小凹蛋白-1对RAW264.7巨噬细胞衰老相关分泌表型表达的抑制作用及其机制

Inhibitory effect of caveolin-1 on expression of senescence-associated secretory phenotype of RAW264.7 macrophages and its mechanism

目的探讨小凹蛋白-1对oxLDL诱导的衰老巨噬细胞分泌基质金属蛋白酶(MMPs)、组织因子(TF)、白介素-6(IL-6)、白介素-8(IL-8)、肿瘤坏死因子-α(TNF-α)等衰老相关分泌表型(SASP)的影响及可能机制。方法选取Ctrl shRNA或Cav1 shRNA感染的巨噬细胞,用oxLDL处理或加入等量的PBS处理24 h后,以β-半乳糖苷酶染色试剂盒鉴定细胞衰老情况;以Transwell实验评价巨噬细胞迁移能力,Western blotting检测细胞MMPs、TF蛋白表达,ELISA检测细胞上清液中IL-6、IL-8、TNF-α的分泌量。通过Cav1 shRNA感染或与促分裂原活化蛋白激酶p38(p38MAPK)特异性激动剂P79350共同处理巨噬细胞,Western blotting检测p38磷酸化蛋白水平。结果oxLDL可诱导巨噬细胞衰老增加,激活p38MAPK通路,使细胞迁移数增多。病毒感染技术特异性下调小凹蛋白-1的表达后,p38MAPK通路被抑制,巨噬细胞衰老减少,细胞迁移能力下降,细胞MMP9、MMP2、TF的蛋白表达量下降,细胞上清液IL-6、IL-8、TNF-α含量减少,差异均有统计学意义(P均<0.05)。而p38激动剂P79350处理可以逆转下调小凹蛋白-1后SASP分泌减少这一效应。结论小凹蛋白-1可能通过p38MAPK通路减弱衰老巨噬细胞的迁移能力,抑制炎症因子释放,减少巨噬细胞SASP的形成。

Objective To explore the effects and mechanism of caveolin-1 on the senescence-associated secretory phenotype(SASP),such as matrix metalloproteinase(MMPs),tissue factor(TF),interleukin-6(IL-6),interleukin-8(IL-8),and tumor necrosis factor-α(TNF-α)in senescent macrophages induced by oxidized low-density lipoprotein(oxLDL).Methods The macrophages infected with Ctrl shRNA or Cav1 shRNA were treated with oxLDL or equal volume of PBS for 24 h,andβ-galactosidase staining was used to assess the cell senescence.Transwell assay was used to observe cell migration ability.The expression levels of matrix metalloproteinases(MMPs)and TF protein in macrophages were detected by Western blotting.The secretion content of IL-6,IL-8,and TNF-αin supernatants was measured by ELISA.The macrophages were infected with Cav1 shRNA or cotreated with mitogen-activated protein kinase p38(p38MAPK)specific agonist P79350,and the expression of p-p38 phosphorylation protein in macrophages was assayed by Western blotting.Results OxLDL induced the senescence of macrophages and activated p38 MAPK signaling pathway,with the increase of cell migration.After down-regulating the expression of caveolin-1 virus infection,the p38 MAPK signaling pathway was inhibited,the cell senescence decreased,the expression of MMP-9,MMP-2,and TF protein in cells decreased and the content of IL-6,IL-8,and TNF-αin supernatants decreased,with statistically significant differences(all P<0.05).Treatment with the p38 MAPK specific agonist P79350 reversed the effect of the decrease in SASP following down-regulation of caveolin-1.Conclusions Caveolin-1 may attenuate migration of the senescent macrophages,inhibit the release of inflammatory cytokines,and decrease the formation of SASP via activating the p38 MAPK signaling pathway.