锌离子在腹膜透析相关的腹膜纤维化中的作用与机制研究

Zn deficiency has been suggested to be associated with patients undergoing peritoneal dialysis (PD). Several lines of evidence demonstrated that Zn deficiency may induce lung fibrosis, liver fibrosis, etc. However, the effect of Zn deficiency on the peritoneal fibrogenesis and its underlying mechanism is still not clarified. We recently documented that Zn might inhibit high glucose (HG)-induced epithelial-to-mesenchymal transition in rat peritoneal mesothelial cells (RPMC) by inhibiting oxidative stress. In addation, the expression of ZnT5 and ZnT7 was significantly upregulated by HG, indicating that ZnT5 and ZnT7 may invove in the pathogeneis of peritoneal fibrosis (PF). Considering a crucial role of ZnT5 and ZnT7 in the delivery of zinc into a newly synthesized zinc-requiring enzymes and proteins, we speculated that the action of ZnT5 and ZnT7, regulating of the activity and expression of fibrosis associated factors, may be an important mechanism of zinc restraining the development of peritoneal fibrosis. The purpose of this study is to validate the hypothesis mentioned above from several aspects, mainly including the regulation of the activation status of oxidative stress and its related signaling pathways as well as the expression and activity of transporter, antioxidant enzymes and critical fibrosis factors. The elluciation of the precise mechanism of zinc against PF will undoutedly provide a potential therapeutic target for PF therapy and guide the use of zinc ions in the prevention and treatment of PD in clinical practice.

腹膜透析(PD)的患者均存在不同程度的锌缺乏。研究表明，锌缺乏与肺、肝等纤维化密切相关。但其对PD相关的腹膜纤维化(PF)的影响尚不清楚。在前期研究工作中，我们通过体外实验证实锌补充能够阻抑大鼠腹膜间皮细胞（RPMC）的转分化，其主要机制可能与抑制氧化应激有关。此外，我们还发现高糖（HG）刺激的RPMC中ZnT5、ZnT7表达显著增高，提示它们可能参与了PF的进程。由于ZnT5、ZnT7是参与含锌蛋白合成的重要锌转运体，我们推测ZnT5、ZnT7对纤维化相关因子的活性与表达变化的调控是锌抑制PF的另一重要机制。本研究拟从氧化应激和相关信号通路活化状态、转运体、抗氧化酶的表达调控、关键纤维化因子的表达水平等不同层面、不同角度来验证上述假设。本项目的研究结果将为揭示锌离子预防和治疗PF的机制，寻找PF的治疗靶点，以及指导锌离子类药物在PD中的应用提供新的科学依据。

腹膜纤维化所致超滤衰竭是患者退出腹膜透析的主要原因。锌是机体不可或缺的一种微量元素，锌缺乏可促进腹膜透析相关性腹膜纤维化的发生和发展，其机制尚未完全阐明。本实验旨在研究锌离子在腹膜透析相关的腹膜纤维化中的作用与机制。本研究通过建立大鼠腹膜透析相关的腹膜纤维化模型和体外细胞实验，采用苏木精-伊红染色法和Masson三色染色观察腹膜纤维化程度，利用 Transwell共培养系统观察HPMCs的迁移功能，应用TSQ探针方法检测细胞内锌含量的变化，应用Real-time PCR技术、免疫组织化学技术、Western Blot技术、免疫荧光技术、ELISA技术等技术检测TGF-β、E-cadherin、Vimentin、锌离子及锌转运体等基因、蛋白表达对HG诱导的腹膜间皮细胞EMT的影响及相关机制。. 体内研究结果：高糖腹膜透析液可导致大鼠腹膜组织病理学、形态学及功能异常，锌螯合剂加重上述改变，而锌补充可减轻腹膜组织纤维化；HG刺激腹膜组织导致MAPK、TGF-β/Smad和Nrf-2信号通路的激活，加入ZnSO4后，发现MAPK、TGF-β/Smad信号通路关键蛋白表达下调，Nrf-2及重要靶基因HO-1和NQO1的蛋白表达上调；在高糖腹膜透析液作用下，大鼠腹膜组织ZnT5的mRNA水平表达明显增加，而锌离子表达减少。. 体外研究结果：进一步证实高糖可刺激人腹膜间皮细胞发生EMT改变，而锌补充可阻抑腹膜间皮细胞的EMT改变；HG刺激腹膜间皮细胞导致TGF-β分泌及ROS的表达增加，而锌螯合剂导致其表达进一步增加，加入ZnSO4后，发现TGF-β分泌及ROS的表达下降，主要通过上调Nrf-2信号通路关键因子Nrf-2及重要靶基因HO-1和NQO1的蛋白表达水平，减轻腹膜间皮细胞的氧化应激和损伤；高糖通过改变ZnT5在mRNA和蛋白表达水平，介导高尔基体锌离子内流，通过TGF-β/Smad通路而调节纤维化和下游信号转导通路关键蛋白的合成、组装和修饰，最终促进腹膜纤维化的形成。. 上述结果从新的视角提示了锌缺乏促进腹膜透析相关性腹膜纤维化的作用和相关机制，为有效干预腹膜纤维化的关键环节与分子靶点提供新的科学依据，对于锌离子在腹膜透析液中的应用具有理论意义和实际应用价值。