温和灸足三里穴对自然衰老大鼠肾组织p16 DNA甲基化的影响

Effect of mild moxibustion at Zusanli(ST36) on p16 DNA methylation in the kidney tissue of naturally aging rats

目的 观察温和灸足三里穴对自然衰老大鼠肾组织p16 DNA甲基化的调控作用及潜在机制。方法 将Sprague-Dawley(SD)大鼠随机分为12月龄组、24月龄组和艾灸组。24月龄组和艾灸组制备自然衰老模型。12月龄组和24月龄组大鼠不予治疗,待相应时间取材;艾灸组采用温和灸足三里穴至24月龄取材。采用免疫荧光法观察3组大鼠肾组织衰老相关标志物β-半乳糖苷酶的表达。采用酶联免疫吸附法和免疫组织化学法检测3组大鼠肾组织衰老相关p16蛋白及甲基转移酶[甲基转移酶3a(DNA methyltransferase 3a,Dnmt3a)和甲基转移酶3b(DNA methyltransferase 3b,Dnmt3b)]蛋白的表达。采用定量反转录聚合酶连锁反应(quantitative real time-polymerase chain reaction,qRT-PCR)法检测3组大鼠肾组织端粒长度、肾组织衰老相关p16 mRNA及甲基转移酶(Dnmt3a和Dnmt3b) mRNA表达。采用重亚硫酸盐测序技术检测3组大鼠肾组织p16 DNA CpG岛甲基化程度。结果 与12月龄组比较,24月龄组大鼠肾组织β-半乳糖苷酶表达、端粒长度的损耗及衰老相关p16蛋白和mRNA表达水平升高(P<0.05),24月龄组大鼠肾组织Dnmt3a和Dnmt3b蛋白及mRNA表达水平降低(P<0.05)。与24月龄组比较,艾灸组大鼠肾组织β-半乳糖苷酶表达、端粒长度的损耗及肾组织衰老相关p16蛋白和mRNA表达显著降低(P<0.05),艾灸组大鼠肾组织Dnmt3a和Dnmt3b蛋白及mRNA表达显著升高(P<0.05)。结论温和灸可通过影响自然衰老大鼠肾组织衰老相关甲基转移酶的表达调控p16 DNA CpG岛整体甲基化水平,降低肾组织p16蛋白及mRNA在衰老机体肾组织的表达,延缓肾组织衰老进程。

Objective To observe the regulation effect of mild moxibustion at Zusanli(ST36) on p16 DNA methylation in the kidney tissue of naturally aging rats and discuss the potential mechanism.Method The Sprague-Dawley(SD) rats were randomized into a 12-month-old group,a 24-month-old group,and a moxibustion group,with 10 rats in each group.The 24-month-old and the moxibustion groups were prepared as naturally aging models.Rats in the 12-month-old and the 24-month-old groups did not receive any interventions before sample collection,and the moxibustion group received mild moxibustion at Zusanli till 24 months old before sample collection.The expression of aging marker β-galactosidase in the kidney tissue of each group of rats was observed using immunofluorescence.The protein expression of aging-related p16and methyltransferases [DNA methyltransferase 3A(Dnmt3a) and DNA methyltransferase 3B(Dnmt3b)] in the kidney tissue of rats was detected using enzyme-linked immunosorbent assay(ELISA) and immunohistochemistry.The aging-related telomere length and mRNA expressions of p16 and methyltransferases(Dnmt3a and Dnmt3B) in the rat kidney tissue were detected by quantitative real time-polymerase chain reaction(q RT-PCR).The degree of p16DNA CpG island methylation in the rat kidney tissue was determined using the bisulfite sequencing technique.Result Compared with the 12-month-old group,the expression level of aging marker β-galactosidase,telomere loss,and expression levels of aging-related p16 protein and mRNA in the rat kidney tissue were increased in the 24-month-old group(P<0.05),and the expression levels of Dnmt3a and Dnmt3b proteins and mRNAs in the rat kidney tissue were reduced in the 24-month-old group(P<0.05).Compared with the 24-month-old group,the expression level of aging marker β-galactosidase,telomere loss,and expression levels of aging-related p16 protein and mRNA in the rat kidney tissue were significantly reduced in the moxibustion group(P<0.05),and the expression levels of Dnmt3a and Dnmt3b proteins and mRNAs in the rat kidney tissue were increased in the moxibustion group(P<0.05).Conclusion By influencing the expression of aging-related methyltransferases in the rat kidney tissue,moxibustion can regulate the overall methylation level of p16 DNA CpG island,reduce the expression levels of renal p16 mRNA and protein in senile kidney tissue,and delay the aging process of kidney tissue.