基于TGF-β信号通路积雪草防治早期糖尿病肾病的分子机制研究

Diabetic kidney disease is one of the most serious microvascular complications of diabetes mellitus,and TGF-β singnal pathway plays an important role in the course of occurance and development. Centella asiatica is commonly used clinical chinese herb, which can eliminate heat, lead to diuresis, and reduce swelling and detoxify. In modern clinical practice it is often widely used in the treatment and prevention of scar proliferations caused by burns, trauma, surgery etc. Clinical practice has proved that centella asiatica is effective in controlling early early diabetic kidney disease's occurring and developing, but its role mechanism and pathway has not been clear. The group's early studies about the herb have proved that centella asiatica could effectively suppress renal tubulointerstitial fibrosis by blocking TGF-beta signal pathway, based on this result, we think that centella asiatica may inhabit kidney fibrosis in early diabetic kidney disease by blocking TGF-beta signal pathway, and therefore it could be used for prevention and treatment on early diabetic kidney disease. The light microscope,the electron microscope, the immunohistochemistry,RT-PCR and western-blot, the gene transfection technologies and so on will be used to conduct the research around TGF-beta signal pathway in this topics study, and on the foundation of studies in the body the exsomatize cell raise research will also be conducted, to seek the herb's role way and target points of preventing and curing early diabetic kidney disease, explore its molecular mechanism and further provide powerful evidence for centella asiatica's wide application in the treatment on the early diabetic kidney disease.

糖尿病肾病是糖尿病最严重的微血管并发症，TGF-β信号通路在早期糖尿病肾病的发生发展过程中起着重要作用。积雪草是临床常用中草药，具有清热利湿，解毒消肿的功效，现代临床常用于治疗及预防烧伤、外伤、手术等所致瘢痕增生。临床实践证明积雪草能够有效地防治早期糖尿病肾病的发生发展，但其作用机制和途径并不清楚.本课题组前期研究证明积雪草通过阻断TGF-β信号通路能够有效地抑制肾小管间质纤维化，基于此我们认为积雪草能够通过阻断TGF-β信号通路，抑制早期糖尿病肾病肾脏纤维化，进而防治糖尿病肾病。本课题围绕TGF-β信号通路，利用光镜、电镜、免疫组化、RT-PCR及western-blot、基因转染等技术进行研究，并在在体研究的基础上进行离体细胞培养研究，寻找积雪草防治早期糖尿病肾病的作用途径和作用靶点，阐明积雪草防治早期糖尿病肾病的分子机制，为积雪草应用于糖尿病肾病的治疗提供有力的佐证。

项目背景：糖尿病肾病（DN）是糖尿病最常见和严重的微血管并发症之一，也是导致糖尿病患者死亡的主要原因。研究证实TGF-β1信号通路在早期糖尿病肾病的发生发展中起着重要作用。临床实践证明积雪草能够有效地防治早期糖尿病肾病的发生发展，但其作用机制和途径并不清楚。.主要研究内容：本课题围绕TGF-β1信号通路，利用生化分析、光镜、电镜、RT-PCR、Western blot、Elisa等技术进行研究，并在在体研究的基础上进行离体细胞培养、基因转染，寻找积雪草防治早期糖尿病肾病的作用途径和作用靶点，阐明积雪草防治早期糖尿病肾病的分子机制。.重要结果及关键数据：动物实验研究证实动物福辛普利和高剂量CA能倒转DN引起的TGF-β1、TGFβRⅠ、TGFβRⅡ、smad2/3 mRNA和蛋白表达及smad2/3磷酸化水平显著降低、smad7 mRNA和蛋白表达增加。离体实验研究证实福辛普利和高剂量积雪草含药血清均可使Smad7的蛋白表达水平显著回升，几乎达到对照组水平；中、低剂量的含药血清几乎没有作用。 .转染细胞的离体实验部分：一、CA可显著降低MC细胞中TGF-β1、Smad2/3的mRNA和蛋白表达水平及smad2/3的磷酸化水平；TGF-β1稳定表达的MC细胞中的smad7 mRNA和蛋白水平显著降低，而积雪草能使smad7的mRNA和蛋白水平显著升高。二、转染了smad2的MC细胞培养液中的胶原蛋白水平显著升高，而积雪草含药血清能显著降低转染smad2的MC细胞培养液中的胶原蛋白Ⅳ水平；稳定表达smad2的MC细胞中smad2、Timp2的mRNA和蛋白水平显著升高，而积雪草含药血清能显著降低smad2、Timp2的mRNA和蛋白表达；转染smad2可使MMP2的 mRNA和蛋白水平显著降低，而积雪草含药血清能拮抗smad2的作用。.科学意义：本课题从整体、细胞、分子水平探讨了积雪草防治早期糖尿病肾病的作用途径和作用靶点，阐明积雪草防治早期糖尿病肾病的分子机制，为积雪草应用于糖尿病肾病的治疗提供理论依据，并有助于积雪草的进一步开发利用。