P38MAPK信号通路抑制剂SB203580对肝细胞缺氧再灌注影响的实验研究

The effects of P38MAPK signaling pathway inhibitor SB203580 on hypoxia-reperfusion of liver cells

目的观察P38MAPK抑制剂SB203580在人肝癌细胞系HepG2细胞和人正常细胞系LO2细胞缺氧再灌注过程中的作用。方法实验分为正常对照组、缺氧对照组、SB203580+正常培养组、SB203580+缺氧培养组,缺氧培养24 h、复氧1 h后,分别应用Western Blot、MTT、划痕实验、Transwell实验、AnnexinV-FITC/PI双染实验检测细胞中P38蛋白表达情况和细胞增殖、迁移、侵袭和凋亡的变化。结果与正常对照组相比,缺氧培养组HepG2细胞的凋亡率、P38MAPK磷酸化水平增加;SB203580+缺氧培养组HepG2细胞的凋亡率相对于缺氧培养组增加,P38MAPK磷酸化水平降低;划痕实验48 h后,缺氧对照组HepG2细胞明显向划痕的中央迁移,而SB203580+缺氧培养组HepG2细胞迁移受到抑制;侵袭实验24 h后,SB203580+缺氧培养组HepG2细胞的侵袭能力较缺氧对照组降低(P<0.05);AnnexinV-FITC/PI双染实验显示与缺氧对照组相比,LO2细胞SB203580+缺氧培养组凋亡率降低,而HepG2细胞SB203580+缺氧培养组凋亡率则增加(P<0.01);MTT结果显示实验组HepG2细胞和LO2细胞增殖抑制率受到影响,并出现时间浓度依赖关系。结论 SB203580通过特异性阻断P38MAPK信号转导通路,对缺氧培养的正常肝细胞LO2细胞产生保护作用,同时对缺氧培养的肝癌细胞HepG2具有促进凋亡、抑制增殖以及抑制细胞迁移和降低侵袭能力的作用。

Objective To investigate the effects of P38MAPK inhibitor SB203580 on hypoxia-reperfusion process of human hepatoma cell line HepG2 cells and normal human cell lines LO2 cells.Methods Experiment were divided into normal control group,hypoxia control group,SB203580 ＋ normal group and SB203580 ＋ hypoxia group,hypoxia Cultured cells 24h and reoxygen 1h,applied Western Blot,MTT,scratch test,Transwell experiments and AnnexinV-FITC/PI staining to test the expression of P38 protein,cell proliferation,migration,invasion and apoptosis respectively.Results Compared with normal control group,the HepG2 cells apoptosis rate and P38MAPK phosphorylation levels of the hypoxia group were increased,while compared with hypoxia control group,the HepG2 cells apoptosis rate was increased and P38MAPK phosphorylation levels was decreased of the SB203580 ＋ hypoxia group; After 48h in scratch test,the control group of HepG2 cells moved to the center of the scratch,and SB203580 ＋ hypoxia group was inhibited; After 24h in migration assay,SB203580 ＋ hypoxia group of HepG2 cells＇ invasive ability was inhibited compared with the control group （P 〈 0.05） ;In Annexin V-FITC/PI assay,the cell apoptosis rates of the SB203580 ＋ hypoxia group showed a significant increase in HepG2 cells and decrease in LO2 cells when compared with control group （P 〈0.01） ;In MTT assay,SB203580 affected the HepG2 cells and LO2 cells＇ Proliferation inhibition rate,and depended on the time and concentration.Conclusion SB203580 through blocking P38MAPK signal transduction pathway specifically,have a protective effect on LO2 cells and promote the apoptosis,inhibit the proliferation and migration,reduce invasion capabilities on HepG2 cells.