摘要
目的探讨衰老对大鼠骨骼肌组织内质网氧化还原酶-1α(ERO1α)DNA甲基化与内质网应激及凋亡相关蛋白表达的影响。方法以6月龄大鼠为成年组,18月龄大鼠为老年组,每组10只,建立动物模型。采用实时荧光定量PCR(qRT-PCR)和Western blot方法分别检测成年组与老年组大鼠骨骼肌组织中凋亡相关基因Bcl-2、Caspase 3、Caspase 9、Caspase 12和内质网应激相关基因ATF6、CHOP、GRP78、XBP-1的mRNA和蛋白表达水平;qRT-PCR和Western blot检测ERO1α的mRNA和蛋白表达水平;巢式降落式甲基化特异性PCR(nMS-PCR)法检测ERO1αDNA甲基化水平的改变。结果与成年组比较,老年组大鼠骨骼肌组织中Bcl-2 mRNA表达差异无统计学意义(P>0.05),Bcl-2蛋白表达水平降低(P<0.01),Caspase 3、Caspase 9、Caspase 12的mRNA和蛋白表达水平均升高(P均<0.01);老年组内质网应激相关指标ATF6的mRNA表达改变与成年组比较差异无统计学意义(P>0.05),而ATF6蛋白表达水平增加(P<0.001);CHOP、GRP78、XBP-1的mRNA和蛋白表达水平较均成年组升高(P均<0.01);老年组大鼠骨骼肌组织中ERO1αmRNA和蛋白表达水平均降低(P均<0.01),而ERO1αDNA甲基化水平升高(P<0.01)。结论衰老大鼠骨骼肌组织ERO1αDNA高甲基化与内质网应激及凋亡相关蛋白的表达水平改变及骨骼肌细胞凋亡有关。
Objective To investigate the effects of aging on the level of DNA methylation in endoplasmic reticulum oxidoreductase-1α(ERO1α)promoter,and the expression of ERO1αand endoplasmic reticulum stress and apoptosis related protein expression in rat muscle.Methods The six-month rats were used as adult group,eighteen months as elderly group,ten rats in each group.Real-time quantitative PCR(qRT-PCR)and Western blot were used to detect the mRNA and protein expression of the Bcl-2,Caspase 3,Caspase 9 and Caspase 12 both in adult group and elderly group.Meanwhile,qRT-PCR and Western blot were used to detect the mRNA and protein expression of the ATF6,CHOP,GRP78,XBP-1 in the two groups.qRT-PCR and Western blot were used to detect the ERO1αexpression level.Nested landing methylation specific PCR(nMS-PCR)was used to detect the level of DNA methylation in ERO1αpromoter.Results The results showed that compared with the adult group,the Bcl-2 protein expression in elderly group was decreased(P<0.01)and mRNA expression had no significance change(P>0.05),Caspase 3,Caspase 9,Caspase 12 mRNA and protein expression were increased(P all<0.01).Compared with the adult group,the mRNA and protein expression of related parameters of endoplasmic reticulum stress CHOP,GRP78,XBP-1 mRNA were increased in the elderly group(P all<0.01).The ATF6 protein expression in elderly group was increased(P<0.01)and mRNA expression had no significance change(P>0.05).The ERO1αmRNA and protein expression were lower in the elderly group than those in adult group(P all<0.01).The result of nMS-PCR showed that the ERO1αDNA methylation level in the elderly group was increased compared with that in adult group(P<0.01).Conclusion ERO1αDNA hypermethylation in aging rats skeletal muscle was associated with ER stress and apoptosis-related proteins expression changes.
作者
熊建团
杨松昊
张辉
杨安宁
朱光荣
蒋袁絮
杨晓玲
姜怡邓
XIONG Jiantuan;YANG Songhao;ZHANG Hui;YANG Anning;ZHU Guangrong;JIANG Yuanxu;YANG Xiaoling;JIANG Yideng(Basic Medical College,Ningxia Medical University,Yinchuan 750004,China;College of Pharmacy of Ningxia Medical University,Yinchuan 750004,China)
出处
《宁夏医科大学学报》
2019年第5期440-445,共6页
Journal of Ningxia Medical University
基金
国家自然科学基金(81670416
81560242)
宁夏高等学校一流学科建设项目(NXYLXK2017B07)
