Department of Internal medicine, Gil Medical Center, Gachon University College of Medicine, Incheon, Korea.
Gachon Medical Research Institute, Gachon University Gil Medical Center, Incheon, Korea.
J Gastroenterol Hepatol. 2020 Jun;35(6):1078-1087. doi: 10.1111/jgh.14979. Epub 2020 Jan 23.
Exogenous 8-hydroxydeoxyguanosine (8-OHdG) was suggested as an inhibitor of Rac1 and NADPH oxidase (NOX). The aim of this study was to evaluate the effects of the exogenous 8-OHdG on hepatic fibrogenesis in vitro and in vivo model of liver fibrosis.
Adult Sprague-Dawley rats were allocated to sham-operated rats (n = 7), rats that underwent bile duct ligation (BDL) (n = 6), and BDL rats treated with 8-OHdG (60 mg/kg/day by gavage, n = 6). All rats were sacrificed on day 21. Double immunofluorescence staining between either NOX1 or NOX2 and α-smooth muscle actin (SMA) in liver was performed. Hepatic fibrotic contents were assessed by hydroxyproline assay and quantified by Sirius red staining. In vitro, hepatic stellate cell (HSC) line LX-2 and HHSteC cells were stimulated by angiotensin II (10 μM). The reactive oxygen species (ROS) production was measured by confocal microscopy. The expressions of NOX1, NOX2, α-SMA, transforming growth factor (TGF)-β1, and collagen Iα were analyzed by quantitative real-time polymerase chain reaction or immunoblotting.
The 8-OHdG treatment in BDL rats reduced the NOX1 and NOX2 protein expression, which overlapped with α-SMA compared with BDL rats. The 8-OHdG treatment in BDL rats significantly decreased the mRNA expression of NOX1, NOX2, α-SMA, TGF-β1, and collagen Iα, and fibrotic contents. Increases of ROS production, Rac1 activation, NOX1, NOX2, and fibronectin expression induced by angiotensin II in HSCs were attenuated by 8-OHdG.
Rac1 activation and NOX-derived ROS are implicated to liver fibrosis. The 8-OHdG ameliorates liver fibrosis through the inhibition of Rac1 activation and NOX-derived ROS.
外源性 8-羟基脱氧鸟苷（8-OHdG）被认为是 Rac1 和 NADPH 氧化酶（NOX）的抑制剂。本研究旨在评估外源性 8-OHdG 在体外和体内肝纤维化模型中对肝纤维化的影响。
成年 Sprague-Dawley 大鼠分为假手术组（n=7）、胆管结扎组（BDL，n=6）和 BDL 大鼠用 8-OHdG 治疗组（60mg/kg/天灌胃，n=6）。所有大鼠于第 21 天处死。行肝组织中 NOX1 或 NOX2 与α-平滑肌肌动蛋白（α-SMA）的双重免疫荧光染色。羟脯氨酸测定法评估肝纤维化含量，并通过天狼猩红染色进行定量。在体外，用血管紧张素 II（10μM）刺激肝星状细胞（LX-2）和 HHSteC 细胞。通过共聚焦显微镜测量活性氧（ROS）的产生。用定量实时聚合酶链反应或免疫印迹法分析 NOX1、NOX2、α-SMA、转化生长因子（TGF）-β1 和胶原 Iα 的表达。
与 BDL 大鼠相比，8-OHdG 治疗可减少 BDL 大鼠的 NOX1 和 NOX2 蛋白表达，且与 α-SMA 重叠。8-OHdG 治疗可显著降低 BDL 大鼠的 NOX1、NOX2、α-SMA、TGF-β1 和胶原 Iα 的 mRNA 表达和纤维化含量。8-OHdG 可减弱血管紧张素 II 诱导的 HSCs 中 ROS 产生、Rac1 激活、NOX1、NOX2 和纤维连接蛋白表达的增加。
Rac1 激活和 NOX 衍生的 ROS 与肝纤维化有关。8-OHdG 通过抑制 Rac1 激活和 NOX 衍生的 ROS 改善肝纤维化。
