TGR5对糖尿病肾脏纤维化的影响及其相关分子机制研究

Glycolipid metabolism disorder and chronic renal inflammation are critical etiology and pathogenesis of diabetic nephropathy (DN) and renal fibrosis, and we have proved that the activation of SphK1/S1P pathway had close relationships with diabetic renal fibrosis. The bile acid membrane receptor TGR5 is confirmed to significantly regulate glycolipid metabolism and resist inflammation in recent years. Given that TGR5 is also highly expressed in kidney tissues, Whether TGR5 can inhibit renal inflammatory reaction and postpone the pathological process of renal fibrogenesis under the diabetic condition, and its relationship with SphK1/S1P pathway are worthy of investigation. Our preliminary experiments showed that TGR5 activator obviously inhibited the inflammatory and fibrotic components levels in both the cell model and kidney tissues of experimental animal as well as the expression of S1P2 receptor in the kidney tissues. Based on the above mentioned, the current program is undertaken to explore systematically the effects of TGR5 on the inflammatory and fibrotic components, such as ICAM-1, FN in the cell model and the kidney tissues of experimental animal, and to further investigate the molecular mechanism of TGR5 on regulating the SphK1/S1P pathway and modulating the transcriptional activities of nuclear factors NF-κB and AP-1 to provide experimental evidences and theoretical foundations for exploring TGR5 as a new target in the treatment of diabetic renal fibrosis and DN.

糖脂代谢紊乱、肾脏慢性炎性反应是糖尿病肾病（DN）、肾脏纤维化的重要病因病机，我们已证实SphK1/S1P通路的活化与糖尿病肾脏纤维化关系密切。近年来有充分证据表明胆汁酸膜受体TGR5具有显著调节糖脂代谢及抗炎作用。鉴于TGR5亦高表达于肾脏组织，TGR5能否抑制糖尿病状态下的肾脏炎性反应、阻抑肾脏纤维化的病理进程？以及该作用与影响SphK1/S1P通路的关系，值得高度关注。预实验结果显示：TGR5激动剂能明显抑制模型细胞与模型动物肾组织炎性、纤维化成分及S1P2的表达。本项目在此基础上，拟通过系统研究TGR5对模型细胞与模型动物肾组织ICAM-1及FN等炎性、纤维化成分的影响，明确TGR5对糖尿病肾脏纤维化的作用；进一步从影响SphK1/S1P通路、调节核因子NF-κB及AP-1的活性等，探讨其抗糖尿病肾脏纤维化的分子机制。为将TGR5作为抑制糖尿病肾脏纤维化、抗DN新靶点提供实验依据。

项目背景与主要研究内容： 糖脂代谢紊乱、肾脏慢性炎性反应是糖尿病肾病（DN）、肾脏纤维化的重要病因病机，我们已证实SphK1/S1P通路的活化与糖尿病肾脏纤维化关系密切。近年来有充分证据表明胆汁酸膜受体TGR5具有显著调节糖脂代谢及抗炎作用。鉴于TGR5亦高表达于肾脏组织，TGR5能否抑制糖尿病状态下的肾脏炎性反应、阻抑肾脏纤维化的病理进程？以及该作用与影响SphK1/S1P通路的关系，值得高度关注。本项目在前期研究基础上，拟通过系统研究TGR5对模型细胞与模型动物肾组织ICAM-1及FN等炎性、纤维化成分的影响，明确TGR5对糖尿病肾脏纤维化的作用；进一步从影响SphK1/S1P通路、调节核因子NF-κB及AP-1的活性等，探讨其抗糖尿病肾脏纤维化的分子机制。.重要研究结果与意义：. 1. 活化TGR5能够明显抑制HG诱导的GMCs炎症因子（ICAM-1）和转化生长因子（TGF-β1）的表达，细胞外基质主要成分纤维连接蛋白（FN）生成明显减少。进一步结果显示，激活TGR5能够抑制高糖诱导的S1P2表达增多，下调核因子AP-1二聚体中c-Jun/c-Fos的磷酸化水平，降低AP-1的转录活性。同为G蛋白偶联受体家族成员，S1P2与TGR5存在相互作用，并且TGR5激活后能够促进S1P2内化失活。证实激活TGR5能够抑制HG诱导的肾小球系膜细胞中炎症纤维化因ICAM-1、 TGF-β1、 FN的过度生成，其机制可能与TGR5抑制S1P/S1P2通路密切相关。. 2. 在GMCs体外模型中，激动TGR5或者在过表达基础上激动TGR5能有效地抑制HG引起的RhoA/ROCK的活化，细胞质中IκBα的降解以及p65入核转位减少，NF-κB的DNA结合活性降低，激活受到抑制。而干扰TGR5，RhoA/ROCK通路异常活化。另外，使用RhoA持续激活型质粒RhoAQ63L活化RhoA/ROCK，FN、TGF-β1蛋白水平异常升高，IκBα发生降解，p65入核增加，NF-κB与DNA的结合活性增强，通路激活；。而使用PKA特异性抑制剂H-89处理GMCs后，INT-777无法抑制RhoA/ROCK通路在高糖条件下的活化。提示TGR5调节FN、TGF-β1等炎症纤维化因子的表达增加也与其通过PKA抑制RhoA/ROCK通路的活化、进而影响核因子NF-κB的激活密切相关。