Kir2.1在LPS或IL-4诱导的巨噬细胞M1/M2型极化中的作用

Role of Kir2.1 in M1/M2 polarization of macrophages induced by LPS or IL-4

目的:通过激活或阻断内向整流钾通道2.1(inwardly-rectifying potassium channel 2.1,Kir2.1),检测巨噬细胞M1/M2型极化标志物及相关细胞因子的改变,探讨Kir2.1在脂多糖(lipopolysaccharide,LPS)或白细胞介素4(interleukin-4,IL-4)诱导的巨噬细胞M1/M2型极化过程中的具体作用。方法:采用RAW264.7小鼠巨噬细胞系,将未转染慢病毒的RAW264.7细胞分为对照(control)组、LPS组、IL-4组、LPS+zacopride(Kir2.1选择性激动剂)组及IL-4+ML133(Kir2.1选择性阻断剂)组,将转染慢病毒的RAW264.7细胞分为vector组、LPS+vector组、IL-4+vector组、KD-shKir2.1组、LPS+KD-shKir2.1组及IL-4+KD-shKir2.1组。应用ELISA技术检测巨噬细胞培养液上清中IL-1β、IL-6和肿瘤坏死因子α(tumor necrosis factor-α,TNF-α)的浓度;应用实时荧光定量PCR技术检测巨噬细胞中Kir2.1、诱导型一氧化氮合酶(inducible nitric oxide synthase,iNOS)、CD86、CD206、IL-6及TNF-α的mRNA表达;应用细胞免疫荧光技术和Western blot技术检测巨噬细胞中Kir2.1、iNOS、CD86及CD206的定位和表达。结果:Kir2.1在RAW264.7细胞的胞膜及胞质中均有表达,并且LPS干预RAW264.7细胞后Kir2.1的蛋白表达显著下降(P<0.01),IL-4干预巨噬细胞后Kir2.1的蛋白表达显著升高(P<0.01)。激活Kir2.1能够显著减少炎症细胞因子的释放(P<0.05或P<0.01),降低M1型极化标志物iNOS和CD86的mRNA和蛋白表达(P<0.05或P<0.01),而阻断或敲减Kir2.1能够显著增加炎症细胞因子的释放(P<0.05或P<0.01),升高M1型极化标志物iNOS和CD86的mRNA和蛋白表达(P<0.05或P<0.01),降低M2型极化标志物CD206的mRNA和蛋白表达(P<0.05或P<0.01)。结论:激活Kir2.1能够抑制LPS诱导的巨噬细胞向M1型极化;而阻断或敲减Kir2.1能够促进LPS诱导的巨噬细胞向M1型极化,抑制IL-4诱导的巨噬细胞向M2型极化。

AIM:To explore the specific role of inwardly-rectifying potassium channel 2.1(Kir2.1)in the M1/M2 polarization of macrophage induced by lipopolysaccharide(LPS)or interleukin-4(IL-4).METHODS:Mouse RAW264.7 macrophages were used in this study.The RAW264.7 cells were divided into control group,LPS group,IL-4group,LPS+zacopride(a selective agonist)group and IL-4+ML133(a selective blocker of Kir2.1)group.The RAW264.7 cells infected with lentivirus were divided into vector group,LPS+vector group,IL-4+vector group,KDshKir2.1 group,LPS+KD-shKir2.1 group and IL-4+KD-shKir2.1 group.The concentrations of IL-1β,IL-6 and tumor necrosis factor-α(TNF-α)in the supernatant of macrophage medium were detected by ELISA.The mRNA levels of Kir2.1,inducible nitric oxide synthase(iNOS),CD86,CD206,IL-6 and TNF-α in the RAW264.7 cells were detected by realtime fluorescence quantitative PCR.The location and protein expression of Kir2.1,iNOS,CD86 and CD206 in the RAW264.7 cells were detected by immunofluorescence and Western blot.RESULTS:The Kir2.1 was mainly expressed in both membrane and cytoplasm of the RAW264.7 cells.The protein expression of Kir2.1 was significantly decreased after LPS intervention and significantly increased after IL-4 intervention in the RAW264.7 cells(P<0.01).Activation of Kir2.1 significantly reduced the release of inflammatory cytokines(P<0.05 or P<0.01),decreased the mRNA and protein expression of iNOS and CD86(P<0.05 or P<0.01).Inhibition of Kir2.1 significantly increased the release of inflammatory cytokines(P<0.05 or P<0.01)and the mRNA and protein levels of iNOS and CD86(P<0.05 or P<0.01),but decreased the mRNA and protein expression of CD206(P<0.05 or P<0.01).CONCLUSION:Activation of Kir2.1 inhibits the M1 polarization of LPS-induced macrophages.Inhibition of Kir2.1 promotes the M1 polarization of LPS-induced macrophages and decreases the M2 polarization of IL-4-induced macrophages.