糖肾清2号调控AMPK信号转导通路抑制DN肾组织免疫炎性病理损伤的分子机制研究

Diabetic nephrosis (DN) is related to various factors. In recent years it is believed that inflammatory mechanism is the key factor inducing abnormality of glucose and lipid metabolism thus mediating renal damage. The core inflammatory mediators, with glucose and lipid metabolic and hemodynamic abnormality, reciprocally affect each other through different signal pathways, making the renal damage deteriorate. Our previous experiments proved that the Chinese herbal medicine Tangshenqing No. 2 (TSQ2) significantly up-regulated the AMPK's (AMP-activated protein kinase) gene and protein expression; and supported by the result of our latest study subsidized by the NSFC (National Natural Science Fund Committee), we conclude that the molecular mechanism of how TSQ2 curbs inflammatory damages may be that TSQ2 mediates and activates AMPK　and its downstream anti-inflammation signaling system. Based on such work, in this project we plan to focus on the transduction mechanism of inflammatory-immuno-signals, and by experiment in vivo (using db/db mice) and in vitro, to study the effect and molecular mechanism of TSQ2 on the AMPK's anti-inflammatory-signaling system, and on the key inflammatory mediators in the development of DN. Probing in the molecular mechanism of how TSQ2 alleviates renal inflammatory-immuno damages can rich the treatment of DN, and lay a foundation for the further study of AMPK's bio-mechanism in the area of inflammatory diseases.

糖尿病肾病（DN）发病机制与多因素相关，近年认为炎症机制是糖脂代谢异常介导肾损害的中心环节。参与DN的核心炎性介质及信号通路因果互动，与紊乱的糖脂代谢及血流动力学环境相伴，不断放大加深损伤效应。前期实验证明：糖肾清（TSQ）2号显著上调DN大鼠肾组织激活腺苷酸活化蛋白激酶（AMPK）基因及蛋白表达。结合已结国家自然基金课题研究结果，可以推论：TSQ2号抑制免疫炎性损伤的主要生物学机制可能由诱导AMPK及下游抗炎信号系统完成。故本项目在原工作基础上，侧重从感染免疫信号转导机制入手，在基因蛋白质水平，采用db/db鼠及细胞体外实验的方式，研究TSQ2号对AMPK抗炎信号系统在DN中表达的影响及分子机制，及其对介导DN的关键炎性介质的干预途径及作用环节。探讨TSQ2号抑制DN免疫炎性损伤的分子机制。为丰富DN的治疗提供依据。为研究AMPK在炎性疾病领域中的生物学机制奠定基础。

DN发病机制与多因素相关，近年认为炎症机制是糖脂代谢异常介导肾损害的中心环节。前期实验证明：TSQ2号上调DN 大鼠肾组织AMPK基因及蛋白表达。由此推论：TSQ2 号抑制免疫炎性损伤的主要生物学机制可能由诱导AMPK 及下游抗炎信号系统完成。故本项目采用体内外实验方式，研究TSQ2 号对AMPK 抗炎信号系统在DN 中表达的影响及分子机制，及其对介导DN 的关键炎性介质的干预途径及作用环节。结果示：（1）、体内实验：①肾功能检测：TSQ2可改善24h尿蛋白、空腹血糖、TG、Scr、BUN、ACR。②形态学检测：光镜示：DN小鼠肾小球增大，系膜基质增生，基底膜增厚；近曲小管上皮细胞空泡变性，肾小管扩张，基底膜增厚；间质炎性细胞浸润，较正常组有显著性差异（P＜0.05）。各用药组肾脏病变均较模型组减轻（P＜0.05）。电镜示：模型组基底膜增厚，足突倒伏、融合，肾小球部分纤维化。各用药组损伤较模型组轻。 ③RT-PCR、Western-blot、ELISA、免疫组化检测：模型组AMPKα1、ADPN、AdipoR1、AdipoR2下调，TLR4、p65、MCP-1、IL-6、IL-1、IL-18、TNF-α、iNOS、RANTES等促炎因子上调；TSQ2组AMPKα1、PPARγ、ADPN上调，各促炎因子下调（P＜0.01，P＜0.05）。（2）体外实验:①巨噬细胞实验：LPS诱导后AMPKα1、PPARγ下调，IL-18、IP-10、TNF-a、MCP-1、IL-1、IL-6、iNOS、VCAM-1、RANTES等促炎因子上调；应用TSQ2后AMPKα1、PPARγ上调，各促炎因子下调（P＜0.05，P＜0.01）。②肾小球系膜细胞实验：高糖环境下AMPKα1、p- AMPKα1、PPARγ等抗炎因子下调，TLR4上调；应用TSQ2后，抗炎因子上调，TLR4下调（P＜0.05，P＜0.01）。研究表明：糖肾清2号可激活AMPK信号转导系统，促进下游抑炎因子的表达，有效抑制促炎因子的表达，从而改善DN肾脏免疫炎性损伤，保护肾脏。

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