染料木素通过TIPE 2负性调控Akt活性促进脂多糖活化的巨噬细胞凋亡

Genistein promotes apoptosis in LPS-activated RAW264.7 cells through regulating Akt by TIPE 2

目的探究染料木素(genistein,GEN)对脂多糖(lipopolysaccharide,LPS)活化的RAW264.7细胞凋亡的影响及其可能的药理学作用机制。方法GEN预孵育RAW264.7细胞或慢病毒介导的肿瘤坏死因子α诱导蛋白8样分子2(tumor necrosis factor-α-induced protein 8-like 2,TIPE 2)过表达细胞2 h,再与LPS共孵育24 h,采用CCK 8试剂盒检测细胞活力,Annexin V-FITC/PI试剂盒检测细胞凋亡水平,qRT-PCR检测TNF-α、IL-6、caspase-8、caspase-3和TIPE 2 mRNA,Western blot检测iNOS、COX-2、caspase-8、caspase-3、TIPE 2、Akt和p-Akt蛋白表达。结果LPS促进RAW264.7细胞TNF-α、IL-6、iNOS、COX-2合成;GEN抑制LPS活化的RAW264.7细胞活力,凋亡细胞增多,并上调caspase-8、caspase-3、TIPE 2 mRNA及蛋白表达;TIPE 2过表达上调活化RAW264.7细胞caspase-8、caspase-3 mRNA及蛋白表达,减少Akt磷酸化,且与GEN具有协同作用。结论GEN可能通过上调TIPE 2抑制Akt活性,激活外源性凋亡途径,促进LPS活化的RAW264.7细胞凋亡。

Aim To investigate the effect of genistein(GEN)on apoptosis of RAW264.7 cells activated by lipopolysaccharide(LPS)and the possible pharmacological mechanisms.Methods RAW264.7 cells and TIPE 2-over expression cells were preincubated with GEN for 2 h,then incubated with LPS for 24 h.CCK 8 kit was used to detect cell viability.Annexin V-FITC/PI kit was used to detect cell apoptotic rate.qRT-PCR was used to detect the level of TNF-α,IL-6,caspase-8,caspase-3 and TIPE 2 mRNA.Western blot was used to detect the expression of iNOS,COX-2,caspase-8,caspase-3,TIPE 2,Akt and p-Akt.Results LPS increased the synthesis of TNF-α,IL-6,iNOS and COX-2 in RAW264.7 cells.GEN inhibited the activity,increased the apoptotic rate and the level of caspase-8,caspase-3 and TIPE 2 of LPS-activated RAW264.7 cells.TIPE 2-over expression up-regulated the level of caspase-8,caspase-3 and reduced the expression of p-Akt,which were further enhanced by GEN in activated macrophage.Conclusions Genistein may promote the apoptosis of LPS-activated RAW264.7 cells through inhibiting Akt activities by up-regulating TIPE 2 and activating the exogenous apoptotic pathway.