基因或特异性抑制剂阻断组蛋白甲基转移酶EZH2调控腹膜纤维化的作用和机制

Peritoneal fibrosis is a major pathophysiological process that contributes to loss of peritoneal dialysis (PD) due to ultrafiltration failure in patients. It has become a critical problem that severely influences the prognosis and long-term survival of PD patients. To date, there is no effective treatment for this complication. It is well known that phenotype transition of peritoneal mesothelial cells is the critical pathophysiological basis of peritoneal fibrosis and is regulated by multiple signaling pathways. Our previous studies have shown that blockade of Histone methyltransferase EZH2 inhibited G2/M phase cell cycle arrest as well as activation of TGF-β/Smad and EGFR signaling pathway, two key pathway of renal fibrosis and attenuated renal fibrogenesis. Moreover, we have recently found that EGFR dephosphorylation alleviates development and progression of peritoneal fibrosis. Inhibition of EZH2 leads to down-regulation of α-smooth muscle actin and Fibronectin in cultured peritoneal mesothelial cells, suggesting that blockade of EZH2 activity can inhibit phenotype transition of peritoneal mesothelial cells. Next, blockade of EZH2 activity inhibits activation of TGF-β/Smad signaling pathway in cultured peritoneal mesothelial cells. Further, we established an animal model of peritoneal fibrosis, peritoneal injury leads to remarkable and persistant activation of EZH2. In this proposal, we will utilize gene transfection, RNA interference and immunoprecipitation techniques to further examine the effect of EZH2 inhibition on activation of cultured peritoneal mesothelial cells, and explore the effect of EZH2 inhibition on activation of the TGF-β/Smad signaling pathway and mechanisms involved. Moreover, we will investigate the role of EZH2 inhibition on production of profibrotic factors and proinflammatory cytokines induced by high glucose, and explore the effect of EZH2 inhibition on activation of the NF-κB signaling pathway and mechanisms involved. In addition, we will assess the therapeutic effect and mechanisms by genetic and special inhibitor blockade of EZH2 on the development and progression of peritoneal fibrosis in a rat model of peritoneal fibrosis. Successful completion of this project will define the anti-peritoneal fibrotic efficacy of blockade of EZH2 activity. And it is also extremely critical to afford pharmacological basis and target for investigation anti-peritoneal fibrosis agent. Further, this will probably provide a novel therapeutic approach for prevention and treatment of ultrafiltration failure in PD patients.

腹膜纤维化（PF）是腹透患者发生超滤衰竭导致腹透治疗失败的重要病理基础。我们新近证实，阻断组蛋白甲基转移酶EZH2能抑制肾脏G2/M期细胞停滞和纤维化关键通路TGF-β/Smad及表皮生长因子受体（EGFR）激活减轻肾脏纤维化。最近我们发现，抑制EGFR可减轻PF，且PF大鼠EZH2表达显著上调，阻断EZH2可抑制腹膜间皮细胞活化及TGF-β/Smad通路激活。因此，阻断EZH2可能成为防治PF的新途径。为验证此假说，本项目拟通过基因转染、干扰RNA打靶等技术，首先明确EZH2在腹膜间皮细胞表型转化中的作用；其次探讨EZH2调控TGF-β/Smad通路活化的机制；再次阐明EZH2对高糖诱导的促纤维化和炎症因子分泌影响及NF-κB通路调控机制；最后揭示基因或药物阻断EZH2在体内延缓PF进展的作用。本研究将为新型抗腹膜纤维化药物研发提供重要依据和靶标，从而为腹透患者超滤衰竭的防治提供新途径。

腹膜透析因其操作简便，费用低、对血流动力学影响小等优势，被广大肾衰竭患者所广泛接受。腹膜纤维化是腹透患者发生超滤衰竭导致腹透治疗失败的重要病理基础，但目前缺乏有效的腹膜纤维化防治措施，最终导致患者退出腹透。因此，揭示腹膜纤维化发生的分子机制，并寻求建立在此基础上的超滤衰竭防治策略已成为目前国内外学者广泛关注的焦点以及临床医生急需解决的瓶颈问题。.本课题组首先收集2017年1月至2019年12月期间在上海市东方医院、上海市第一人民医院宝山分院和上海市松江区中心医院行长期腹膜透析患者的腹膜标本和腹透流出液，检测EZH2在腹膜组织和腹透流出液中的表达并做相关分析；其次，我们建立两个小鼠腹膜纤维化（4.25%高糖腹透液和0.1%葡萄糖氯己定）模型，通过腹腔注射EZH2特异性抑制剂3-DZNeP，观察3-DZNeP对小鼠腹膜纤维化的预防和治疗作用，并研究相关的调控机制；同时我们构建EZH2loxP/loxP: Col1a2-Cre+基因敲除小鼠，从基因层面上靶向抑制EZH2，探讨EZH2在腹膜纤维化进展中的作用；最后，在体外细胞实验中，利用EZH2抑制剂或EZH2小分子干扰RNA处理腹膜间皮细胞，观察EZH2对TGF-β1诱导的腹膜间皮细胞间充质转分化（EMT）、凋亡、迁移的影响和机制。.我们发现使用基因或抑制剂阻断EZH2都能显著抑制小鼠腹膜纤维化的发生，并对已经形成的纤维化病灶也具有一定的治疗作用。机制研究上，我们发现3-DZNeP能够抑制腹膜间皮细胞EMT、迁移和增殖，减少细胞外基质蓄积，阻断TGF-β/Smad3、EGFR和NF-κB信号通路激活，改善腹膜炎症，抑制血管新生，并调控TIMP2/MMPs的动态平衡。在腹膜透析患者的临床标本研究中也证实，EZH2在腹膜炎患者腹膜中高表达，并发现随着腹透龄的增加，EZH2在腹透流出液中的表达水平也逐渐升高，并和TGF-β1、VEGF、IL-6呈正相关，而和CA125呈负相关，这提示EZH2有望成为临床预测腹透患者腹膜功能的生物学标志物。.因此，阻断EZH2活化可能成为防治腹膜纤维化的新途径。本研究将为研发新型抗腹膜纤维化药物提供重要实验依据和靶标，从而为腹透患者超滤衰竭防治提供新途径。