EGCG升高cTnIR193H限制型心肌病模型小鼠未突变cTnI的表达水平
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摘要
目的:研究表没食子儿茶素没食子酸酯(EGCG)升高cTnIR193H限制型心肌病模型小鼠未突变cTnI的表达水平。方法:8周龄野生型C57小鼠及8周龄cTnIR193H限制型心肌病模型小鼠分别随机分为EGCG干预组、DMSO干预组以及未干预组。每周干预5 d,3个月后采集小鼠心脏组织。分别以Western blot与RT-PCR分别检测HDAC1、GATA4及cTnI的mRNA表达水平。组蛋白H3K9乙酰化水平、cTnI基因启动子区域中的GATA4与HDAC1结合水平以ChIP-Q-PCR方式检测。结果:EGCG干预组未突变cTnI蛋白表达水平与mRNA表达水平、GATA4的mRNA表达水平、组蛋白H3K9乙酰化水平、cTnI启动子区域GATA4的结合水平均高于未干预组(P<0.05)。cTnI启动子区域HDAC1结合水平、HDAC1的mRNA表达水平均低于未干预组(P<0.05)。结论:在EGCG干预R193H模型小鼠实验中,EGCG可抑制HDAC1的表达,抑制cTnI启动子区域HDAC1的结合,促进心脏核心转录因子GATA4的表达、组蛋白H3K9的乙酰化、及cTnI启动子区域GATA4的结合,上调心肌的未突变cTnI的表达水平。
AIM: To increase the expression level of normal cTnI in mice with cTnIR193 H restricted cardiomyopathy by EGCG. METHODS: 8 weeks old wild type C57 mice and 8 weeks old cTnIR193 H restricted cardiomyopathy model mice were randomly divided into EGCG intervention, DMSO intervention group and non intervention group. After 5 days of intervention, the heart tissues of mice were collected after 3 months. mRNA expression levels of HDAC1, GATA4 and cTnI were detected by Western blot and RT-PCR respectively. The acetylation level of histone H3 K9, the binding level of GATA4 and HDAC1 in cTnI gene promoter region were detected by ChIP-Q-PCR. RESULTS:The level of protein level in the EGCG intervention group and the expression of normal cTnI at the level of mRNA, the level of mRNA expression of GATA4, the level of histone H3 K9 acetylation, and the binding level of GATA4 were higher than those of the unpretreated group(P<0.05). The level of HDAC1 binding and HDAC1 mRNA expression level in cTnI promoter region were lower than those in the non intervention group(P<0.05). CONCLUSION: In the R193 H model mice, EGCG inhibits the expression of HDAC1 and inhibits the binding of HDAC1 in the promoter region of cTnI, thus promoting the expression of cardiac transcription factor GATA4, the acetylation of histone H3K9, and the combination of GATA4 in the promoter region of cTnI, and up regulation of the cTnI expression of myocardium.
AIM: To increase the expression level of normal cTnI in mice with cTnIR193 H restricted cardiomyopathy by EGCG. METHODS: 8 weeks old wild type C57 mice and 8 weeks old cTnIR193 H restricted cardiomyopathy model mice were randomly divided into EGCG intervention, DMSO intervention group and non intervention group. After 5 days of intervention, the heart tissues of mice were collected after 3 months. mRNA expression levels of HDAC1, GATA4 and cTnI were detected by Western blot and RT-PCR respectively. The acetylation level of histone H3 K9, the binding level of GATA4 and HDAC1 in cTnI gene promoter region were detected by ChIP-Q-PCR. RESULTS:The level of protein level in the EGCG intervention group and the expression of normal cTnI at the level of mRNA, the level of mRNA expression of GATA4, the level of histone H3 K9 acetylation, and the binding level of GATA4 were higher than those of the unpretreated group(P<0.05). The level of HDAC1 binding and HDAC1 mRNA expression level in cTnI promoter region were lower than those in the non intervention group(P<0.05). CONCLUSION: In the R193 H model mice, EGCG inhibits the expression of HDAC1 and inhibits the binding of HDAC1 in the promoter region of cTnI, thus promoting the expression of cardiac transcription factor GATA4, the acetylation of histone H3K9, and the combination of GATA4 in the promoter region of cTnI, and up regulation of the cTnI expression of myocardium.
引文
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[2] Antlanger M,Aschauer S,Kopecky C,et al.Heart failure with preserved and reduced ejection fraction in hemodialysis patients:prevalence,disease prediction and prognosis[J].Kidney Blood Press Res,2017,42(1):165-176.
[3] Maron BJ,Towbin JA,Thiene G,et al.Contemporary definitions and classification of the cardiomyopathies:an american heart association scientific statement from the council on clinical cardiology,heart failure and transplantation committee;quality of care and outcomes research and functional genomics and translational biology interdisciplinary working groups;and council on epidemiology and prevention[J].Circulation,2006,113(14):1807-1816.
[4] Huang X,Pi Y,Lee KJ,et al.Cardiac troponin I gene knockout:a mouse model of myocardial troponin I deficiency[J].Circ Res,1999,84(1):1-8.
[5] Yang CS,Landau JM,Huang MT,et al.Inhibition of carcinogenesis by dietary polyphenolic compounds[J].Annu Rev Nutr,2001,21:381-406.
[6] Zhang L,Nan C,Chen Y,et al.Calcium desensitizer catechin reverses diastolic dysfunction in mice with restrictive cardiomyopathy[J].Arch Biochem Biophys,2015,573:69-76.
[7] Pan B,Quan J,Liu L,et al.Epigallocatechin gallate reverses cTnI-low expression-induced age-related heart diastolic dysfunction through histone acetylation modification[J].J Cell Mol Med,2017,21(10):2481-2490.
[8] Elliott P,Andersson B,Arbustini E,et al.Classification of the cardiomyopathies:a position statement from the European Society of Cardiology Working Group on Myocardial and Pericardial Diseases[J].Eur Heart J,2008,29(2):270-276.
[9] Leone O,Longhi S,Quarta CC,et al.New pathological insights into cardiac amyloidosis:implications for non-invasive diagnosis[J].Amyloid,2012,19(2):99-105.
[10] Gomes AV,Liang J,Potter JD.Mutations in human cardiac troponin I that are associated with restrictive cardiomyopathy affect basal ATPase activity and the calcium sensitivity of force development[J].J Biol Chem,2005,280(35):30909-30915.
[11] van den Wijngaard A,Volders P,Van Tintelen JP,et al.Recurrent and founder mutations in the Netherlands:cardiac Troponin I (TNNI3) gene mutations as a cause of severe forms of hypertrophic and restrictive cardiomyopathy[J].Neth Heart J,2011,19(7/8):344-351.
[12] Webber SA,Lipshultz SE,Sleeper LA,et al.Outcomes of restrictive cardiomyopathy in childhood and the influence of phenotype:a report from the Pediatric Cardiomyopathy Registry[J].Circulation,2012,126(10):1237-1244.
[13] 杨靖亚,王锡昌,刘梅芳,等.表没食子儿茶素没食子酸酯通过下调GTP结合蛋白RhoA的表达抑制癌细胞的浸润和转移(英文)[J].中国临床药理学与治疗学,2006,11(7):729-734.
[14] Mi Y,Qi G,Fan R,et al.EGCG ameliorates diet-induced metabolic syndrome associating with the circadian clock [J].Biochim Biophys Acta,2017,1863(6):1575-1589.
[15] Liu S,Sun Z,Chu P,et al.EGCG protects against homocysteine-induced human umbilical vein endothelial cells apoptosis by modulating mitochondrial-dependent apoptotic signaling and PI3K/Akt/eNOS signaling pathways[J].Apoptosis,2017,22(5):672-680.
[16] McElyea SD,Starbuck JM,Tumbleson-Brink DM,et al.Influence of prenatal EGCG treatment and Dyrk1a dosage reduction on craniofacial features associated with down syndrome[J].Hum Mol Genet,2016,25(22):4856-4869.
[17] Xu Z,Ma G,Zhang Q,et al.Inhibitory mechanism of EGCG on fibrillation and aggregation of amidated human islet amyloid polypeptide[J].Chemphyschem,2017,18(12):1611-1619.
[18] Gallagher SJ,Tiffen JC,Hersey P.Histone Modifications,modifiers and readers in melanoma resistance to targeted and immune therapy[J].Cancers (Basel),2015,7(4):1959-1982.
[19] Wang Y,Han Y,Fan E,et al.Analytical strategies used to identify the readers of histone modifications:A review[J].Anal Chim Acta,2015,891:32-42.
[20] Yun M,Wu J,Workman JL,et al.Readers of histone modifications[J].Cell Res,2011,21(4):564-578.
[21] Wu J,Wang SH,Potter D,et al.Diverse histone modifications on histone 3 lysine 9 and their relation to DNA methylation in specifying gene silencing[J].BMC Genomics,2007,8:131.
[22] 王洋,堵培,高珂琴,等.曲古霉素A对CD14+单核细胞活化及TLR4信号通路的影响[J].中国临床药理学与治疗学,2018,23(8):867-873.
[23] Sun H,Zhu J,Lu T,et al.Curcumin-mediated cardiac defects in mouse is associated with a reduced histone H3 acetylation and reduced expression of cardiac transcription factors[J].Cardiovasc Toxicol,2014,14(2):162-169.
[24] Block DH,Shapira M.GATA transcription factors as tissue-specific master regulators for induced responses[J].Worm,2015,4(4):e1118607.
[25] Zhou T,Guo S,Zhang Y,et al.GATA4 regulates osteoblastic differentiation and bone remodeling via p38-mediated signaling[J].J Mol Histol,2017,48(3):187-197.
[26] Géraud C,Koch PS,Zierow J,et al.GATA4-dependent organ-specific endothelial differentiation controls liver development and embryonic hematopoiesis[J].J Clin Invest,2017,127(3):1099-1114.
[27] Zhou L,Liu J,Xiang M,et al.Gata4 potentiates second heart field proliferation and Hedgehog signaling for cardiac septation[J].Proc Natl Acad Sci U S A,2017,114(8):E1422-E1431.