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目的观察ApoAⅠ对脂多糖(LPS)诱导的THP-1巨噬细胞源性泡沫细胞炎症因子表达的影响,并从ABCA1介导激活的STAT3/TTP信号途径入手探讨其分子机制。方法采用ELISA检测炎症因子表达;构建含肿瘤坏死因子α(TNF-α)启动子的CAT报告基因,转染THP-1细胞,采用ELISA检测TNF-α启动子活性;定量PCR检测TNF-α等炎症因子mRNA表达;免疫印迹技术检测pSTAT3、TTP和HuR等蛋白表达;RNA-EMSA检测TTP与RNA复合物;构建TTP、STAT3 siRNA,转染细胞。结果用10μg/L LPS和/或10 mg/L ApoAⅠ处理THP-1源性泡沫细胞4 h,结果显示ApoAⅠ明显抑制LPS诱导的TNF-α、IL-1β和IL-6的表达;CAT报告基因显示,ApoAⅠ并不明显影响LPS诱导的TNF-α启动子活性;LPS处理细胞3 h,加入放线菌素D(Act D)终止转录,检测TNF-αmRNA表达量(0、30、60及120 min),结果显示ApoAⅠ明显增加LPS作用下TNF-α的mRNA降解;ApoAⅠ明显促进LPS作用下TTP的表达,而对HuR的表达没有明显影响,TTP siRNA处理后明显抑制ApoAⅠ促进TNF-αmRNA降解的作用;RNA-EMSA结果显示,ApoAⅠ明显促进胞内蛋白与ARE探针形成复合物,加入TTP抗体形成su-pershift;ApoAⅠ处理THP-1细胞后迅速(15 min)磷酸化STAT3,核内STAT3表达增加,STAT1和STAT6不受影响;STAT3 siR-NA处理后明显抑制TTP的表达及TNF-αmRNA的降解;采用siRNA下调ABCA1的表达,明显抑制ApoAⅠ作用下TTP的表达及TNF-αmRNA的降解。结论 ApoAⅠ通过转录后调控方式抑制LPS诱导的炎症因子表达;ApoAⅠ促进TTP表达,并通过与炎症因子3′UTR ARE序列结合促进其mRNA降解;ApoAⅠ通过激活STAT3促进TTP表达,该效应直接受ABCA1介导。
Objective To investigate the effect of Apo AI on the expression of inflammatory cytokines induced by lipopolysaccharide (LPS) in THP-1 macrophage-derived foam cells, and to explore its molecular mechanism from ABCA1-mediated STAT3 / TTP signaling pathway. Methods The expression of inflammatory cytokines was detected by ELISA. The CAT reporter gene containing tumor necrosis factor α (TNF-α) promoter was constructed and transfected into THP-1 cells. The activity of TNF-α promoter was detected by ELISA. The expression of inflammatory cytokines mRNA was detected by Western blotting. The protein expressions of pSTAT3, TTP and HuR were detected by Western blotting. The complexes of TTP and RNA were detected by RNA-EMSA. TTP and STAT3 siRNA were constructed. Results THP-1 derived foam cells were treated with 10 μg / L LPS and / or 10 mg / L ApoAⅠ for 4 h. The results showed that ApoA Ⅰ significantly inhibited the LPS-induced TNF-α, IL-1β and IL- The results showed that ApoAⅠhad no significant effect on LPS-induced TNF-α promoter activity. LPS treated cells for 3 h, and then treated with Act D to stop the transcription. The expression of TNF-αmRNA (0, 30, 60 and 120 min. The results showed that ApoAI significantly increased the mRNA degradation of TNF-αin LPS group. ApoAI significantly promoted the expression of TTP under the action of LPS, but had no significant effect on the expression of HuR. TTP siRNA significantly inhibited the degradation of TNF-α mRNA by ApoAⅠ The results of RNA-EMSA showed that ApoA Ⅰ promoted the formation of complex of intracellular protein and ARE probe by adding TTP antibody to form su-pershift. After ApoAⅠ treatment of THP-1 cells, phosphorylated STAT3 was rapidly (15 min) STAT1 and STAT6 were not affected. STAT3 siR-NA significantly inhibited the expression of TTP and the degradation of TNF-αmRNA. The down-regulation of ABCA1 expression by siRNA inhibited the expression of TTP and the degradation of TNF-αmRNA. Conclusion ApoA Ⅰ inhibits LPS-induced inflammatory cytokines by post-transcriptional regulation. ApoA Ⅰ promotes TTP expression and promotes its mRNA degradation by binding to 3’UTR ARE sequence of inflammatory cytokines. ApoAⅠ promotes TTP expression by activating STAT3, which is directly regulated by ABCA1 mediated guide.