糖耐康干预糖尿病肾病肾小管上皮细胞转分化研究

【作者】 吴丽丽；

【导师】 刘铜华；

【作者基本信息】 北京中医药大学 ， 中医内科学， 2012， 博士

【摘要】 糖尿病肾病(diabetic nephropathy, DN)是糖尿病最常见的慢性微血管并发症之一,是糖尿病患者的主要死亡原因之一。近年来,DN已成为一个严重威胁人们生命健康的疾病,寻找预防和治疗DN的有效药物和治疗方法已成为世界范围的研究热点。肾小管上皮细胞转分化(tubular epithelial to mesenchymal transdifferentiation, TEMT)是肾间质纤维化发生和发展的重要机制之一。TEMT过程中TGF-β1/Smad信号转导途径起了重要作用。中医药在DN治疗方面积累了丰富的经验,特别是在治疗早期DN方面取得了较好的临床疗效,但是对中医药治疗DN的作用机制研究较少,有待于进一步深入研究。本论文首先对近年来中西医对DN的认识和防治进展进行了综述,拟在前期研究工作基础上,进一步从整体、组织、蛋白和基因分子水平探讨了中药复方糖耐康通过调节TGF-β1/Smad信号通路来抑制TEMT,以此来治疗DN的作用机制。1文献综述参照古今中医有关文献,对DN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DN肾小管上皮转分化研究所取得的进展进行了综述。2实验研究目的：紧紧围绕肾小管上皮细胞转分化机制中有待探索的热点问题TGF-β1/Samds信号转导途径与DN肾间质纤维化的关系,探讨临床有效中药“糖耐康”防治糖尿病肾间质纤维化的作用及机制,为其治疗DN提供实验依据。方法：50只2次随机血糖均大于13.9mmol/L的雄性KKAy小鼠,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、糖耐康低剂量组及缬沙坦组,每组10只；健康雄性C57BL／6J小鼠10只为正常组。糖耐康高、中、低剂量组分别以4.56g/kg.d,2.28g/kg.d,1.14g/kg.d浓度按0.01ml/g体重灌服糖耐康水溶液,缬沙坦组按0.46mg/kg.d灌服缬沙坦水溶液,正常组和模型组灌服等体积0.9%氯化钠溶液。每日1次,连续给药8周。记录小鼠一般情况、体重、血糖,实验周期结束时,测定小鼠血清血糖(glucose, Glu)、胰岛素(Insulin, INS)、甘油三酯(glyceride, TG)、总胆固醇(total cholesterol, TC)、尿素氮(blood urea nitrogen, BUN)和肌酐(serum creatinine, Scr)水平,做肾组织HE染色、Massom染色和PAS染色并进行图像分析；免疫组化法检测肾组织α-平滑肌肌动蛋白(α-smooth muscle actin,α-SMA)、E-钙粘蛋白(E-Cadherin)表达；Real-time PCR检测肾TGF-β1mRNA、TGF-βR1mRNA、TGF-βR2mRNA、Collagen I mRNA、Collagen III mRNA和FNmRNA表达；wetern blot检测肾TGF-β1、Smad2, P-smad2, Smad3, P-smad3, Smad7蛋白表达。实验数据用SPSS16.0进行统计学处理。结果：实验一：与同周龄正常组小鼠比较,模型组小鼠体质量显著增加(P<0.01),各给药组小鼠体质量较同期模型组有显著下降(P<0.05,P<0.01)；模型组与正常组比较,快速空腹血糖(fasting blood sugar,FBG)、TG、TC、INS显著升高(P<0.01),糖脂代谢紊乱；24h尿蛋白定量、肾质量升高(P<0.05),但是肾功能无差异(P>0.05)。20周龄模型组小鼠肾小球较正常组增大,细胞外膜基质增加,肾小管管腔扩大,蛋白管型明显,半定量分析肾组织病理损伤评分增加。实验二：病理学检测结果显示,与正常组比较,模型组小鼠肾间质纤维化评分和纤维化面积显著增大(P<0.01)；与模型组比较,各治疗组小鼠肾间质纤维化评分和纤维化面积显著减小(P<0.05)；免疫组化结果显示,模型组小鼠肾组织中a-SMA蛋白表达呈现较强阳性。与模型组比较,缬沙坦组、糖耐康低、中、高剂量组a-SMA蛋白表达显著减少(P<0.01)；模型组小鼠肾组织中E-cadherin蛋白表达弱阳性。与模型组比较,缬沙坦组、糖耐康低、中、高剂量组E-cadherin蛋白表达明显增加(P<0.01)；wstern blot结果显示,与正常组小鼠比较,KKAy小鼠肾脏组织TGF-β1蛋白表达明显升高(P<0.01)。与模型组比较,缬沙坦组、糖耐康低、中和高剂量组TGF-β1蛋白表达含量都明显降低(P<0.01),其中糖耐康高剂量组与缬沙坦组相比较,TGF-β1显著降低(P<0.01)。实验三：Real-time PCR结果显示,与正常组比较,KKay小鼠TGF-β1 mRNA. Collagen I mRNA、Collagen III mRNA和FNmRNA相对表达量显著升高(P<0.05), TGFβ-R1 mRNA和TGFβ-R2 mRNA无显著变化(P>0.05)；与模型组比较,缬沙坦组和糖耐康高剂量组TGF-β1 mRNA、Collagen III mRNA和FN mRNA的降低具有统计学意义(P<0.05)；western blot结果显示,与正常组小鼠比较,KKAy小鼠肾smad2蛋白、p-smad-2蛋白、smad-3蛋白、p-smad-3蛋白表达均显著升高(P<0.01),smad-7蛋白表达显著降低(P<0.01),与模型组比较,缬沙坦组、糖耐康各治疗组上述蛋白表达有不同的改变(P<0.05)。结论：1、中药糖耐康对自发性KKAy小鼠有显著的降糖和降脂作用,能减少尿蛋白量,降低DN肾脏病理损伤评分,抑制糖原等细胞外基质的沉积,改善早期肾脏病变,与缬沙坦疗效相当,且在降低体质量、肾重、FBG、血清INS和TC等方面优于缬沙坦,对DN有一定的保护作用；2、TEMT过程存在于KKAy小鼠肾组织,参与了DN肾纤维化,而缬沙坦和糖耐康具有一定的抑制TEMT的作用；3、糖耐康可干预TGF-β1/smads信号转导途径的多个位点,主要机制可能是增加smad7表达从而抑制TEMT过程,这可能是其治疗DN的重要靶点。更多还原

【Abstract】 Diabetic nephropathy is one of the serious complications of diabetes which is the chief of cause death. Nowadays, diabetic nephropathy torment people and also threaten people’s health and life, so it is urgent for researchers to find effective treatment methods or medicine for patients with diabetic nephropathy worldwild. Renal tubular epithelial-mesenchymal transition brings an important impact in tubulointerstitial fibrosis, and TGF-β1/Smad signaling pathway palys the main role in this procession. Traditional Chinese medicine has accumulated rich experiences in treats diabetic nephropathy, especially for early stage, it has better curative effect. But there is less researches of the mechanism of action of herbs. This paper gives an overview of understanding and treatment for diabetic nephropathy based on Chinese medicine and Western medicine at the beginning of paper, then to analyze the mechanism of Tang Nai Kang treats diabetic nephropathy through TGF-β1/Smad signaling pathway.1 Reference reviewThe reference review discussed the recent research progresses in prevention and treatment of diabetic nephropathy both from the aspects of Chinese medicine and modern medicine. Reference to the literature of traditional Chinese medicine, Chinese medicine on the diabetic nephropathy name, pathogenesis, diagnosis and treatment, clinical and experimental research are reviewd, while made in a summary of modern medicine on the diabetic nephropathy progresses, emphatically summarized pathogenesis, diagnosis, and treatment.2 Experimental studyObjective:Using spontaneously KKAy mice as the diabetic nephropathy model, in order to discuss the effect mechanism of Tang Nai Kang on diabetic nephropathy, which provides the foundation for for treating diabetic nephropathy.Methods:Using spontaneously KKAy mice as diabetic nephropathy model, divided the mice into normal group, model group, control group, small dose of Tang Nai Kang, Tang Nai Kang middle dose group, Tang Nai Kang high dose groups. We tested mice blood glucose, the level of insulin, TC, TG, BUN, and Scr, kidney tissues HE staining and electron microscope observation. Immunohistochemical detection ofα-SMA、E-cadherin protein expression; Using real-time fluorescent quantitatie polymerase chain reaction (PCR) method to test kidney TGF-β1 mRNA, TGF-βR1 mRNA、TGF-βR2 mRNA、Collagen I mRNA、CollagenⅢmRNA and Fn mRNA gene, western blot tested TGF-β1, Smad2, P-smad2, Smad3, P-smad3, Smad7 protin expression. All data were analyzed by SPSS 16.0 statistical package.Results:Experiment 1:Compared with the normal group, the weight of model group’s mice has been significantly increased with the same ages (P<0.01). Groups with Tang Nai Kang weight gets down. Tang Nai Kang could decrease the level of fasting blood sugar and serum TC, TG, INS,24-hours proteinuria, compared to that of the model group remarkably, but there is no difference in renal function test between the groups. HE staining showed that enlargement of glomerulus and tubular, increased extra-cellular matrix, renal tubule was filled with the protein and cells in control group at 20 weeks, the treatment group pathological changes of mice kidney morfology were improved compared with model group. Experiment 2:Compared with the normal group, the model group showed a significant renal fibrosis (P <0.01), each Tang, Nai Kang group have a better condition than model group. Wstern blot showed that there is a high level of TGF-β1 in model group.In addition,compared with control group Tang Nai Kang high does group has a better treatment effect. Experiment 3:Real-time PCR showed that model group’s TGF-β1 mRNA, Collagen I mRNA, CollagenⅢmRNA, FN mRNA were high than normal group. For TGFβ-R1 mRNA and TGFβ-R2 mRNA express, there is no difference between any groups.Western blot showed that smad2, p-smad-2, smad-3, p-smad-3 have a high level in model group (P< 0.01).Conclusion:1, Tang Nai Kang can decrease diabetic nephropathy mice weigh, blood glucose, and urine protein,, which means that it can prevente the occurrence of early diabetic nephropathy and development, and protect the renal function; 2, Tangnai-Kang can reduce renal fibosis and better than valsartan.3 Tangnai-Kang can improve the renal function,its proper mechanism that may inhibit and block TGF-β1/smads signaling pathway更多还原