邻苯二甲酸二乙基己酯(DEHP)通过调控Wnt/β-catenin信号通路加重肝纤维化的机制研究  

Study on the mechanism of di-(2-ethylhexyl)phthalate(DEHP)aggravating liver fibrosis by regulating Wnt/β-catenin signaling pathway

作　　者：赵宗彪 李向阳 王法财 李鹏飞 徐维平[2] 沈炳香 ZHAO Zongbiao;LI Xiangyang;WANG Facai;LI Pengfei;XU Weiping;SHEN Bingxiang(Department of Pharmacy,the Lu'an Hospital Affiliated to Anhui Medical University(the Lu'an People's Hospital),Lu'an 237005;The First Affiliated Hospital of University of Science and Technology of China,Hefei 230000)

机构地区：[1]安徽医科大学附属六安医院(六安市人民医院)药学部,六安237005 [2]中国科学技术大学附属第一医院,合肥230000

出　　处：《环境科学学报》2022年第5期465-474,共10页Acta Scientiae Circumstantiae

基　　金：2016年国家自然科学基金(No.51672004);2016年安徽省自然科学基金(No.1608085MH176)。

摘　　要：基于Wnt/β-catenin信号通路探究邻苯二甲酸二乙基己酯(DEHP)在加重肝纤维化中的调控机制.选取60只雄性SD大鼠,随机分成6组:正常组、CCl_(4)造模组、CCl_(4)+DEHP(0.05、5和500 mg·kg^(-1))暴露组和单独DEHP暴露(500 mg·kg^(-1))对照组.通过皮下注射50%CCl_(4)(0.1 m L·100 g^(-1))诱导肝纤维化模型,每周2次,同时从造模开始给予DEHP灌胃,共9周.采用HE和MASSON染色观察肝脏组织病理学变化和胶原沉积情况;采用ELISA法检测血清中透明质酸(HA)、层粘连蛋白(LN)、三型前胶原(PCⅢ)和Ⅳ型胶原(Ⅳ-C)的水平;免疫组化法和Western Blot法检测肝脏组织中Wnt/β-catenin信号通路蛋白的表达水平;体外将人肝星状细胞(LX-2)分为正常组,DEHP刺激组(50μmol·L_(-1)),DEHP刺激组(100μmol·L_(-1))及DEHP(100μmol·L_(-1))+FH535(1μmol·L_(-1))(Wnt/β-catenin信号通路阻断剂)组,共培养24 h,RT-PCR法检测各组细胞中β-catenin和α-SMA m RNA的表达水平.体内实验结果显示,与正常组相比,CCl_(4)模型组中肝纤维化指标(HA、LN、PCⅢ、Ⅳ-C)的水平、肝组织病理损伤、胶原沉积以及肝组织中Wnt1、β-catenin蛋白的表达水平明显升高.与CCl_(4)模型组相比,随着DEHP暴露剂量的增加,肝纤维指标(HA、LN、PCⅢ、Ⅳ-C)及肝脏的病理损伤显著加重,并上调Wnt1、β-catenin的表达,尤其在CCl_(4)+DEHP(500 mg·kg^(-1))组中较为明显;体外细胞实验结果显示,与正常组相比,DEHP(50、100μmol·L_(-1))两个剂量组均能显著刺激LX-2细胞中β-catenin和α-SMA m RNA的表达,给予FH535(1μmol·L_(-1))(Wnt/β-catenin信号通路阻断剂)后,β-catenin和α-SMA m RNA表达水平明显降低,差异具有统计学意义,p<0.05.DEHP能显著加重肝纤维化大鼠的病理损伤和纤维化的形成,其机制可能与介导的Wnt/β-catenin信号通路有关.To explore the regulatory mechanism of di-(2-ethylhexyl)phthalate(DEHP)in aggravating liver fibrosis based on Wnt/β-catenin signaling pathway.Sixty male SD rats were selected and randomly divided into six groups:normal group,CCl_(4)model,CCl_(4)+DEHP(0.05,5 and 500 mg·kg^(-1))exposure group and DEHP alone(500 mg·kg^(-1))exposure control group.The liver fibrosis model was induced by subcutaneous injection of 50%CCl_(4)(0.1 m L·100g^(-1))twice a week,and DEHP was administered intragastrically for 9 weeks from the beginning of the model establishment.HE and MASSON staining were used to observe liver histopathological changes and collagen deposition;ELISA was used to detect the level of serum hyaluronic acid(HA),laminin(LN),type III procollagen(PCⅢ)and type IV collagen(Ⅳ-C);Immunohistochemical method and Western Blot were applied to explore the proteins expression of Wnt/β-catenin signaling pathway in liver tissue;In vitro experiment,human hepatic stellate cells(LX-2)were divided into normal group and DEHP stimulation group(50μmol·L_(-1)),DEHP stimulation group(100μmol·L_(-1))and DEHP(100μmol·L_(-1))+FH535(1μmol·L_(-1))(Wnt/β-catenin signaling pathway blocker),then cells were co-cultured for 24 h respectively.The m RNA expression ofβ-catenin andα-SMA in each group was detected by RT-PCR.In vivo results showed that compared with the normal group,the levels of liver fibrosis indexes(HA,LN,PCⅢ,Ⅳ-C),liver pathological damage,collagen deposition,and the expression levels of Wnt1 andβ-catenin in liver tissue were significantly increased in the CCl_(4)model group.Compared with CCl_(4)model group,liver fiber indexes(HA,LN,PCⅢ,Ⅳ-C)and liver pathological damage were significantly aggravated with the increase of DEHP exposure dose,and the expression of Wnt1 andβ-catenin was up-raised,especially in CCl_(4)+DEHP(500 mg·kg^(-1))group;in vitro cell experiment results showed that compared with the normal group,DEHP 50μmol·L_(-1)and 100μmol·L_(-1)could both significantly stimulate the expression of�

关 键 词：邻苯二甲酸二乙基己酯(DEHP) 肝纤维化 WNT/Β-CATENIN 

分 类 号：X171.5[环境科学与工程—环境科学] X592