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大黄素靶向Notch通路抗腹膜透析大鼠腹膜纤维化

王慧超, 林旭红, 房晓鹏, 穆小云, 李铁军, 刘建林

河南大学第一附属医院肾内科，开封 475000；河南大学淮河医院检验科，河南大学淮河医院转化医学中心，开封 475000

摘要

长期腹膜透析治疗经常并发腹膜纤维化，本文旨在探讨大黄素对腹膜透析相关性腹膜纤维化大鼠的治疗作用及其相关的细胞、分子机制。在体内实验中，以腹腔注射4.25%葡萄糖腹膜透析液(100 mL/kg)诱发大鼠腹膜透析相关性腹膜纤维化动物模型，于2周、4周、6周时进行模型大鼠腹膜平衡实验，并测量腹膜厚度、应用HE染色和Masson染色进行组织病理学评估，ELISA法测定血浆中III 型前胶原氨基端肽(PIIINP)水平。选取6周模型大鼠，用大黄素腹腔注射进行治疗，观察治疗后上述指标的变化，同时以Real-time PCR方法检测大鼠腹膜Notch1、Jagged-1、Hes-1基因表达水平，Western blot方法检测大鼠腹膜组织Notch1、Jagged-1、Hes-1、Notch胞内结构域(Notch intracellular domain, NICD)蛋白表达。在体外实验中，培养大鼠腹膜间皮细胞，使其过表达或沉默Notch1，Western blot检测大黄素对腹膜间皮细胞Hes-1、Hey的诱导表达作用。HE染色显示：随着造模时间的延长，间皮细胞减少、部分脱落，间质有炎细胞浸润，6周时Masson染色显示模型组腹膜明显增厚(P < 0.01)，可见大量胶原沉积。与对照组相比，6周模型组大鼠血浆PIIINP水平显著升高(P < 0.01)。与6周模型组相比，大黄素处理可增加模型大鼠腹膜组织中间皮细胞数量，抑制腹膜增厚(P < 0.01)，改善胶原沉积，降低血浆PIIINP水平(P < 0.05)，并在基因、蛋白水平下调Notch1、Jagged-1、Hes-1的表达(P < 0.05或P < 0.01)，且下调NICD蛋白水平(P < 0.01)；体外实验显示：与正常细胞大黄素处理组相比，大黄素对过表达Notch1间皮细胞的Hes-1、Hey表达具有抑制作用(P < 0.05)，而对Notch1沉默间皮细胞的Hes-1、Hey表达无明显影响(P > 0.05)。以上结果表明，在高浓度葡萄糖腹膜透析液所致的大鼠腹膜纤维化模型中有Notch通路的活化，大黄素可能通过抑制Notch通路改善腹膜纤维化程度。

关键词： 大黄素; 腹膜透析; 腹膜纤维化; Notch

分类号：R363.2

[Emodin ameliorates the peritoneal dialysis-related peritoneal fibrosis via inhibiting the activation of Notch pathway.] [Article in Chinese]

WANG Hui-Chao, LIN Xu-Hong, FANG Xiao-Peng, MU Xiao-Yun, LI Tie-Jun, LIU Jian-Lin

Department of Nephrology, First Affiliated Hospital of Henan University, Kaifeng 475000, China； Department of Clinical Laboratory, Translational Medicine Center, Huaihe Hospital Affiliated to Henan University, Kaifeng 475000, China

Abstract

Long term peritoneal dialysis (PD) is often associated with peritoneal fibrosis. The aim of this study was to explore the effect of emodin on PD-related peritoneal fibrosis and its related cellular and molecular mechanism. PD-related peritoneal fibrosis rats and cultured rat peritoneal mesothelial cells were recruited in the experiment. PD-related peritoneal fibrosis was induced by intraperitoneal injection of lactate-buffered solution containing 4.25% glucose. The peritoneal equilibrium test (PET) was performed at the end of 2 weeks, 4 weeks, and 6 weeks, respectively. HE staining and Masson staining were used for histopathological evaluation. Enzyme linked immunosorbent assay (ELISA) was used to measure the plasma N-terminal procollagen III propeptide (PIIINP) level. Real-time PCR technique was used to detect the mRNA levels of Notch1, Jagged-1, and Hes-1 in peritoneal tissue. Western blot was applied to identify the protein levels of Notch1, Jagged-1, Hes-1, and Notch intracellular domain (NICD). In vitro, Notch1 overexpressing or knockdown rat peritoneal mesothelial cells were established and Western blot was used to examine the effect of emodin on the expressions of Hes-1 and Hey. Compared with the control group, HE staining revealed that PD rats suffered from decreasing in mesothelial cells, or detaching from surface of parietal peritoneum, accompanied by infiltration of inflammatory cells; Masson staining result showed thickened peritonea (P < 0.01), and the collagen deposition in the parietal peritoneum was increased; also, PIIINP level in plasma was elevated (P < 0.01). Treatment of the PD rats with emodin increased mesothelial cells in peritoneal tissue, and decreased the peritoneal thickness (P < 0.01), collagen depositions, as well as the plasma PIIINP level (P < 0.05). The expressions of Notch1, Jagged-1, Hes-1 and NICD in peritoneal tissue were also attenuated (P < 0.05 or P < 0.01). In cultured rat peritoneal mesothelial cells, compared with emodin group, emodin further inhibited the expressions of Hes-1 and Hey induced by Notch1-overexpression (P < 0.05), but not the expressions of Hes-1 and Hey induced by Notch1-knockdown (P > 0.05). Therefore, the activation of Notch pathway may be involved in the pathological process of PD-induced peritoneal fibrosis. Emodin may ameliorate the PD-related peritoneal fibrosis through inhibiting the activation of Notch pathway.

Key words: emodin; peritoneal dialysis; peritoneal fibrosis; Notch

收稿日期：2016-05-03　　录用日期：2016-08-29

通讯作者：林旭红　　E-mail: lxh80726@126.com

引用本文：

王慧超, 林旭红, 房晓鹏, 穆小云, 李铁军, 刘建林. 大黄素靶向Notch通路抗腹膜透析大鼠腹膜纤维化[J]. 生理学报 2016; 68 (6): 747-756.

WANG Hui-Chao, LIN Xu-Hong, FANG Xiao-Peng, MU Xiao-Yun, LI Tie-Jun, LIU Jian-Lin. [Emodin ameliorates the peritoneal dialysis-related peritoneal fibrosis via inhibiting the activation of Notch pathway.] [Article in Chinese]. Acta Physiol Sin 2016; 68 (6): 747-756 (in Chinese with English abstract).