血清和糖皮质激素调节蛋白激酶1通过核因子-КB抑制Toll样受体4介导的炎性反应

Serum- and glucocorticoid-regulated kinase 1 suppresses inflammatory responses mediated by toll like receptor 4 via nuclear factor-κB

目的探讨血清和糖皮质激素调节蛋白激酶（SGK）1在TLR介导的炎性反应中的作用。方法将小鼠给予腹腔内注射SGK1抑制剂EMD638683 10mg／kg或不预处理，2h后注射大肠埃希菌脂多糖1mg／kg，同时设立对照组（只注射PBS或EMD638683），注射脂多糖3、24h后收取小鼠血清，使用ELISA检测小鼠IL-6、IL-12、TNF—α含量。同时取注射脂多糖后6、24h的肝、肺组织，OCT包埋，HE染色。分离人PBMC后，使用SGK1或P13K抑制剂预处理或不预处理，并给予脂多糖刺激，使用ELISA检测细胞因子IL-6、IL-12、TNF-α水平，使用Western印迹法检测细胞IKKα／β、IKBα和核因子-κB p65蛋白的表达。采用单因素方差分析。结果在脂多糖诱导的小鼠脓毒血症体内实验中，SGK1抑制剂增加了小鼠炎性因子IL-6（t=3．007）、IL-12（t=4．413）、TNF-α（t=5．403）的产生（均P〈0．05），并且在早期（6h）增加了小鼠重要脏器肝、肺炎性细胞的浸润，晚期（24h）加重了器官损伤，引起肺泡组织塌陷与肝脏脂肪变性。在人PBMC中，SGK1抑制剂EMD638683增加了脂多糖诱导的炎性因子IL-6（t=18．540）、IL-12（t=16．520）、TNF-α（t=34．880）水平（均P〈0．01），同时抑制了IKKα／β、IKBα和核因子-κB p65的磷酸化。结论SGK1通过核因子-κB负向调节TLR4介导的炎性反应。

Objective To investigate the role of serum and glucocorficoid regulated protein kinase （SGK） 1 in the inflammatory responses mediated by toll like receptors. Methods Mice were injected with lipopolysaccharide （LPS, 1 mg/kg） 2 h after the pretreatment of EMD638683 （10 mg/kg） or phosphate buffered saline （PBS） as control. At the time points of 3 and 24 h, pro-inflammatory cytokines （interleukin [IL]-6, IL-12 and tumor necrosis factor [TNF]-α） in serum were measured using enzymelinked immunosorbent assay （ELISA）. Livers and lung were harvested at 6 h and 24 h after the injection of LPS, embedded by optimum cutting temperature （OCT） and then stained with hematoxylin and eosin （HE）. Peripheral blood mononuclear cell （PBMC） were isolated and stimulated by LPS with or without the pretreatment of EMD or LY294002. Cytokines （IL-6, IL-12 and TNF-α） were measured using ELISA. IKKα/β, IKBα and nuclear factor （NF）-κB p65 were detected by Western bolt. Data were analyzed by one way analysis of variance. Results In the model of LPS-induced endotoxin sepsis, inhibition of SGK1 induced secretion of pro-inflammatory cytokine （IL-6 [t=3. 007, P〈0.05], IL-12[t=4. 413, P〈0.05] and TNF-α[t=5. 403, P〈0. 05]）, increased inflammatory ceils infiltration into the liver and lung within 6 h, and induced serious multiple organ damage with collapse of alveoli and fatty degeneration of liver. After 24 h, pharmacological inhibition of SGK1 with EMD638683 increased pro- inflammatory cytokine （IL-6 [t= 18. 540, P〈0. 01], IL-12[t=16. 520, P〈0.01] and TNF-α[t=34. 880,P〈0. 01]） production in human PBMC upon LPS stimulation and inhibited the phosphorylation of IKKα/ β/IKBα and nuclear factor （NF）-κB p65. Conclusions SGK1 suppresses the toll like receptor 4 mediated inflammatory responses via NF-κB.