电针治疗单纯性肥胖的下丘脑TSC1-mTOR信号通路表观遗传机制研究

The incidence of simple obesity is increasing year by year. Simple obesity, which has been a threat to human health , results in decline quality of life and leads to serious diseases. Acupuncture as an effective non-drug treatment method, has aroused a high attention at home and abroad. But unclear mechanism hinders the promotion and application of the effective and unique Chinese acupuncture usage. Epigenetic research about simple obesity finds a new breakthrough in mechanism. Our clinical trial has proved the validity of acupuncture of treating obesity. On the basis of pre-clinical study and pre-experiment, in the guide of traditional Chinese medicine theory, with the obesity rat model as the research object, we propose hypothese that the hypothalamus DNA methyltransferase 1 ( Dnmt1) activity increases in simple obesity, acupuncture makes TSC1 demethylation through negative regulation of Dnmt1, and inhibits mTOR activity, thereby ameliorates the obese state. From the TSC1-TOR signaling pathway, which is considered as key link of simple obesity onset, with the use of sodium bisulfite modification gene clone sequencing technique, we observe the effect and mechanism of electro-acupuncture treatment of simple obesity with"ST25", "SP6", " CV12/RN12" and " ST36" in the perspective of epigenetics, which will provide a solid scientific basis for acupuncture treatment of simple obesity in clinical practice.

单纯性肥胖的发病率逐年上升，已明显威胁人类健康导致生活质量下降，并引发相关的严重疾病，针刺作为一种切实有效的非药物的治疗手段，引起了国内外的高度重视。但是针刺治疗该病的机理不明阻碍了这一有效而独特的中医疗法的推广应用。目前开展的单纯性肥胖的表观遗传研究为深入的机制探讨找到了新的突破口；本课题在针灸治疗肥胖有效的前期临床研究和预实验基础上，以中医理论为指导，以单纯性肥胖大鼠模型为研究对象，基于单纯性肥胖者下丘脑DNA甲基化酶转移酶1（Dnmt1）活性增加，针刺通过负调控Dnmt1的活性，使TSC1去甲基化，抑制mTOR活性，从而改善肥胖状态的假说，从单纯性肥胖发病的关键环节-下丘脑TSC1-mTOR信号通路入手，运用亚硫酸氢钠修饰后克隆测序等技术，从表观遗传学的角度观察电针天枢、三阴交、中脘、足三里等穴治疗肥胖的效应及发生机理，为针刺治疗单纯性肥胖的临床实践提供坚实的科学依据。

针刺是治疗单纯性肥胖的切实有效的非药物的手段，表观遗传可能参与了单纯性肥胖的发生。前期研究发现Tsc1启动子区甲基化参与了肥胖的发病。在此基础上，本课题采用高脂饲料建立肥胖大鼠模型，使用电针、DNA甲基化转移酶（Dnmt）抑制剂进行干预， 观察干预措施对大鼠体质量、腹腔内脂肪含量的影响，采用亚硫酸氢钠修饰后克隆测序、免疫印迹（Western-blot）、实时荧光定量PCR、免疫组化等技术检测大鼠下丘脑Tsc1甲基化水平、mTOR/mTORC1、Dnmt1、Dnmt2、Dnmt3a、Dnmt3b，食欲相关肽AgRP/NPY等基因的表达。我们发现食源性肥胖（DIO）大鼠下丘脑Tsc1基因启动子区存在可被甲基化位点，该组大鼠的甲基化率、mTOR基因表达高于正常饲料喂养组（对照组），Dnmt1基因没有差异，Dnmt2、Dnmt3a、Dnmt3b表达增加，刺鼠基因相关蛋白（AgRP）/神经肽Y（NPY）表达增加。电针干预治疗可减轻DIO大鼠体重，下调下丘脑Dnmt2、Dnmt3a、Dnmt3b表达和Tsc1启动子甲基化程度，降低DIO大鼠下丘脑mTOR及mTORC1的水平，下调AgRP/NPY的表达。下丘脑微量注射Dnmt抑制剂5-氮杂-2-脱氧胞苷也可降低DIO大鼠下丘脑mTOR、mTORC1的水平，下调AgRP/NPY表达；使用电针和5-氮杂-2-脱氧胞苷同时进行干预也观察到了类似的结果。因此我们得出结论电针可下调下丘脑Dnmt2、Dnmt3a、Dnmt3b的活性，使Tsc1基因启动子甲基化水平下降，抑制mTOR/mTORC1的水平，通过抑制AgRP/NPY基因的表达参与减轻体重，从而为针刺治疗单纯性肥胖的临床实践提供了科学依据。