NADPH氧化酶介导血管紧张素Ⅱ诱导的腹膜间皮细胞转分化及细胞外基质积聚

目的 探讨NADPH氧化酶在血管紧张素(Ang)Ⅱ诱导的腹膜间皮细胞转分化以及细胞外基质积聚中的作用。 方法 体外培养SD大鼠原代腹膜间皮细胞，静止24 h后，随机分为以下4组：正常对照组，AngⅡ（10^-7 mol/L）组，AngⅡ+ Los(洛沙坦,10 μmol/L)组及AngⅡ+DPI（NADPH氧化酶活性抑制剂，10 μmol/L）组。应用荧光染料（DCF）及激光共聚焦显微镜检测细胞内活性氧（ROS）的产生。RT-PCR检测NADPH氧化酶亚单位p47phox以及纤溶酶原激活物抑制剂（PAI）1、α平滑肌肌动蛋白（SMA）、E钙黏蛋白（cadherin） mRNA的表达。Western印迹检测p47phox、α-SMA的蛋白表达。 结果 （1）外源性AngⅡ可显著增加大鼠腹膜间皮细胞ROS的产生，刺激15 min后，ROS 的表达较对照组上升了(3.64±0.53)倍。DPI和洛沙坦可显著抑制AngⅡ刺激后ROS的产生（P < 0.05）。（2）AngⅡ刺激腹膜间皮细胞后， NADPH氧化酶亚单位p47phox mRNA和蛋白的表达均呈上升趋势。洛沙坦和DPI可阻断由AngⅡ诱导的p47phox表达上调（P < 0.05）。（3） AngⅡ诱导α-SMA表达的上调以及 E-cadherin mRNA的下调, 洛沙坦和DPI可部分逆转AngⅡ的这种作用。（4）AngⅡ刺激8 h后可明显上调PAI-1的mRNA表达，为正常对照组的（3.06±0.77）倍。 洛沙坦和DPI可明显阻断PAI-1的表达上调（P < 0.05）。 结论 NADPH氧化酶依赖产生的ROS介导了AngⅡ诱导的腹膜间皮细胞转分化及细胞外基质积聚。阻断AngⅡ的作用及抑制NADPH氧化酶的表达和活性可作为防治腹膜纤维化的潜在治疗靶点。

NADPH oxidase mediates angiotensin Ⅱ-induced epithelial-mesenchymal transition and accumulation of extracellular matrix

Objective To investigate the role of NADPH oxidase-dependent reactive oxygen species(ROS) in angiotensin (Ang)Ⅱ-induced epithelial-mesenchymal transition (EMT) and accumulation of extracellular matrix(ECM) in rat peritoneal mesothelial cells (RPMC). Methods Primary rat peritoneal mesothelial cells were cultured in vitro. After synchronization for 24 hours, the cells were randomly assigned to four groups: the control group, the AngⅡ(10^-7 mol/L) group,the AngII type Ⅰ receptor antagonist losartan (10 μmol/L) treatment group(AngⅡ+losartan), and the NADPH oxidase inhibitor diphehylendieodonium (DPI) (10 μmol/L) treatment group (AngⅡ+DPI). The DCF-sensitive cellular ROS was measured by fluorometric assay and confocal microscopy. RT-PCR was employed to detect the mRNA expression for NADPH oxidase subunit p47phox and PAI-1, E-cadherin. α-smooth muscle actin and p47phox protein expression were examined by Western blot. Results (1)AngⅡ significantly induced the production of intracellular ROS by (3.64±0.53) folds compared with control (P < 0.05). DPI and losartan inhibited AngⅡ-induced ROS generation (P < 0.05). (2)AngⅡ stimulated NADPH oxidase subunit p47phox mRNA and protein overexpression. Both losartan and DPI inhibited the up-regulation of p47phox mRNA overexpression. (3)AngⅡ stimulated α-SMA mRNA and protein synthesis in RPMC and down-regulated E-cadherin mRNA expression, which were ameliorated by losartan and DPI. (4)AngⅡ significantly up-regulated PAI-1 mRNA expression after 8 h stimulation by (3.06±0.77) folds compared with control (P < 0.05), which was significantly down-regulated by losartan and DPI (P < 0.05). Conclusions NADPH oxidase-dependent ROS mediates EMT and the accumulation of ECM induced by AngⅡ in peritoneal mesothelial cells. AngⅡ and NADPH oxidase may be the potential therapeutic targets in the progress of peritoneal fibrosis.