从NLRP3炎症小体介导细胞焦亡探讨急性肾损伤纤维化进展的机制及丹酚酸B的干预

The mean pathological basis of renal fibrosis is death of renal tubular epithelial cell in chronic kidney disease progressed by acute kidney injury. Pyroptosis is associated with death of renal tubular epithelial cell in acute kidney injury (AKI). Reduction of pyroptosis plays a key role in AKI treatment. Salvianolic Acid B (SalB) is effective in restraining NLRP3, alleviating AKI, improving renal fibrosis and reversing pyroptosis induced by oxygen deficit in HK2, which has been proved in our previous study. Caspase-1 is a classical active pathway in pyroptosis which could be activated by NLRP3 and related with ROS. Simultaneously, salB is known as an activator of Nrf2, which is also connected with ROS. To this end, we hypothesize that salB could inhibit ROS so that depress the activation of NLPR3 and alleviate pyroptosis accordingly. For the sake of hypothesis testing, laser confocal, flow cytometry, westen blot and so on are used in mice suffered from AKI induced renal fibrosis. To further explore the treatment mechanism of salB in pyroptosis inhibition associated with Nrf2 and NLRP3, gene silencingand Co-immunoprecipitation are applicated in vitro. In general, this study commits to providing molecular mechanism of the treatment effect of salB and illuminating the role of pyroptosis in AKI progressing renal fibrosis.

急性肾损伤（AKI）发展为慢性肾脏病（CKD）中，肾小管上皮细胞死亡是其纤维化的主要病理基础，而焦亡是此上皮细胞死亡的重要路径，所以减轻焦亡是改善AKI预后的关键。前期研究发现丹酚酸（SalB）能抑制NLRP3炎症小体，减轻AKI后的肾纤维化，且可抑制缺氧诱导的肾小管上皮细胞焦亡。焦亡经典激活途径中，NLRP3炎症小体活化caspase-1可诱导焦亡发生，并与ROS相关。由于SalB可激活抗氧化应激转录因子Nrf2，故推测其可通过抑制ROS对NLRP3的激活，减轻细胞焦亡。因此拟利用AKI后肾纤维化模型和激光共聚焦、流式细胞术、分子印迹等手段，探讨SalB通过抗焦亡改善AKI后纤维化的机制，并应用基因沉默、免疫共沉淀等技术在细胞水平进一步考察Nrf2通过NLRP3减轻焦亡的分子机制，为SalB治疗AKI后纤维化提供证据，同时可以加深焦亡在AKI肾纤维化中的认识。

项目背景：急性肾损伤-慢性化（AKI-CKD）是目前肾脏病领域的热点及难点，而肾小管上皮细胞大量死亡是致肾功能短期内迅速下降的主要因素。因此，针对肾小管上皮细胞死亡的靶向治疗成为防治AKI向CKD纤维化进展的重要策略。已有研究报道，焦亡作为一种与炎症密切相关的新型死亡形式，可能在AKI肾小管上皮细胞死亡中具有重要作用，而我们前期的研究也发现丹酚酸B（SalB）可抑制NLRP3/caspase-1通路，减轻AKI损伤。因此，本项目拟建立AKI后肾纤维化模型，以激光共聚焦、流式细胞术、蛋白印迹等手段，明确SalB抑制焦亡、改善AKI后纤维化的作用，并在细胞水平，应用基因沉默等技术进一步考察其调控Nrf2、抑制NLRP3活化，减轻焦亡的分子机制，为SalB治疗AKI后纤维化提供证据，明确抑制焦亡对肾纤维化防治的意义。. 主要研究内容：①明确丹酚酸B对AKI肾纤维化小鼠的改善作用；②考察焦亡在AKI向纤维化进展过程中的变化，探讨丹酚酸B对焦亡的抑制作用；③探索丹酚酸B调控Nrf2/NLRP3炎症小体减轻焦亡，改善AKI后肾纤维化的分子机制。. 结果：小鼠AKI后21d可见肾组织纤维化样改变，且AKI向CKD进展过程中有焦亡发生。丹酚酸B可减轻焦亡，改善小鼠肾纤维化，其机制与激活Nrf2，抑制ROS/NLRP3/capase-1通路有关。. 科学意义：基于Nrf2/NLRP3/caspase-1通路，阐明丹酚酸B抑制焦亡改善AKI后纤维化的分子机制，为其防治AKI后肾纤维化提供研究基础和依据。