中介素对脂多糖诱导巨噬细胞核苷酸结合寡聚化结构域样受体蛋白3炎症小体活化及细胞焦亡的影响

Effect of intermedin on activation of nucleotide-binding oligomerization domain-like receptor protein 3 and pyroptosis in lipopolysaccharide induced macrophages

目的利用脂多糖刺激的小鼠巨噬细胞系RAW 264.7,研究中介素对腺嘌呤核苷三磷酸(ATP)诱导的核苷酸结合寡聚化结构域样受体蛋白3(NLRP3)炎症小体活化和细胞焦亡的影响及其机制。方法细胞分组为:对照组,脂多糖组,中介素处理组,磷脂酰肌醇-3-激酶(PI3K)/蛋白激酶B(Akt)阻滞剂LY294002组。实时荧光定量PCR和蛋白质印迹法检测细胞NLRP3炎症小体的激活情况及炎症因子IL-1β和IL-18表达,碘化丙锭(PI)染色法检测细胞焦亡的改变。计量资料以±s表示,组间差异比较采用方差分析,以P<0.05为差异有统计学意义。结果与对照组[(0.83±0.09),(0.49±0.04)]比较,脂多糖组磷酸化(p)-PI3K/PI3K(0.44±0.05),p-Akt/Akt(0.27±0.06)比值均显著降低,与脂多糖组相比,脂多糖+中介素组细胞中p-PI3K/PI3K(1.22±0.18),pAkt/Akt(0.83±0.09)比值均显著升高(F=31.40,P<0.001;F=50.88,P<0.001)。与对照组比较,脂多糖组(脂多糖和ATP)可刺激RAW264.7细胞炎症因子IL-1β、IL-18和NLRP3炎症小体(NLRP3,caspase-1,ASC)的mRNA及蛋白表达上调;与脂多糖组相比,中介素处理可抑制炎症因子IL-1β、IL-18和NLRP3炎症小体表达,这种效应可被PI3K/Akt通路的阻滞剂LY294002所阻断。Real-time PCR结果显示,IL-1βmRNA相对表达量:对照组为(1.00±0.11),脂多糖组为(8.32±0.61),脂多糖+中介素组为(8.32±0.55),脂多糖+中介素+LY组为(7.23±0.41)(F=15.42,P<0.001);IL-18 mRNA相对表达量:对照组为(1.00±0.17),脂多糖组为(1.82±0.21),脂多糖+中介素组为(1.14±0.15),脂多糖+中介素+LY组为(1.53±0.11)(F=18.16,P<0.001);NLRP3 mRNA相对表达量:对照组为(1.00±0.13),脂多糖组为(2.58±0.18),脂多糖+中介素组为(1.07±0.17),脂多糖+中介素+LY组为(1.33±0.32)(F=15.98,P<0.001);caspase-1 mRNA相对表达量:对照组为(1.00±0.09),脂多糖组为(6.20±0.19),脂多糖+中介素组为(3.43±0.06),脂多糖+中介素+LY组为(5.50±0.45)(F=18.39,P<0.001);ASC mRNA相对表达量:对照组为(1.00±0.21),脂多糖组为(4.58±0.48),脂多糖+中介素组为(2.07±0.51),脂多糖+中介素+LY组为(3.33±0.32)(F=15.19,P<0.001)。蛋白质免疫印迹结果显示,IL-1β蛋白相对表达量:对照组为(100%),脂多糖组为[(188±14)%],脂多糖+中介素组为[(112±11)%],脂多糖+中介素+LY组为[(171±27)%](F=21.25,P<0.001);IL-18蛋白相对表达量:对照组为(100%),脂多糖组为[(183±16)%],脂多糖+中介素组为[(115±19)%],脂多糖+中介素+LY组为[(179±23)%](F=19.62,P<0.001);NLRP3蛋白相对表达量:对照组为(100%),脂多糖组为[(149±15)%],脂多糖+中介素组为[(106±10)%],脂多糖+中介素+LY组为[(144±15)%](F=14.35,P<0.001);ASC蛋白相对表达量:对照组为(100%),脂多糖组为[(188±12)%)],脂多糖+中介素组为[(110±18)%],脂多糖+中介素+LY组为(192±8)%(F=15.79,P<0.001)。结论中介素通过调节PI3K/Akt活性,抑制NLRP3炎症小体的活化与细胞焦亡。

Objective The effect of intermedin(IMD)on ATP-induced activation of inflammatory bodies and pyroptosis of cells and its mechanism were studied using lipopolysaccharide(LPS)-sensitized mouse macrophage line RAW 264.7.Methods The cells were divided into the control groups,the LPS groups,LPS+IMD groups,and LPS+IMD+LY294002 groups.The expression of interleukin(IL)-1βand IL-18 and the activation of nucleotide-binding oligomerization domain-like receptor protein 3(NLRP3)inflammatory cells were detected by real-time PCR and western blotting,and the pyroptosis of cells was detected by propidium iodide(PI)staining.The measurement data was represented by MS±SD,and the inter-group difference was compared with ANOVA calculations,and P<0.05 represented the difference with statistical significance.Results Compared with the control group[(0.83±0.09)vs(0.49±0.04)],the ratio of phosphorylated phosphatidylinositol-3-kinase,p-PI3K)/phosphatidylinositol-3-kinase(PI3K)(0.44±0.05)and p-Akt/Akt(0.27±0.06)in the LPS group was significantly decreased.The ratios of p-PI3K/PI3K(1.22±0.18)and pAkt/Akt(0.83±0.09)in LPS+IMD group was significantly increased(F=31.40,P<0.001;F=50.88,P<0.001).Compared with the control group,the mRNA and protein expressions of IL-1β,IL-18 and NLRP3 inflammasome(NLRP3,caspase-1,ASC)in RAW264.7 cells were up-regulated in the LPS group(LPS and ATP).Compared with LPS group,IMD treatment inhibited the expression of inflammatory cytokines IL-1β,IL-18 and NLRP3 inflammasome,which was blocked by LY294002,a blocker of PI3K/Akt pathway.The results of real-time PCR showed that the relative expression of IL-1βmRNA was(1.00±0.11)in the control group,(8.32±0.61)in the LPS group,(8.32±0.55)in the LPS+IMD group,and(7.23±0.41)in the LPS+IMD+LY group(F=15.42,P<0.001).The relative expression of IL-18 mRNA in the control group was(1.00±0.17),(1.82±0.21)in the LPS group,(1.14±0.15)in the LPS+IMD group,and(1.53±0.11)in the LPS+IMD+LY group respectively(F=18.16,P<0.001).The relative expression of NLRP3 mRNA in the control group was(1.00±0.13),(2.58±0.18)in the LPS group,(1.07±0.17)in the LPS+IMD group,and(1.33±0.32)in the LPS+IMD+LY group respectively(F=15.98,P<0.001);The relative expression of caspase-1 mRNA in the control group was(1.00±0.09),(6.20±0.19)in the LPS group,(3.43±0.06)in the LPS+IMD group,and(5.50±0.45)in the LPS+IMD+LY group respectively(F=18.39,P<0.001).The relative expression of ASC mRNA in the control group was(1.00±0.21),(4.58±0.48)in the LPS group,(2.07±0.51)in the LPS+IMD group,and(3.33±0.32)in the LPS+IMD+LY group respectively(F=15.19,P<0.001).Western blotting results showed that the relative expression of IL-1βprotein was as follows:(100%)in the control group,[(188±14)%]in the LPS group,[(112±11)%]in the LPS+IMD group,and[(171±27)%]in the LPS+IMD+LY group respectively(F=21.25,P<0.001).The relative expression of IL-18 protein in the control group was 100%,[(183±16)%]in the LPS group,[(115±19)%]in the LPS+IMD group,and[(179±23)%]in the LPS+IMD+LY group respectively(F=19.62,P<0.001).The relative expression of NLRP3 protein was 100%in the control group,[(149±15)%]in the LPS group,[(106±10)%]in the LPS+IMD group,and[(144±15)%]in LPS+IMD+LY group respectively(F=14.35,P<0.001).The relative expression of ASC protein was 100%in the control group,[(188±12)%]in the LPS group,[(110±18)%]in the LPS+IMD group,and[(192±8)%]in the LPS+IMD+LY group(F=15.79,P<0.001).Conclusion IMD inhibits the activation of NLRP3 inflammasome and cell pyroptosis by regulating PI3K/Akt activity.