“浊阴凝痞”视角下基于内质网应激-细胞自噬途径探讨祛湿通浊化瘀方干预非酒精性脂肪肝的分子机制研究

As a metabolic disease,Nonalcoholic fatty liver disease (NAFLD) has an increasingly high incidence rate in China,which has become a serious public health problem.Traditional Chinese Medicine has shown great advantage and potentiality in the prevention and treatment of NAFLD. Based on study over 10 years,we put forward that chronic liver diseases evolved from dampness to stagnation, meridian diseases to collateral diseases and the therapy of clearing away heat ,expelling dampness and activating blood according to the theory of “dampness-heat in initial stage of diseases is at meridians,and stagnant heat invaded into collaterals”.Based on the previous research,with the breakthrough point of endoplasmic reticulum stress-cell autophagy,we build NAFLD rat models and HePG2 steatosis models induced by FFA and study effects of clearing away heat ,expelling dampness and activating blood therapy on the expression of endoplasmic reticulum stress and autophagy related protein,signaling pathway changes of nuclear transcription factor as well as liver apoptosis induced by it. What’s more ,the project will discuss moderating effect of the therapy on imbalance of endoplasmic reticulum stress-cell autophagy steady state and clarifies its molecular mechanism so as to provide theory and scientific evidence for TCM treatment and prevention of NAFLD.

非酒精性脂肪肝（NAFLD）作为一个代谢性疾病，其发病率越来越高，已成为一个严重的公共健康问题。中医药在NAFLD防治中已显示出较大优势和潜力，本课题组在前期研究基础上，根据叶天士“初病湿热在经，久则瘀热入络”及“阳气窒闭，浊阴凝痞”理论提出‘湿热是NAFLD始动因素，其发展是由湿热至痰浊瘀滞，由经病至络病的病变过程，湿热、痰浊和瘀滞等“浊阴凝痞”是其关键病理因素’的科学假说，并制定祛湿通浊化瘀方。项目拟在前期研究基础上，以内质网应激-细胞自噬为切入点，通过建立NAFLD大鼠模型和FFA诱导的HePG2细胞脂肪变性模型，从体内外两个途径研究祛湿通浊化瘀方对内质网应激和细胞自噬相关蛋白的表达、信号通路的变化及其介导细胞凋亡的影响，探讨祛湿通浊化瘀对NAFLD发展过程中内质网应激-细胞自噬稳态失衡的调节作用，阐明该治法干预NAFLD的分子机制，为NAFLD的中医药防治提供多种治法理论依据。

NAFLD是临床常见且发病机制复杂的病理综合征，中医药在防治本病上突显特色优势。内质网应激与细胞自噬均和NAFLD发生发展密切相关，但中医药特色治法对此二者调控NAFLD的机制国内外尚无报道。本项目针对NAFLD发病特点，根据叶天士经典理论，以祛湿通浊化瘀治法干预NAFLD，探讨了叶天士“初病湿热在经，久则瘀热入络”理论的科学内涵，证实了“湿热是NAFLD始动因素，其发展是由湿热至痰浊瘀滞，由经病至络病的病变过程，湿热、痰浊和瘀滞等‘浊阴凝痞’是其关键病理因素”的科学假说。通过体内干预高脂饲料诱导的NAFLD大鼠模型，体外含药血清干预FFA诱导的HepG2细胞模型，采用实时荧光定量PCR、Western blot、免疫组化、油红O染色等技术和方法，从分子生物学和形态学水平，检测内质网应激、细胞自噬相关指标，观察肝脏组织细胞形态变化，探讨中医药特色治法有效干预NAFLD的作用机制。结果显示：体内实验中，祛湿通浊化瘀方明显降低NAFLD大鼠血清肝酶、血脂，减轻肝细胞脂肪变性、炎性浸润、坏死程度，具有一定的保护肝脏、改善脂质代谢紊乱的作用；可抑制ATF4、eIF2α、IRE1α、JNK1等内质网应激、细胞自噬相关因子的蛋白含量及mRNA表达，从而发挥对NAFLD的治疗作用。在体外实验中，祛湿通浊化瘀方含药血清能够减轻肝细胞内脂质过度堆积和肝损伤程度，并且明显上调HepG2细胞内自噬相关基因Beclin1、LC3-II蛋白和mRNA表达。由此可见，祛湿通浊化瘀方能够有效调节内质网应激-细胞自噬稳态失衡，从而有效干预NAFLD，为临床上中医药多种治法防治NAFLD提供科学依据。