天王补心丹加减通过orexin A/OX1R对慢性睡眠剥夺小鼠糖脂代谢的干预作用

Intervention Effect of Modified Tianwang Buxindan on Glucose and Lipid Metabolism in Mice with Chronic Sleep Deprivation Through Orexin A/OX1R

目的:从食欲素A(orexin A)/食欲素受体1(OX1R)信号通路探讨天王补心丹加减改善慢性睡眠剥夺小鼠糖脂代谢异常的作用机制。方法:6周龄雄性C57BL/6小鼠50只,随机分为空白组,模型组,艾司唑仑组及天王补心丹加减低、高剂量组,每组10只。对除空白组外采用多平台水环境法睡眠剥夺8周,后4周予天王补心丹水煎剂(8.5,17 g·kg^(-1))与艾司唑仑溶液(9.1 mg·kg^(-1))灌胃,空白、模型组灌服等体积纯水。每周测量2次小鼠摄食量与体质量;第49天尾静脉采血进行葡萄糖耐受实验(GTT),第52天尾静脉采血进行胰岛素耐受实验(ITT);应用全自动生化分析仪检测血清中总胆固醇(TCH),甘油三酯(TG)与游离脂肪酸(FFA)表达;采用酶联免疫吸附测定(ELISA)检测血清及下丘脑中orexin A表达量;采用实时荧光定量聚合酶链式反应(Real-time PCR)和蛋白免疫印迹法(Western blot)检测下丘脑中OX1R mRNA和蛋白表达水平。结果:给药后各组小鼠的食物摄入量出现差异;与空白组比较,模型组体质量明显降低(P<0.05),糖耐量出现明显异常,TCH,TG,FFA水平显著升高(P<0.01),同时血清及下丘脑中orexin A表达量显著升高(P<0.01),下丘脑中OX1R mRNA和蛋白表达水平显著升高(P<0.01);与模型组比较,天王补心丹加减各组体质量明显升高(P<0.05),具有更好的葡萄糖耐受性和胰岛素敏感性,TCH,TG,FFA水平均明显降低(P<0.05,P<0.01),并伴有血清及下丘脑中orexin A表达量明显下降(P<0.05,P<0.01),OX1R的mRNA和蛋白表达水平明显降低(P<0.05,P<0.01)。结论:天王补心丹加减可以保护慢性睡眠剥夺诱导的小鼠糖脂代谢异常,推测其机制可能与orexin A/OX1R信号表达下调有关。

Objective: to explore the mechanism of modified Tianwang Buxindan in improving abnormal glucose and lipid metabolism in mice with chronic sleep deprivation from the signal pathway of orexin A/orexin receptor 1(OX1 R). Method: The 50 6-week-old male C57 BL/6 mice were randomly divided into blank group,model group,estazolam group and Tianwang Buxindan low and high dose groups,for ten mice of each group. Except the blank group,rats were deprived of sleep for 8 weeks by the method of multi-platform water environment. In the last 4 weeks,Tianwang Buxindan(8.5,17 g·kg^(-1))and estazolam solution(9.1 mg·kg^(-1)) were given to the stomach,and the blank group and the model group were fed with pure water of the same volume. The food intake and body weight of mice were measured twice a week,on the 49 thday,blood samples were collected from the tail vein for glucose tolerance test(GTT),on the 52 ndday for insulin tolerance test(ITT),was used to detect the expression of total cholesterol(TCH),triglyceride(TG)and free fatty acid(FFA)in serum,and enzyme-linked immunosorbent assay(ELISA)was used to detect the expression of orexin A in serum and hypothalamus. Real-time fluorescence quantitative polymerase chain reaction(Real-time PCR)and Western blot were used to detect the m RNA and protein expression of OX1 R in hypothalamus. Result: After administration,the food intake of mice in each group was different,compared with the blank group,the body weight of model group was significantly reduced(P<0.05),the glucose tolerance was significantly abnormal,and the TCH,TG,FFA values were significantly increased(P<0.01). The expression of orexin A in serum and hypothalamus increased significantly(P<0.01),and the m RNA and protein expression levels of OX1 R in hypothalamus increased significantly(P<0.01). Compared with the model group,the body weight of each group of Tianwang Buxindan was significantly increased(P<0.05), with better glucose tolerance and insulin sensitivity,TCH,TG,FFA values were significantly reduced(P<0.05,P<0.01),accompanied by serum and the expression of orexin A in the hypothalamus was significantly decreased(P<0.05,P<0.01),the m RNA and protein expression levels of OX1 R were significantly decreased(P<0.05,P<0.01). Conclusion: Tianwang Buxindan can protect mice from abnormal glucose and lipid metabolism induced by chronic sleep deprivation,and its mechanism may be related to the down-regulation of orexin A/OX1 R signal expression.