糖耐康对糖尿病大鼠肾脏保护作用及机制研究

【作者】 王志程；

【导师】 刘铜华；

【作者基本信息】 北京中医药大学 ， 中医内科学， 2009， 博士

【摘要】 糖尿病肾病是糖尿病常见的慢性微血管并发症之一,是导致慢性肾衰的重要原因。在一些国家和地区大约50%的DN病人最终发展成为终末期肾衰。因此,保护糖尿病病人的肾脏,延缓和阻止糖尿病肾脏损害的病程进展,提高DM患者的生存质量,成为国内外医学界关注的热点。本论文首先对近年来中西医对DN的认识和防治进行了较为全面的概述,然后在前期研究工作的基础上,进一步通过体内和体外实验,从整体、细胞和分子等不同水平探讨了中药糖耐康对糖尿病肾脏保护的相关作用机制。1文献综述参照古今中医有关文献,对糖尿病肾病的中医病名、病因病机、辨证论治、临床及实验研究进行了概述,同时对现代医学在糖尿病肾病研究中所取得的进展进行了总结,明确了糖尿病肾病的发病机制、诊断和治疗。本论文同时总结了导师刘铜华教授辨治糖尿病肾病的方法和学术观点,强调治疗早期糖尿病肾病从气阴两虚入手,瘀血痰浊贯穿始终的学术思想。2实验研究2.1体内实验目的:观察糖耐康对自发性2型糖尿病模型GK大鼠肾脏的保护作用并探讨其作用机理。方法:将SPF级GK大鼠50只按随机血糖值,随机分为5组:模型组、糖耐康高剂量组、糖耐康中剂量组、糖耐康低剂量组、吡格列酮组,每组10只,并将普通wistar大鼠10只作为正常对照组。各组大鼠连续灌胃给药60天。每周测量大鼠血糖、摄食量及饮水量,并于60天后称取各组大鼠体重、肾重,计算肾重/体重,测定血糖、24h尿白蛋白（ALB）、β2微球蛋白（β2-MG）,同时对各组大鼠进行HE、PAS染色以及透射电镜（TEM）观察肾脏病理学变化。结果:糖耐康组动物一般状况包括饮水量、摄食量等较模型组有一定改善,但无明显差异。第8周末糖耐康组的血糖值、肾重/体重较模型组显著降低（P<0.05）;模型组24hALB和β2-MG较正常组明显升高,经过糖耐康各浓度组和吡格列酮组治疗后,效果较模型组明显（P<0.05）;PAS染色和TEM观察,模型组肾脏组织病变明显,各治疗组均较模型组有所改善。2.2体外实验目的:观察高糖对大鼠肾小球系膜细胞（MCs）增殖和周期的影响和糖耐康含药血清对其的干预作用;观察糖耐康含药血清对MCs的外基质分泌及细胞内ERK信号途径相关位点的影响并探讨其作用机制。方法:用30mmol/L高糖诱导大鼠肾脏MCs,同时制备糖耐康和吡格列酮含药血清及空白鼠血清,对MCs进行分组,包括空白组、模型组（高糖组）、吡格列酮含药血清组、糖耐康高（0.80g/ml）、中（0.47g/ml）、低含药血清组（0.28g/ml）共6组。作用24h后,MTT法检测细胞增殖,流式细胞仪检测细胞周期变化,ELISA法检测细胞上清纤维连接蛋白（FN）、转化生长因子β1（TGF-β1）、Ⅳ型胶原蛋白（IV-C）含量;用RT-PCR法测定细胞上清中纤溶酶原激活物抑制物-1（PAI-1）和胞浆磷脂酶（cPLA）、ras mRNA的表达;免疫印迹杂交法（Western blot）测定细胞外信号调节激酶的磷酸化（p-ERK）表达及蛋白激酶C（PKC）-α、核内转录因子c-fos的蛋白表达含量。结果:高糖组在48h内刺激系膜细胞增殖,在48h后却抑制系膜细胞增殖。糖耐康及吡格列酮含药血清对MCs的增殖有较好的抑制作用。高糖组G0/G1期细胞显著性减少,而S期及G2/M期细胞显著性增加,吡格列酮组及糖耐康组G0/G1期细胞显著性增加,S期细胞显著性减少,而G2/M差距不明显。与高糖模型组比较,各给药组中MCs上清中的FN、TGF-β1、Ⅳ-C都有非常明显改善（P<0.01）;糖耐康治疗组p-ERK、PKC-α、c-fos的蛋白表达及PAI-1、cPLA₂、ras mRNA都较高糖模型组有明显改善（P<0.01或P<0.05）,与西药吡格列酮含药血清组作用效果相似。2.3结论本论文通过实验说明,中药糖耐康能够改善2型糖尿病模型GK大鼠的血糖,改善尿蛋白的排泄,具有改善肾脏病理,保护肾脏功能,从而起到防治DM肾脏病变、延缓肾小球硬化进程的作用。细胞实验进一步显示糖耐康对DN的保护作用机制之一可能是:①通过有效的阻止ERK信号转导通路的激活,减缓了肾脏系膜细胞的肥大、增厚;②调节异常的细胞因子、生长因子的分泌等作用,改善或延缓DN肾小球的硬化。更多还原

【Abstract】 Diabetic nephropathy(DN) is an important microvascular complication of diabetic mellitus(DM) as well as the common reason resulting in chronic renal failure(CRF),and half of which is caused by DN in some countries or areas. So protecting renal function,reducing the rate of renal damage and improving quality of life becomes to the focus of international researches’ attention. On the former study,systematic research on prevention and treatment of DN by Tangnai-Kang concoction has been performed from the whole,organ,cell and molecule levels in this essay which was composed of the reference review and experimental study.1 Reference reviewThe reference review discussed the recent research progresses in prevention and treatment of DN both from the aspects of Chinese medicine and modern medicine. In the theoretical research,we have referred to the related ancient and modern TCM literature in order to explore the source of disease name of TCM for DN, etiology and pathogenesis of TCM,selection of therapeutic methods by different stages or according to the differential syndrome diagnosis,advance on the clinical and experimental research as well as the problems lying in the research of TCM and corresponding strategies.In the meanwhile,we further exploited the pathologic characteristics,diagnosis and treatment of DN.We also have summarized the tutor’s academic point of view on the differentiation and treatment of DN,and emphasized that deficiency of qi and yin is the basic pathogenesis of DN,blood stasis and turbid phlegm run through the progress of DN.2 Experimental study2.1 experiments in vivoObjective:To study the affection of Tangnai-Kang concoction for protecting for diabetic kidney in spontaneous diabetic animal model GK mouse,and explaining the mechanism of it.Mwthods:Specific pathogen free(SPF) GK mice 50 were divided randomly according to plasma glucose level into 5 groups:Models、TNK-low dosage、TNK-middle dosage、TNK-high dosage、Pioglitazone and normal wistar mice(normal control group). Each group was tested randomly plasma glucose level every week.60 days later, all animals were tested body weight、renal weight、blood glucose、urinary albumin (ALB)、β2 microglobulin(β2-MG) and dying kidney by HE and PAS.At the end, observe the ultrastructure of the kidney through TEM.Results:Compared with Model groups,blood glucose、ALB、β2-MG were all decreased significantly(p＜0.05);the recipe could protected diabetic kidney in some degree and postponed kidney failure,and improve pathological change in diabetic kidney through kidney dying by HE、PAS and TEM.2.2 experiments in vitroObjective:To observe the proliferation of mesangial cells(MCs) in high glucose, and the affection of rat serum containing Tangnai-Kang to it firstly;To study the intervention of the serum in extracellular matrix and extracellular regulated kinases(ERKs) of MCs.Methods:Mesangial cells were cultured,then using 30%high glucose induced it. Divided cells into six groups,such as Models、TNK-low dosage(0.28g/ml) TNK-middle dosage(0.47g/ml)、TNK-high dosage(0.80g/ml)、Rosiglitazone dosage and normal control group(MCs).Treated the cells 24h,test FN、IV-C、TGF-βin the extracellular matrix,and extracted the total RNA and protein.At the end,tested the expression of cPLA and PAI-l mRNA,and test p-ERK、PKC-α、c-fos protein by reverse transcriptase PCR(RT-PCR) and Western blot.Results:Cultured rat MCs exposed to high glucose environment,MCs proliferation was assessed by MTT colorimetric method.Our studies demonstrated that raising ambient glucose concentration results in transient stimulation of growth in 48 hours,and in inhibition after 48.Biapbasic growth response of MCs to high glucose concentration could be reversed by rat serum containing Tangnai-Kang. Compared with Model groups,FN、IV-C、TGF-βwere downregulated in some digree in the supernatant secreted by MCs following the concentration of Tangnai-Kang Compound,and the expression of everyone were downregulated,such as cPLA and PAI-l mRNA、p-ERK、PKC-αand c-fos protein level.The effecting of Rosiglitazone dosage group were similar with TNK dosage.Conclusion:Tang-nai Kang Concoction have the affection of improving blood glucose and decreasing urine protein excretion,and improving renal function, lessen nephridial tissue damage,prevented the hardening of glomerular,which may realized through these links as follows:①Block ERK signal-transduction effectively,controlled the goal proteins or genes expression level,increasing the activity of the goal points in ERK signal-transduction pathway for which decreasing the FPG urine protein and so on.②Through decreasing the secretion of MCs stimulated by high glucose,such as FN、IV-C、TGF-β,relief the interference factors on ERK signal-transduction pathway.On the other hand,the mechanism of the serum containing Tangnai-Kang Compound improving diabetic nephropathy might be down-regulated the expression of key points.更多还原