Objectives: To observe the effect of electroacupuncture (EA) in obese rats with insulin resistance (IR) through regulating intestinal silent information regulator 1 (SIRT1)/Toll-like receptor 4 (TLR4) signaling pathway, so as to explore the underlying mechanism of EA in improving obesity-induced IR.
Methods: A total of 40 Wistar rats were randomly divided into 4 groups, i.e. normal group, model group, EA group and EA combined with inhibitor group, with 10 rats in each group. The obesity-induced IR model was induced by feeding high-fat diet for 8 weeks. EA (2 Hz, 1mA) was applied at "Zhongwan"(CV12), "Guanyuan"(CV4), "Zusanli"(ST36) and "Fenglong" (ST40) for 10 min, 3 times a week for 8 weeks in both EA and EA combined with inhibitor groups. Sirtinol, an inhibitor of SIRT1 was injected into the tail vein (1 mg/kg), 3 times a week for 8 weeks in EA combined with inhibitor group. The body weight, glucose infusion rate (GIR) of rats in each group were recorded. The contents of serum C-reactive protein (CRP), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and lipopolysaccharide (LPS) were detected by ELISA. Mucosal morphological changes in the small intestine was observed by HE staining and was graded using Chiu's score. The protein relative expression levels of SIRT1 and TLR4 and the co-labeling of SIRT1 with TLR4 in the small intestine was detected by Western blot and double immunofluorescence staining, separately.
Results: Compared with the normal group, the body weight, serum contents of CRP, TNF-α, IL-6, LPS, Chiu's score, TLR4 protein relative expression level and percentage of TLR4 positive expression area were increased (P<0.01, P<0.05), while the GIR, SIRT1 protein expression, percentage of SIRT1 positive expression area and SIRT1/TLR4 were decreased (P<0.01) in the model group. The pathological injury of small intestine mucosa was severe, accompanied with inflammatory cell infiltration in the model group. Following interventions, the body weight, serum contents of CRP, TNF-α and LPS, Chiu's score, TLR4 protein relative expression level and percentage of TLR4 positive expression area were decreased(P<0.01, P<0.05), and the GIR was increased (P<0.01), the pathological injury and inflammatory cell infiltration of small intestine mucosa were reduced in both EA and EA combined with inhibitor groups in contrast to the model group. Compared with the model group, the serum IL-6 content was significantly decreased (P<0.01), and the SIRT1 protein relative expression level and percentage of positive expression area, SIRT1/TLR4 were increased (P<0.01, P<0.05) in the EA group. Compared with the EA group, EA combined with inhibitor group showed the body weight, serum CRP, IL-6, LPS, Chiu's score, TLR4 protein relative expression level and TLR4 positive expression area were increased (P<0.01, P<0.05), and the GIR level , SIRT1 relative expression level, SIRT1/TLR4 ratio were decreased (P<0.05, P<0.01).
Conclusions: EA can reduce the body weight and ameliorate peripheral insulin sensitivity in IR obese rats, which may be related with its function in regulating intestinal SIRT1/TLR4 signaling pathway to reduce inflammatory response.
目的: 观察电针对肥胖胰岛素抵抗（IR）大鼠小肠沉默信息调节因子1（SIRT1）/Toll样受体4（TLR4）炎性信号通路的影响，探讨电针改善肥胖IR的机制。方法: Wistar大鼠随机分为正常组、模型组、电针组、电针联合抑制剂组，每组10只。采用高脂饮食饲养8周建立肥胖IR大鼠模型。电针组大鼠予“中脘”“关元”“足三里”“丰隆”电针治疗，10 min/次，3次/周，共8周；电针联合抑制剂组大鼠在电针治疗的基础上，予尾静脉注射SIRT1抑制剂Sirtinol（1 mg/kg），隔日1次，3次/周，共8周。观察并记录各组大鼠体质量、葡萄糖输注速率（GIR）；ELISA法检测血清C反应蛋白（CRP）、肿瘤坏死因子-α（TNF-α）和白细胞介素-6（IL-6）、脂多糖（LPS）的含量；HE染色观察小肠组织的黏膜形态并用Chiu’s评分法评价小肠黏膜损伤情况；Western blot法检测小肠组织SIRT1、TLR4的蛋白相对表达量；免疫荧光双标法检测SIRT1和TLR4在小肠组织中的共表达情况。结果: 与正常组比较，模型组大鼠体质量显著升高（P<0.01），GIR显著降低（P<0.01），血清CRP、TNF-α、IL-6、LPS含量和小肠Chiu’s评分均显著升高（P<0.01，P<0.05），小肠组织中SIRT1蛋白相对表达量、阳性表达面积百分比和SIRT1/TLR4均显著降低（P<0.01），而TLR4蛋白相对表达量和阳性表达面积百分比均显著升高（P<0.01），小肠黏膜炎性细胞浸润和病理损伤严重。干预后，与模型组比较，电针组和电针联合抑制剂组大鼠体质量，血清CRP、TNF-α、LPS含量和小肠Chiu’s评分均显著降低（P<0.01，P<0.05），GIR显著升高（P<0.01），TLR4蛋白相对表达量和阳性表达面积百分比显著降低（P<0.01，P<0.05），小肠黏膜病理损伤和炎性细胞浸润显著减轻；电针组血清IL-6含量显著降低（P<0.01），小肠组织中SIRT1蛋白相对表达量、阳性表达面积百分比和SIRT1/TLR4显著升高（P<0.01，P<0.05）。与电针组比较，电针联合抑制剂组大鼠体质量、小肠Chiu’s评分显著升高（P<0.01），GIR显著降低（P<0.05），血清CRP、IL-6、LPS含量均显著升高（P<0.05），小肠组织中SIRT1蛋白相对表达量显著降低（P<0.01）、TLR4蛋白相对表达量和阳性表达面积百分比升高（P<0.01），SIRT1/TLR4显著降低（P<0.05）。结论: 电针可以改善肥胖IR大鼠体质量，提高外周胰岛素敏感性，其机制可能与调控小肠组织中SIRT1/TLR4信号通路，减少炎性因子释放有关。.
Keywords: Electroacupuncture; Insulin resistance; Obesity; Silent information regulator 1; Toll-like receptor 4.