高渗盐水影响小胶质细胞释放单核细胞趋化蛋白1的研究

Hypertonic saline decreases monocyte chemotactic protein 1 expression in microglia may be through inhibition of p38 MAPK signaling pathway

目的 在体外细胞实验中探讨高渗盐水（hypertonic saline,HS）是否影响小胶质细胞的激活及释放单核细胞趋化蛋白-1 （monocyte chemotactic protein 1,MCP-1）,以及其潜在的机制.方法 体外混合培养原代胶质细胞,利用摇床震摇的方法纯化分离小胶质细胞,分为对照组、缺氧＋无糖培养基组（简称缺氧组）、缺氧＋无糖培养基＋ 100 mmol/L HS组（简称HS组）及SB203580组（p38信号通路特异抑制剂）.除对照组外,其他各组均进行氧糖剥夺（oxygen glucose deprivation,OGD）,利用免疫荧光及ELISA方法检测HS对小胶质细胞生成及释放MCP-1的影响,并使用Western blot方法检测HS是否可影响p38丝裂原活化蛋白激酶（p38 MAPK）蛋白的磷酸化水平.数据采用单因素方差分析,用LSD法进行组间两两比较,P＜0.05为差异具有统计学意义.结果 免疫荧光实验显示缺氧组与对照组比较,MCP-1表达明显增高;HS与缺氧组比较,MCP-1表达明显减少;ELISA检测发现缺氧4h后,SB203580组及HS组与缺氧组比较,细胞培养基上清中MCP-1含量（pg/mL）明显减少;SB203580组与HS组间差异无统计学意义（对照组：107.956＆#177;8.921;缺氧组：173.467＆#177;9.027; HS组：145.957＆#177;10.952; SB203580组：136.279＆#177;15.63; P＜0.05）;western blot显示HS组各时间点与缺氧组各相应时间点相比,p38磷酸化水平均明显降低（对照组：0.590＆#177;0.105;缺氧组：30 min,1.553＆#177;0.215;1 h,1.212＆#177;0.161;2 h,1.179＆#177;0.105;4 h,1.178＆#177;0.122.HS组：30 min,1.028＆#177;0.168;1 h,0.852＆#177;0.112;2 h,0.927＆#177;0.121; 4h,0.815＆#177;0.107; P＜0.05）.结论 100 mmol/L HS可能通过抑制p38信号通路的激活来抑制小胶质细胞的激活,从而减少小胶质细胞释放趋化因子MCP-1.

Objective To explore whether hypertonic saline （HS） could affect the microglial activation and release of monocyte chemotactic protein 1 （MCP-1） in vitro experiments,and to study the mechanism.Methods Microglia isolated from primary mixed glia cells by shaking method were divided into four groups：control group,hypoxia ＋ glucose free medium group （hypoxia group）,100 mmol/L HS ＋ hypoxia ＋ glucose free medium group （HS group） and SB203580 （a specific inhibitor of p38 MAPK） group.Oxygen-glucose deprivation （OGD） was performed in all experiment groups but control group.Double immunofluorescence and ELISA were used to determine whether HS could decrease the production of MCP-1 after OGD,and western blot was employed to determine the effect of HS on the level of phosphorylated p38 MAPK.Results Double immunofluorescence showed that 100 mmol/L HS attenuated MCP-1 level in HS group after treatment with 100 mmol/L HS compared with hypoxia group,and the level of MCP-1 in cultured medium released by microglia was significantly decreased after 100 mmol/L HS or SB203580 treatment for 4 h.There was no significant difference in MCP-1 between HS group and SB203580 group （P 〉 0.05）,but compared with control group （107.956 ＆#177; 8.921）,there were higher levels of MCP-1 in hypoxia group （173.467 ＆#177;9.027）,HS group：（145.957 ＆#177; 10.952）,and SB203580 group （136.279 ＆#177; 15.63）,P 〈0.05.At 30 min,1 h,2 h and 4 h after hypoxia,western blot showed that the levels of phosphorylated p38 MAPK markedly decreased in HS group as compared to the hypoxia group at corresponding intervals （control group：0.590＆#177;0.105.Hypoxia group：30 min,1.553 ＆#177;0.215; 1 h,1.212＆#177;0.161; 2 h,1.179 ＆#177; 0.105; 4 h,1.178 ＆#177;0.122.HS group：30 min,1.028 ＆#177;0.168; 1 h,0.852 ＆#177;0.112; 2 h,0.927 ＆#177;0.121; 4 h,0.815 ＆#177; 0.107; P 〈 0.05）.Conclusions HS decreasing MCP-1 level in microglia after hypoxia may be attributed to the inhibition of p38 MAPK signaling pathway.