组蛋白去乙酰化酶6在腹膜纤维化中的作用和机制研究

Peritoneal dialysis is an effective treatment for patients with end stage renal disease. However, it frequently causes peritoneal fibrosis. Currently, there is no available therapy for prevention and treatment of peritoneal fibrosis. Recently, we have demonstrated that histone deacetylase 6 (HDAC6) is highly expressed in cultured peritoneal mesothelial cells with epithelial-to-mesenchymal transition (EMT) and murine peritoneal membrane with fibrosis. Furthermore, pharmaceutical inhibition of HDAC6 could significantly attenuate TGF-β1 induced phosphorylation of Smad3 and the EMT in mesothelial cells. This suggests that HDAC6 may regulate development and progression of peritoneal fibrosis via TGF-β1/Smad3 signaling pathway. By HDAC6 inhibition via HDAC6 inhibitors, HDAC6 knockout and HDAC6 siRNA in a murine model of peritoneal fibrosis and EMT peritoneal mesothelial cells induced by high glucose peritoneal dialysate fluid (PDF), we will first evaluate the effect of HDAC6 inhibition on the pathogenesis of peritoneal fibrosis；second, we will elucidate the role of HDAC6 in the activation of TGF-β1/Smad3 signaling pathway; finally, we will define the molecular mechanisms by which HDAC6 regulates Smad3 activation. This study will lay the ground work for understanding the pathophysiology of peritoneal fibrosis and identify the novel therapeutic target for prevention and treatment of peritoneal fibrosis.

腹膜透析是终末期肾病患者的有效治疗方案，但长期腹透易导致腹膜纤维化，目前尚无有效防治措施。我们前期研究发现：组蛋白去乙酰化酶6(HDAC6)在间皮-间充质转分化(EMT)的腹膜间皮细胞和纤维化的小鼠腹膜组织中高表达。药物抑制HDAC6可显著缓解TGF-β1引起的腹膜间皮细胞Smad3磷酸化及细胞EMT。提示HDAC6可能通过TGF-β1/Smad3信号通路调控腹膜纤维化发生发展。本课题将在高糖腹膜透析液(PDF)致腹膜纤维化小鼠模型和EMT腹膜间皮细胞上，通过药物抑制剂、基因敲除、siRNA抑制HDAC6，首先评估HDAC6抑制对高糖PDF致腹膜纤维化的影响；然后阐明HDAC6在调控TGF-β1/Smad3信号通路活化中的作用；最后明确HDAC6调控Smad3活化的具体分子机制。本研究将为揭示腹膜纤维化的病理生理机制，探索新的防治靶点奠定基础。

腹膜透析是终末期肾病患者的有效替代治疗方案，但长期腹透易导致腹膜纤维化，目前尚无有效防治措施。我们的前期研究发现：组蛋白去乙酰化酶6 (histone deacetylase 6, HDAC6)在腹膜纤维化过程中起到了关键作用。然而，其确切机制尚未完全阐明。在本研究中，我们进一步利用葡萄糖氯己定(chlorhexidine gluconate, CG)诱导的小鼠腹膜纤维化模型，发现用tubastatin A (TA)抑制HDAC6活性可显著缓解腹膜的病理性改变并减少细胞外基质制沉积。CG损伤导致TGF-β/Smad3信号通路活化，并显著上调两个上皮-间质转分化(epithelial -mesenchymal transition, EMT)相关的转录因子，Snail和Twist。而TA处理可有效阻断上述反应。细胞实验显示，通过TA抑制或siRNA沉默HDAC6可阻断白细胞介素-6 (interleukin-6, IL-6)诱导的人腹膜间皮细胞(human peritoneal mesothelial cell, HPMC)的EMT和增殖。此外，TA能够通过抑制TGF-β/Smad3和Akt信号通路阻断纤维化腹膜中的巨噬细胞及白细胞介素-4 (IL-4)诱导的Raw264.7巨噬细胞M2极化。表现为M2标志物Arginase-1 与CD163的下调。最后，通过TA抑制或siRNA沉默HDAC6可抑制Wnt1/β-catenin 及STAT3信号通路激活从而减少IL-6刺激引起的HPMC中血管内皮生长因子(vascular endothelial growth factor, VEGF)表达上调及人脐静脉内皮细胞(human umbilical vein endothelial cell, HUVEC)的增殖及管腔形成。综上，HDAC6通过激活多条信号通路促进腹膜间皮细胞EMT和增殖，巨噬细胞M2极化及血管内皮细胞管腔形成引发的血管新生，从而促进腹膜纤维化的进展。我们的研究成果提示，HDAC6可作为防治腹膜纤维化的新靶点。