丹皮酚干预咪喹莫特诱导的巨噬细胞死亡和自噬的作用研究

Effect of paeonol on imiquimod-induced macrophage death and autophagy

目的探讨咪喹莫特诱导Raw264.7巨噬细胞自噬和死亡的发生及丹皮酚的干预保护作用。方法将Raw264.7巨噬细胞分为5组:正常组、模型组和低、中、高3个剂量实验组。正常组加DMEM培养液;模型组用终浓度为10μg·mL^-1咪喹莫特处理RAW264.7巨噬细胞24 h;低、中、高3个剂量实验组分别用终浓度为1,10,100μmol·L^-1的丹皮酚预先加入2 h后,再加入10μg·mL^-1咪喹莫特处理24 h。用CCK8法检测细胞存活率,以蛋白质印迹法检测自噬相关蛋白[微管相关蛋白1轻链3-Ⅰ(LC3-Ⅰ)、微管相关蛋白1轻链3-Ⅱ(LC3-Ⅱ)、自噬相关蛋白5(ATG5)]的表达。结果空白组、模型组和低、中、高3个剂量实验组的细胞存活率分别为(100.00±0.04)%,(38.68±0.03)%,(47.02±0.06)%,(80.48±0.06)%和(54.12±0.04)%;模型组与空白组相比或者低、中、高剂量3个实验组与模型组相比,差异均有统计学意义(均P<0.05),其中中剂量实验组效果最佳。空白组、模型组和中剂量实验组的LC3Ⅱ/LC3Ⅰ的比值分别为0.88±0.15,1.27±0.04和0.97±0.03;这3组的ATG5的相对表达量分别为0.65±0.02,0.95±0.02和0.46±0.06;模型组与正常组相比,差异均有统计学意义(均P<0.05);中剂量实验组与模型组相比,差异均有统计学意义(P<0.05,P<0.01)。结论丹皮酚对咪喹莫特诱导的RAW264.7巨噬细胞自噬和死亡具有保护作用。

Objective To investigate the occurrence of autophagy and death of Raw264.7 macrophages induced by imiquimod and the protective effect of paeonol.Methods Raw264.7 macrophages were divided into five groups:normal group,model group and experimental-L,-M,-H groups.The normal group was treated with DMEM,and the model group was treated with 10μg·mL^-1 imiquimod for 24 h with Raw264.7 macrophages;for experimental-L,-M,-H,groups,paeonol with a final concentration of 1,10,100μmol·L-1 was pre-added for 2 h and then 10μg·mL^-1 imiquimod was added for 24 h.CCK8 method was used to detect the survival rate.Western blot method was used to detect autophagy related proteins[microtubule-associated proteins light chain 3-Ⅰ(LC3-Ⅰ),microtubule-associated proteins light chain 3-Ⅱ(LC3-Ⅱ),autophagy related gene 5(ATG5)]expression.Results The cell survival rates in normal group,model group and experimental-L,-M,-H groups were respectively(100.00±0.04)%,(38.68±0.03)%,(47.02±0.06)%,(80.48±0.06)%,(54.12±0.04)%.Compared between model group and normal group or experimental-L,-M,-H groups and model group,the differences were significant(P<0.05).Among experimental groups,the expression-M group was the best.Ratios of LC3Ⅱ/LC3Ⅰproteins in normal group,model group and experimental-M group were 0.88±0.15,1.27±0.04,and 0.97±0.03;the relative expression of ATG5 in the three groups were 0.65±0.02,0.95±0.02,and 0.46±0.06,respectively.Compared between model group and normal group,the differences of the factors were significant(all P<0.05);compared between experimental-M group and model group,the difference of the factors were significant(P<0.05,P<0.01).Conclusion Paeonol can protect Raw264.7 macrophages from autophagy and death induced by imiquimod.