咯利普兰通过NF-κB抑制MRP8/14在RAW264.7细胞中促炎性因子的释放  

Rolipram represses MRP8/14-induced pro-inflammatory cytokine releases in RAW264.7 cells through NF-κB activation

作　　者：杨晨[1] 王俊[1] 黄林[1] 罗海华[2] 姜勇[2] 刘爱华[1] 

机构地区：[1]南方医科大学南方医院呼吸内科,广东广州510515 [2]南方医科大学病理生理学教研室和广东省蛋白质组学重点实验室,广东广州510515

出　　处：《中国病理生理杂志》2017年第6期1098-1103,共6页Chinese Journal of Pathophysiology

基　　金：国家自然科学基金资助项目(No.81372030);国家自然科学基金-广东省联合基金重点项目(No.U1601225);广东省科技计划项目(No.2016A020216015)

摘　　要：目的:探讨PDE4抑制剂咯利普兰抑制髓样相关蛋白8/14(MRP8/14)对小鼠巨噬细胞系RAW264.7中促炎性因子释放的刺激作用,并研究核因子κB(NF-κB)在其中的作用。方法:MRP8/14刺激RAW264.7细胞,采用液相芯片技术和实时荧光定量PCR(q PCR)技术分别检测肿瘤坏死因子α(TNF-α)和白细胞介素6(IL-6)的蛋白和mRNA水平;咯利普兰预处理RAW264.7细胞后,MRP8/14刺激细胞,采用液相芯片技术和q PCR技术分别检测TNF-α和IL-6的蛋白水平和mRNA水平;Western blot法检测NF-κB P65蛋白磷酸化水平;间接免疫荧光法检测RAW264.7细胞内NF-κB P65蛋白核移位情况。结果:液相芯片及q PCR结果显示MRP8/14具有很强的促炎性因子TNF-α和IL-6释放的作用(P<0.01),而咯利普兰可以显著减弱MRP8/14促炎性因子释放的作用(P<0.05)。此外,MRP8/14能够诱导NF-κB P65蛋白发生磷酸化,胞核中P65蛋白增加;而咯利普兰显著减弱NF-κB P65蛋白磷酸化(P<0.05)。结论:咯利普兰可能通过NF-κB途径显著减弱MRP8/14的促炎作用。AIM: To investigate the inhibitory effect of PDE4 inhibitor rolipram on the releases of inflammatory cytokines in mouse macrophages( RAW264. 7 cells) induced by myeloid-related protein 8/14( MRP8/14),and to explore the role of nuclear factor-κB( NF-κB) in this process. METHODS: RAW264. 7 cells were treated with MRP8/14. The inflammatory cytokines TNF-α and IL-6 were determined by Liqui Chip and q PCR. In contrast,RAW264. 7 cells were pretreated with rolipram prior to MRP8/14 exposure. After MRP8/14 challenge,the inflammatory cytokines TNF-α and IL-6were determined by Liqui Chip and q PCR. Moreover,the phosphorylation level of NF-κB P65 was determined by Western blot. The nuclear translocation of NF-κB P65 in RAW264. 7 cells was detected by indirect immunofluorescence. RESULTS: The results of Liqui Chip and q PCR showed that MRP8/14 enhanced the expression of TNF-α and IL-6( P <0. 01),while pretreatment with rolipram markedly down-regulated the level of inflammatory cytokines induced by MRP8/14( P < 0. 05). Meanwhile,MRP8/14 significantly activated the phosphorylation of NF-κB P65,and the protein expression of p65 in the nucleus was increased,while pretreatment with rolipram suppressed the phosphorylation of NF-κB P65 induced by MRP8/14. CONCLUSION: Rolipram may significantly reduce the releases of the inflammatory cytokines induced by MRP8/14 through the NF-κB signaling.

关 键 词：急性肺损伤 咯利普兰 髓样相关蛋白8/14 炎性因子 核因子ΚB 

分 类 号：R363.21[医药卫生—病理学] R965[医药卫生—基础医学]