温肾健脾化痰方调控单纯性肥胖大鼠脂代谢及瘦素抵抗的机制研究

发布时间：2018-08-19 16:35

【摘要】：目的本研究采用高脂饮食建立单纯性肥胖大鼠模型,以奥利司他胶囊为西药对照,分别观察温肾健脾化痰方对肥胖大鼠体重、Lee's指数、血脂、血清游离脂肪酸、肝脏及脂肪组织病理形态、脂肪因子瘦素(Leptin)、chemerin、产热调节因子UCP3及AMPK-ACC信号通路中相关蛋白和基因表达的影响,探讨温肾健脾化痰方改善单纯性肥胖大鼠脂代谢和瘦素抵抗的可能作用机制。方法SPF级雄性SD大鼠80只(体重120±20g),随机抽取10只作为正常组,给予普通饲料喂养;其余70只大鼠采用高脂饲料喂养。喂养8周末,筛选出体重超过正常组大鼠平均体重20%者作为单纯性肥胖大鼠模型。按照随机数字表将造模成功的肥胖大鼠分为5组:肥胖模型组(即模型组)、西药奥利司他治疗组(即西药组)、温肾健脾化痰方低剂量组(即低剂量组)、温肾健脾化痰方中剂量组(即中剂量组)、温肾健脾化痰方高剂量组(即高剂量组),每组大鼠各10只。于第9周开始灌胃,给药量如下,低剂量组为3.47g/(kg·d)、中剂量组为6.93g/(kg·d)、高剂量组为13.86g/(kg·d)、西药组为37.8 mg/(kg·d),正常组和模型组以等体积的蒸馏水灌胃,各组按lml/100g的标准灌胃,连续给药6周。实验期间,每周测量两次大鼠的体重及体长。至实验第14周末,从腹主动脉取血,分离血清,检测各组大鼠血清总胆固醇、甘油三酯、高密度脂蛋白、低密度脂蛋白及血清游离脂肪酸水平;ELISA法测定血清Leptin及肝脏chemerin水平;切取少许大鼠肝脏组织和腹部脂肪组织,光镜下观察其肝脏及脂肪组织病理形态;采用Westernblot法检测各组大鼠肝脏组织OB-R、AMPK、P-AMPK、ACC、CPT1的蛋白表达水平及骨骼肌中UCP3的蛋白表达水平,采用实时荧光定量RT-PCR法检测各组大鼠肝脏OB-R、AMPK、ACC、CPT1m RNA表达量及骨骼肌中UCP3 m RNA表达量。结果1.温肾健脾化痰方对单纯性肥胖大鼠脂代谢的影响:(1)高脂喂养8周后,造模组体重、Lee's指数与正常组比较均显著增加(p0.01),提示单纯性肥胖大鼠造模成功;(2)温肾健脾化痰方能降低单纯性肥胖大鼠体重及Lee's指数,用药6周末,高、中剂量组体重与模型组相比均有显著性差异(p0.01),高剂量组Lee's指数与模型组比较有显著性差异(p0.01);(3)与模型组比较,高、中剂量组血清TG、TC、LDL-C、FFA的含量显著降低(p0.01),且低剂量组TG水平相比模型组有统计学意义(p0.05),高剂量组HDL-C的含量较模型组有统计学差异(p0.05)。2.温肾健脾化痰方对单纯性肥胖大鼠脂肪及肝脏组织病理形态的影响:(1)脂肪组织病理改变:中、高剂量组大鼠脂肪细胞面积较模型组减小,单位视野内脂肪细胞数目增多;(2)肝脏组织病理改变:与模型组比较,高剂量组大鼠肝细胞排列较为均匀有序,胞浆内脂滴变小,细胞水肿及细胞脂肪变性有明显好转,存在少量炎性细胞浸润。3.温肾健脾化痰方对单纯性肥胖大鼠脂肪因子Leptin、chemerin的影响:(1)与空白组相比,模型组血清Leptin显著升高(p0.01),高、中剂量组较模型组血清Leptin均降低(p0.01,p0.05);(2)与正常组比较,模型组血清及肝脏chemerin明显升高(p0.01),高、中剂量组血清及肝脏chemerin含量较模型组均下降(p0.01,p0.05)。4.温肾健脾化痰方对单纯性肥胖大鼠骨骼肌组织UCP3的影响:模型组较正常组大鼠骨骼肌的UCP3m RNA和蛋白表达均显著降低(p0.01);高、中剂量组较模型组大鼠骨骼肌的UCP3m RNA和蛋白表达水平升高(p0.01)。5.温肾健脾化痰方对单纯性肥胖大鼠肝脏AMPK-ACC-CPT1通路的影响:(1)模型组大鼠较正常组大鼠肝脏OB-R m RNA及蛋白表达均显著降低(p0.01),与模型组比较,高、中剂量组大鼠肝脏OB-R m RNA及蛋白表达升高(p0.05);(2)与正常组比较,模型组大鼠肝脏AMPK、p-AMPK、CPT1的蛋白含量显著降低(p0.01),模型组大鼠肝脏AMPKm RNA和CPT1 m RNA表达也降低(p0.01);与模型组相比,高剂量组大鼠肝脏AMPK蛋白表达量增加(p0.05),高、中剂量组大鼠肝脏p-AMPK蛋白表达量增高(p0.01,p0.05);高、中剂量组大鼠肝脏CPT1的蛋白含量升高(p0.05),高、中剂量组大鼠肝脏AMPKm RNA和CPT1 m RNA表达量也增加(p0.01,p0.05);(3)模型组较正常组大鼠肝脏ACC蛋白和基因表达水平升高(p0.01),与模型组相比,高、中剂量组大鼠肝脏的ACCm RNA及蛋白表达水平均降低(p0.05)。结论温肾健脾化痰方的减肥作用机制可能是通过增加肝脏OB-R的蛋白表达量,增加OB-R与Leptin的结合力,提高瘦素敏感性,从而激活肝脏Leptin介导的AMPK-ACC-CPT1信号通路,并通过增加解偶联作用提高基础代谢率,促进脂肪酸氧化分解、降低FFA水平、减少脂质沉积、改善脂质代谢紊乱及瘦素抵抗。
[Abstract]:Objective To observe the effects of Wenshen Jianpi Huatan Recipe on body weight, Lee's index, blood lipids, serum free fatty acids, pathological morphology of liver and adipose tissue, fat factor leptin (Leptin), chemerin, heat-producing regulators UCP3 and AMPK in obese rats. To explore the possible mechanism of Wenshen Jianpi Huatan Recipe on improving lipid metabolism and leptin resistance in simple obese rats. Methods Eighty male SD rats of SPF grade (weighing 120 + 20g) were randomly selected as normal group and fed with normal diet. The other 70 rats were fed with high-fat diet. The obese rats were divided into five groups according to the random number table: obesity model group (i.e. model group), western medicine olista treatment group (i.e. western medicine group), warm kidney and spleen-invigorating and phlegm-resolving prescription low-dose group (i.e. low-dose group). The dosage of Shenjianpi Huatan Recipe was 3.47 g / (kg d), 6.93 g / (kg d), 13.86 g / (kg During the experiment period, the body weight and length of rats were measured twice a week. At the end of the 14th week, the blood was taken from abdominal aorta and the serum was separated. The total cholesterol, triglyceride, high density lipoprotein, low density lipoprotein and serum free fat were detected in each group. The levels of serum Leptin and liver Chemerin were measured by ELISA, the pathological changes of liver and abdominal adipose tissue were observed by light microscope, and the expression of OB-R, AMPK, P-AMPK, ACC, CPT1 and UCP3 protein in skeletal muscle were detected by Western blot. The expression of OB-R, AMPK, ACC, CPT1m RNA in liver and UCP3 m RNA in skeletal muscle were detected by real-time fluorescence quantitative RT-PCR. Results 1. Effect of Wenshen Jianpi Huatan Recipe on lipid metabolism in simple obese rats: (1) After 8 weeks of high-fat feeding, the body weight and Lee's index of model group increased significantly compared with normal group (p0.01), suggesting that (2) Wenshen Jianpi Huatan Decoction can reduce the body weight and Lee's index of simple obese rats. At the end of 6 weeks, the body weight of high and middle dose group was significantly different from that of model group (p0.01). The Lee's index of high dose group was significantly different from that of model group (p0.01). (3) Compared with model group, high and middle dose group was significantly different. The levels of serum TG, TC, LDL-C and FFA were significantly lower in low dose group than in model group (p0.05). The content of HDL-C in high dose group was significantly higher than that in model group (p0.05). 2. The effect of Wenshen Jianpi Huatan Recipe on the pathological morphology of adipose tissue and liver tissue in simple obese rats: (1) Pathological changes of adipose tissue: in middle Compared with the model group, the area of adipocytes in the high dose group was smaller and the number of adipocytes in the unit field of vision was increased. (2) Pathological changes of liver tissue: Compared with the model group, the hepatocytes in the high dose group were arranged more uniformly and orderly, the lipid droplets in the cytoplasm were smaller, the cell edema and steatosis were obviously improved, and there was a small amount of inflammatory cell infiltration. Effects of Wenshen Jianpi Huatan Recipe on Fat Factor Leptin and Chemerin in Simple Obesity Rats: (1) Compared with the blank group, serum Leptin in the model group was significantly increased (p0.01), while that in the middle dose group was significantly lower than that in the model group (p0.01, p0.05); (2) Compared with the normal group, serum and liver Chemerin in the model group were significantly increased (p0.01). The content of Chemerin in serum and liver was lower than that in model group (p0.01, p0.05). 4. Effect of Wenshen Jianpi Huatan Recipe on UCP3 in skeletal muscle of simple obese rats: The expression of UCP3 mRNA and protein in skeletal muscle of model group was significantly lower than that of normal group (p0.01); the expression of UCP3m RNA and protein in middle dose group was significantly lower than that of model group (p0.01). Effect of Wenshen Jianpi Huatan Recipe on AMPK-ACC-CPT1 Pathway in Liver of Simple Obesity Rats: (1) The expression of OB-R m RNA and protein in model group was significantly lower than that in normal group (p0.01). Compared with model group, the expression of OB-R m RNA and protein in liver of high dose group was higher (p0.05); (2) Compared with normal group, the expression of OB-R m RNA and protein in model group was significantly lower. The expression of AMPK, p-AMPK and CPT1 protein in the liver of model group decreased significantly (p0.01), and the expression of AMPK m RNA and CPT1 m RNA in the liver of model group decreased (p0.01); compared with model group, the expression of AMPK protein in the liver of high dose group increased (p0.05), while the expression of p-AMPK protein in the liver of middle dose group increased (p0.01, p0.05). The content of CPT1 protein in rat liver increased (p0.05), and the expression of AMPKm RNA and CPT1 m RNA in rat liver increased (p0.01, p0.05); (3) The level of ACC protein and gene expression in model group was higher than that in normal group (p0.01). Compared with model group, the level of ACC m RNA and protein expression in rat liver in middle dose group were decreased (p0.01). 05). Conclusion The mechanism of Wenshen Jianpi Huatan Formula in reducing weight may be through increasing the expression of OB-R protein in liver, increasing the binding power of OB-R and Leptin, increasing the sensitivity of leptin, activating the AMPK-ACC-CPT1 signaling pathway mediated by liver Leptin, increasing the basal metabolic rate, promoting the oxidative decomposition of fatty acids, and decreasing the leptin sensitivity. Low FFA level, reduce lipid deposition, improve lipid metabolism and leptin resistance.
【学位授予单位】：湖北中医药大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R285.5

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