虎杖活性成分白藜芦醇影响SphK-1/S1P信号通路抗糖尿病肾病的作用机制研究

Renal fibrosis, results from mesangial extracellular matrix accumulation with long term and chronic inflammation, is the main pathological character of diabetic nephropathy. Our previous studies have demonstrated that SphK-1/S1P pathway is a crucial regulator in renal fibrosis. There is no specific medicine for diabetic nephropathy in clinical treatment as yet. The traditional Chinese medicine “Polygonum cuspidatum Sieb. et Zucc”, with notable efficiency, is considered as the basic reagent in treatment of diabetic nephropathy. The active component extracted from Polygonum cuspidatum Sieb. et Zucc, named resveratrol, is confirmed to have protective effect in diabetic nephropathy by inhibiting the oxidative stress and renal inflammation. However, the molecular mechanism remains further elucidation. The newest studies have shown that resveratrol could inhibit the cell proliferation by modulating the activity of SphK-1, the content of S1P and then producing an effect on NF-κB activation. So we surmised that SphK-1/S1P pathway may be a new target of resveratrol in treatment for diabetic nephropathy. Based on these foundations, the current project is performed to confirm the effect and mechanism of resveratrol in diabetic nephropathy through mediating the regulation of SphK-1/S1P pathway with the exploition of in vitro cell models, targeted binding experiments and in vivo animal models. Taken together, this work will provide objective experimental evidence for revealing new mechanisms of resveratrol in diabetic nephropathy and developing new specific target to be used in clinical treatment for delaying the progress of diabetic nephropathy.

肾小球细胞外基质积聚及长期伴随的炎症反应所导致的肾脏纤维化是糖尿病肾病(DN)主要的病理特征。我们前期研究发现，SphK-1/S1P信号通路对糖尿病肾脏纤维化起关键调节作用。目前糖尿病肾病(DN)的临床治疗缺乏特效药物，我国传统中药“虎杖”常被作为主药用于DN的治疗，其功效显著。研究表明其活性成分“白藜芦醇”通过抑制氧化应激及肾脏炎症反应而发挥抗DN的作用，但确切的分子机制尚不明确。最新研究发现白藜芦醇通过调节SphK-1活性、S1P表达进而影响NF-κB活性起到抑制细胞增殖的作用。我们推测抑制SphK-1/S1P信号通路有可能成为白藜芦醇抗DN的一个新靶点。因此本项目拟通过体外细胞实验、靶向结合实验及体内动物实验，旨在系统研究并确证白藜芦醇影响SphK-1/S1P信号通路发挥抗DN的分子作用机制，为明确白藜芦醇抗DN特异性作用靶点及进一步研发为新药用于临床提供有利的实验依据。

虎杖活性成分白藜芦醇(RSV)具有抗糖尿病肾病（DN）的作用，而其作用机制是否与影响SphK-1/S1P信号通路有关目前国内外尚未见报道。本项目是基于前期DN与SphK- 1/S1P信号通路之间的一些研究，拟从体外细胞实验和体内动物实验，系统观察RSV对GMC细胞/模型肾组织纤维化成分及炎症介质表达的影响，同时探讨其影响SphK- 1/S1P 信号通路的分子机制。. 其重要的研究结果及科学意义：. 1.体内动物实验证实，RSV具有抗糖尿病肾病的作用，表现在RSV组大鼠相对于模型大鼠组，虽不能显著降低DN大鼠空腹血糖水平，但可显著降低DN大鼠体重、肾脏指数、尿素氮、肌酐及24h尿蛋白等这些指标的水平。. 2.肾小球细胞外基质积聚及长期伴随的炎症反应所导致的肾脏纤维化是DN主要的病理特征。我们的研究结果显示：在高糖诱导的GMC模型、LPS诱导的炎症细胞模型以及过表达SphK-1的GMC模型中，炎症因子（如ICAM-1、iNOS）及纤维化成分(如FN、TGF-β1)等蛋白表达明显增加，而RSV可明显降低上述蛋白的表达，抑制GMC细胞增殖。说明白藜芦醇可能通过下调纤维化成分及炎症因子的表达而影响糖尿病肾脏纤维化的进程。. 3. RSV对GMC模型中核转录因子AP-1及NF-kB的影响：高糖、LPS等刺激因素可激活AP-1及NF-kB等核转录因子，进而调控下游相关基因表达。我们的研究结果显示：RSV可降低这些转录因子与DNA的结合活性，同时抑制相关的蛋白p-c-jun、p-c-fos及胞核p65的表达。说明RSV抑制GMC纤维化成分及炎症介质的表达可能与抑制AP-1及NF-kB活化及其相关蛋白表达密切相关。. 4. RSV对GMC模型中SphK1/S1P2通路的影响：高糖、LPS等刺激下，GMC中SphK1活性、SphK1蛋白、S1P含量及S1P2蛋白表达明显升高，而RSV、SK-II、JTE-013均可显著抑制上述刺激所引起的这些异常变化。高浓度RSV与抑制剂JTE-013作用更为接近，且抑制剂JTE-013作用较抑制剂SK-II更强，说明RSV发挥作用可能与S1P2关系更密切。我们通过转染实验进一步发现，RSV可不依赖其降糖、抗炎抗氧化应激的效应而发挥对SphK1/S1P2的相对特异性的抑制作用。