摘要：
Objective To investigate whether interleukin-1 receptor-associated kinase M (IRAK-M) regulates p22phox-mediated lipopolysaccharide (LPS)-induced macrophage respiratory burst. Methods Mouse peritoneal macrophages were isolated and cultured in votro. The cells were divided into endotoxin tolerance (ET) group and control group (NC group). The ET group was stimulated with 10 μg/L lipopolysaccharide (LPS) for 2 h, then with 100 μg/L LPS re-stimulation. The NC group was not pretreated, directly stimulated by 100 μg/L LPS. Supernatant was collected 3 h later, TNF-α and IL-6 levels were detected by enzyme-linked immunosorbent assay (ELISA). H2O2 and O2-levels were also detected. Western blot was used to detect the expression levels of IRAK-M and p22phox proteins. IRAK-M small interfering RNA (siRNA) was used to interfere with IRAK-M. Groups are set as follows: NC-control group, the interfering with IRAK-M+LPS stimulation group (NC-si-IRAK-M group), the interfering with endotoxin tolerance group (ET-control group), and the interfering with endotoxin tolerance group (ET-si-IRAK-M group). The interference efficiency was detected by polymerase chain reaction (PCR). The protein expression levels of IRAK-M and p22phox were also measured. After successful IRAK-M interference, p22phox was inhibited by apocynin. The groups were: dimethyl sulfoxide (DMSO)+endotoxin tolerance group (control- placebo group), apocynin+endotoxin tolerance group (control-apocynin group), DMSO+interference IRAK-M+ endotoxin t olerance group (si-IRAK-M-placebo group) and interfering with IRAK-M+apocynin+endotoxin tolerance group (si-IRAK-M-apocynin group). The supernatant was taken for detection of TNF-α, IL-6, H2O2 and O2- levels. Results Comparing with ET-control group, after interference with IRAK-M, the protein content of ET-si-IRAK-M was significantly decreased. In contrast, the protein content of p22phox was significantly increased (P<0.05); after interfering with IRAK-M and inhibiting p22phox, the levels of TNF-α, IL-6 and H2O2 O2-in the IRAK-M-apocynin group were significantly lower than the levels of these proteins in si-IRAK-M-placebo group (P<0.05). Compared with NC group, TNF-α, IL-6, H2O2 and O2-levels were decreased in ET group. The ET group has low expression of p22phox protein and high expression of IRAK-M protein (P<0.05). Compared with the ET-control group, the expression of IRAK-M in the ET-si-IRAK-M group was significantly decreased while the expression of p22phox was significantly increased after interference IRAK-M (P<0.05). Compared with the si-IRAK-M-placebo group, the levels of TNF-α, IL-6 and H2O2, O2-in the si-IRAK-M-apocynin group were significantly decreased after interfering with IRAK-M and inhibiting p22phox (P<0.05). Conclusions IRAK-M exerts a negative regulatory effect on endotoxin-induced respiratory outbursts of macrophages in mice by inhibiting p22phox.%目的 研究白细胞介素-1受体相关激酶M(interleukin-1 receptor-associated kinase M, IRAK-M)是否调控p22phox介导脂多糖(lipopolysaccharides, LPS)诱导的巨噬细胞呼吸爆发. 方法 分离、培养小鼠腹腔巨噬细胞,细胞贴壁后分为内毒素耐受(endotoxin tolerance, ET)组和对照组(NC组). ET组预先10 μg/L LPS刺激2 h后,予100 μg/L LPS再刺激,NC组不作预先处理,直接100 μg/L LPS刺激,3 h后收集上清液,ELISA法检测TNF-α和IL-6水平,试剂盒检测过氧化氢(H2O2)、超氧阴离子(O2-)水平;细胞裂解后提取蛋白,Western blot法检测IRAK-M和p22phox蛋白表达.采用IRAK-M 小干扰RNA(small interfering RNA, siRNA)干扰IRAK-M,分组为:LPS刺激组(NC-control组)、干扰IRAK-M+LPS刺激组(NC-si-IRAK-M组)、内毒素耐受组(ET-control组)和干扰IRAK-M+内毒素耐受组(ET-si-IRAK-M组),PCR检测干扰效率并再次检测IRAK-M和p22phox的蛋白表达.IRAK-M干扰成功后,分组为:二甲基亚砜(dimethyl sulfoxide, DMSO)+内毒素耐受组(control-placebo组)、夹竹桃麻素(apocynin)+内毒素耐受组(control-apocynin组)、DMSO+干扰IRAK-M+内毒素耐受组(si-IRAK-M-placebo组)和干扰IRAK-M+apocynin+内毒素耐受组(si-IRAK-M-apocynin组),采用apocynin抑制p22phox,再次取上清液检测TNF-α、IL-6以及H2O2、O2-水平. 结果 与NC组比较,ET组TNF-α、IL-6水平和H2O2、O2-水平均降低,p22phox表达水平降低,而IRAK-M表达水平升高(P<0.05);干扰IRAK-M后,与ET-control组比较,ET-si-IRAK-M组蛋白水平明显降低,p22phox的蛋白水平明显升高(P<0.05);干扰IRAK-M并抑制p22phox后,与si-IRAK-M-placebo组比较,si-IRAK-M-apocynin组TNF-α、IL-6和H2O2、O2-的水平明显降低(P<0.05). 结论 IRAK-M通过抑制p22phox对内毒素诱导的小鼠巨噬细胞呼吸爆发产生负向调控作用.
