PLA2R与IMN脾肾阳虚证的关联及温阳利水法作用机制的研究

Optimum treatment of idiopathic membranous nephropathy (IMN) plays a pivot role in reducing the risk of end-stage renal disease. The progression of IMN is due to Spleen and Kidney Yang deficiency in TCM theory. At the centre of the pathogenesis of IMN is the damage of a podocyte. On a molecular level, podocyte-membrane proteins are prone to serve as targets for immune-complex formation, which first requires identification of the pathogenic antigen(s). Recently, researchers proved the M-type phospholipase A2 receptor (PLA2R) as the target autoantigen of IMN. However, there is no specific medication for PLA2R. Our early studies suggest that the therapy of Warming Yang and Diuresis would be prospective for the disease. This project mainly characterized by investigating the relationship between PLA2R and syndrom of Spleen and Kidney Yang deficiency of IMN and the mechanism of Warming Yang and Diuresis therapy. Using data from clinical cases, the study is to detect the relevance between PLA2R and Syndrome of Spleen and Kidney Yang deficiency, to clarify the pathogeny of TCM microscopically , and to broaden options of TCM therapy based on modern evidence. By molecular biological methods, we investigate the mechanism of Patient's serum extracted from syndrom of Spleen and Kidney Yang deficiency of IMN affecting the structure and apoptosis of podocytes, together with machanism of Warming Yang and Diuresis therapy. These lab evidence would support more specific target based therapy and possible TCM approaches for IMN prevention and treatment.

有效防治特发性膜性肾病(IMN)，是减少本病进展至终末期肾病的最关键策略。本病发病焦点在中医病机与脾肾阳虚有关；在组织学与肾小球足细胞受损伤有关；在分子生物学与足细胞的抗原决定簇暴露成为自身抗原致抗原抗体复合物反应有关。目前研究发现M型磷脂酶A2受体（PLA2R）是IMN的靶抗原，而本病治疗尚无特效药，前期研究结果显示中医温阳利水法疗效好。本项目旨在探讨PLA2R与IMN脾肾阳虚证的关联及温阳利水法的作用机制。通过临床研究探讨IMN患者脾肾阳虚证与PLA2R的关联，有利于中医客观地微观地认识IMN的实质，为中医药辨证论治IMN开创治疗思路和提供理论依据。又借助细胞分子生物学探讨脾肾阳虚证IMN患者血清对足细胞结构和凋亡的影响及温阳利水法的作用机制，为寻找靶向性更强的治疗方法提供实验依据，为进一步开展中医药防治本病奠定基础。

成人特发性膜性肾病(idiopathic membranous nephropathy, IMN)的主要靶抗原为M型磷脂酶A2受体(M-type phospholipase A2 receptor, PLA2R)，在组织学与肾小球足细胞受损有关。目前，尚未发现本病治疗的特效药。我们前期研究结果提示IMN与脾肾阳虚密切相关，且应用温阳利水法取得较好疗效。为进一步探讨PLA2R与IMN脾肾阳虚证的关联，明确温阳利水法的作用机制，本研究通过收集IMN脾肾阳虚证和IMN非脾肾阳虚证病例，检测血清anti-PLA2R、生化等指标并行组间比较，以探讨PLA2R与IMN脾肾阳虚证的关联；基础研究部分，利用体外培养小鼠永生化足细胞，借助细胞分子生物学方法探讨脾肾阳虚型IMN患者血清对足细胞结构和凋亡的影响以及温阳利水法的作用机制。临床研究部分，脾肾阳虚组患者31例；非脾肾阳虚组患者9例。脾肾阳虚组患者血清anti-PLA2R阳性率为70.97%，非脾肾阳虚组为33.33%，差异无统计学意义。脾肾阳虚组患者年龄、血清anti-PLA2R光密度、血清球蛋白水平均高于非脾肾阳虚组，差异均有统计学意义(P＜0.05)；两组性别、病程、平均动脉压、ALB、Scr、UA、TG、CHOL、HDL-C、LDL-C、PT、APTT、UTP、RBC(HPF)比较，差异均无统计学意义。两组肾间质炎症细胞浸润构成比差异有统计学意义(P＜0.05)。脾肾阳虚组血清anti-PLA2R光密度与血清球蛋白呈正相关。基础研究部分，构建10%脾肾阳虚型IMN患者血清损伤足细胞模型，根据CCK-8检测结果，设置正常对照组、模型组、2mg/ml中药组、4mg/ml中药组和8mg/ml中药组共5组，对应予10%健康人血清+RPMI-1640培养基、10%脾肾阳虚型IMN患者血清+RPMI-1640培养基、10%脾肾阳虚型IMN患者血清+RPMI-1640培养基+相应浓度温阳利水方水提物，分别共孵育24h和48h。结果示该水提物可能通过抑制p53的表达、上调Bcl-2的水平，稳定细胞骨架蛋白F-actin结构，减少脾肾阳虚型IMN患者血清致足细胞凋亡的发生。本项目验证了anti-PLA2R对IMN诊治的指导作用，阐明了anti-PLA2R与IMN脾肾阳虚证的关联，证实了温阳利水法治疗IMN的作用及分子机制。