脂氧素对巨噬细胞RAW264.7的抗炎实验研究

The preliminary study of LX A4 on the LPS-induced RAW264.7 cell line

目的研究脂氧素(LX)A4和叔丁氧羟基-苯丙氨酸-亮氨酸-苯丙氨酸-亮氨酸-苯丙氨酸(BOC-2)对LPS作用巨噬细胞RAW264.7存活的影响。方法取对数生长期的巨噬细胞RAW264.7为研究载体,实验用不同浓度的脂多糖(LPS)在不同时间点处理细胞,观察LX A4和BOC-2对LPS作用巨噬细胞RAW264.7后的存活率。CCK-8法观察LPS对各组巨噬细胞RAW264.7的不良反应,Western blot法检测LX A4和BOC-2对LPS处理后巨噬细胞RAW264.7的Toll样受体4(TLR4)和pNF-κB p65蛋白水平,酶联免疫吸附(ELISA)法检测LX A4和BOC-2对LPS处理后巨噬细胞RAW264.7培养上清液中白细胞介素6(IL-6)水平。结果在1 000ng/mL浓度LPS组,作用时间6h,巨噬细胞RAW264.7内TLR4蛋白水平和pNF-κB p65蛋白水平显著高于其余各组(P<0.05)。在LPS作用下,LX A4组细胞存活率显著高于对照组(P<0.05);BOC-2组在LPS作用后巨噬细胞RAW264.7的存活率显著低于无LPS作用(P<0.05)。在LPS作用下,LX A4组pNF-κB p65蛋白水平低于对照组及BOC-2组(P<0.05),BOC-2组pNF-κB p65蛋白水平高于其余各组(P<0.05)。在LPS作用下,LX A4组IL-6的水平低于对照组及BOC-2组(P<0.05)。结论 LX A4能够抑制LPS对巨噬细胞RAW264.7的作用及TLR4/NF-κB信号通路的激活,有助减轻炎性反应。

Objective To examine the effect of lipoxins（LX）A4and its antagonist（BOC-2）on the survival rate of RAW264.7macrophage.Methods RAW264.7were treated with different concentrations of LPS,and the cells were collected after2,4,6hrespectively,then we observed the effect of LX A4 and BOC-2on the survival rate of those cells.Cytotoxicity were detected by CCK-8method.The expression of TLR4 and pNF-κB p65 in RAW264.7were detected by Western blot.The expression of IL-6in cell supernatant was measured by ELISA.Results The protein expression of TLR4 and pNF-κB p65 treated with LPS for 6hin1 000ng/mL of LPS group were higher than those of other groups（P〈0.05）.In the present of LPS,the survival rate of macrophage in the LX A4 group was significantly higher than that of the control group（P〈0.05）.Meanwhile,in the BOC-2group,the survival rate of macrophage stimulated with LPS was significantly lower than the that of corresponding non-LPS group（P〈0.05）.Stimulated with LPS,the protein expression of pNF-κB p65 in the LX A4 group was significantly lower than those of the control group and the BOC-2group（P〈0.05）,and the protein expression of pNF-κB p65 in the BOC-2group was significantly higher than those of other groups（P〈0.05）.In the present of LPS,the concentration of IL-6in the LX A4 group was significantly lower than those of the control group and the BOC-2group（P〈0.05）.Conclusion LX A4 could inhibit the cytotoxicity of LPS and the activation of TLR4/NF-κB signaling pathway,then reduce the inflammation.