自噬在缺氧条件下视网膜色素上皮细胞表达趋化因子IL-8、MCP-1中的作用

Role of autophagy in expression of IL-8 and MCP-1 in retinal pigment epithelium cells under hypoxia

目的研究自噬在缺氧条件下视网膜色素上皮(RPE)细胞表达趋化因子白细胞介素8(IL-8)和单核细胞趋化蛋白-1(MCP-1)中发挥的作用。方法将培养良好的ARPE-19细胞分为对照组、缺氧组、3-甲基腺嘌呤(3-MA)+缺氧组和氯喹(CQ)+缺氧组。对照组细胞在常氧条件下培养;缺氧组细胞在缺氧箱中培养;3-MA+缺氧组和CQ+缺氧组分别先用10 mM 3-MA或50μM CQ预处理细胞2 h,然后置于缺氧箱中继续培养。24 h后采用ELISA法检测细胞上清液中趋化因子IL-8、MCP-1的浓度;采用Western blot法检测细胞的自噬关键蛋白微管相关蛋白1轻链3 B(LC3B)、Beclin-1及p62的相对表达水平。对4组细胞表达的IL-8、MCP-1浓度和LC3B-II/I、Beclin-1及p62水平进行统计分析。结果4组中IL-8、MCP-1浓度组间比较,差异均有统计学意义(IL-8:F=539.27,P=0.000;MCP-1:F=56.16,P=0.000);两两比较发现,缺氧组的IL-8、MCP-1浓度明显高于其它3组,差异均有统计学意义(IL-8:均P=0.000;MCP-1:缺氧组vs.对照组P=0.000;缺氧组vs.3-MA组P=0.001;缺氧组vs.CQ组P=0.000)。4组细胞中的LC3B-II/I、Beclin-1及p62的相对蛋白表达量组间比较,差异均有统计学意义(LC3B-II/I:F=80.65,P=0.000;Beclin-1:F=139.29,P=0.000;p62:F=70.64,P=0.000)。两两比较发现,缺氧组细胞中LC3B-II/I的相对蛋白表达量明显高于对照组和3-MA组(缺氧组vs.对照组P=0.000;缺氧组vs.3-MA组P=0.001);缺氧组中LC3B-II/I的相对蛋白表达量低于CQ组,差异有统计学意义(P=0.000);缺氧组细胞中Beclin-1的相对蛋白表达量均明显高于其它3组(缺氧组vs.对照组P=0.000;缺氧组vs.3-MA组P=0.001;缺氧组vs.CQ组P=0.000);缺氧组细胞中p62相对蛋白表达量明显低于其它3组,差异均有统计学意义(P<0.05)。结论在缺氧条件下,自噬水平升高可促进RPE细胞趋化因子IL-8和MCP-1的表达。

OBJECTIVE To investigate the role of autophagy in the expression of interleukin(IL)-8 and monocyte chemokine protein(MCP)-1 in retinal pigment epithelium(RPE)cells under the hypoxia conditions.METHODS Well cultured ARPE-19 cells were divided into control group,hypoxia group,3-methyladenine(3-MA)+hypoxia group and chloroquine(CQ)+hypoxia group.The cells in the control group were cultured under the normoxia conditions,the cells in the hypoxia group were cultured in the hypoxia box.The cells in the 3-MA+hypoxia group and the CQ+hypoxia group were pretreated with 10 mM 3-MA or 50μM CQ for two hours respectively,and then were placed in the hypoxic box for further culture.After 24 hours,the concentrations of IL-8 and MCP-1 in the supernatant were detected by ELISA.The relative expression levels of autophagy-associated key proteins,which included microtubule associated protein 1 light chain 3B(LC3B),Beclin-1 and p62,were detected by Western blot method.The concentrations of IL-8,MCP-1 as well as the level of LC3B-II/I,Beclin-1and p62 expressed in the four groups were statistically analyzed.RESULTS The concentration of IL-8 and MCP-1 among the four groups were compared in general,and the differences were statistically significant(IL-8:F=539.27,P=0.000;MCP-1:F=56.16,P=0.000).By pairwise comparison,the concentration of IL-8 and MCP-1 in the hypoxia group was significantly higher than those in other three groups,and the differences were statistically significant(IL-8:all P=0.000;MCP-1:hypoxia group vs control group P=0.000;hypoxia group vs.3-MA group P=0.001;hypoxia group vs.CQ group P=0.000).The relative protein expression of LC3B-II/I and Beclin-1 among the four groups of cells were compared in general,and the differences were statistically significant(LC3B-II/I:F=80.65,P=0.000;Beclin-1:F=139.29,P=0.000;p62:F=70.64,P=0.000).By pairwise comparison,the relative protein expression of LC3B-II/I in the hypoxia group was significantly higher than that in control group and 3-MA group(hypoxia group vs.control group P=0.000,hypoxia group vs.3-MA group P=0.001).The relative protein expression of LC3B-II/I in the hypoxia group was lower than that in CQ group,the differences were statistically significant(P=0.000).The relative protein expression of Beclin-1 in the hypoxia group of cells was significantly higher than that in the other three groups(hypoxia group vs.control group P=0.000,hypoxia group vs.3-MA group P=0.001;hypoxia group vs.CQ group P=0.000).The relative protein expression of p62 in the hypoxia group of cells was significantly lower than that in other three groups,and the differences were statistically significant(P<0.05).CONCLUSIONS Under the condition of hypoxia,elevated autophagy level could promote the expression of chemokines IL-8 and MCP-1 in RPE cells.