【作者】 卞尧尧；

【导师】 周春祥；

【作者基本信息】 南京中医药大学 ， 中医临床基础， 2013， 博士

【摘要】 代谢综合征metabolic syndrome, MS)作为一组与代谢相关的症候群,近年来成为医学界多学科共同研究的热点。胰岛素抵抗(insulin resistance, IR)是MS病理核心环节,肥胖(obesity)是MS重要诱导因素,炎症(inflammation)是MS重要病理特点。代谢综合征病机常表现为津液输布失调、气化失常、气机失调、痰湿郁滞等,而这与脏腑三焦生理功能异常有着密切联系[4]。审症求因,审因论治,针对代谢综合征病机特点,故从脏腑三焦论治代谢综合征。柴胡加龙骨牡蛎汤是调达三焦的经典方剂,具有抗炎、降血脂、降血糖等作用。因此,柴胡加龙骨牡蛎汤治疗代谢综合征值得深入探讨。目的：观察柴胡加龙骨牡蛎汤对代谢综合征大鼠的治疗作用并探讨其机制,为柴胡加龙骨牡蛎汤治疗代谢综合征提供理论和实验支撑,从而进一步探索中医药防治代谢综合征方法和手段。方法：高脂高糖高盐饲料喂养SD大鼠16周建立代谢综合征模型。模型大鼠随机分为模型组,柴胡加龙骨牡蛎汤高、中、低剂量组,阳性药组,饮食组。模型建立后模型组继续高脂高糖高盐饲料喂养,其余各组予普通饲料喂养。阳性药予二甲双胍,饮食组不予药物干预。各组共干预4周。实验包括五部分：1.代谢综合征模型大鼠的建立。通过高脂高糖高盐饲料喂养16周建立模型。通过测定大鼠空腹血糖(fasting blood glucose, FBG)、血脂(serum lipid)、空腹血清胰岛素(fasting serum insulin, FISN)、胰岛素抵抗稳态模型评估(homeostasis model assessment of insulin resistance, HOMA-IR)以及Lee’s指数(Lee’s index)等判断模型是否成功。2.柴胡加龙骨牡蛎汤对模型大鼠糖脂代谢的影响。通过观察实验大鼠各组间空腹血糖、总胆固醇(total cholesterol, TC)、甘油三酯(triglyceride, TG)、高密度脂蛋白(high density lipoprotein, HDL-C)、低密度脂蛋白(low density lipoprotein, LDL-C)以及HOMA-IR等指标的差异,探讨柴胡加龙骨牡蛎汤对代谢综合征治疗作用。3.柴胡加龙骨牡蛎汤对模型大鼠炎性因子的影响。通过观察实验大鼠各组间肿瘤坏死因子-α(tumor necrosis factor-a, TNF-a)及白介素-6(interleukin-6, IL-6)的差异,探讨柴胡加龙骨牡蛎汤抗炎作用及机制。4.柴胡加龙骨牡蛎汤对模型大鼠PI3K通路的影响。检测大鼠膈肌胰岛素受体底物蛋白1(insulin receptor substrate1, IRS1)和葡萄糖转运蛋白4(glucose transporter4, GLUT4)的表达,通过观察大鼠各组间蛋白表达的差异,探讨柴胡加龙骨牡蛎汤治疗代谢综合征机制。5.柴胡加龙骨牡蛎汤毒性探讨。检测大鼠血铅含量、肝肾功能、肝肾切片HE染色,探讨柴胡加龙骨牡蛎汤毒性。结果1.代谢综合征模型大鼠造模成功。结果显示,造模组大鼠与空白组相比,体重(P<0.01)、空腹血糖(P<0.01)、收缩压(P<0.05)、TG (P<0.05)、LDL-C (P<0.05)、TC(P<0.01)、空腹血清胰岛素(P<0.01)、HOMA-IR (P＜0.01)、Lee’s指数(P<0.01)升高。说明造模组大鼠具有代谢综合征特征,符合模型要求。2.柴胡加龙骨牡蛎汤高剂量、中剂量和阳性药组大鼠空腹血糖出现不同程度下降,与模型组比较三组皆有显著性差异(P<0.01)。空腹血清胰岛素和HOMA-IR各组数据显示阳性药组、柴胡加龙骨牡蛎汤高剂量组以及中剂量组与模型组有显著性差异(P<0.01),柴胡加龙骨牡蛎汤低剂量组空腹血清胰岛素水平较模型组降低,差异有统计学意义(P<0.05)。柴胡加龙骨牡蛎汤高剂量TG值与模型组相比,有显著性差异(P<0.01)。柴胡加龙骨牡蛎汤中剂量组大鼠TG值与模型组相比差异有统计学意义(P<0.05)。阳性药组、柴胡加龙骨牡蛎汤高中低剂量各组都能降低大鼠TC值,柴胡加龙骨牡蛎汤高剂量组、中剂量组与模型组相比,有显著性差异(P<0.01)；阳性药组、低剂量组与模型组比较差异有统计学意义(P<0.05)。柴胡加龙骨牡蛎汤高剂量组LDL-C值、HDL-C值与模型组相比降低,差异有统计学意义(P<0.05)。3.柴胡加龙骨牡蛎汤高剂量组、中剂量组TNF-α和IL-6值与模型组相比有显著性差异(P<0.01)。柴胡加龙骨牡蛎汤低剂量组TNF-α值与模型组相比差异有统计学意义(P<0.05)。阳性药组、柴胡加龙骨牡蛎汤低剂量组IL-6值与模型组相比,IL-6降低,有显著性差异(P<0.01)。饮食组IL-6值与模型组比较下降,有统计学意义(P<0.05)。4.蛋白免疫印迹(western blot, WB)实验结果：阳性药组、柴胡加龙骨牡蛎汤高剂量组能升高大鼠膈肌组织内的IRS1的表达,与模型组比较有显著性差异(P<0.01)。柴胡加龙骨牡蛎汤中剂量组亦能上调模型大鼠膈肌组织内的IRS1的表达,与模型组比较差异有统计学意义(P<0.05)。阳性药组、柴胡加龙骨牡蛎汤高剂量组和中剂量能升高大鼠膈肌组织内的GLUT4的表达,与模型组比较有显著性差异(P<0.01)。免疫组织化学染色实验结果显示：膈肌有较多IRS1和GLUT4阳性细胞表达,阳性药组、柴胡加龙骨牡蛎汤高剂量组、中剂量组和低剂量组IRS1和GLUT4累积光密度值显著高于模型组,有显著性差异(P<0.01)。饮食干预组IRS1累积光密度值高于模型组,差异有统计学意义(P<0.05)。5.柴胡加龙骨牡蛎汤干预后,各组大鼠血铅含量有差异,但在正常范围之内；肾功能、肾HE染色正常,肝功能、肝HE染色见异常,但空白组未见异常。结论1.高脂高糖高盐饲料喂养SD大鼠能够较好的造成代谢综合征大鼠模型。2.柴胡加龙骨牡蛎汤能调控代谢综合征大鼠空腹血糖、血脂等各项指标。说明柴胡加龙骨牡蛎汤对代谢综合征大鼠糖脂代谢紊乱有较好的治疗效果。3.柴胡加龙骨牡蛎汤能降低代谢综合征大鼠TNF-a和IL-6水平。说明柴胡加龙骨牡蛎汤对代谢综合征大鼠炎症状态有较好的治疗效果。4.柴胡加龙骨牡蛎汤能升高大鼠IRS1和GLUT4表达。说明柴胡加龙骨牡蛎汤在蛋白质水平上调控PI3K信号通路,治疗代谢综合征。5.柴胡加龙骨牡蛎汤治疗代谢综合征较安全,肝脏病理变化考虑由代谢综合征引发。6.柴胡加龙骨牡蛎汤治疗代谢综合征理论可行,实验支持。更多还原

【Abstract】 Metabolic syndrome (MS), as a group of symptoms associated with the metabolism has recently become a mutual hot topic in medical multidiscipline research and is generally considered to have insulin resistance as the pathological core area, obesity an important inducing factors, and inflammation an important pathological feature. The pathogenesis of MS is usually manifested by abnormal aerification, qi activity disorder, body fluid distribution, as well as dampness and phlegm stagnation, which is closely related to the physical functions of triple-jiao. In line with the determination of etiologic factors based on differentiation and treatment based on etiology, and according to the pathogenesis features of MS, one should therefore treat MS from the aspect of triple-jiao. Chaihu Modified Longgu Muli Decoction is a classic formula for discharging triple-jiao and the modern pharmacological studies have also indicated that the main ingredients within show good effects in anti-inflammation, decreasing blood lipid and sugar. Hence, the effects of Chaihu Modified Longgu Muli Decoction in the management of MS is worth being further studied and analyzed.ObjectivesTo observe the protective effects of Chaihu Modified Longgu Muli Decoction on MS model rats and to probe into its mechanism with molecular biology methods and to provide theories and experimental foundations for the prevention of MS with Chaihu Modified Longgu Muli Decoction, so as to further explore ways and methods to prevent and manage MS.MethodsAfter MS rats modeling, Chaihu Modified Longgu Muli Decoction was used as an intervention. Five steps as follows were included in the experiment:1.The MS rat models were established after administration of high sugar, lipid and salt diets for16wks. The levels of fasting blood glucose, lipid, serum insulin and HOMA-IR, as well as Lee’s index were determined to evaluate rats modeling successful or not.2.The effects of Chaihu Modified Longgu Muli Decoction on MS model rats glucose and lipid metabolism were observed thorough differences indicated in levels of fasting blood glucose, TG, TC, HDL-C ad HOMA-IR of rats from varied groups, so as to explore its prevention and management of MS and the relevant mechanism.3.The effects of Chaihu Modified Longgu Muli Decoction on model rats inflammatory factors were observed by differences between TNF-a and IL-6in each group in order to figure out its anti-inflammatory mechanism and to analyze the interventions. 4.The effects of Chaihu Modified Longgu Muli Decoction on model rats PI3K pathway were evaluated thorough observing differences in model rats diaphragm IRS-1protein and GLUT4protein expression from each group with Western blot so to explore Chaihu Modified Longgu Muli Decoction’s intervention on MS.5.The toxicity of Chaihu Modified Longgu Muli Decoction was determined with model rats blood lead levels, hepatorenal functions and biopsy HE staining.Results1.The MS model rats were established successfully. The model rats showed significant increase in body weight(P<0.01), length(P<0.01), FBG(P<0.01), diastolic pressure(P<0.05), serum insulin(P<0.01), TC(P<0.01), TG(P<0.05), HDL-C(P<0.05), Lee’s index(P<0.01), and HOMA-IR values(P<0.01),which met the MS characteristics and was in line with modelling requirement.2.The FBGs in model rats from high dose, middle dose and positive drug groups decreased obviously, with significant difference with model group (P<0.01). The levels of serum insulin and HOMA-IR in positive drug, high does and middle dose groups showed significant difference with model group (P<0.01); serum insulin of the low dose group decreased significantly with comparison to model group (P<0.05). TG index of high dose group increased significantly with comparison to model group (P<0.01); TG index of the middle dose group showed statistic difference with model group (P<0.05). TC index in positive drug, high dose, middle dose and low dose groups decreased with significant differences between the high, middle doses groups and model groups (P<0.01), positive drug, low dose and model groups showing statistic difference (P<0.05). LDL-C and HDL-C levels of high groups decreased significantly with comparison to the model group(P<0.05).3.TNF-a and IL-6levels in high and middle dose groups showed significant differences with comparison to model group (P<0.01), low dose group showing statistic difference with model group (P<0.05). IL-6in low dose group decreased significantly with comparison to model group(P<0.01), diet group showing statistic difference with model group (P<0.05).4.Western blot (WB) demonstrated that:rats IRS1expression in positive, high dose groups was increased, with significant difference with model group (P<0.01); rats IRS1expression in middle dose group was also improved with statistic difference to model group (P<0.05). GLUT4expression in positive, high dose and middle dose groups was increased with significant difference to model group (P<0.01). Immunohistochemical staining demonstrated that active IRS1and GLUT4expression in positive, high dose, middle dose and low dose groups, with statistic differences to model group (P<0.01); diet group also showed statistic difference in IRS1integrated optical density with comparison to model group (P<0.05).5.It was also demonstrated that lead content varied respectively in each group with Chaihu Modified Longgu Muli Decoction intervention but within the normal range; renal function and HE staining were normal; liver function and HE staining showed differences with no significant difference in control group.Conclusion1. A high lipid, sugar and salt diet intervention on SD rats can contribute to the MS model rats.2. Chaihu Modified Longgu Muli Decoction can regulate indicators of fasting blood glucose and lipids of MS model rats, suggesting a good intervention effects on sugar and lipid metabolism of MS model rats.3. Chaihu Modified Longgu Muli Decoction can decrease TNF-a and IL-6levels and effectively inhibit the release of inflammatory factors, indicating its good intervention functions on the inflammatory state of MS model rats4. Chaihu Modified Longgu Muli Decoction can improve IRS-1and GLUT4protein expression, showing its PI3K signal pathway intervention at protein level so as to prevent and manage MS.5. Chaihu Modified Longgu Muli Decoction is safe and the pathological changes in liver is considered resulted from MS.6. The management of MS with Chaihu Modified Longgu Muli Decoction is practical in theory and has been supported by experiments.更多还原