父代饮酒致子代小鼠心脏发育异常的组蛋白乙酰化修饰机制
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摘要
目的:探讨父代饮酒对子代心脏发育的影响,以及组蛋白乙酰化修饰机制。方法:雄性C57小鼠给予连续酒精(体积分数40%,每天10 mL/kg)暴露6周,分别设空白对照组(无处理)及阴性对照组(生理盐水灌胃)。干预后雌雄小鼠合笼,收集新生子代小鼠心脏组织。测量新生小鼠出生体重;电镜检测心肌细胞超微结构;TUNEL法检测心肌细胞凋亡水平;qPCR检测caspase-3及Bcl-2的mRNA表达水平;Western blot法检测active caspase-3、caspase-3及Bcl-2蛋白表达水平;ChIP-qPCR法检测caspase-3及Bcl-2启动子上组蛋白H3K9及H3K27乙酰化水平。结果:父代饮酒小鼠子代出生体重明显较对照组降低(P<0.05);电镜下见酒精组新生小鼠心肌细胞出现肌丝溶解、肌浆网扩大和肌丝排列紊乱;同时其凋亡细胞较对照组明显增多;酒精组caspase-3 mRNA表达较对照组显著升高(P<0.05),active caspase-3蛋白水平较对照组显著升高(P<0.05),Bcl-2 mRNA及蛋白均较对照组显著降低(P<0.05);caspase-3基因启动子组蛋白H3K9乙酰化水平较对照组显著升高(P<0.05);Bcl-2启动子组蛋白H3K27乙酰化水平较对照组显著降低(P<0.05)。结论:父代饮酒可以引起子代小鼠心脏发育异常。组蛋白乙酰化修饰可能介导了子代小鼠心肌细胞凋亡过程。
AIM: To investigate the role of histone acetylation modification in paternal alcohol consumption-induced heart development abnormalities of offspring. METHODS: Male C57 mice were exposed to 40% alcohol for 6 weeks at a dose of 10 mL·kg~(-1)·d~(-1). Normal saline was used as negative control and non-treatment was for blank control. Heart samples of the newborn offspring mice were collected. Birth weight was measured. Cardiac ultrastructure was observed by transmission electron microscope(TEM). The apoptosis of cardiomyocytes was measured by TUNEL method. The mRNA levels of caspase-3 and Bcl-2 were detected by qPCR. The protein levels of active caspase-3 and Bcl-2 were determined by Western blot. ChIP-qPCR was employed to analyze the levels of acH3 K9 and acH3 K27 on the promoters of caspase-3 and Bcl-2. RESULTS: Low birth weight was found in the mice of alcohol exposure group. TEM observation showed broad existence of sarcomere dissolution, enlargement of sarcoplasmic reticulum and disorder of sarcomere arrangement. The results of TUNEL displayed increased cardiac apoptosis in alcohol group compared with the controls. The mRNA levels of caspase-3 and the protein levels of active caspase-3 were increased compared with control group(P<0.05). The protein level of active caspase-3 was decreased compared with the controls(P<0.05). Both mRNA and protein levels of Bcl-2 were decreased compared with the controls(P<0.05). The acH3 K9 on the promoter of caspase-3 went up significantly(P<0.05), and acH3 K27 on the promoter of Bcl-2 was decreased(P<0.05). CONCLUSION: Paternal alcohol exposure causes heart development abnormalities in offspring mice. Histone acetylation may play an important role in the apoptosis of cardiomyocytes.
AIM: To investigate the role of histone acetylation modification in paternal alcohol consumption-induced heart development abnormalities of offspring. METHODS: Male C57 mice were exposed to 40% alcohol for 6 weeks at a dose of 10 mL·kg~(-1)·d~(-1). Normal saline was used as negative control and non-treatment was for blank control. Heart samples of the newborn offspring mice were collected. Birth weight was measured. Cardiac ultrastructure was observed by transmission electron microscope(TEM). The apoptosis of cardiomyocytes was measured by TUNEL method. The mRNA levels of caspase-3 and Bcl-2 were detected by qPCR. The protein levels of active caspase-3 and Bcl-2 were determined by Western blot. ChIP-qPCR was employed to analyze the levels of acH3 K9 and acH3 K27 on the promoters of caspase-3 and Bcl-2. RESULTS: Low birth weight was found in the mice of alcohol exposure group. TEM observation showed broad existence of sarcomere dissolution, enlargement of sarcoplasmic reticulum and disorder of sarcomere arrangement. The results of TUNEL displayed increased cardiac apoptosis in alcohol group compared with the controls. The mRNA levels of caspase-3 and the protein levels of active caspase-3 were increased compared with control group(P<0.05). The protein level of active caspase-3 was decreased compared with the controls(P<0.05). Both mRNA and protein levels of Bcl-2 were decreased compared with the controls(P<0.05). The acH3 K9 on the promoter of caspase-3 went up significantly(P<0.05), and acH3 K27 on the promoter of Bcl-2 was decreased(P<0.05). CONCLUSION: Paternal alcohol exposure causes heart development abnormalities in offspring mice. Histone acetylation may play an important role in the apoptosis of cardiomyocytes.
引文
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[15] Naru Z,Zubing C,Ronghua W,et al.Dynamic changes of histone H3 lysine 27 acetylation in pre-implantational pig embryos derived from somatic cell nuclear transfer[J].Anim Reprod Sci,2014,148(3-4):153-163.
[2] Day J,Savani S,Krempley BD,et al.Influence of paternal preconception exposures on their offspring:through epigenetics to phenotype[J].Am J Stem Cells,2016,5(1):11-18.
[3] Xia R,Jin L,Li D,et al.Association between paternal alcohol consumption before conception and anogenital distance of offspring[J].Alcohol Clin Exp Res,2018,42(4):735-742.
[4] Ouko LA,Shantikumar K,Knezovich J,et al.Effect of alcohol consumption on CpG methylation in the differen-tially methylated regions of H19 and IG-DMR in male ga-metes:implications for fetal alcohol spectrum disorders[J].Alcohol Clin Exp Res,2009,33(9):1615-1627.
[5] Liang F,Diao L,Jiang N,et al.Chronic exposure to ethanol in male mice may be associated with hearing loss in offspring[J].Asian J Androl,2015,17(6):985-990.
[6] Lange S,Rehm J,Popova S.Implications of higher than expected prevalence of fetal alcohol spectrum disorders[J].JAMA,2018,319(5):448-449.
[7] Pan B,Zhu J,Lv T,et al.Alcohol consumption during gestation causes histone3 lysine9 hyperacetylation and an alternation of expression of heart development-related genes in mice[J].Alcohol Clin Exp Res,2015,38(9):2396-2402.
[8] Abel E.Paternal contribution to fetal alcohol syndrome[J].Addict Biol,2004,9(2):127-133.
[9] Lee HJ,Ryu JS,Choi NY,et al.Transgenerational effects of paternal alcohol exposure in mouse offspring[J].Anim Cells Syst,2013,17(6):429-434.
[10] 刘淑珍,尤鑫,熊小栓,等.心肌再灌注对抑郁大鼠心肌细胞凋亡以及和表达的影响[J].中国病理生理杂志,2012,28(3):453-458.
[11] 毛挺挺,郑亚华,方红波,等.Bcl-2和PCNA表达在大鼠成肌细胞氧化应激损伤中的作用[J].中国病理生理杂志,2017,33(5):935-938.
[12] Bose DA,Donahue G,Reinberg D,et al.RNA binding to CBP stimulates histone acetylation and transcription[J].Cell,2017,168(1-2):135-149.
[13] Pradeepa MM.Causal role of histone acetylations in enhancer function[J].Transcription,2017,8(1):40-47.
[14] Rollo C,Li Y,Jin XL,et al.Histone 3 lysine 9 acetylation is a biomarker of the effects of culture on zygotes[J].Reproduction,2017,154(4):375-385.
[15] Naru Z,Zubing C,Ronghua W,et al.Dynamic changes of histone H3 lysine 27 acetylation in pre-implantational pig embryos derived from somatic cell nuclear transfer[J].Anim Reprod Sci,2014,148(3-4):153-163.