基因或药物阻断表皮生长因子受体酪氨酸磷酸化调控腹膜纤维化的作用

Peritoneal fibrosis is a major pathophysiological process that contributes to loss of peritoneal dialysis due to ultrafiltration failure in patients. Our previous studies have shown that blockade of epidermal growth factor receptor (EGFR) inhibited phosphorylation of Smad3, a key mediator of renal fibrosis and attenuated renal fibrogenesis. Moreover, we have recently found that EGFR phosphorylation was increased in the peritoneum in a rat model of peritoneal fibrosis. Further, we found that EGFR dephosphorylation led to down-regulation of α-smooth muscle actin and collagen I in cultured peritoneal mesothelial cells, suggesting that EGFR dephosphorylation can inhibit activation of peritoneal mesothelial cells. And Blockade of EGFR also can inhibit excretion of transforming growth factor-β1(TGF-β1) and activation of NF-κB signaling pathway. These data suggest that EGFR plays an important role in the development and progression of peritoneal fibrosis. Blockade of EGFR may provide a novel therapeutic approach for prevention and treatment of peritoneal fibrosis.To test this hypothesis, we will first utilize gene transfection, RNA interference and other molecular techniques to clarify the role of EGFR in regulating phenotype transition of peritoneal mesothelial cells. Next, we will explore the mechanism by which EGFR regulates high glucose-induced activation of the TGF-β/Smad signaling pathway.Third, we will elucidate the role of EGFR in mediating high glucose-induced production of profibrotic factors /proinflammatory cytokines as well as activation of the NF-κB signaling pathway and mechanisms involved. Finally, we will assess the therapeutic effect of genetic and pharmacologic EGFR blockade on the development of peritoneal fibrosis and define the mechanism involved in a rat model of peritoneal fibrosis. Successful completion of this project will lay the ground work for the development of anti-peritoneal fibrotic treatments and may provide a novel therapeutic approach for prevention and treatment of ultrafiltration failure in PD patients.

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。本课题组前期研究发现，阻断表皮生长因子受体（EGFR）活化能抑制纤维化通路中的关键信号蛋白Smad3的激活。最近我们发现，腹膜纤维化大鼠EGFR磷酸化水平显著上调，阻断EGFR可抑制腹膜间皮细胞活化，抑制其TGF-β分泌和核转录因子NF-κB激活。因此，阻断EGFR活化可能成为防治腹膜纤维化的新途径。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EGFR在腹膜间皮细胞表型转化中的作用；其次探讨EGFR调控高糖诱导的TGF-β/Smad信号通路活化的机制；再次阐明EGFR对高糖诱导的促纤维化和炎症因子分泌的影响及NF-κB通路激活的机制；最后揭示基因或药物阻断EGFR在体内延缓腹膜纤维化进展的作用和调控机制。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。本课题组研究显示，表皮生长因子受体（EGFR）在纤维化腹膜中高表达；并且伴随腹膜纤维化程度加重，EGFR激活会显著增强。CG或高糖腹透液腹腔注射构建腹膜纤维化大鼠模型，吉非替尼腹腔注射，发现抑制EGFR活化同时，胶原沉积明显减少，腹膜厚度变薄，免疫印迹方法检测发现α-SMA和I型胶原表达显著下调，免疫组化技术检测发现靶向抑制EGFR，I型胶原表达显著下降，与免疫印迹结果一致，证实抑制EGFR可明显减轻CG或高糖腹透液诱导的腹膜纤维化的发生。此外，CG连续腹腔注射3周，构建腹膜纤维化大鼠模型，吉非替尼腹腔注射2周延期治疗，发现吉非替尼延期治疗，可显著抑制腹膜纤维化的进展；吉非替尼延期干预，胶原沉积明显减少，腹膜厚度变薄，免疫印迹方法检测发现α-SMA和I型胶原表达显著下调，免疫组化技术检测发现靶向抑制EGFR，I型胶原表达显著下降，与免疫印迹结果一致，证实吉非替尼延期治疗可明显延缓CG诱导的腹膜纤维化进展，吉非替尼可能成为腹膜纤维化治疗的潜在研究药物。机制探索发现：阻断表皮生长因子受体（EGFR）活化能抑制纤维化通路中的关键信号通路TGF-β/Smad3和NF-κB激活；阻断EGFR可抑制腹膜间皮细胞活化，抑制高糖诱导的促纤维化和炎症因子分泌，抑制巨噬细胞浸润；抑制STAT3磷酸化激活和血管增生（下调VEGF和CD31表达）。因此，阻断EGFR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的防治提供新途径。