摘要/Abstract

摘要： 目的·利用HepG2细胞建立D-氨基半乳糖（D-galactosamine，D-GalN）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，rhIL-1Ra）对肝细胞的保护作用。方法·建立D-GalN诱导HepG2细胞损伤模型，将HepG2细胞放置于含有不同浓度的D-GalN培养基中（0.02、0.2、2、4 mg/mL），在第1、2和3日分别观察细胞形态和活力变化，优化D-GalN浓度和处理时间；研究不同IL-1Ra浓度（10、20、50 μg/mL）处理的D-GalN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，qRT-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 mRNA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/mL浓度的D-GalN处理HepG2细胞2 d后，细胞活力显著下降；rhIL-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-GalN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·rhIL-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。
关键词: HepG2细胞, 肝损伤, rhIL-1Ra, D-GalN, 活性氧簇, ERK1/2
Abstract:
Objective · To evaluate protective effects of recombinant human IL-1Ra (rhIL-1Ra) on acute liver injury in vitrousing D-galactosamine (D-GalN) and HepG2 cells to establish the D-galactosamine (D-GalN)-induced HepG2 cells injury models. Methods · Models of HepG2 cells injury inducedD-GalN was established. HepG2 cells were maintained in mediums which contained different concentration of D-GalN (0.02, 0.2, 2 or 4 mg/mL) for different time (1, 2 or 3 d). Optimized concentration and time of D-GalN were used to analyze cell viability and morphology. A serial dose of rhIL-1Ra (10, 20 or 50 μg/mL) was used to treat HepG2 cells which were challenged with D-GalN. 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 mRNA 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-1Ra protecting hepatocytes or not. Results · Cell viability was significantly decreasedD-GalN whose concentration was 4 mg/mL in HepG2 cells after 2 d. Compared with the control group, rhIL-1Ra could significantly improve cell survival and down-regulate the level of ROS in the cells. RhIL-Ra also could suppress of pro-apoptotic cytokines factors (IL-1β, IL-6 and TNF-α) inducedD-GalN in HepG2 cells. The results also showed that erk1/2 signaling pathways have certain effect on mediating the injury of rhIL-1Ra to protect hepatocytes. Conclusion · RhIL-1Ra can protect hepatocytes toxinsdirectly targeting hepatocytes and inhibit cells apoptosisactivating ERK1/2 pathway in HepG2 cells.
Key words: HepG2, liver injury, rhIL-1Ra, D-GalN, reactive oxygen species, ERK1/2


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