摘要
目的探讨华支睾吸虫溶血磷脂酶(Cs LysoPLA)对肝星状细胞的作用。方法实验分为4组:PBS对照组为PBS孵育肝星状细胞系LX-2细胞24 h;Cs LysoPLA单独刺激组为Cs LysoPLA(10μg/mL)单独孵育LX-2细胞24 h;联合刺激组为不同浓度Cs LysoPLA(1、5、10μg/mL)孵育LX-2细胞2 h后,IL-13(20 ng/mL)孵育24 h;IL-13单独刺激组为IL-13(20 ng/mL)单独孵育LX-2细胞24 h;逆转录荧光定量PCR(qRT-PCR)检测星状细胞活化指标分子α-平滑肌肌动蛋白(α-SMA)以及纤维化相关分子胶原蛋白Ⅰ型(Collagen-Ⅰ)、胶原蛋白Ⅲ型(Collagen-Ⅲ)以及促纤维化分子转化生长因子β1(TGF-β1)、血小板源性生长因子(PDGF)的mRNA水平。Western Blotting检测不同浓度Cs LysoPLA(1、5、10μg/mL)联合IL-13(20 ng/mL)刺激时LX-2细胞α-SMA蛋白表达量。结果Cs LysoPLA(10μg/mL)可直接抑制LX-2细胞α-SMA、Collagen-Ⅲ的转录,与PBS对照组相比差异有统计学意义(P=0.0142、0.0027);并且Cs LysoPLA(10μg/mL)可刺激LX-2细胞TGF-β1、PDGF基因转录升高,与PBS对照组相比差异有统计学意义(P=0.0091、0.0034);Cs LysoPLA(10μg/mL)可以下调IL-13引起的α-SMA、Collagen-Ⅰ、Collagen-Ⅲ、TGF-β1的mRNA水平的升高,与单独IL-13刺激组相比差异有统计学意义(P=0.0030、0.0119、0.0430、0.0256);Western Bloting结果显示,不同浓度Cs LysoPLA(1、5、10μg/mL)均可以下调IL-13引起的星状细胞活化分子α-SMA的表达,与单独IL-13刺激组相比差异有统计学意义(P=0.0080、0.0005、0.0002)。结论Cs LysoPLA(10μg/mL)在高浓度下既可以直接抑制LX-2细胞纤维化,又可以抑制IL-13引起的LX-2细胞纤维化,可能在华支睾吸虫严重感染所致纤维化过程中发挥了抑制纤维化的作用。
Objective To investigate the in vitro effects of Clonorchis sinensis lysophospholipase(Cs LysoPLA)on hepatic stellate cell line LX-2.Methods There were four groups,LX-2 cells incubated with PBS for 24 h,LX-2 cells treated with Cs LysoPLA(10μg/mL)for 24 h,LX-2 cells administrated with Cs LysoPLA(1,5,or 10μg/mL)for 2 h followed with interleukin-13(20 ng/mL)for 24 h,LX-2 cells stimulated with interleukin-13(20 ng/mL)for 24 h.The relative mRNA levels of active marker of hepatic stellate cell,alpha smooth muscle actin(α-SMA),and hepatic fibrosis related molecules such as type I collagen(CollagenⅠ),typeⅢcollagen(CollagenⅢ),transforming growth factor-beta1(TGF-β1)and platelet-derived growth factor(PDGF)were quantified by real-time fluorescence quantitative polymerase chain reaction(qRT-PCR).Expression ofα-SMA was semi-quantified by western blotting.Results qRT-PCR showed that mRNA levels ofα-SMA and Collagen-Ⅲin LX-2 cells were down regulated by 10μg/mL of Cs LysoPLA compared with PBS treated group(P=0.0142,P=0.0027).In addition,Cs LysoPLA(10μg/mL)could stimulate transcriptions of TGF-β1 and PDGF LX-2,compared with PBS incubated group(P=0.0091,P=0.0034).Cs LysoPLA(10μg/mL)could down-regulate mRNA levels ofα-SMA,CollagenⅠ,CollagenⅢand TGF-β1 induced by IL-13(P=0.0030,P=0.0119,P=0.0430,and P=0.0256,respectively).Cs LysoPLA(1,5,and 10μg/mL)could down-regulate the protein level ofα-SMA(P=0.0080,P=0.0005,and P=0.0002,respectively).Conclusions Cs LysoPLA could directly suppress activation of LX-2 and levels of hepatic fibrosis related molecules in LX-2.Moreover,Cs LysoPLA could down-regulate the activation of LX-2 and levels of hepatic fibrosis related molecules in LX-2 induced by IL-13.
作者
侯珠秀
黄艳
余新炳
HOU Zhu xiu;HUANG Yan;YU Xin bing(Department of Parasitology of Zhongshan School of Medicine,Sun Yat sen University,Guangzhou,Guangdong 510080;Key Laboratory of Tropical Disease Control(SYSU),Ministry of Education,Guangzhou,Guangdong 510080;Provincial Engineering Technology Research Center for Biological Vector Control,Guangzhou,Guangdong 510080,China)
出处
《热带医学杂志》
CAS
2019年第6期674-677,694,共5页
Journal of Tropical Medicine
基金
国家自然科学基金(81772212).
关键词
华支睾吸虫
溶血磷脂酶
白细胞介素-13
肝纤维化
Clonorchis sinensis
Lysophospholipase
Interleukin-13
Hepatic fibrosis related molecules
作者简介
侯珠秀(1992-),女,硕士研究生,研究方向为华支睾吸虫致病机制;通讯作者:余新炳,E mail:yuxb@mail.sysu.edu.cn;通讯作者:黄艳,E mail:huang66@mail.sysu.edu.cn
