ox-LDL诱导的Raw264.7细胞炎症因子表达变化及PPARγSer273磷酸化调控作用观察

Expression changes of inflammatory cytokines in mouse monocytic macrophage leukemia cell line Raw264.7 induced by oxidized low-density lipoprotein and regulation of PPARγSer273 phosphorylation

目的观察氧化低密度脂蛋白(ox-LDL)诱导的小鼠单核巨噬细胞白血病细胞株Raw264.7炎症因子的表达变化及PPARγSer273磷酸化的调控作用,探讨PPARγSer273磷酸化在动脉粥样硬化(AS)中的作用。方法取Raw264.7细胞并分为ox-LDL组和对照组,ox-LDL组以50 mg/L ox-LDL诱导,对照组不予处理,采用ELISA法检测两组细胞培养液中的TNF-α、IL-1β,Western blot法检测细胞中过氧化物酶体增殖物激活受体γ(PPARγ)和PPARγSer273磷酸化蛋白,计算PPARγSer273磷酸化与PPARγ蛋白相对表达量的比值。取缺陷型Raw264.7细胞并分为4组,S273A+ox-LDL组细胞经PPARγSer273突变型质粒转染,并以50 mg/L ox-LDL诱导;S273A组细胞经PPARγSer273突变型质粒转染,但不予50 mg/L ox-LDL诱导;WT+ox-LDL组经PPARγ野生型质粒转染,并以50 mg/L ox-LDL诱导;WT组经PPARγ野生型质粒转染,但不予50 mg/L ox-LDL诱导;采用ELISA法检测细胞培养液中TNF-α和IL-1β分泌量,实时荧光定量PCR法检测细胞中TNF-α和IL-1βmRNA。结果ox-LDL组和对照组TNF-α的分泌量分别为229.43±10.92、186.85±16.12,IL-1β分泌量分别为69.68±6.87、54.80±3.27,PPARγSer273磷酸化与PPARγ蛋白相对表达量的比值为0.67±0.06、0.55±0.01,两组比较,P均<0.05。S273A+ox-LDL组、S273A组、WT+ox-LDL组、WT组TNF-α分泌量分别为333.05±41.44、251.47±24.73、445.68±31.63、373.64±37.59,TNF-αmRNA相对表达量分别为1.35±0.28、0.63±0.12、2.37±0.21、1.00±0.00,IL-1β分泌量分别为83.25±4.74、67.56±6.14、110.73±10.01、94.47±7.48,IL-1βmRNA相对表达量分别为1.18±0.09、0.67±0.13、1.55±0.15、1.00±0.00,S273A+ox-LDL组与WT+ox-LDL组比较,S273A组与WT组比较,P均<0.05。结论PPARγSer273磷酸化通过促进ox-LDL诱导的Raw264.7细胞炎症因子TNF-α和IL-1β的分泌和表达,促进了AS进程。

Objective To observe the changes of inflammatory factors in mouse monocyte macrophage leukemia cell line Raw264.7 induced by oxidized low-density lipoprotein(ox-LDL)and the regulation of PPARγSer273 phosphorylation in order to explore the role of PPARγSer273 phosphorylation in atherosclerosis.Methods The mouse monocyte macrophage leukemia cell line Raw264.7 were divided into the ox-LDL group and control group.The cells in the ox-LDL group were induced by 50 mg/L ox-LDL,while the cells in the control group were not treated.The concentrations of inflammatory cytokines tumor necrosis factor-α(TNF-α)and interleukin-1β(IL-1β)in the cell culture medium of the two groups were detected by ELISA.The relative expression levels of peroxisome proliferator-activated receptorγ(PPARγ)and PPARγSer273 phosphorylated protein was detected by Western blotting.The ratio of relative expression of PPARγSer273 phosphorylation to PPARγprotein was calculated.The PPARγSer273 mutant and PPARγwild type plasmids were transferred into PPARγdeficient Raw264.7 cells and were divided into the S273 A+ox-LDL group,S273 A group,WT+ox-LDL group,and WT group.Cells in the S273 A+ox-LDL group and WT+ox-LDL group were induced by 50 mg/Lox-LDL,while cells in the S273 A group and WT group were not treated.The concentrations of inflammatory cytokines TNF-αand IL-1βin cell culture medium of the above four groups were detected by ELISA,and the relative mRNA expression levels of TNF-αand IL-1βin cells were detected by real-time fluorescence quantitative PCR.Results The secretion of TNF-αin the ox-LDL group and control group was 229.43±10.92 and 186.85±16.12,respectively;the secretion of IL-1βwas 69.68±6.87 and 54.80±3.27,respectively;the ratios of relative expression of PPARγSer273 phosphorylation to PPARγprotein were 0.67±0.06 and 0.55±0.02,respectively(both P<0.05),with statistically significant difference between these two groups(all P<0.05).The secretion of TNF-αin the S273 A+ox-LDL group,S273 A group,WT+ox-LDL group,and WT group was 333.05±41.44,251.47±24.73,445.68±31.63,and 373.64±37.59,respectively;the relative expression levels of TNF-αmRNA were 1.35±0.28,0.63±0.12,2.37±0.21,and 1.00±0.00,respectively;the secretion of IL-1βwas 83.25±4.74,67.56±6.14,110.73±10.01,and 94.47±7.48,respectively;the relative expression levels of IL-1βwere 1.18±0.09,0.67±0.13,1.55±0.15,and 1.00±0.00,respectively;significant difference was found between the Ser273 A+ox-LDL group and WT+ox-LDLgroup,and between the Ser273 A group and WT group(all P<0.05).Conclusion PPARγSer273 phosphorylation promotes atherosclerosis by promoting the secretion and expression of inflammatory cytokines TNF-αand IL-1βin Raw264.7 cells induced by ox-LDL.