牛葛合剂对早期糖尿病肾病氧化应激和炎症差异协同作用的机制研究

The incidence of diabetic nephropathy (DN) has been dramatically increasing during last several years and is ranked now as 2nd cause of ESRD in China. Regardless of the extensive research, the treatment option of DN is still very limited. Treatment of patients with early DN with Traditional Chinese Medicine (TCM) has been shown to halt or slow the progression of DN providing us an attractive alternative therapy for DN. Puerarin and Arctigenin are the “ruler drugs” in the TCM formula for treatment of DN which were selected based on the accumulated practice experience of well-known senior TCM practitioners and some clinical and basic science research performed by our team. The applicant has already shown that Puerarin or Arctigenin alone reduced proteinuria in diabetic mice and the combination therapy with these two compounds had an additive effect. In addition, our preliminary data suggest that Purerarin and Arctigenin have some anti-oxidative stress and anti-inflammatory effects, which are two major pathways mediating the progression of DN. However, the exact mechanism of these effects remains unclear. In the current proposal we will use both cultured mouse podocytes and an animal model of DN (eNOS-/--STZ) to study mitochondrial function, different pathways of ROS generation, and expression of pro-inflammatory genes in kidney cells. We will also investigate the role of SIRT1 in regulation of acetylation of PGC1a, a key co-factor for mitochondrial function, and NF-kB and STAT3, two major transcription factors that mediate inflammation in DN. The proposed studies will help us to understand the molecular mechanism by which Puerarin and Arctigenin improve early DN through a synergic effect against oxidative stress and inflammation. These studies will provide us a scientific basis for our future clinical studies and application of these two drugs in patients with early DN.

糖尿病肾病的发病率近年来迅速上升，在终末期肾病中已跃居第二。虽然开展了大量研究，但治疗手段仍十分有限。中医药在早期能有效阻断或延缓本病进展，运用前景广阔。牛葛合剂（葛根素、牛蒡苷元）由名老中医治疗早期糖尿病肾病（EDN）经验方中君药的主要有效成分组成，经课题组多年临床和基础研究筛选形成。申请人已证实其能降低小鼠蛋白尿，比单药作用明显，且与EDN的重要发病机制氧化应激和炎症相关，但深入机制仍不明确。此次我们采用小鼠永生型足细胞和eNOS-/--STZ小鼠动物模型，通过评价线粒体功能、产生ROS的不同途径和促炎症因子的表达水平；观察SIRT1去乙酰化PGC-1α（线粒体功能的关键协同因子）和NF-kB/STAT3（介导炎症的两个主要转录因子）对下游的影响，来帮助我们发现并明确两药在抗氧化、抗炎上各自侧重但又互相协同的作用机制特色，为以后的临床研究和运用奠定科学的基础。

背景：糖尿病肾病的发病率近年来迅速上升，在终末期肾病中已跃居第二。虽然开展了大量研究 ，但治疗手段仍十分有限。中医药在早期能有效阻断或延缓本病进展，运用前景广阔。牛葛合剂（葛根素、牛蒡苷元（ATG））由名老中医治疗早期糖尿病肾病（EDN）经验方中的君药的主要有效成分组成，经课题组多年临床和基础研究筛选形成。.主要研究内容：（1）证实高糖环境下 SIRT1 对线粒体损伤的保护作用。（2）证实高糖环境下，炎症和氧化应激通过修饰STAT3和NF-κB激活下游相关因子，导致足细胞损伤。（3）通过体内和体外实验，证实牛葛合剂(葛根素、牛蒡苷元)通过抗氧化、抗炎，各有侧重但又互相补充的保护足细胞，延缓EDN肾脏损伤。.重要结果：1）SIRT1去乙酰化PGC-1α在足细胞中的表达，减少线粒体损伤。.2）阻断P65磷酸化可以抑制HO-1减少，但阻断STAT3磷酸化不能抑制HO-1减少。阻断STAT3和P65磷酸化对iNOS高表达均有抑制作用。.3）乙酰化p65和STAT3可以活化通路，刺激下游炎症相关因子过表达。.4）细胞实验证实，葛根素通过去乙酰化PGC-1α、P65和STAT3来干预高糖环境下足细胞的线粒体损伤和氧化应激水平；ATG在高糖环境下可以去磷酸化P65和STAT3发挥抗炎作用；葛根素和ATG对P65和STAT3信号通路下游的相关炎症因子存在差异性抑制作用。.5）动物实验证实葛根素和ATG具有抗炎和抗氧化的协同作用。.科学意义：本项目首次探讨牛蒡苷元和葛根素组分配伍的协同作用机制。为优化治疗DKD的处方奠定基础。