基于TLR5-肠道菌群途径探讨电针对营养型肥胖小鼠的作用机制

Effect of Electroacupuncture on Nutritional Obese Mice Based on TLR5-Gut Microbiota Pathway

目的 基于TLR5-肠道菌群探讨电针对营养型肥胖小鼠的作用机制。方法 选取60只SPF级C57BL/6小鼠,其中10只空白鼠为正常组,另外50只通过高脂饮食诱导营养型肥胖模型,筛选得到营养型肥胖模型鼠20只,随机均分成模型组和电针组,每组10只。电针组针刺中脘、天枢、关元、丰隆四穴,得气后接通电针仪,连续21 d进行电针处理。观察各组实验鼠一般指标(体重、Lee’s指数)、组织指标(肾周脂肪组织质量)、血清指标[胆固醇(CHO)、三酰甘油(TG)、高密度脂蛋白胆固醇(HDL-C)、低密度脂蛋白胆固醇(LDL-C)、血糖]、分子指标[瘦素(LEP)、脂联素(ADPN)、白介素-6(IL-6)、白介素-1β(IL-1β)、肿瘤坏死因子-α(TNF-α)、脂多糖结合蛋白(LBP)和胆囊收缩素(CCK)]等差异变化;运用免疫组织化学方法检测Toll样受体5(TLR5)蛋白分布差异变化,使用RT-PCR法检测TLR5基因表达的差异变化;采用16S rDNA二代基因测序技术分析肠道菌群结构的差异变化。结果 模型组的体重、Lee’s指数、肾脏周围脂肪质量、血清CHO、TG、HDL-C含量、脂肪细胞因子LEP及游离脂肪酸(free fatty acid, FFA)含量、肠黏膜组织中IL-6、IL-1β、TNF-α、LBP和CCK含量显著高于正常组,脂肪细胞因子ADPN含量显著低于正常组,经电针治疗后,均明显改善。模型组肠黏膜上TLR5的蛋白和基因表达显著高于正常组,经电针干预后明显下调。模型组肠道菌群的种类显著少于正常组,经电针干预后明显增加;模型组拟杆菌纲(Bacteroidia)、变形菌纲(Gammaproteobacteria)、迟缓埃格特菌科(Eggerthellacrae)、疣微菌科(Sutterellaceae)比例明显升高,芽孢杆菌纲(Bacilli)、梭菌纲(Clostridia)、梭菌科(Clostridiaceae)、放线菌纲(Actinobacteria)比例明显下降,经电针干预后明显改善。结论 电针具有较好治疗营养型肥胖症的作用,通过平衡肠道菌群的结构,良性调节肠黏膜上TLR5蛋白的分布与表达,降低肠黏膜慢性炎症反应,促进机体脂质代谢,达到减肥降脂目的。

Objective To investigate the effect of electroacupuncture on nutritional obese mice through TLR5-gut microbio-ta pathway,and the underlying mechanism.Methods A total of 60 SPF C57BL/6 mice were selected.Among them,10 blank mice were assigned to normal group,and the nutritive obesity model was established in the other 50 mice,which was induced by high-fat diet.Twenty nutritive obesity model mice were screened and randomly divided into model group and electroacupuncture group,with 10 mice in each group.The electroacupuncture group was needled at Zhongwan,Tianshu,Guanyuan and Fenglong points.And after obtaining Qi,electroacupuncture treatment was conducted on those mice for 21 days continuously.The change in differences of general indexes(body weight,Lee's index),tissue indexes(perirenal adipose-tissue mass),serum indexes[cho-lesterol(CHO),triglyceride(TG),high-density lipoprotein(HDL-C),low-density lipoprotein(LDL-C),blood glucose,molecular indexesleptin(LEP),adiponectin(ADPN),interleukin-6(IL-6),interleukin-1β(IL-1β),tumor necrosis factor-α(TNF-α),lipopo-lysaccharide-binding protein(LBP)and cholecystokinin(CCK)]were observed.TLR5 protein distribution was detected by immu-nohistochemical reaction,and TLR5 gene expression was detected by RT-PCR.Structural differences of intestinal flora was ana-lyzed by second generation 16S rDNA gene sequencing technology.Results The body weight,Lee's index,fat mass around kid-ney,serum contents of CHO,TG,HDL-C,the contents of LEP and FFA in fat tissue,and contents of IL-6,IL-1β,TNF-α,LBP in intestinal mucosa tissue of model group were significantly higher than that of the normal group,and the content of adipocyto-kine ADP was significantly lower.After electroacupuncture treatment,those indicators were significantly improved.The protein distribution and gene expression of TLR5 in the intestinal mucosa of the model group were significantly higher than those of the normal group,and were significantly down-regulated after electroacupuncture intervention.The number of intestinal flora spe-cies in the model group was significantly lower than those in the normal group,and were significantly increased after electroacu-puncture intervention.Proportions of Bacteroidia,Gammaproteobacteria,Eggerthellacrae and Sutterellaceae were significantly increased,while proportions of Bacilli,Clostridia,Clostridiaceae and Actinobacteria were significantly decreased.Changes in gut flora were significantly reversed after electroacupuncture intervention.Conclusion Electroacupuncture has a satisfying effect in treatment of nutritional obesity.It can regulate the distribution and expression of TLR5 protein in intestinal mucosa by balancing the structure of intestinal flora,reduce the chronic inflammatory reaction of intestinal mucosa,promote lipid metabo-lism of the body,and achieve the purpose of weight loss and lipid reduction.