温肾健脾化痰方治疗肥胖症的理论及实验研究

发布时间：2018-03-20 23:35

  本文选题：肥胖症　切入点：大鼠　出处：《湖北中医药大学》2016年博士论文　论文类型：学位论文

【摘要】：目的通过中医药理论及临床实践研究,探讨肥胖症的中西医发病机制,为临床应用中医理论治疗肥胖症提供理论指导,通过高脂饮食诱导肥胖动物模型,探讨温肾健脾化痰方对肥胖症大鼠的影响,从分子水平及药理学方面研究温肾健脾化痰对肥胖症的作用机制,从而为温肾健脾化痰法临床治疗肥胖症提供理论依据。方法本研究从理论研究和实验研究两个方面进行。理论研究:从肥胖症的诊断、分类和病因病机以及辨证论治方面进行了系统的论述,并从理、法、方、药四个方面对温肾健脾化痰方治疗肥胖症的机制进行了理论探讨。实验研究:将60只体重100~150g的健康雄性SD大鼠随机分为2组,一组为正常对照组10只,给予普通饲料喂养,自由取食饮水;其余50只给予高脂饲料喂养,自由取食饮水。每周测量体重、身长2次,喂养4周后选取体质量增加大于正常组平均体质量15%且Lee's指数比正常组平均水平增加大于1.5%的大鼠,作为高脂饮食诱导肥胖造模成功大鼠,选取40只造模成功的大鼠随机分为4组,分别为模型组、低剂量组、中剂量组和高剂量组,每组10只。正常组和模型组大鼠给予生理盐水1 ml/100g·d灌胃;低、中、高剂量组大鼠分别给予低、中、高剂量温肾健脾化痰方1ml/100g·d灌胃。正常组继续喂以普通饲料,其他4组继续予高脂饲料喂养,实验过程中每周测量体重2次,并根据体重调整给药量,连续治疗10周。观察各组大鼠一般情况和体质量的变化,采用全自动生化分析仪检测各组大鼠血脂及肝功能。运用HE染色观察各组大鼠肝脏组织和脂肪组织的病理形态学改变。采用RT-PCR法观察温肾健脾化痰方对肝脏组织SOCS3m RNA表达的影响,免疫组织化学法观察其对各组大鼠脂肪组织Ghrelin水平的影响。结果1)各组大鼠体质量的变化:温肾健脾化痰方低、中、高剂量均能明显控制肥胖症大鼠体重的增加,使体重增加的速度明显变缓,与模型组相比具有统计学意义(P0.01),且中剂量组和高剂量组效果明显优于低剂量组并具有统计学意义(P0.01),而中、高剂量组之间差异无统计学意义。2)各组大鼠肝脏组织及脂肪组织病理学变化:正常组肝细胞形态排列正常,肝小叶规则,细胞质均匀;模型组均出现程度不等的弥漫性肝细胞脂肪变性,肝细胞体积增大,胞浆内可见数量不等、大小不一的圆形脂滴或脂肪空泡,胞核被推向周边;低剂量组脂变肝细胞与模型组相当;中、高剂量组脂变肝细胞较模型组明显减少,可见少量脂肪空泡。与正常组大鼠相比,模型组大鼠脂肪细胞明显增大,单位视野内脂肪细胞数明显减少;低剂量组大鼠脂肪细胞与模型组相当;中、高剂量组大鼠脂肪细胞较模型组明显减小,单位视野内脂肪细胞数明显增多,且中剂量组的变化更为显著。3)各组大鼠肝功能比较:与正常组相比,模型组与低、中、高剂量组的ALT,AST均显著升高(P0.01,P0.05);与模型组相比,温肾健脾化痰方低、中、高剂量均可显著降低ALT,AST水平(P0.01),且中、高剂量组效果显著优于低剂量组(P0.01),高剂量组在降低AST水平方面又显著优于中剂量组(P0.01),但在降低ALT水平方面则无明显区别(P0.05)。4)各组大鼠血脂比较:与正常组相比,模型组与低、中、高剂量组的TC,TG,LDL-C均显著性升高(P0.05),HDL-C显著性降低(P0.05,提示肥胖症大鼠模型存在明显的血脂紊乱。与模型组相比,温肾健脾化痰方低、中、高剂量组均可显著性降低TC,TG以及LDL-C(P0.01),升高HDL-C(P0.01),且三组之间比较也有显著性差异(P0.01)。5)各组大鼠肝脏组织SOCS3m RNA表达:模型组大鼠肝脏组织内SOCS3m RNA表达量显著升高(P0.01),经温肾健脾化痰方治疗后,与模型组比较,药物治疗组SOCS3m RNA表达均显著下降(P0.01),其中高剂量组与低、中剂量组相比有显著性差异(P0.01),与正常组相比无明显区别(P0.05)。6)各组大鼠脂肪组织Ghrelin基因表达:模型组大鼠脂肪组织内Ghrelin基因表达量显著升高(P0.05),经温肾健脾化痰方治疗后,与模型组比较,药物治疗组Ghrelin基因表达均显著下降(P0.01),其中高、中剂量组相比无明显区别(P0.05),但与低剂量组相比有显著性差异(P0.01)各组大鼠脂肪组织Ghrelin免疫组化检测结果:光镜下脂肪细胞的细胞核呈棕黄色或棕褐色的为Ghrelin阳性细胞,阴性细胞核为蓝色或蓝紫色。观察各组大鼠的脂肪细胞:模型组大鼠脂肪细胞内Ghrelin阳性细胞明显较正常组增多,经温肾健脾化痰方药物治疗的大鼠脂肪细胞内Ghrelin阳性细胞较模型组明显减少,以中、高剂量组较明显。IPP分析结果也显示:与正常组相比,模型组、低、中、高剂量组Ghrelin阳性表达率明显升高(P0.05),经治疗后,低、中、高剂量组大鼠脂肪Ghrelin阳性表达率较模型组显著下降(P0.01)。结论理论研究:肥胖症的病机为本虚标实,表现以脾肾亏虚为本,以痰湿、血瘀等为标。温肾健脾化痰方的组方体现了肥胖症的辨证论治,为运用其治疗肥胖症提供了理论依据。实验研究:1)温肾健脾化痰方在控制肥胖模型大鼠体重增加,减轻肝功能损害,减少腹内脂肪,降低血清胆固醇及甘油三酯减轻肝脏脂肪变性方面有良好作用。2)温肾健脾化痰方可调控食欲,增加能量代谢,抑制脂肪合成,改善糖、脂代谢,减轻肥胖,可能与其通过调节肥胖症模型大鼠的SOCS3m RNA水平来实现的。3)温肾健脾化痰方可调节肥胖症模型大鼠Ghrelin基因的表达,而调节这些基因表达不仅可直接影响食欲和饮食,提高能量代谢效率,还能通过调节瘦素等脂肪因子,达到治疗肥胖症的目的。
[Abstract]:Objective to study the TCM theory and clinical practice of traditional Chinese medicine and Western medicine, to explore the pathogenesis of obesity, and provide theoretical guidance for the clinical application of the theory of traditional Chinese medicine for the treatment of obesity, the high fat diet induced obesity animal model to investigate the effects of Wenshen Jianpi Huatan Decoction on obese rats, at the molecular level and pharmacology research on obesity and phlegm of warming kidney and invigorating spleen the mechanism of action, so as to provide a theoretical basis for the clinical treatment of warming kidney and invigorating spleen and resolving phlegm obesity. Methods this study from two aspects of theoretical research and experimental research. The theoretical research: from obesity diagnosis, classification and pathogenesis and syndrome differentiation were discussed, and the principle, method, party. The mechanism of the four aspects of medicine Wenshen Jianpi Huatan Decoction in the treatment of obesity are discussed. Experimental study: 60 healthy male SD rats weighing 100~150g random divided into 2 groups As a group, 10 rats in normal control group, given normal diet, free feeding, drinking water; the remaining 50 rats were given high fat diet, feeding free drinking water. Weekly measurement of body weight, body length 2 times, after 4 weeks of feeding selection of body weight increased more than the normal group average body mass index increased more than 1.5% and 15% Lee's in rats compared with normal group average, as the high fat diet induced obese rats, 40 rats were randomly divided into 4 groups of successful modeling, respectively, model group, low dose group, middle dose group and high dose group, 10 rats in each group. The normal group and model group were given saline 1 ml/100g D Ig; low, high dose group rats were given low, high dose of Wenshen Jianpi Huatan Decoction 1ml/100g d by gavage. The normal group fed with normal diet, the other 4 groups continue to be fed with high-fat diet during the experiment, body weight was measured weekly for 2 times, and according to the weight adjustment The whole dosage, continuous treatment for 10 weeks. To observe the changes of general condition and body weight of the rats were detected by automatic biochemistry analyzer rats blood lipid and liver function. Using HE staining to observe the pathologic liver tissue of rats and adipose tissue changes. Observation of Wenshen Jianpi Huatan Decoction on expression of SOCS3m RNA in liver organized by RT-PCR method, its impact on the level of Ghrelin in adipose tissue of rats were observed by immunohistochemistry. Results: 1) the body weight of the rats in each group: Wenshen Jianpi Huatan Decoction in low, high dose could significantly increase the weight control in obese rats, the body weight increased significantly slow speed compared with the model group, with statistical significance (P0.01), and the effect of middle dose group and high dose group was significantly better than the low dose group and had statistical significance (P0.01), and between the high dose group, the difference was statistically significant. 2) liver histology of rats and adipose tissue pathology: normal hepatic cell morphology were normal, uniform rules, lobular cytoplasm; model group showed different degrees of diffuse fatty degeneration of liver cells, liver cell volume increases, cytoplasm number ranging from the size of the circular lipid or fat vacuoles, nucleus is pushed to the periphery; low dose group of fat liver cell and model group; high dose group, fatty degeneration of liver cells was significantly reduced compared with the model, a small amount of fat vacuoles. Compared with normal rats, fat cells of rats in the model group increased obviously, number of fat cells in the field units significantly reduced; low dose group rat adipocytes with the model group, high dose group; rat fat cells decreased significantly compared with the model group, the number of fat cells in the field of units increased significantly, and the change in dose group is more significant.3) liver function of rats 鑳芥瘮杈，

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