基于解毒通络探讨柴胡多糖阻断HMGB1-TLR2/4信号通路防治糖尿病肾病

Diabetic Nephropathy(DN), a type of capillary vessel syndrome, is one of the most common and severe chronic complications of diabetes mellitus. Diabetic nephropathy is the leading cause of end-stage renal disease. There is no effective drug for treating DN. Diabetic nephropathy (DN) is an inflammatory disease. HMGB1 is an endogenous ligand of TLR2/4. HMGB1 mediated TLR2/4 signaling pathway has been confirmed to involve in the pathogenesis of DN. According to integrated traditional Chinese and western medicine theory, JieDuTongLuo and inhibition of inflammation is a new strategy for the treatment of DN. Our previous study found that Bupleurm polysaccharides（BPs）inhibited TLR4 signaling pathway and decreased the level of inflammatory cytokines. BPs improved kidney damage and reduced the expression of HMGB1 in renal tubules in DN mice. We hypothesized that the therapeutic effect of BPs on DN resulted from blocking the HMGB1 mediated TLR2/4 signaling pathway. In this study, both spontaneous and induced DN mice will be used to determine the effect of BPs on DN. We will focus on the effect of BPs on HMGB1 structure, expression, secretion and TLR2/4 binding activity. It is evaluated that HMGB1 might be a new target for DN therapy. To determine whether BPs independent of TLR2/4 signaling pathway exerts its anti-inflammatory activity, the translocation of NF-κB to nuclear is observed. Through siRNA silencing TLR2 and antagonize the effects of TLR4, domination position is evaluated between effect of TLR2 and TLR 4 in DN development. The realization of the mechanism of BPs will be coincidence with the theory of traditional Chinese medicine. This work proposed some potential suggestions for future research and the direction of the investigation. The established reseaching stage will be helpful for new drug discovery by screening from traditional Chinese medicinal materials. Exploring the Inflammatory mechanism of DN by the combination of the traditional and Western medicine, is conducive to give full taking the advantages of traditional Chinese Medicine.

糖尿病肾病(DN)是糖尿病的严重并发症，也是终末期肾病的主要原因，目前尚无有效药物。TLR2/4识别HMGB1而启动炎症反应与DN发生发展密切相关。解毒通络抑制炎症是防治DN的重要策略。前期研究发现，柴胡多糖抑制TLR4通路而降低炎症水平；改善STZ诱导的DN小鼠肾损伤，降低HMGB1表达。由此推测：柴胡多糖通过阻断HMGB1-TLR2/4信号通路治疗DN。本课题拟采用自发和诱导两种DN模型进一步观察柴胡多糖的药效及机制，在分子和细胞水平上，重点观察柴胡多糖对HMGB1表达、分泌及活性的影响，探讨HMGB1作为治疗DN新靶点的可能性；观察柴胡多糖对NF-κB入核的影响，判断其是否独立于对TLR2/4的作用；通过对TLR2 siRNA沉默和对TLR4作用的拮抗，观察柴胡多糖对TLR2和TLR4作用的差异。明确柴胡多糖治疗DN的作用机制，能够为在中药中挖掘DN治疗药物提供新的思路和研究方向。

糖尿病肾病（DKD）的发生发展与肾组织炎症程度密切相关，其中TLR2/4信号通路是导致糖尿病肾脏损伤的重要机制之一。HMGB1是一种DNA结合蛋白，主要稳定核小体的结构并调节基因转录。作为TLR2/4内源性配体，在糖尿病状态下受损的肾实质细胞释放HMGB1至细胞外，激活HMGB1- TLR2/4信号通路，促使炎症因子和趋化因子的分泌；反之，TNF-α、IL-1等炎症因子又可以激活单核-巨噬细胞，主动分泌HMGB1，再一次进入炎症通路，构成正反馈，增强炎症反应。此外，HMGB1还可促进巨噬细胞迁移，导致肾内皮细胞活化，趋化因子产生，加强其释放炎症介质和细胞因子的能力，最终形成肾脏炎症的持续状态，导致肾脏损伤及纤维化。因此，阻断HMGB1-TLR2/4通路有可能减弱肾脏炎症并缓解组织损伤。在动物水平上，我们优化了STZ诱导的糖尿病肾病模型，证实在此模型中糖尿病小鼠中肾脏有明显的功能损伤，而HMGB1通过激活TLR2/4通路参与了糖尿病肾脏损伤的发生发展。同时糖尿病小鼠肠道的机械、免疫和生物屏障均出现损伤，肠道炎症因子IL-6、TNF-α等的分泌增加，并伴有肠道菌群多样性和特征菌的异常。柴胡是我国的传统中药和常用中药，常被用于治疗各种肾病，且疗效显著，而多糖是柴胡重要的有效成分。我们研究发现，柴胡多糖能够显著改善肾脏功能，抑制肾脏炎症反应，可能通过抑制HMGB1/TLR4/NF-κB信号通路发挥对糖尿病小鼠肾脏的保护作用。此外，我们还发现，柴胡多糖能够改善肠道的炎症进展，修复高血糖造成的肠屏障损伤，恢复肠道菌群的多样性和益生菌丰度。其机制可能是基于肠-肾轴通过调节肠道屏障实现肾脏损伤的改善作用。本项目的研究结果不仅为柴胡多糖作为糖尿病肾病治疗药物提供了实验依据，有利于柴胡多糖的进一步研究开发奠定了基础，也有助于丰富肠-肾轴的调控机制，为多糖及大分子物质的机体调控机制研究提供了证据及研究方向。