复方贞术调脂方通过TLR4/NF-κB通路改善p120-catenin敲低所致肝脏炎症性糖脂代谢紊乱的机制研究

Inflammation is one of the important reasons for the glycolipid metabolic disorders, but its mechanisms are not clear yet. Traditional Chinese Medicine believes that the pathogenesis of the glycolipid metabolic disorders is "depression of the liver, decrease of circulation of the spleen, and the internal obstruction by dampness, phlegm, stasis and turbidity ", which is closely related to inflammation. The "Fufang Zhenzhu Tiaozhi Capsule，FTZ", developed by our research center, has the effects of reducing blood glucose, lowering lipid and anti-inflammation, which indicating that FTZ may improve inflammation-caused glycolipid metabolic disorders. Our previous studies have shown that p120 gene silencing in HepG2 cells could activate TLR4/NF-κB signaling pathway of inflammation, and cause the abnormal expression of metabolism related genes such as CYR7A1 and AdipoR1; we also found that FTZ could significantly improve the inflammatory response and abnormal glycolipid metabolisms caused by the p120 RNAi in the HepG2 cells. Based on these results, we put forward the scientific hypothesis of this project: FTZ may improve the inflammation and abnormal glucose and lipid metabolisms induced by p120 knockdown via inhibiting TLR4/NF-κB inflammatory pathway, and achieve its anti-inflammatory and glycolipid-lowering effects. In this project, we will further elucidate the mechanisms of FTZ in improving the inflammatory glycolipid metabolic disorders of liver based on the mouse model of hepatic inflammatory glycolipid metabolic disorders which we have successfully established, providing scientific evidence for the treatment of inflammatory glycolipid metabolic diseases by FTZ.

研究发现炎症是导致糖脂代谢紊乱的重要原因之一，但其机制尚不完全清楚。中医认为糖脂代谢紊乱的病机为“肝郁脾枢失运，湿痰瘀浊内阻”，与炎症密切相关。本研究中心研制的“复方贞术调脂方（FTZ）”具有降糖、降脂及抗炎等疗效，表明FTZ可能具有改善炎症性糖脂代谢紊乱的作用。前期研究发现，在HepG2细胞中沉默p120基因，能够激活TLR4/NF-κB炎症信号通路，并引起CYR7A1、AdipoR1等代谢相关基因表达异常，同时发现FTZ可明显改善沉默p120基因导致的炎症反应和糖脂代谢异常。我们由此提出本项目的科学假说：FTZ可能通过抑制TLR4/NF-κB炎症通路，改善p120敲低所致的炎症性糖脂代谢紊乱，发挥降糖和降脂的作用。本项目拟利用已建立的肝脏炎症性糖脂代谢紊乱的小鼠模型，进一步阐明FTZ改善肝脏炎症性糖脂代谢紊乱的作用机制，通过本项目研究，可为FTZ治疗炎症性糖脂代谢疾病提供科学依据。

糖脂代谢紊乱性疾病包括非酒精性脂肪肝、2型糖尿病等多个病种，以胰岛素抵抗为其重要特征之一，发病机制复杂，单靶点治疗效果很不理想。炎症、内质网应激和脂质异位积累是导致肝脏等重要代谢器官发生胰岛素抵抗的原因。p120蛋白为细胞粘附连接的重要构成分子，参与细胞的连接、分裂、信号传导等多种生物学功能。研究发现p120表达水平的降低或缺失会导致肺、肠等发生炎症反应。前期研究发现，敲低HepG2肝细胞系中的p120基因导致HepG2细胞发生炎症反应和糖脂代谢相关基因的表达异常，但具体分子机制尚不清楚。研究发现复方贞术调脂方（FTZ）通过多靶点作用，具有较好的降脂、降糖功能。前期研究表明，FTZ也具有抗炎功能。为了探明p120在调控肝脏糖脂代谢中的功能和FTZ改善肝脏糖脂代谢紊乱的分子机制，通过AAV8-p120 shRNA、p120 siRNA等技术构建p120基因敲低的小鼠和细胞模型，同时利用LPS、衣霉素、葡萄糖胺等化学诱导法和高脂饮食诱导法建立糖脂代谢紊乱的小鼠模型和肝细胞模型，给予FTZ及其活性成分黄连素和特女贞苷等处理，利用形态学和组织学、生物化学和分子生物学、肠道菌群16S rDNA测序分析等技术检测小鼠和细胞的表型与基因表达水平变化。敲低p120基因导致小鼠肝脏、HepG2细胞和Hepa 1-6细胞发生细胞自主性的炎症反应，促炎因子相关基因表达水平显著升高，炎症相关的p38和ERK1/2等信号通路被显著激活。FTZ能够有效改善低浓度LPS所致的系统性慢性炎症和糖脂代谢紊乱。FTZ活性成分黄连素能够减轻衣霉素诱导的肝脏内质网应激、脂质代谢紊乱和胰岛素抵抗，同时调节小鼠肠道菌群的结构和功能。葡萄糖胺引发HepG2细胞内的内质网应激进而导致胰岛素抵抗，FTZ活性成分特女贞苷能够有效改善其胰岛素抵抗。同时发现特女贞苷能够通过减轻高脂饮食诱导的2型糖尿病小鼠肝脏炎症反应及调节肠道菌群改善其肝脏胰岛素抵抗。这些结果证明了p120在肝脏糖脂代谢中的重要功能，确认了FTZ及其活性成分改善肝脏糖脂代谢紊乱的分子机制，为探讨糖脂代谢紊乱疾病的发病机制和药物治疗提供了实验和理论依据。