【作者】 张梁；

【导师】 许能贵；

【作者基本信息】 广州中医药大学 ， 针灸推拿学， 2010， 博士

【摘要】 胰岛素抵抗与高血压、糖尿病、血脂异常、心脑血管疾病、肥胖、感染等多种疾病密切相关,是导致这些疾病的“共同土壤”。大量的临床研究报道证实,针刺在这些疾病的治疗中具有不可替代的优势,其作用机理与改善这些疾病中存在的胰岛素抵抗现象密切相关。通过本课题组的前期研究,我们发现针刺能有效调整胰岛素的敏感性,并认为针刺主要从受体及受体后水平调整和改善胰岛素抵抗状态。本研究是在课题组前期研究的基础上,以高糖高脂高盐饮食复制胰岛素抵抗动物实验模型,针对胰岛素抵抗主要的信号转导途径——PI3K途径来进行系统研究：以空腹血糖(FPG)、血浆胰岛素(FINS)、C肽、胰岛素敏感指数(ISI)为指标来观察针刺对胰岛素抵抗改善的效应；通过QT-PCR、Western blotting等检测手段,观察针刺前后胰岛素抵抗模型大鼠骨骼肌PI3K-p85、PDK1、Akt2、PKCλ/ζ的mRNA和蛋白表达的影响,从分子水平进一步探讨针刺调整胰岛素抵抗的机理。全文分文献研究、实验研究和小结三大部分进行。1文献研究通过对大量关于胰岛素抵抗的文献综合研究,从胰岛素抵抗的概念、胰岛素抵抗的主要信号转导途径及其各级组成蛋白的具体作用和相互联系、中医药防治胰岛素抵抗的研究现状、胰岛素的动物模型选择等几个方面阐述了胰岛素抵抗的最新研究进展,并进行了分析和评述,提出了今后的研究方向,认为针刺是改善胰岛素抵抗的有效方法之一,应加强对其作用机理的深入研究和探讨。2实验研究2.1目的观察针刺对胰岛素抵抗模型大鼠骨骼肌PI3K-p85、PDK1、Akt2、PKCλ/ζ的mRNA和蛋白表达的影响,从分子水平进一步探讨针刺调整胰岛素抵抗的机理。2.2方法将24只雄性SD大鼠(180-220g)按体重随机分为3组,每组8只,分别为空白组、模型组和针刺组。空白组以普通饲料喂养,其余各组则以高糖高脂高盐饲料喂养。从第6周开始,每隔1周对空白组和模型组大鼠从眼眶静脉窦采血1次,检测空腹血糖(FPG)、血浆胰岛素(FINS),并计算胰岛素敏感性指数(ISI)进行对比,当模型组大鼠ISI与空白组相比明显降低(P<0.01)时为造模成功。第12周造模成功后,空白组继续以普通饲料喂养3周,模型组继续以高脂高糖高盐饲料喂养3周,针刺组继续以高脂高糖高盐饲料喂养3周,同时给予电针治疗,穴选“内关”、“足三里”、“三阴交”和“肾俞”,1次／日。各组大鼠于治疗结束后禁食12h过夜,次晨眼眶静脉窦采血检测FPG, FINS、C-P等指标,FPG测定采用葡萄糖氧化酶法,FINS、C-P测定采用ELISA法,ISI用1/(CFPG×JFINS)公式计算；胰岛素灌注后处死,取股四头肌,用QT-PCR、Western blotting等检测手段,观察针刺前后胰岛素抵抗模型大鼠骨骼肌PI3K-p85、PDK1、Akt2、PKCλ/ζ的mRNA和蛋白表达情况。2.3结果给予高脂高糖高盐饲料喂养12周后,模型组大鼠和针刺组大鼠的FPG、FINS均较空白组显著升高(P<0.01),ISI显著下降(P<0.01),表明造模成功。经过3周针刺治疗后,针刺组的FPG、FINS、C-P较模型组显著降低(P<0.01,P<0.05),ISI较模型组显著升高(P<0.01)。模型组大鼠骨骼肌的PI3K-p85αmRNA和PI3K-p85蛋白表达均较空白组显著升高(P<0.01)；针刺组大鼠骨骼肌PI3K-p85a mRNA和PI3K-p85蛋白表达均较模型组显著降低(P<0.01)。三组大鼠骨骼肌PDK1蛋白表达的差异无统计学意义(P>0.05)。模型组大鼠骨骼肌Akt2 mRNA表达较空白组显著降低(P<0.05)；针刺组大鼠骨骼肌Akt2 mRNA表达较模型组和空白组均显著升高(P<0.01)。模型组大鼠骨骼肌的phospho-PKCζ/λmRNA与空白组相比均有明显下降(P<0.01),其中针刺对phospho-PKCζmRNA有显著的上调作用(P<0.01),而对phospho-PKCλmRNA无明显影响(P>0.05)；胰岛素抵抗模型组大鼠的phospho-PKCζ/λ蛋白表达与空白组相比无明显变化(P>0.05),亦未发现针刺对其有明显影响(P>0.05)。2.4结论研究表明,针刺可通过影响PI3K通路的信号转导,主要是降低PI3K-p85αmRNA and PI3K-p85蛋白的过度表达、增加Akt2 mRNA和phospho-PKCζmRNA的表达,以调整和改善胰岛素抵抗状态,其具体的作用形式和途径尚未阐明,有待今后进一步探讨。更多还原

【Abstract】 ObjectiveOur aim was to observe the effects of acupuncture on PI3K-p85, PDK1, Akt2 and VKCλ/ζof insulin resistance model rats’femoris muscle, so as to further explore its underlying mechanism.MethodsTwenty four male SD rats were divided randomly into three groups:the blank group, the model group and the acupuncture group. We induced insulin resistance model through the high glucose, high salt and high fat diet. From the sixth week, we tested FPG and FINS in the rat’s blood every week, then figured out the ISI. When the ISI of the model group and the blank group was statistically significant (P<0.01), the model was success. Then in the next three weeks, the rats of the blank group were fed by normal diet, while the rats of the model group and the acupuncture group were fed by the high glucose, high salt and high fat diet. The rats of the acupuncture group were treated by electroacupuncture at the same time, twenty minutes per day. We chose four points:Neiguan, Zusanli, Sanyinjiao and Shenshu. After three weeks of treatment, all rats were drew blood, preparing for testing FPG, FINS and C-P. Then they were perfused by insulin before being killed. Their quadriceps femoris muscle was cut off. Finally PI3K-p85, PDK1, Akt2 and PKCλ/ζwas tested by methods such as QT-PCR, western blotting and so on.ResultsInsulin resistance rat model was built successfully. FPG and FINS of the model group rose evidently(P<0.01), ISI of of the model group declined remarkably(P＜0.01). After treatment, FPG, FINS, ISI and C-P of the acupuncture group improved evidently(P<0.01,P<0.05).PI3K-p85a mRNA and PI3K-p85 protein of the model group over expressed(P<0.01), acupuncture made it descend to normal level.PDK1 protein expression of three groups was not statistically significant(P>0.05). Compare to the model group and the blank group, Akt2 mRNA of the acupuncture group rose significantly(P＜0.01).Phospho-PKCζ/λmRNA of the model group reduced remarkably(P<0.01). The acupuncture group’s phospho-PKCζmRNA rose(P<0.01), but its phospho-PKCλmRNA had not changed(P>0.05). Phospho-PKCζ/λ. protein expression of three groups was not statistically significant(P>0.05).ConclusionIn this study, we found acupucture’s regulating action to PI3K route. We presume acupuncture treat insulin resisitance model rats through reducing PI3K-p85a mRNA and PI3K-p85 protein expression, increase Akt2 mRNA and phospho-PKCζmRNA expression. The action mode and pathway of these protein were not clear yet, which need us to study further.更多还原