Punicalagin And Ellagic Acid Prevents Diabetic Myocardium Injury Through Promoting DNA Demethylation

Backgroud and Objective:Diabetic cardiovascular disease is one of the common complications of diabetes.Diabetic myocardium injury(DMI)is a pathological state caused by diabetes,with abnormal cardiac structure and function.DMI is the early clinical stage of diabetic cardiomyopathy,which significantly increases the risk of heart failure and death in diabetic patients.At present,there are no therapeutic drugs for DMI,and the prevention is still focus on the prevention and treatment of DMI.Therefore,on the basis of understanding the pathogenesis of DMI,moving the prevention and control threshold forward and actively searching for natural products that can prevent DMI has become one of the current research hotspots.DNA epigenetic disorders are one of the important reasons for the occurrence and development of DMI.When the DNA demethylation process is inhibited,the levels of 5-hydroxymethylcytosine(5hm C)in the promoter region of key structural and functional genes in the heart are significantly reduced,leading to myocardium injury.The process of DNA demethylation is mainly regulated by the α-ketoglutaric acid-dependent 10-11 translocation protein(TET1/2/3)family.The intermediate products of the tricarboxylic acid cycle,fumaric acid and succinic acid competes α-ketoglutarate acid,thus inhibits TET enzyme activity.In addition,AMP-activated protein kinase(AMPK)maintains the protein stability of TET2 by phosphorylating the serine 97 and 99 sites.Previous studies have found that punicalagin(PU)and ellagic acid(EA)have a certain protective effect on the heart,and the molecular mechanism is related to activating AMPK and improving mitochondrial function.However,no research reports on whether PU and EA prevent DMI by promoting DNA demethylation.This study consisted of two parts.The first part revealed the relationship between the levels of 5-methylcytosine(5m C)and 5hm C in peripheral blood DNA and cardiac function through a case-control study.The second part explored whether PU and EA prevented DMI by promoting DNA demethylation and the molecular mechanisms through animal experiments.Method:1.The population study:(1)This study used a case-control study to recruit 153 volunteers in Licha Town,Jiaozhou City,including 50 healthy(HL)volunteers,49 abnormal glucose metabolism(AGM)volunteers,and 54 abnormal glucose metabolism and myocardium injury(AGM+MI)volunteers.(2)Collect the basic information of the research volunteers through questionnaire survey and physical examination.The serum concentrations of fasting blood glucose(FBG),triglyceride(TG),total cholesterol(TC),high-density lipoprotein cholesterol(HDL-C)and low-density lipoprotein cholesterol(LDL-C),and the activities of serum markers of myocardium injury including aspartate aminotransferase(AST),lactate dehydrogenase(LDH),and creatine kinase(CK),were all measured by automatic biochemical analyzer.Dot-blot was used to detect the levels of 5-methylcytosine(5m C)and 5-hydroxymethylcytosine(5hm C)in peripheral blood DNA of volunteers.2.The animal study:(1)Male C57BL/6J mice were randomly divided into 6 groups after adaptive feeding for 1 week(n=10 in each group): normal control group(NC),diabetes mellitus group(DM),resveratrol group(RS,100 mg/kg/day),low-dose of PU group(LP,50mg/kg/day),high-dose of PU group(HP,100 mg/kg/day)and EA group(EA,100mg/kg/day).The NC group was fed normal diet with a fat to energy of 10%,while the other groups were fed high-fat diet with a fat to energy of 60%.After 8 weeks of intervention,intraperitoneal glucose tolerance tests were carried out in mice.Subsequently,the mice rested for 5 days,fasted overnight,and were sacrificed.Blood and cardiac tissue were collected for subsequent index detection.(2)The serum concentrations of TG,TC,HDL-C,LDL-C and serum activities of AST and CK were detected by automatic biochemical analyzer.Enzyme-linked immunosorbent assay was used to detect the serum concentrations of fasting insulin(FINS)in mice.The levels of total antioxidant capacity(T-AOC),superoxide dismutase(SOD),glutathione(GSH)and catalase(CAT)in cardiac tissue of mice were detected by biochemical kit.Dot-blot was used to detect the levels of DNA 5m C,DNA 5hm C and DNA 5-formylcytosine(5f C)in mice.Western blot was used to detect the protein expression levels of myosin heavy chain 7B(Myh7B),Xin actin-binding repeatcontaining protein 2(Xirp2),DNA methyltransferases(DNMTs),TET,AMPK-Acetyl Co A carboxylase(ACC),mitochondrial respiratory chain complex enzymes and tricarboxylic acid cycle related enzymes.Reverse transcription-polymerase chain reaction was used to detect the m RNA levels of TET and mitochondrial DNA copy number in mice.The contents of tricarboxylic acid cycle metabolites in mice hearts were detected by gas chromatography-mass spectrometry.Results:1.The population study:(1)Analysis of the basic characteristics of volunteers found that,there were no significant differences in age,gender,educational level,diastolic blood pressure level,hip circumference,waist circumference,waist to hip ratio,height,weight,and body mass index among the HL,AGM and AGM+MI groups.However,the levels of systolic blood pressure in the AGM+MI group was significantly higher than the HL group.(2)The analysis of serum biochemical indicators of volunteers found that,there were no statistically differences in the serum concentrations of TC,HDL-C,LDL-C,and the activities of AST and LDH among the HL,AGM and AGM+MI groups.The concentrations of FBG in the AGM+MI group and the AGM group were significantly higher than that in the HL group.While,the serum concentration of TG in the AGM group was significantly higher than that in the HL group.In addition,the serum activity of CK in the AGM+MI group was significantly higher than in the HL and AGM group.(3)The results of dot-blot showed that there was no statistically difference in DNA5 m C levels among the HL,AGM and AGM+MI groups.The levels of DNA 5hm C in the AGM+MI group were significantly lower than in HL and AGM group.The ratio of5 m C/5hm C in AGM+MI group were significantly higher than in HL and AGM group.(4)Correlation analysis showed that there was no significant correlation between DNA 5m C level and serum activities of AST,CK,LDH.There was a significant negative correlation between the levels of DNA 5hm C and the serum activities of AST(r =-0.4357,P < 0.001),CK(r =-0.5325,P < 0.001)and LDH(r =-0.6291,P < 0.001).2.The animal study:(1)After 2 weeks of intervention,the body weight of mice in DM group was significantly higher than in other groups.Compared with the DM group,the supplementation of PU and EA significantly reduced the heart weight,heart weight/body weight,blood glucose levels,area under the curve,FINS levels,serum activities of AST and CK,and improved the morphology of mice hearts and myocardial cells in mice.Under the supplemented with PU and EA,the protein expression levels of Myh7 B in mice were significantly higher than that in the DM group.The protein expression levels of Xirp2 in the EA group were significantly higher than that in the DM group.(2)There was no significant difference in DNA 5m C levels among all groups.Compared with the DM group,the supplementation of PU and EA increased the levels of DNA 5hm C and DNA 5f C in mice hearts.Compared with the DM group,there were no statistical difference in the protein expression levels of DNMT 1/3A/3B,TET1 and m RNA levels of TET in LP,HP and EA groups.The protein expression levels of TET2 in LP,HP and EA groups were significantly higher than that in DM group.The protein expression levels of TET3 in HP and EA groups were significantly higher than that in DM group.(3)The protein expression levels of p-AMPK/AMPK in LP group were significantly higher than that in DM group.Compared with the DM group,the supplementation of PU and EA increased the levels of T-AOC,SOD,GSH,CAT,mitochondrial DNA copy number and the protein expression levels of complexes II/III/V,and also increased the protein expression levels of aconitase 2,isocitrate dehydrogenase 2,α-ketoglutarate dehydrogenase,succinate dehydrogenase B and fumarate hydratase.The contents of fumaric acid and succinic acid in mice hearts with the PU and EA supplemented were significantly lower than those in the DM group.Moreover,the ratio of(fumaric acid+succinic acid)/ α-ketoglutarate acid was significantly higher in DM group than in the other groups.ConclusionThe case control studies found,there was a close correlation between the inhibition of DNA demethylation and the occurrence of DMI.Promoting DNA demethylation was expected to be a potential therapeutic strategy to prevent or delay DMI.Animal experiments found that PU and EA alleviated mitochondrial dysfunction,especially improved the ratio of α-ketoglutaric acid/(succinic acid+ fumaric acid),up-regulated the activities of TETs,promoted DNA demethylation,thus prevented the occurrence of DMI.