基于Tsc1-mTOR表观遗传修饰介导自噬探讨电针减肥效应差异化机制研究

Acupuncture has a certain effect on weight loss, but individual differences exist, which limit the clinical application to a certain degree. This topic in before a Based on the previous National Natural Science Foundation Youth Science Fund Project and clinical research, the facts of the hypothalamus Tsc1-mTOR methylation modification regulating autophagy is closely related to the development of obesity and pre-experiment found that different effects of electroacupuncture intervention of obesity were connected with hypothalamic Tsc1 promoter methylation modification and mTOR gene expression, we put forward the hypothesis that "Tsc1-mTOR epigenetic modification in regulation of autophagy is the mechanism of the individualization effect of electroacupuncture on reducing weight”. Under the guidance of the theory of Traditional Chinese Medicine, with the obesity rat model as the research object, on the basis of clinical practice and individualization effect of electroacupuncture on reducing weight, our research consider the Tsc1-mTOR epigenetic modification in regulation of autophagy as the target, use sodium bisulfite modification gene clone sequencing technique, RT-PCR, Western blot, Immunofluorescence and other molecular biology methods and techniques, probe the internal mechanism of individualization effect of acupuncture in lowering weight, which will provide a solid scientific basis for acupuncture in the treatment of obesity in clinical practice.

针刺减肥疗效肯定但存在个体差异，在一定程度上限制了临床应用。本课题在前一个国家自然科学基金青年项目和前期临床研究的基础上，基于“下丘脑Tsc1-mTOR甲基化表观遗传修饰调控自噬与肥胖发生密切相关，预实验发现电针干预肥胖大鼠不同效应个体下丘脑Tsc1启动子甲基化修饰和mTOR基因表达存在差异”的基本事实，提出“电针通过调控下丘脑Tsc1-mTOR表观遗传修饰介导的自噬是其减肥疗效个体差异重要机制”的研究假说。本项目在中医针灸经穴理论指导下，以临床实践为基础，食源性肥胖大鼠为载体，围绕针刺减肥效应的个体差异，以Tsc1-mTOR表观遗传修饰介导的自噬作为研究切入点，运用亚硫酸氢钠法依赖的基因测序、RT-PCR、Western blot、免疫荧光等分子生物学的方法和技术，从整体—组织-细胞分子水平进行研究，探讨针刺减肥疗效响应差异性的内在机制，将为促进针刺减肥在临床上的推广应用提供科学依据。

针刺减肥疗效肯定但存在个体差异，在一定程度上限制了临床应用。本课题在前期研究基础上提出“电针通过调控下丘脑Tsc1-mTOR表观遗传修饰介导的自噬是其减肥疗效个体差异重要机制”的研究假说。本课题通过构建饮食诱导肥胖（diet induced obesity，DIO）大鼠模型，建立下丘脑微量注射系统，并进行电针干预，探讨了针刺干预DIO大鼠的疗效差异及其脂肪、下丘脑效应机制。结果显示肥胖大鼠体重、附睾脂肪量、下丘脑TSC1甲基化率增加， mTOR、p-mTOR蛋白质量上调，自噬相关基因Beclin-1表达量降低，p62增加。5-AZA干预可降低肥胖大鼠下丘脑TSC1甲基化率，5-AZA、RAPA可减轻大鼠体重和附睾脂肪量，下调下丘脑mTOR、p-mTOR表达，增加Beclin-1蛋白质量，降低p62的表达，提示DIO肥胖的发生机制可能与下丘脑TSC1甲基化修饰调节mTOR信号介导的自噬有关。电针干预肥胖大鼠存在效应差异，针刺有效大鼠附睾脂肪组织mTOR、p70S6K mRNA 表达水平显著升高，Beclin-1、LC3 mRNA 表达水平显著降低，p62 mRNA 表达水平显著升高。针刺无效和假针刺大鼠脂肪组织mTOR、p70S6K mRNA 表达下降，Beclin-1、LC3 mRNA 表达水平显著增加，p62 mRNA下降。肥胖大鼠下丘脑TSC1存在高的甲基化率，p-mTOR高表达，自噬相关基因Beclin-1表达下降，p62蛋白上调，针刺有效大鼠的下丘脑TSC1甲基化率下调，p-mTOR表达减弱，自噬相关基因Beclin-1增加，p62蛋白下降，LC3B免疫荧光阳性与beclin-1基因表达有相似的趋势，针刺无效和假针刺组大鼠上述相关基因表达与肥胖组相比无显著差异。因此我们得出结论：针刺减重的效应差异机制可能与附睾脂肪组织mTOR介导的自噬、下丘脑Tsc1-mTOR表观遗传修饰介导的自噬机制有关。本项目从表观遗传-自噬角度探讨了针刺减肥疗效响应差异性的内在可能机制，为促进针刺减肥在临床上的推广应用提供了一定的科学依据。