摘要
目的·利用HepG2细胞建立D-氨基半乳糖(D-galactosamine,D-Gal N)体外肝细胞损伤模型,研究重组人白介素1受体拮抗剂(recombinant human IL-1Ra,rh IL-1Ra)对肝细胞的保护作用。方法·建立D-Gal N诱导HepG2细胞损伤模型,将HepG2细胞放置于含有不同浓度的D-Gal N培养基中(0.02、0.2、2、4 mg/m L),在第1、2和3日分别观察细胞形态和活力变化,优化D-Gal N浓度和处理时间;研究不同IL-1Ra浓度(10、20、50μg/m L)处理的D-Gal N损伤模型细胞形态变化,测定细胞活力;分析各组细胞凋亡情况和各组胞内活性氧簇(reactive oxygen species,ROS)水平,运用荧光实时定量PCR(quantitative real-time PCR,q RT-PCR)检测各组细胞中凋亡诱导相关细胞因子白介素1β(interleukin-1β,IL-1β)、IL-6 (interleukin-6,IL-6)和肿瘤坏死因子α(tumor necrosis factor-α,TNF-α)的m RNA水平,并采用ERK1/2通路抑制剂(SCH772984)检测IL-1Ra是否是通过促进肝损伤细胞ERK1/2磷酸化的方式来保护肝细胞。结果·采用4 mg/m L浓度的D-Gal N处理HepG2细胞2 d后,细胞活力显著下降;rh IL-1Ra显著提高损伤模型细胞的存活率,减少细胞中ROS的生成,显著抑制D-Gal N诱导的细胞内凋亡诱导相关细胞因子的表达;ERK1/2信号途径在介导IL-1Ra保护肝细胞的损伤过程中有一定的作用。结论·rh IL-1Ra可直接靶向肝细胞,并通过在细胞内上调ERK1/2信号通路的活化水平,抑制细胞凋亡,发挥对肝损伤细胞的保护作用。
Objective To evaluate protective effects of recombinant human IL-1 Ra(rh IL-1 Ra) on acute liver injury in vitro by using D-galactosamine(D-Gal N) and HepG2 cells to establish the D-galactosamine(D-Gal N)-induced HepG2 cells injury models. Methods Models of HepG2 cells injury induced by D-Gal N was established. HepG2 cells were maintained in mediums which contained different concentration of D-Gal N(0.02, 0.2, 2 or 4 mg/m L) for different time(1, 2 or 3 d). Optimized concentration and time of D-Gal N were used to analyze cell viability and morphology. A serial dose of rh IL-1 Ra(10, 20 or 50 μg/m L) was used to treat HepG2 cells which were challenged with D-Gal N. Cell apoptosis and the levels of intracellular reactive oxygen species(ROS) were analyzed in different treatment groups. Real-time PCR was employed to analyze the m RNA levels of IL-1β, IL-6 and TNF-α in cells. ERK1/2 inhibitor(SCH772984) was used to confirm whether ERK1/2 phosphorylation played a critical role in IL-1 Ra protecting hepatocytes or not. Results Cell viability was significantly decreased by D-Gal N whose concentration was 4 mg/m L in HepG2 cells after 2 d. Compared with the control group, rh IL-1 Ra could significantly improve cell survival and down-regulate the level of ROS in the cells. Rh IL-Ra also could suppress expression of pro-apoptotic cytokines factors(IL-1β, IL-6 and TNF-α) induced by D-Gal N in HepG2 cells. The results also showed that erk1/2 signaling pathways have certain effect on mediating the injury of rh IL-1 Ra to protect hepatocytes. Conclusion Rh IL-1 Ra can protect hepatocytes from toxins by directly targeting hepatocytes and inhibit cells apoptosis by activating ERK1/2 pathway in HepG2 cells.
作者
郑滢
肖欣怡
杨卓一
周美琪
陈会
袁运生
ZHENG Ying;XIAO Xin-yi;YANG Zhuo-yi;ZHOU Mei-qi;CHEN Hui;YUAN Yun-sheng(Engineering Research Center of Cell and Therapeutic Antibody,School of Pharmacy,Shanghai Jiao Tong University,Shanghai 200240,China)
出处
《上海交通大学学报（医学版）》
CAS
CSCD
北大核心
2019年第10期1115-1121,共7页
Journal of Shanghai Jiao tong University:Medical Science
基金
国家自然科学基金（31671388,81302825）
上海市浦江人才计划（16PJ1405000）
上海交通大学医工交叉项目（YG2015MS64）~~
