【作者】 孙璐；

【导师】 范冠杰；

【作者基本信息】 广州中医药大学 ， 中医内科学， 2012， 博士

【摘要】 目前我国20岁以上的成年人糖尿病患病率为9.7%,我国成人糖尿病总数达9240万,约90%为2型糖尿病(type2diabetes mellitus, T2DM),糖尿病已经成为影响我国人民健康的主要公共卫生问题。中医学早在《灵枢·五变篇》即有“肾虚则善病消瘅”的记载,认为本病的发生于肾虚有密切关系,或曰肾虚是消渴发病的关键因素。在西医学的认识中近年来糖尿病的炎症学说得到越来越多业内专家学者的认可,本学说认为T2DM是一种慢性、亚临床性的炎症疾病,许多促炎症因子、双链RNA和病毒等通过作用于IKKβ而引起胰岛素抵抗(insulin resistance,IR)和胰岛β细胞凋亡,导致T2DM。IKKβ已成为防治T2DM研究的新的药理学作用靶点,抑制IKKβ的活性可以阻止T2DM及其并发症的发生发展。前期的研究表明,中药复方降糖补肾方有补肾健脾,养阴清热之功效,临床治疗T2DM疗效较好,并证实该方能抑制炎症反应。本研究旨在了解2型糖尿病的临床特点及代谢特点,总结2型糖尿病的证候规律,对中医证候与炎症指标及其他实验室指标进行相关性分析,了解中医证候的物质基础。并在前期临床研究的基础上,深入探讨降糖补肾方治疗T2DM的作用机理,从炎症细胞因子作用的源头IKKβ/NF-κB通路入手,观察降糖补肾方对此通路的关键环节IKKβ表达及活性的干预作用。这一研究可能明确降糖补肾方具体作用环节,研究结果将为针对T2DM炎症生物效应靶点可能开发出预防和治疗T2DM的中药新药,从而产生客观的经济和社会效益。目的：1.通过2型糖尿病的相关文献研究,了解目前中西医对2型糖尿病炎症学说的认识,系统评价国内生物医学类期刊发表的补肾活血类中药复方治疗2型糖尿病的临床疗效及安全性。2.通过临床观察了解2型糖尿病的临床特点及代谢特点,总结2型糖尿病的证候规律,了解2型糖尿病中医证候的物质基础。3.通过实验研究了解中药复方降糖补肾方的疗效及具体作用环节。方法：1.文献研究对近年来中西医对2型糖尿病炎症学说的认识进行文献综述,通过计算机检索建库-2011年5月国内生物医学类期刊发表的有关中医药补肾活血法治疗2型糖尿病的临床研究文献并对其临床疗效及安全性进行系统评价。2.临床研究广东省中医院内分泌科2011年3月至2011年6月门诊和内分泌科病房的2型糖尿病患者60例,搜集患者的一般人口学资料、西医相关指标、炎症因子指标、并发症及伴发疾病情况及中医证候积分,采用EPIdata3.1软件包建立数据库,spss17.0进行统计分析。3.实验研究采用高脂饲料喂养+小剂量链脲佐菌素腹腔注射的方法制备糖尿病大鼠模型,成模大鼠随机分组,并对各组大鼠予相应干预,检测各组大鼠的血糖水平、血清炎症因子水平、糖尿病大鼠肌肉组织的IKK β mRNA表达及糖尿病大鼠肌肉组织的InsR磷酸化蛋白水平、IRS-1酪氨酸磷酸化蛋白水平,采用spssl7.0软件包进行统计分析。结果：1.临床研究结果(1)60例2型糖尿病患者的平均年龄为61.20±12.95岁,糖尿病病史为8.81±5.97年,体重指数26.24±2.16kg/m2,血常规、肝功能、肾功能、尿酸在正常范围,TNF-α、IL-6、CRP均有不同程度的升高。(2)本研究60例2型糖尿病患者其并发症及伴发疾病前3位为高血压病(40%),血脂异常、糖尿病神经病变、糖尿病眼病(均占16.7%)及糖尿病肾病(10%)。(3)本研究2型糖尿病患者证候发生率依次为：肾虚证、气阴两虚、血脉瘀阻证、肝气郁结、湿热内蕴、心神失养、肺胃燥热、血分郁热。(4)中医证候与实验室指标的相关性分析：X5(胆固醇)与Y8(血脉瘀阻证)二者有相关性,X1(BMI)与Y5(肺胃燥热证)二者有相关性,X6(TNF-α)与Yl(肾虚证)二者有相关性。2.实验研究结果(1)糖尿病大鼠血糖：降糖补肾方能降低糖尿病大鼠的空腹血糖,降糖补肾方高剂量组降糖效果与阿司匹林组相当(P>0.05)。(2)糖尿病大鼠血清炎症因子：降糖补肾方高、中、低剂量治疗组均能明显降低血清TNF-α水平,并与阿司匹林组相比差异有统计学意义(P<0.05)；降糖补肾方高、中剂量治疗组能明显降低血清IL-6水平,并与阿司匹林组效果相当；降糖补肾方高、中、低剂量治疗组均能明显降低血清CRP平,并与阿司匹林组相比差异有统计学意义(P<0.01)。(3)糖尿病大鼠肌肉组织InsR磷酸化、IRS-1酪氨酸磷酸化蛋白印迹检测结果：模型组糖尿病大鼠肌肉组织InsR的磷酸化水平下降,与空白对照组比较差异有统计学意义(P<0.01),降糖补肾低剂量组与阿司匹林组相比P>0.05,说明在改善糖尿病大鼠肌肉组织InsR磷酸化水平方面这两组效果相当。模型组糖尿病大鼠肌肉组织IRS-1的酪氨酸磷酸化下降,与空白对照组比较差异有统计学意义(P<0.01),阿司匹林组、降糖补肾高剂量组与空白对照组相比(P>0.05),说明阿司匹林组、降糖补肾高剂量组能显著改善IRS-1酪氨酸磷酸化水平,两组效果相当(P>0.05)。(4)糖尿病大鼠大腿肌肉组织IKK β mRNA表达水平：模型组IKK β mRNA表达明显高于空白对照组,是空白对照组的5.0983倍,阿司匹林组、降糖补肾方高剂量组能明显降低IKK β mRNA表达,分别是空白对照组的2.8144、3.2464倍；降糖补肾方中、低剂量组能降低IKK β mRNA表达,分别是空白对照组的3.7705、4.974倍。结论：1.临床研究2型糖尿病患者以中老年多见,大多体形肥胖、血脂紊乱。2型糖尿病并发症及伴发疾病多见高血压病、血脂异常、糖尿病神经病变、糖尿病眼病及糖尿病肾病。2型糖尿病患者血清TNF-α、IL-6、CRP均有不同程度的升高。2型糖尿病为本虚标实之证,本虚证以肾虚及气阴两虚为主要表现,标实证以血瘀、气郁、湿热为主。在2型糖尿病实验室指标与中医证候研究中发现,胆固醇与血脉瘀阻证二者有相关性,BMI与肺胃燥热证二有相关性；TNF-α与肾虚证二者有相关性。2.实验研究降糖补肾方可显著降低糖尿病模型大鼠的空腹血糖及血清TNF-α、IL-6、CRP水平,可以抑制糖尿病大鼠肌肉组织IKK β mRNA表达,增加糖尿病大鼠肌肉组织InsR磷酸化、IRS-1酪氨酸磷酸化白蛋表达,其机制可能与降糖补肾方抑制了炎症信号通路的转导,从而发挥其改善胰岛素抵抗及治疗糖尿病的作用有关。更多还原

【Abstract】 BackgroundPrevalence of adult diabetes in our country over the age of20was9.7%, the total number of adults with diabetes in China reached92.4million, approximately90%for type2diabetes mellitus (T2DM).Diabetes has become a major public health problem. Chinese medicine as early as the LingSHU(?)WuBianPian Kidney good disease elimination hate the record,that this disease occurred in the kidney is closely related to, or said that the kidney is a key factor in diabetes incidence.Inflammation hypothesis of diabetes are recognized by more and more industry experts and scholars in recent years in the understanding of Western medicine. T2DM is a chronic, subclinical inflammatory disease,many pro-inflammatory factor, double-stranded RNA and viruses by acting on the IKKβ caused by insulin resistance(IR) and islet β-cell apoptosis,leading to T2DM. IKKβ has become a new pharmacological target of prevention and treatment of T2DM study, inhibition of IKKβ activity can prevent the development of T2DM and its complications.Previous studies showed that traditional Chinese medicine compound the Jiangtang Bushen Recipe has the effect of clearing away heat and efficacy of clinical treatment of T2DM, and confirmed the order to inhibit the inflammatory response. The purpose of this study was to understand the clinical features of type2diabetes and metabolic characteristics,summary of type2diabetes Syndromes TCM syndromes and analysis the correlation of inflammatory markers and other laboratory parameters, to understand the material basis of traditional Chinese medicine syndrome. And on the basis of pre-clinical studies,in-depth to explore the mechanism of the Jiangtang Bushen Recipe treatment of T2DM,start from the source of the role of inflammatory cytokines IKKβ/NF-κ B pathway to observe the the hypoglycemic kidney side pathway links IKKβ expression and activity in rats. The research may clear hypoglycemic specific role of Jiangtang Bushen Recipe, the results will be possible to develop for T2DM inflammation of the biological effects of target prevention and treatment of T2DM in Chinese medicine new drugs,resulting in the objective economic and social benefits.Objectives1.Type2diabetes-related literature to understand the present Western understanding of the doctrine of inflammation in type2diabetes,Systematic reviews of clinical efficacy and safety of the domestic biomedical journals published Bushenhuoxue class traditional Chinese medicine treatment of type2diabetes.2. Clinical surveillance to the clinical features of type2diabetes and metabolic characteristics,and summarize the Syndromes of type2diabetes, understanding the material basis of traditional Chinese medicine with type2diabetes syndrome.3.Experimental study to understand the efficacy of traditional Chinese medicine hypoglycemic kidney side and the specific role of the link.Methods1.Literature researchWestern medicine in recent years,the understanding of type2diabetes inflammation hypothesis literature review, by computer search of the clinical research literature database～the domestic biomedical journals published in May2011for the treatment of type2diabetes Chinese medicine Bushenhuoxue law and its clinical efficacy and safety of the system evaluation.2. Clinical research60cases,the Guangdong Provincial Hospital of Endocrinology outpatient and endocrine ward patients with type2diabetes in March2011to June2011, to collect the patient’s general demographic data, and Western-related indicators,indicator of inflammatory factors,complications and concomitant diseases and TCM syndrome score, to EPIdata3.1package to establish a database for statistical analysis, spss17.0.3. Experimental researchPrepared diabetic rats with high fat diet+small dose of streptozotocin by intraperitoneal injection into a mold in rats randomly assigned to the appropriate intervention, and the rats in each group, detect blood glucose levels of rats in each group, serum levels of inflammatory factors, muscle tissue IKKβ mRNA expression and protein levels of InsR phosphorylation of IRS-1tyrosine phosphorylation protein levels, with spss17.0packages for statistical analysis.Results1.The result of clinical research(1)60patients with type2diabetes patients with an average age of61.20±12.95years of age,history of diabetes was8.81±5.97years,body mass index26.24±2.16kg/m2,blood, liver function, renal function, uric acid in the normal range, the TNF-α,IL-6, CRP increased in varying degrees.(2)60cases of type2diabetes patients with complications and diseases associated with the first three is hypertension(40%),dyslipidemia, diabetic neuropathy, diabetic eye disease (account for16.7%)and diabetic nephropathy(10%).(3)In this study, patients with type2diabetes syndrome incidence as follows:kidney deficiency syndrome,Qi and Yin deficiency blood stasis,Liver Qi Stagnation, damp, mind dystrophy, lung and stomach hot,Blood swelter.(4)Canonical analysis of TCM Syndrome Correlation with laboratory indexes:the X5(cholesterol)and Y8(blood stasis)related,X1(BMI) and Y5(hot lung and stomach)between correlation;X6(TNF-alpha)and Y1(kidney deficiency syndrome)between the correlation.2. The result of experimental research(1)The result of fasting blood glucose (FBG) showed that the Jangtang Bushen Recipe can reduce FBG of diabetic rats,the FBG of high-dose of Jiangtang Bushen Recipe group and aspirin group showed a no difference (P>0.05).(2)The Jangtang Bushen Recipe of high,medium and low-dose group can significantly reduce serum TNF-α levels, and significant difference(P<0.05) compared with the aspirin group;the Jangtang Bushen Recipe of high and medium dose group can significantly reduce the serum IL-6level,with the aspirin group and the effect is quite;the Jangtang Bushen Recipe of high, medium and low-dose group could significantly reduce serum CRP levels and a significant difference(P<0.01) compared with the aspirin group (3) The result of Western blotting method showed that protein activity of InsR phosphorylation in muscle tissue of diabetic rats comparison between the model group and control group showed a significant difference(P<0.01), the protein activity of InsR phosphorylation in muscle tissue of diabetic rats comparison between low-dose of Jiangtang Bushen Recipe group and aspirin group showed a no difference (P>0.05),and the2groups are on the same level.The protein activity of IRS-1tyrosine phosphorylation in muscle tissue of diabetic rats comparison between the model group and control group showed a significant difference (P<0.01),the protein activity of IRS-1tyrosine phosphorylation in muscle tissue of diabetic rats comparison with high-dose of Jiangtang Bushen Recipe group, aspirin group and control group showed a no difference (P>0.05),the high-dose of Jiangtang Bushen Recipe group and aspirin group are on the same level (P>0.05)(4) RT-PCR shows that the model group is5.098times higher than blank control group in improving expression of IKKβ mRNA (P<0.01), but the group of Aspirin is2.814times,high-dose group of Jiangtang Bushen recipe is3.246times,medium-dose group of Jiangtang Bushen recipe is3.770times higher than the blank control group in decreasing.compared to model group P<0.01; low-dose group of Jiangtang Bushen recipe is4.974times higher than the blank control group in decreasing.compared to model group P>0.05.Conclusion1.Clinical researchType2diabetes is more common in middle-aged, mostly body obesity, dyslipidemia. Type2diabetes complications and concomitant diseases prevalent hypertension, dyslipidemia, diabetic neuropathy, diabetic eye disease and diabetic nephropathy.Type2diabetic patients serum levels of TNF-alpha, IL-6, CRP, in varying degrees rise.Type2diabetes is deficiency of Ben and repletion of Biao, this deficiency in order to kidney and Qiyinliangxu, mainly standard empirical blood stasis, qi,damp, mainly. In the laboratory markers of type2diabetes and TCM syndrome research found that cholesterol and blood stasis,BMI and lung and stomach hot, TNF-α and kidney deficiency syndrome both correlation.2.Experimental researchJangtang Bushen Recipe significantly reduce fasting blood glucose and serum TNF-alpha, IL-6, CRP levels in diabetic rats,can inhibit muscle tissue of diabetic rats IKKβmRNA expression, increase in muscle tissue of diabetic rats InsR phosphorylation of IRS-1tyrosine phosphorylation in the expression of white eggs, its mechanism may be inhibition of the inflammatory signal transduction pathway with hypoglycemic kidney side, to play to the role of improving insulin resistance and treatment of diabetes.更多还原