FoxO1/p38 MAPK信号通路在LPS致急性肺损伤中的作用研究

Role of FoxO1/p38MAPK signaling pathway in acute lung injury induced by LPS

目的探讨叉头状转录因子O1(forkhead transcription factors of O class1,FoxO1)在脂多糖(lipopolysaccharide,LPS)致急性肺损伤(acute lung injury,ALI)中的作用及其调控机制。方法采用LPS模拟诱导ALI模型。HE染色法观察肺组织的病理变化;ELISA法检测肺组织中肿瘤坏死因子α(TNF-α)和白细胞介素6(IL-6)的含量;免疫组化染色观察FoxO1在小鼠肺组织的表达;Western blot检测FoxO1、DNA甲基转移酶和p38 MAPK的磷酸化水平;qRT-PCR检测FoxO1、IL-6、TNF-α和DNA甲基转移酶的mRNA水平;巢式甲基化特异性PCR(nMS-PCR)检测肺组织中FoxO1启动子区DNA甲基化的水平变化;培养肺血管内皮细胞(pulmonary vascular endothelial cells,PVECs)并转染FoxO1 siRNA,Western blot检测p38 MAPK的磷酸化水平;采用Pearson方法分析FoxO1甲基化水平与炎症因子的相关性。结果与Control组相比,LPS组小鼠肺泡炎性细胞明显增多,肺组织水肿、充血明显;TNF-α和IL-6的水平分别升高52.2%和150.4%(P<0.05);p38 MAPK磷酸化水平及FoxO1表达分别增加134.1%和61.8%(P<0.05),而FoxO1启动子区DNA甲基化水平降低17.2%(P<0.05),体外转染FoxO1 siRNA后,p38磷酸化水平降低,Pearson分析显示,FoxO1甲基化水平与炎症因子成负相关。结论FoxO1启动子区低甲基化调控FoxO1/p38 MAPK信号通路是LPS诱导急性肺损伤的重要机制。

Aim To investigate the effect of forkhead transcription factors of O class1(FoxO1)on lipopolysaccharide(LPS)-induced acute lung injury and its regulatory mechanism.Methods The model of acute lung injury(ALI)was simulated by LPS.HE staining was used to observe the pathological changes of lung tissues.The contents of tumor necrosis factorα(TNF-α)and interleukin-6(IL-6)in lung tissues were determined by ELISA.The expression of FoxO1 in mouse lung tissues was observed by immunohistochemical staining.The phosphorylation levels of FoxO1,DNA methyltransferase and p38 MAPK were detected by Western blot.The mRNA levels of FoxO1,IL-6,TNF-αand DNA methyltransferase were detected by qRT-PCR.DNA methylation in FoxO1 promoter region in lung tissues was detected by nested methylation specific PCR(nMS-PCR).Pulmonary vascular endothelial cells(PVECs)were cultured and transfected with FoxO1 siRNA,and the phosphorylation of p38 MAPK was detected by Western blot.The correlation between FoxO1 methylation level and inflammatory factors was analyzed by Pearson method.Results Compared with control group,alveolar inflammatory cells increased significantly in LPS group,and pulmonary edema and hyperemia were obvious.TNF-αand IL-6 levels increased by 52.2%and 150.4%(P<0.05),respectively.The phosphorylation level of p38 MAPK and FoxO1 expression increased by 134.1%and 61.8%(P<0.05),respectively,while the DNA methylation level of FoxO1 promoter region decreased by 17.2%(P<0.05).After transfection of FoxO1 siRNA in vitro,the phosphorylation level of p38 decreased.Pearson analysis showed that FoxO1 methylation level was negatively correlated with inflammatory factors.Conclusion The regulation of FoxO1/p38 MAPK signaling pathway by hypomethylation of FoxO1 promoter is an important mechanism of LPS-induced acute lung injury.