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电针对胰岛素抵抗模型大鼠肝脏AMPK、ACC的干预机制研究

发布时间:2018-05-17 03:37

  本文选题:胰岛素抵抗 + 针刺 ; 参考:《广州中医药大学》2016年博士论文


【摘要】:第一部分文献研究随着生活水平提高、饮食架构的变化,人类饮食相关疾病成为发展中国家及发达国家的潜在威胁。营养过剩的饮食习惯及以静坐为主的工作模式导致现代人代谢类疾病的频繁发展,最明显的就是糖尿病(DM)。胰岛素抵抗(IR)作为糖尿病的前驱状态,受到了现代研究的高度关注。而且作为高血压、脂肪肝、多囊卵巢综合症等疾病的“共同土壤”,胰岛素抵抗同时受到广泛关注。目前研究表明,糖尿病发病率的增长趋势正在逐年增长,严重危害人类健康。目前为止,对现代医学对2型糖尿病(T2DM)的发病机制尚未完全剖析清楚,胰岛素抵抗和胰岛素β细胞功能缺陷是其基本特征,而现代生活营养过剩所引起的肥胖、高血压、糖尿病与胰岛素抵抗关系密切。胰岛素抵抗作为T2DM发生的核心环节,是治疗T2DM及胰岛素抵抗相关疾病的关键。因此,调节胰岛素信号通路异常,从而改善胰岛素抵抗,以此为突破口,是治疗T2DM的关键所在。中医又将糖尿病称为消渴病,历史记载由来已久,因此历代医家及后世学者都在治疗消渴病的过程中积累了丰富的临床经验,或以之撰写医案分享临床心得,或以之进行实验深化研究,都为人类的医学事业作出了伟大的贡献。大量文献表明,针刺治疗胰岛素抵抗相关疾病过程中存在不可取代的优势,其作用机理与改善胰岛素抵抗密切相关。本课题组通过既往研究发现针刺可通过受体及受体后水平改善胰岛素抵抗。为了进一步完善针刺改善胰岛素抵抗的机理,本课题选择胰岛素抵抗发生、发展过程中的脂代谢紊乱作为切入点,以脂联素所激发的AMPK-ACC信号转导通路为研究方向,通过检测相关指标观察针刺对胰岛素抵抗从宏观到微观的整体性理论,进一步证明其作用机理与治疗作用。第二部分实验研究目的:以高脂饮食诱导胰岛素抵抗模型大鼠为研究对象,以噻唑烷二酮类药物吡格列酮为对照,系统观察针刺对胰岛素抵抗大鼠血清脂联素、游离脂肪酸含量的影响,肝脏AMPK、ACC蛋白表达的影响,以及肝脏超微结构形态学的改变,从而多层次、多水平诠释针刺对胰岛素抵抗脂质代谢紊乱的调节效应,为其强化胰岛敏感功能提供新的理论基础。方法:将52只雄性SPF级SD大鼠(180-220g)普通饲料适应性饲养5天,随机抽出10只为正常组,喂以普通饲料;余下大鼠改用高脂饲料喂养3个月为造模组。从第8周开始,每隔一周对所有大鼠进行眼眶静脉窦取血,检测空腹血糖(FPG)、血浆胰岛素(FINS),并计算胰岛素敏感指标(ISI、HOMA-IR),当模型组大鼠ISI比空白组明显降低(P0.05),HOMA-IR明显升高(P0.05)时,即进行高胰岛素正葡萄糖钳夹试验,当葡萄糖输注率(GIR)与空白组相比明显降低(P0.05)时为造模成功。第12周造模成功后,采用分层随机法分为3组:模型组(11只)、西药组(13只)、电针组(12只)。空白组继续以普通饲料饲养2周,模型组继续以高脂饲料饲养2周。电针组继续以高脂饲料饲养2周,同时给予电针治疗,穴取“丰隆”、“三阴交”,1次/日。西药组续以高脂饲料饲养2周,同时给予吡格列酮悬浊液10mg/kg灌胃,1次/日。各组大鼠于治疗结束后眼眶静脉窦采血检测FINS. ADP. FFA等指标,FPG测定采用葡萄糖氧化酶法,FINS. ADP. FFA测定采用ELISA法。取肝脏,使用蛋白印迹检测手段,观察针刺前后胰岛素抵抗模型大鼠肝脏AMPK. ACC蛋白表达情况。使用透射电镜观察针刺后胰岛素抵抗模型大鼠肝脏组织超微结构形态学的改变。成果:1.高脂饲料饲养12周后,模型组、西药组、电针组大鼠的GIR均较空白组显著降低(P0.01),表明造模成功。经过2周治疗后,西药组、电针组的GIR较模型组显著升高(P0.01)。2.透射电镜观察:空白组干细胞中细胞器结构完好,线粒体、粗面内质网丰富,胞浆内无脂滴及脱颗粒现象,线粒体膜、嵴结构完整,排列规则,粗面内质网排列整齐;模型组肝细胞内可见大量脂滴分布,线粒体呈现不同程度肿胀,线粒体嵴变少变短,甚至出现空泡化。通过治疗,西药组、电针组肝细胞胞浆中脂滴较模型组减少,线粒体肿胀减轻,内外膜部分融合。3.模型组血清中ADP较空白组显著性降低(R0.01), FFA较空白组显著性升高(P0.01)。经治疗后,西药组、电针组的ADP较模型组显著性升高(R0.01, R0.05), FFA较模型组显著性降低(P0.01,P0.05)。4.模型组大鼠肝脏的AMPK蛋白水平较空白组显著降低(P0.01), ACC蛋白水平显著性升高(P0.05)。经治疗后,西药组、电针组AMPK蛋白水平较模型组显著升高(P0.01,P0.05);西药组、电针组ACC蛋白水平较模型组显著降低(P0.01)。结论:研究表明,电针可通过激活脂联素介导的信号通路,上调AMPK蛋白表达,进而下调ACC蛋白表达,以调整机体脂质代谢紊乱从而改善胰岛素抵抗状态。这可能是针刺治疗胰岛素抵抗的机制之一。
[Abstract]:The first part of the literature study is a potential threat to developing countries and developed countries with the improvement of living standards, dietary patterns and human diet related diseases. The dietary habits of excess nutrition and the mode of sitting mainly lead to the frequent development of modern metabolic diseases. The most obvious is diabetes mellitus (DM). IR, as a precursor of diabetes, is highly concerned by modern research. And as a "common soil" for hypertension, fatty liver, polycystic ovary syndrome and other diseases, insulin resistance is widely concerned. The current research shows that the trend of diabetes incidence is increasing year by year, seriously endangering human health. So far, the pathogenesis of type 2 diabetes (T2DM) has not been completely dissect. Insulin resistance and insulin beta cell dysfunction are the basic characteristics of modern medicine. Obesity, hypertension, diabetes and insulin resistance are closely related to the excess nutrition in modern life. Insulin resistance is the core link in the occurrence of T2DM. The key to T2DM and insulin resistance related diseases is to adjust the abnormal insulin signaling pathway to improve insulin resistance, which is the key to the treatment of T2DM. Great contributions have been made to the medical cause of human being, with rich clinical experience, or by writing medical cases to share clinical experiences, or to carry out experimental deepening research. A large number of literatures show that acupuncture treatment of insulin resistance related diseases has an important potential in the process of insulin resistance, and its mechanism is closely related to the improvement of insulin resistance. Through previous studies, we found that acupuncture can improve insulin resistance through receptor and receptor level. In order to further improve the mechanism of acupuncture to improve insulin resistance, this topic chooses insulin resistance, the disorder of lipid metabolism in the development process as a breakthrough point, AMPK-ACC signal transduction pathway induced by adiponectin as a signal transduction pathway. Research direction, through the detection of relevant indicators to observe the theory of acupuncture on insulin resistance from macro to microcosmic, and further prove its mechanism and therapeutic effect. The second part of the experimental study: using high fat diet induced insulin resistance model rats as the research object, with the thiazolidane two ketone drug pioglitazone as the control, The effects of acupuncture on serum adiponectin, free fatty acid content, the expression of AMPK and ACC in the liver, and the changes in the ultrastructural morphology of the liver were observed. The effects of acupuncture on the lipid metabolism disorder of insulin resistance were explained at a multi-level and multilevel level, and a new theory was provided for the enhancement of the islet sensitive function. Methods: 52 male SPF grade SD rats (180-220g) common feed was fed for 5 days, 10 normal groups were randomly selected as normal diet, and the remaining rats were fed with high fat feed for 3 months. From the eighth week, all rats were taken blood from the orbital venous sinus every other week to detect the fasting blood glucose (FPG) and plasma Insulin (FINS), and the insulin sensitivity index (ISI, HOMA-IR), when the model group was significantly lower (P0.05) than the blank group (P0.05), HOMA-IR significantly increased (P0.05), that is, the high insulin positive glucose clamp test, when the glucose infusion rate (GIR) was significantly lower than the empty white group (P0.05) was a successful model. After twelfth weeks of successful model production, harvest Stratified random method was divided into 3 groups: model group (11), western medicine group (13), electroacupuncture group (12). The blank group continued to feed with ordinary feed for 2 weeks, the model group continued to feed with high fat feed for 2 weeks. The electroacupuncture Group continued to feed high fat feed for 2 weeks, and the acupuncture treatment was given, "Feng long", "Three Yin Jiao", and 1 times. For 2 weeks, pioglitazone suspension was given at the same time with 10mg/kg gavage, 1 times. The rats in each group were used to detect FINS. ADP. FFA in the orbital venous sinus after the end of the treatment. The FPG was measured by the glucose oxidase method and the FINS. ADP. FFA was measured by ELISA. The liver was taken and the insulin resistance was observed before and after the acupuncture. The expression of AMPK. ACC protein in the liver of the model rats was observed. The ultrastructural morphological changes of the liver tissues of the rats were observed by transmission electron microscope. Results: after 12 weeks of feeding of 1. high fat diet, the GIR of the rats in the model group, the western medicine group and the electroacupuncture group were significantly lower than those in the blank group (P0.01), indicating that the model was successful after 2 weeks of treatment. After that, the GIR in the western medicine group and the electroacupuncture group were significantly higher than the model group (P0.01).2. transmission electron microscope observation: the cell organelle structure in the blank group was intact, the mitochondria, the rough endoplasmic reticulum rich, the cytoplasm no fat drop and degranulation phenomenon, the mitochondria membrane, the ridge structure complete, the arrangement rules, the rough endoplasmic reticulum arranged neatly; the model group liver cells can be seen big inside. After treatment, the lipid droplets in the cytoplasm of liver cells in the western medicine group and electroacupuncture group decreased compared with the model group, the swelling of mitochondria decreased, and the serum ADP in the.3. model group was significantly lower than that in the blank group (R0.01), and the FFA was more significant than that in the blank group. After treatment, the ADP in the western medicine group and the electroacupuncture group was significantly higher than the model group (R0.01, R0.05), and the FFA in the model group was significantly lower than that in the model group (P0.01, P0.05). The level of AMPK protein in the liver of the rat liver was significantly lower than that in the blank group (P0.01), and the ACC protein was significantly increased (P0.05). After treatment, the western medicine group and the electroacupuncture group had a protein water. Compared with the model group (P0.01, P0.05), the level of ACC protein in the western medicine group and the electroacupuncture group was significantly lower than that in the model group (P0.01). Conclusion: the study showed that the electroacupuncture could increase the expression of AMPK protein by activating the signal pathway mediated by adiponectin, and then down the expression of ACC protein in order to adjust the disorder of lipid metabolism and improve the insulin resistance. This may be one of the mechanisms of acupuncture for insulin resistance.
【学位授予单位】:广州中医药大学
【学位级别】:博士
【学位授予年份】:2016
【分类号】:R245

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