高盐诱导下以TonEBP/NF-κB通路为基础的小鼠巨噬细胞表型偏移机制的研究

Mechanisms of macrophage phenotype polarization based on the TonEBP/NF-κB pathway under high salt intervention

目的探讨高盐刺激下巨噬细胞表型偏移的可能机制,为相关心血管疾病的防治提供新的实验依据。方法选取Raw264.7小鼠巨噬细胞为研究对象,构建TonEBP慢病毒干扰稳定细胞株,利用Real-time PCR技术检测TonEBP干扰效率,选取干扰效率最高的感染组细胞株用于后续实验。给予各组相应干预后同步培养24 h,利用Real-time PCR检测各组细胞TonEBP、NF-κB及其M1、M2型巨噬细胞表型标志物mRNA的表达水平;Western blot法检测各组TonEBP、NF-κB、pNF-κB、pIKKα/β的蛋白表达水平。结果成功构建TonEBP-shRNA稳定干扰细胞株,与Control组相比其TonEBP干扰效率达70%;Real-time PCR结果显示单纯高盐干预下Raw264.7细胞TonEBP、NF-κB和M1型巨噬细胞表型标志物的mRNA表达水平显著上调(P<0.05),M2型巨噬细胞表型标志物mRNA表达水平显著下调(P<0.05);高盐刺激下分别干扰TonEBP、NF-κB的表达后,M1型巨噬细胞表型标志物的mRNA表达水平显著下调,M2型巨噬细胞表型标志物mRNA表达水平显著上调。Western blot结果显示单纯高盐刺激下Raw264.7细胞TonEBP、p-IKKα/β、NF-κB、p-NF-κB的蛋白表达水平均明显上调,NF-κB,p-NF-κB,p-NF-κB与NF-κB比值增加;高盐刺激下分别干扰TonEBP、NF-κB的表达后,p-IKKα/β、p-NF-κB蛋白表达水平均显著下调。结论TonEBP/NF-κB信号通路的激活是高盐刺激下Raw264.7细胞向M1型巨噬细胞发生偏移的可能机制;调节TonEBP/NF-κB信号通路,可改变高盐诱导下Raw264.7细胞表型偏移方向。

Objective To investigate the possible mechanism of macrophage phenotypic polarization under hypersaline stimulation so as to provide data for future research into the prevention and treatment of salt-sensitive hypertension and other macrophage related cardiovascular diseases.Methods Using Raw264.7 mouse macrophages as the subjects,a TonEBP lentivirus interference stable cell line was constructed.The interference efficiency of TonEBP gene was detected by real-time PCR,and the cell lines of the infected group with the highest interference efficiency were selected for subsequent experiments.The mRNA expression levels of TonEBP、NF-κB、M1-type and M2-type macrophage phenotypic markers in each group were detected by real-time PCR after twenty hours of corresponding intervention.Western blot was used to detect the protein expression levels of TonEBP、NF-κB、p-NF-κB and p-IKKα/β.Results Stable interfering cell lines of TonEBP-shRNA were constructed,and the TonEBP interfering efficiency was over 70%compared with the control group(P<0.05).The mRNA expression levels of TonEBP、NF-κB andM1 macrophage phenotypic markers in the HS group were significantly up-regulated,(P<0.05),while the mRNA expression levels of M2 type macrophage phenotypic markers were significantly down-regulated(P<0.05).mRNA expression of M1-type macrophage phenotypic markers was significantly down-regulated,(P<0.05)while mRNA expression of M2-type macrophage phenotypic markers was significantly up-regulated by interfering with the expressions of TonEBP and NF-κB respectively under high salt stimulation(P<0.05).Compared with the control group,the protein expression levels of TonEBP、p-IKKα/β、NF-κB and p-NF-κB in the HS group were significantly up-regulated(P<0.05),and the ratio of p-NF-κB to NF-κB was increased(P<0.05).Compared with the HS group,protein expression levels of p-IKKα/β、NF-κB and NF-κB were significantly decreased after interference with the expression of TonEBP and NF-κB respectively under high salt stimulation(P<0.05),but difference in the ratio of p-NF-κB to NF-κB was of no statistical significance(P>0.05).Conclusions Activation of TonEBP/NF-κB signaling pathway is a possible mechanism of Raw264.7 cell shifting to M1 phenotype under high salt stimulation.The regulation of TonEBP/NF-κB signaling pathway can change the direction in which the phenotypes of Raw264.7 cells are polarized under high salt induction.