【作者】 吴洪阳；

【导师】 王华；

【作者基本信息】 湖北中医药大学 ， 针灸推拿学， 2013， 博士

【摘要】 目的近年来,2型糖尿病的发病率呈现快速增长趋势,由糖尿病引起的死亡人数仅次于心脑血管疾病、恶性肿瘤。胰岛素抵抗是2型糖尿病早期重要的发病机制,2型糖尿病发病前会经历多年的胰岛素抵抗状态,因此在胰岛素抵抗状态即采取措施提高胰岛素敏感性,是治疗2型糖尿病的关键,对于预防糖尿病及其相关并发症具有重要意义。目前治疗糖尿病的主要手段包括口服降糖药、注射胰岛素与控制饮食等。虽然这些方法对于改善血糖以及胰岛素敏感性等有较好疗效,但均存在一定毒副作用。随着时间的延长,始终无法避免胰岛p细胞的损伤,而且控制饮食后人的生活质量将大大降低。因此寻找疗效好、稳定持久、无毒副作用的治疗方法,是摆在医学界面前的一项重大课题。针刺越来越广泛地被用于胰岛素抵抗相关性疾病的防治,且疗效明显,在临床研究中逐步得到证实。为了进一步探讨针刺治疗胰岛素抵抗的作用机制,本课题通过高脂饲料喂养Wistar大鼠,造成胰岛素抵抗模型。本研究以中医针灸“治未病”理论为指导,采用“标本配穴”电针法,观察胰岛素靶器官骨骼肌中SIRT1去乙酰化激活PGC-1α情况及其下游相关基因与蛋白表达水平,并重点观察线粒体生物合成及氧化相关基因和蛋白表达水平,分析这些变化与胰岛素敏感性升高的关系。旨在深入研究电针治疗胰岛素抵抗在线粒体葡萄糖和脂肪酸利用环节的科学内涵与重要机制,为临床运用针刺治疗胰岛素抵抗及其相关疾病提供理论支持和科学依据。方法将48只8周龄雄性Wistar大鼠按随机数字法随机分4组：正常组(n=12)、模型组(n-12)、电针组(n=12),白藜芦醇组(n-12)。正常组,喂基础饲料(总热量为3.8kcal/g,含70%碳水化合物,20%蛋白质,10%脂肪)；其余3组,给予高脂饲料(总热量为5.4kcal/g,含38.5%碳水化合物,15%蛋白质,46.5%脂肪)。喂养8周获得胰岛素抵抗大鼠模型。大鼠喂养8周后,每组随机抽2只Wistar大鼠行高胰岛素-正葡萄糖钳夹术测定全身胰岛素敏感性,结果以葡萄糖输注率GIR表示。治疗期各组大鼠均喂基础饲料,自由摄食和饮水。电针组,采用0.30mm×15mm不锈钢毫针,选取“足三里”和“丰隆”直刺3-5mm,左右交替,“关元”向剑突方向和“中脘”向耻骨联合方向斜刺3-5mm;捻转1min后,连接HANS LH202H型电针治疗仪,连续波,频率2Hz,强度1mA,通电10min,每日1次,每周5次,总疗程为8周。同侧“足三里”和“丰隆”连接同一输出的两个电极,“关元”和“中脘”连接另一输出的两个电极,电针时用特制的鼠服将大鼠固定。正常组和模型组不予治疗；但两组大鼠相同时间、相同方法抓取固定。白藜芦醇组大鼠每天10：00给予白藜芦醇100mg/kg灌胃,连续进行8周。治疗8周后,观察各组大鼠骨骼肌胰岛素敏感性,采用Real-time PCR检测SIRT1mRNA、PGC-1αmRNA和NRF-1mRNA表达,免疫印迹法检测各组大鼠股四头肌细胞核中PGC-1α和NRF-1蛋白的表达,采用比色法检测CPT-1, MCAD和LCAD的活性,对上述结果进行综合统计分析。结果1.各组大鼠基本生化资料：各组大鼠的空腹血糖水平无明显差异。模型组的空腹血浆胰岛素水平已明显高于正常组(P<0.05),葡萄糖平均输注速率GIR60～120(～20%,P<0.05)已显著低于正常组,提示模型组大鼠已出现明显的系统胰岛素抵抗。大鼠经过治疗8周后,电针和白藜芦醇干预后空腹血浆胰岛素水平明显减低,GIR60-120显著增加(P<0.05),提示电针和白藜芦醇均可改善高脂饮食诱导的系统胰岛素敏感性。此外,模型组的空腹血脂如FFA和TG均高于正常组(P<0.05)。电针治疗后其FFA和TG明显降低,趋于正常值水平,与模型组比较,有显著性差异(P<0.05)；而白藜芦醇干预后,对血脂水平无明显影响。2.各组大鼠骨骼肌SIRT1mRNA表达的影响：治疗8周后,电针组大鼠股四头肌细胞核SIRT1mRNA表达水平明显增加,与模型组相比,具有显著性差异(P<0.05)。白藜芦醇组大鼠股四头肌细胞核SIRT1mRNA表达水平明显增加,与模型组相比,具有显著性差异(P<0.05)。电针组与白藜芦醇组比较,差异无统计学意义。3.各组大鼠骨骼肌PGC-1α蛋白表达：与正常组比较,模型组骨骼肌PGC-1α蛋白表达下降,差异有极显著性意义(P<0.01)；电针组与白藜芦醇组骨骼肌PGC-1α蛋白表达较模型组明显上升,差异有极显著性意义(P<0.01)；电针组与白藜芦醇组骨骼肌PGC-1α蛋白表达无明显差异。4.各组大鼠骨骼肌NRF-1蛋白表达：与正常组比较,模型组骨骼肌NRF-1蛋白表达下降,差异有统计学意义(P<0.05)；电针组与白藜芦醇组骨骼肌NRF-1蛋白表达均较模型组升高,经统计学处理,均有显著性差异(P<0.05)；电针组与白藜芦醇组比较,无显著性差异。5.各组大鼠骨骼肌PGC-1αmRNA表达：与正常组比较,模型组骨骼肌PGC-1αmRNA表达下降,差异具有统计学意义(P<0.05)；电针组与白藜芦醇组骨骼肌PGC-1αmRNA表达均较模型组升高,差异有统计学意义(P<0.05)；电针组与白藜芦醇组比较,无显著性差异。6.各组大鼠骨骼肌NRF-1mRNA表达：与正常组比较,模型组骨骼肌NRF-1mRNA表达下降,差异具有统计学意义(P<0.05)；电针组与白藜芦醇组骨骼肌NRF-1mRNA表达均较模型组升高,差异有统计学意义(P<0.05)；电针组与白藜芦醇组比较,无显著性差异。7.各组线粒体CPT-1、MCAD、LCAD活性检测：治疗8周后,电针组大鼠骨骼肌单位组织蛋白的SS线粒体CPT-1、MCAD和LCAD的活性显著增加,与模型组相比,具有显著性差异(P<0.05)。白藜芦醇组大鼠骨骼肌单位组织蛋白的SS线粒体CPT-1、MCAD和LCAD的活性显著增加,与模型组相比,具有显著性差异(P<0.05)。电针组与白藜芦醇组比较,差异无统计学意义。结论1.电针能升高胰岛素靶器官SIRT1表达及其活性,通过去乙酰化激活PGC-1α。2.电针激活SIRT1/PGC-1α后,诱导与线粒体生物合成和氧化相关的蛋白和酶体的表达,增加线粒体氧化能力,从而减轻或控制胰岛素抵抗。3.电针可纠正骨骼肌脂质转运和ss线粒体脂肪酸氧化的平衡,减轻脂质异位沉积,从而改善高脂饮食大鼠骨骼肌的胰岛素抵抗。更多还原

【Abstract】 Objective:In recent years, the incidence of type2diabetes mellitus presents the fast growth trend, and the death toll caused by diabetes mellitus ranks only after cardiac-cerebral vascular diseases and malignant tumor. Diabetes related complications such as coronary heart disease, diabetic nephropathy, diabetic retinopathy and diabetic peripheral neuropathy are the main reasons for death and disability, which threatened human health seriously. Because insulin resistance is an important pathogenesis of type2diabetes mellitus in early stage and there should be many years of insulin resistance before the onset of type2diabetes mellitus, it is the key point that taking measures to improve insulin sensitivity in insulin resistance stage to treat type2diabetes mellitus and prevent diabetes and related complications.The main methods to treat diabetes mellitus are oral medications, insulin injection, diet control and etc. Although those methods have good curative effect to improve blood sugar content and insulin sensitivity, there are some toxic and side effects. But over time, the damage to islet β cells cannot be avoided, and the quality of life in diet will be greatly reduced. Therefore, looking for a therapy with good curative effect, stability and non-toxic side effects is a major subject in front of the medical professionals.Acupuncture has been more and more widely used in preventing and controlling insulin resistance related diseases with obvious curative effect, which is gradually confirmed in clinical researches. In order to further explore the mechanisms of acupuncture treatment on insulin resistance, this project focused on "" electropuncture on insulin resistance model of Wistar rats after high-fat diet, under the guidance of the theory of traditional Chinese medicine as "preventive treatment of disease", by observing SIRT1activating PGC-1α and its downstream related genes and proteins expression in skeletal muscle, a insulin target organ, by deacetylation. Priority observations were made on mitochondria biosynthesis and oxidative related gene and protein expression and the relations between these changes and increased insulin sensitivity. This project will further reveal the function of "" electroacupuncture on insulin resistance in mitochondrial glucose and fatty acid utilization to provide the theoretical support and scientific basis for clinical use of acupuncture treatment on insulin resistance and related diseases in the future.Methods:Forty-eight male Wistar rats at8weeks of age were randomly divided into4groups:normal group (n=12), model group (n=12), electroacupuncture group (EA, n=12) and resveratrol group (n=12). For those in normal group, the basal feed (total heat is3.8kcal/g, including70%carbohydrate,20%protein and10%fat) was given; while high-fat diet (total heat is5.4kcal/g, including38.5%carbohydrate,15%protein and46.5%fat) for8weeks to establish the rat model with insulin resistance. After8weeks’ feeding,2Wistar rats of each group were randomly selected to detect whole body insulin sensitivity which was presented with glucose infusion rate (GIR) by high insulin-glucose clamps. During treatment, all the rats were fed with basal feed and free access to food and water. For EA group, Zusanli (ST36) and Fenglong (ST40) were inserted perpendicularly with3-5mm, while Guanyuan(CV4) was inserted obliquely towards processus xiphoideus and Zhongwan(CV12) towards symphysis pubis with3-5mm. After twirling the needles for lmin, HANS LH202H electric acupuncture therapeutic apparatus was used with continuous wave, frequency of2Hz and intensity of1mA for10min, once per day, five times per week for8weeks. One pair of electrodes on the homolateral Zusanli (ST36) and Fenglong (ST40) and the other on Guanyuan(CV4) and Zhongwan(CV12). Purpose-made clothes were used to fix the rats during the treatment. For resveratrol group, the resveratrol (100mg/kg) was given by intragastric dministration on10:00am everyday for8weeks. And then, insulin sensitivity in skeletal muscle was detected. The expression of SIRT1mRNA, PGC-1αmRNA and NRF-1mRNA were detected with real-time PCR, protein expression of PGC-1α and NRF-1with western blot, and the activity of CPT-1, MCAD and LCAD were detected with colorimetric method. All the data were given statistic analysis.Results:1. Basic biochemical information of the rats in each group:fasting blood glucose levels of the rats in each group has no obvious difference. Fasting plasma insulin level in model group was significantly higher than that in normal group (p<0.05), an average glucose infusion rate GIR60～120(～20%, P<0.05) was significantly lower than normal group, suggesting that rats in the model group has a definite system insulin resistance. After8weeks treatment with EA or resveratrol intervention, fasting plasma insulin levels significantly reduced, GIR60～120increased significantly (p<0.05), suggesting both EA and resveratrol could improve insulin sensitivity induced by the high-fat diet system. In addition, the fasting blood lipids such as TG and FFA in model group were higher than that in normal group (p<0.05). TG and FFA significantly reduced after EA, tending to normal level. Compared with model group, there was significant difference (p<0.05); while there was no obvious effect after the intervention of resveratrol.2. The expression of SIRT1mRNA in skeletal muscle of the rats in ach group:the expression level of quadriceps nucleus SIRT1mRNA in EA group increased significantly after8weeks treatment. Compared with model group, there was significant difference (P<0.05). The expression level of quadriceps nucleus SIRT1mRNA in resveratrol group rats increased significantly. Compared with model group, there was significant difference (P<0.05). There was no statistically significant difference between resveratrol group and EA group.3. The protein expression of PGC-la in skeletal muscle of the rats in each group:compared with normal group, the protein expression of PGC-1α decreased in the skeletal muscle of model group with significant difference significance (P<0.01); protein expression of PGC-la in the skeletal muscle of resveratrol and EA groups increased more significantly than that in model group with extremely significant (P<0.01); The protein expression of PGC-1α in skeletal muscle between EA and resveratrol group has no obvious difference.4. The protein expression of NRF-1in skeletal muscle of the rats in each group:compared with normal group, the expression of skeletal muscle protein NRF-1in model group decreased with statistically significant (P<0.05); the expression of NRF-1in resveratrol and EA groups were higher than that in model group with significant difference (P<0.05); there was no significant difference between EA and resveratrol group.5. The expression of PGC-lamRNA in skeletal muscle of the rats in each group:compared with normal group, the expression of skeletal muscle PGC-1αmRNA in model group decreased with significant difference (P<0.05); the expression of PGC-1αmRNA in resveratrol and EA groups were higher than that in model group with significant difference (P<0.05); there was no significant difference between EA and resveratrol group.6. The expression of NRF-1mRNA in skeletal muscle of the rats in each group:compared with normal group, the expression of skeletal muscle NRF-1mRNA in model group decreased with significant difference (P<0.05); the expression of NRF-1mRNA in resveratrol and EA groups were higher than that in model group with significant difference (P<0.05); there was no significant difference between EA and resveratrol group.7. The activity detection of CPT-1, MCAD and LCAD in mitochondria of the rats in each group:after8weeks’ treatment, the activity of SS mitochondrial CPT-1, MCAD and LCAD in the rats skeletal muscle of EA group increased significantly with significant difference compared with that of model group (P<0.05). the activity of SS mitochondrial CPT-1, MCAD and LCAD in the rats skeletal muscle of resveratrol group increased significantly with significant difference compared with that of model group (P<0.05). There was no significant difference between EA and resveratrol group.Conclusion:1. Electroacupuncture may increase the expression and activity of SIRT1in insulin target organ and activate PGC-1α via deacetylation.2. Electroacupuncture may reduce or control insulin resistance by inducing the proteins and enzymes related with mitochondria biosynthesis and oxidative and enhance the oxidizability of mitochondria following the activation of SIRT1/PGC-1α.3. Electroacupuncture may improve insulin resistance in skeletal muscle of the rats fed with high fat diet by correcting the balance between lipid transport and fatty acid oxidation in SS mitochondria of skeletal muscle so as to reduce the ectopic deposition of lipids.更多还原