柴黄益肾颗粒通过调节OATs减少肾小管上皮细胞尿毒素积聚治疗糖尿病肾病机制研究

Chaihuangyishen(CHYS) granules a new TCM preparation which was developed based on our previous several funds.Our recent studies found that CHYS could ameliorate renal injury and urinary protein in diabetic rats. Further study revealed that in renal tubular epithelial cell of early diabetic model rats, the accumulation of organic anion(OA) toxins has been occurred, which is associated with tubulointerstitial injury and could be effectively reduced by CHYS granules. OA transports in renal tubular epithelial cell is the important pathway of OA uremic toxins excreted from kidney. Based on previous literatures, we speculated that the abnormality of OATs in diabetic condition could mediate the early accumulation of uremic toxins in renal tubular epithelial cell which subsequently induced the kidney damage. We further hypothesize that CHYS granules could improve DN by adjusting this pathway. We plan an experiment to clarify the abnormality of OATS in renal tubular epithelial cell/uremic toxins accumulation pathway in the development of DN, to study the pharmacological mechanism of CHYS granules mediating this pathway, to explore the target of CHYS, to provide the scientific basis for modern TCM treating diabetic nephropathy.

柴黄益肾颗粒是我们在既往基金资助下开发的一个六类中药制剂。药效学结果表明，该药能显著减少糖尿病肾病（DN）模型大鼠肾损伤和尿蛋白排泄。进一步研究发现，模型大鼠早期有机阴离子（OA）尿毒素已经在肾小管上皮细胞显著积聚，且与小管间质损伤程度相关，而柴黄益肾颗粒可以有效降低其积聚水平。肾小管上皮细胞有机阴离子转运蛋白（OATs）是OA尿毒素经肾排泄的重要途径。结合大量文献报道，我们推测糖尿病状态下OATs的异常可能介导了肾小管上皮细胞尿毒素的早期积聚，进而引起DN肾损伤，而柴黄益肾颗粒可能通过调节该途径治疗DN。本课题拟结合体内外实验，阐明肾小管上皮细胞膜上OATs异常/尿毒素积聚途径在DN发生发展中作用，研究柴黄益肾颗粒调控此途径的作用及相关机制，探讨柴黄益肾颗粒治疗DN的作用靶点，为治疗DN的现代中药研究提供科学依据。

多种尿毒素分子的异常升高已证实与糖尿病肾病(DN)密切相关，有机阴离子分子转运蛋白（OATs）是尿毒素经肾排泄的关键途径。柴黄益肾颗粒治疗DN药效确切，并能显著降低尿毒素水平。基于此项目组提出柴黄益肾颗粒通过调节OATs介导的尿毒素积聚治疗DN的研究假说。项目组首先利用UPLC-qTOF-MS技术对柴黄益肾颗粒物质基础进行了分析，鉴定了108种成分，结合网络药理学研究发现调控尿毒素转运的OAT1和OAT3是柴黄益肾颗粒治疗DN的重要靶标，柴胡皂苷C、D、Makisterone A和6''-Acetylsaikosaponin D是作用于OAT1和OAT3的关键成分。进而，利用体外研究证实尿毒素分子马尿酸和吲哚磺酸能诱导OAT1和OAT3表达，加重肾小管上皮细胞的氧化应激、凋亡与纤维化，揭示了尿毒素介导肾小管损伤的机制，在此基础上，结合体内外研究明确了柴黄益肾颗粒能上调OAT1、OAT2及OAT3表达，降低肾脏尿毒素UA、HUA、IS、PCS及IAA水平，活化Nrf2抗氧化通路减轻氧化应激，抑制尿毒素受体AhR介导NF-κB炎症通路的活化，抑制了细胞凋亡与FN1、Collagen III及Collagen IV等多种纤维化标志分子表达，从而改善了糖尿病肾损伤。通过本研究阐释了柴黄益肾颗粒调控OATs介导肾脏尿毒素积聚改善DN的机制，有助于揭示柴黄益肾颗粒治疗DN科学内涵与物质基础，为其临床用于DN的治疗提供了实验证据与科学支撑。