatment reversed Ang Ⅱ-induced those changes in the Nrf2 promoter region.Ang Ⅱ caused an increase in protein expression and activity of DNMTs and HDACs in cardiac tissues,whereas SFN significantly inhibited the increase in the expression and activity of those enzymes induced by Ang Ⅱ.SFN up-regulated the expression of acetylated histone H3/H4(Ac-H3/H4).Conclusion 1.Ang Ⅱ caused increased expression of myocardial inflammatory factors,oxidative damage and myocardial fibrosis,leading to cardiac hypertrophy and cardiac dysfunction,and SFN effectively improved Ang Ⅱ-induced cardiac damage.2.SFN could prevent Ang Ⅱ-induced cardiomyopathy by activation of cardiac Nrf2,which was through inhibiting the expression and activity of DNMTs and HDACs,reducing the methylation of the first 15 Cp G islands,and promoting the acetylation of histone H3 in the Nrf2 promoter region to up-regulating the transcription of Nrf2.