脂多糖诱导小鼠腹腔巨噬细胞炎性反应中前列腺素E2/前列腺素E受体4调控高迁移率族蛋白1的机制

Mechanisms for the regulatory effect of prostaglandin E2/prostaglandin E receptor 4 on high mobility group box-1 protein in lipopolysaccharide-induced sepsis in mouse peritoneal macrophage

目的:探讨前列腺素E2(prostaglandin E2,PGE2)对脂多糖(lipopolysaccharide,LPS)诱导的小鼠腹腔巨噬细胞高迁移率族蛋白1(high mobility group box-1 protein,HMGB1)表达的影响及其可能的机制。方法:运用常规方法提取、培养小鼠腹腔巨噬细胞;采用LPS诱导小鼠腹腔巨噬细胞构建炎症模型;使用PGE2和前列腺素E受体(prostaglandin E receptor,EP)激动剂、RNAi技术下调巨噬细胞EP4受体表达、抑制性表达DN.CREB质粒处理LPS诱导的小鼠腹腔巨噬细胞,并通过Western印迹测定HMGB1表达水平。结果:与单纯应用LPS处理巨噬细胞比较,PGE2联合LPS共孵育小鼠腹腔巨噬细胞24 h后,HMGB1蛋白的表达水平明显上升,且EP4受体激动剂联合LPS处理小鼠腹腔巨噬细胞24 h后HMGB1蛋白的表达亦增加(均P<0.05);与PGE2+LPS联合处理比较,应用RNAi技术可下调小鼠腹腔巨噬细胞EP4受体表达,再给予外源性PGE2加LPS处理,HMGB1水平下降(P<0.05);使用DN.CREB质粒抑制c AMP结合元件结合蛋白(c AMP respondse element bound protein,CREB)后再加上EP4受体激动剂联合LPS处理,与EP4受体激动剂+LPS处理比较,HMGB1水平差异无统计学意义(P>0.05);与单纯应用LPS比较,EP4受体激动剂联合LPS处理小鼠腹腔巨噬细胞可上调表皮生长因子受体(epidermal growth factor receptor,EGFR)及蛋白激酶B(protein kinase B,PKB,亦称Akt)thr308磷酸化水平(均P<0.05);使用EGFR抑制剂预处理细胞后,Akt thr308磷酸化水平及HMGB1表达均降低(均P<0.05);采用Akt特异性抑制剂预处理细胞后,HMGB1表达降低(P<0.05)。结论:PGE2可以通过EP4受体上调LPS诱导的小鼠腹腔巨噬细胞HMGB1的表达,且这一作用与激活EGFR/磷脂酰肌醇3激酶(PI3K)/Akt信号通路有关。

Objective： To explore the effect of prostaglandin E2（PGE2） on the expression of high mobility group box-1 protein（HMGB1） in peritoneal macrophages of septic mice and its possible mechanisms.Methods：The mouse peritoneal macrophages were isolated and cultured by conventional methods.The model of inflammation was established by using lipopolysaccharide（LPS）to incubate with mouse peritoneal macrophages.The PGE2,prostaglandin E receptor（EP）4 agonist,EP4 RNAi,and DN.CREB inhibitory plasmid were used to interfere with the LPS-treated mouse peritoneal macrophage.The levels of HMGB1 was determined by Western blot.Results：Compared with LPS alone treatment,the expression of HMGB1 in peritoneal macrophages was increased obviously after 24 h by treatment with PGE2 and LPS,and it was also increased after the combined treatment of EP4 receptor agonist with LPS for 24 h（both P〈0.05）;compared with the PGE2＋LPS treatment,the level of HMGB1 was decreased after knockdown of EP4 receptor expression（P〈0.05）;compared with EP4 receptor agonist＋LPS treatment,there was no difference in HMGB1 levels in mice after the treatment with DN.CREB plasmid to suppress CREB function（P〈0.05）;compared with LPS alone treatment,the combined treatment of EP4receptor agonist with LPS for 24 h could up-regulate the phosphorylation of epidermal growth factor receptor（EGFR）and protein kinase B（Akt）thr308（P〈0.05）,which were blocked by EGFR inhibitor.Once Akt specific inhibitor was used before EP4 and LPS treatment,the expression of HMGB1 was declined（P〈0.05）.Conclusion：PGE2 can up-regulate the expression of HMGB1 in sepsis of peritoneal macrophages through EP4 receptor,which may be related to the activation of EGFR/PI3K/Akt signaling pathway.