【摘要】：目的本研究采用高脂飲食建立單純性肥胖大鼠模型,以?shī)W利司他膠囊為西藥對(duì)照,分別觀察溫腎健脾化痰方對(duì)肥胖大鼠體重、Lee's指數(shù)、血脂、血清游離脂肪酸、肝臟及脂肪組織病理形態(tài)、脂肪因子瘦素(Leptin)、chemerin、產(chǎn)熱調(diào)節(jié)因子UCP3及AMPK-ACC信號(hào)通路中相關(guān)蛋白和基因表達(dá)的影響,探討溫腎健脾化痰方改善單純性肥胖大鼠脂代謝和瘦素抵抗的可能作用機(jī)制。方法SPF級(jí)雄性SD大鼠80只(體重120±20g),隨機(jī)抽取10只作為正常組,給予普通飼料喂養(yǎng);其余70只大鼠采用高脂飼料喂養(yǎng)。喂養(yǎng)8周末,篩選出體重超過(guò)正常組大鼠平均體重20%者作為單純性肥胖大鼠模型。按照隨機(jī)數(shù)字表將造模成功的肥胖大鼠分為5組:肥胖模型組(即模型組)、西藥奧利司他治療組(即西藥組)、溫腎健脾化痰方低劑量組(即低劑量組)、溫腎健脾化痰方中劑量組(即中劑量組)、溫腎健脾化痰方高劑量組(即高劑量組),每組大鼠各10只。于第9周開(kāi)始灌胃,給藥量如下,低劑量組為3.47g/(kg·d)、中劑量組為6.93g/(kg·d)、高劑量組為13.86g/(kg·d)、西藥組為37.8 mg/(kg·d),正常組和模型組以等體積的蒸餾水灌胃,各組按lml/100g的標(biāo)準(zhǔn)灌胃,連續(xù)給藥6周。實(shí)驗(yàn)期間,每周測(cè)量?jī)纱未笫蟮捏w重及體長(zhǎng)。至實(shí)驗(yàn)第14周末,從腹主動(dòng)脈取血,分離血清,檢測(cè)各組大鼠血清總膽固醇、甘油三酯、高密度脂蛋白、低密度脂蛋白及血清游離脂肪酸水平;ELISA法測(cè)定血清Leptin及肝臟chemerin水平;切取少許大鼠肝臟組織和腹部脂肪組織,光鏡下觀察其肝臟及脂肪組織病理形態(tài);采用Westernblot法檢測(cè)各組大鼠肝臟組織OB-R、AMPK、P-AMPK、ACC、CPT1的蛋白表達(dá)水平及骨骼肌中UCP3的蛋白表達(dá)水平,采用實(shí)時(shí)熒光定量RT-PCR法檢測(cè)各組大鼠肝臟OB-R、AMPK、ACC、CPT1m RNA表達(dá)量及骨骼肌中UCP3 m RNA表達(dá)量。結(jié)果1.溫腎健脾化痰方對(duì)單純性肥胖大鼠脂代謝的影響:(1)高脂喂養(yǎng)8周后,造模組體重、Lee's指數(shù)與正常組比較均顯著增加(p0.01),提示單純性肥胖大鼠造模成功;(2)溫腎健脾化痰方能降低單純性肥胖大鼠體重及Lee's指數(shù),用藥6周末,高、中劑量組體重與模型組相比均有顯著性差異(p0.01),高劑量組Lee's指數(shù)與模型組比較有顯著性差異(p0.01);(3)與模型組比較,高、中劑量組血清TG、TC、LDL-C、FFA的含量顯著降低(p0.01),且低劑量組TG水平相比模型組有統(tǒng)計(jì)學(xué)意義(p0.05),高劑量組HDL-C的含量較模型組有統(tǒng)計(jì)學(xué)差異(p0.05)。2.溫腎健脾化痰方對(duì)單純性肥胖大鼠脂肪及肝臟組織病理形態(tài)的影響:(1)脂肪組織病理改變:中、高劑量組大鼠脂肪細(xì)胞面積較模型組減小,單位視野內(nèi)脂肪細(xì)胞數(shù)目增多;(2)肝臟組織病理改變:與模型組比較,高劑量組大鼠肝細(xì)胞排列較為均勻有序,胞漿內(nèi)脂滴變小,細(xì)胞水腫及細(xì)胞脂肪變性有明顯好轉(zhuǎn),存在少量炎性細(xì)胞浸潤(rùn)。3.溫腎健脾化痰方對(duì)單純性肥胖大鼠脂肪因子Leptin、chemerin的影響:(1)與空白組相比,模型組血清Leptin顯著升高(p0.01),高、中劑量組較模型組血清Leptin均降低(p0.01,p0.05);(2)與正常組比較,模型組血清及肝臟chemerin明顯升高(p0.01),高、中劑量組血清及肝臟chemerin含量較模型組均下降(p0.01,p0.05)。4.溫腎健脾化痰方對(duì)單純性肥胖大鼠骨骼肌組織UCP3的影響:模型組較正常組大鼠骨骼肌的UCP3m RNA和蛋白表達(dá)均顯著降低(p0.01);高、中劑量組較模型組大鼠骨骼肌的UCP3m RNA和蛋白表達(dá)水平升高(p0.01)。5.溫腎健脾化痰方對(duì)單純性肥胖大鼠肝臟AMPK-ACC-CPT1通路的影響:(1)模型組大鼠較正常組大鼠肝臟OB-R m RNA及蛋白表達(dá)均顯著降低(p0.01),與模型組比較,高、中劑量組大鼠肝臟OB-R m RNA及蛋白表達(dá)升高(p0.05);(2)與正常組比較,模型組大鼠肝臟AMPK、p-AMPK、CPT1的蛋白含量顯著降低(p0.01),模型組大鼠肝臟AMPKm RNA和CPT1 m RNA表達(dá)也降低(p0.01);與模型組相比,高劑量組大鼠肝臟AMPK蛋白表達(dá)量增加(p0.05),高、中劑量組大鼠肝臟p-AMPK蛋白表達(dá)量增高(p0.01,p0.05);高、中劑量組大鼠肝臟CPT1的蛋白含量升高(p0.05),高、中劑量組大鼠肝臟AMPKm RNA和CPT1 m RNA表達(dá)量也增加(p0.01,p0.05);(3)模型組較正常組大鼠肝臟ACC蛋白和基因表達(dá)水平升高(p0.01),與模型組相比,高、中劑量組大鼠肝臟的ACCm RNA及蛋白表達(dá)水平均降低(p0.05)。結(jié)論溫腎健脾化痰方的減肥作用機(jī)制可能是通過(guò)增加肝臟OB-R的蛋白表達(dá)量,增加OB-R與Leptin的結(jié)合力,提高瘦素敏感性,從而激活肝臟Leptin介導(dǎo)的AMPK-ACC-CPT1信號(hào)通路,并通過(guò)增加解偶聯(lián)作用提高基礎(chǔ)代謝率,促進(jìn)脂肪酸氧化分解、降低FFA水平、減少脂質(zhì)沉積、改善脂質(zhì)代謝紊亂及瘦素抵抗。
[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.
【學(xué)位授予單位】：湖北中醫(yī)藥大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R285.5

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