肺炎支原体LAMPs经PI3K/Nrf2诱导人单核细胞表达HO-1

目的 探讨肺炎支原体(Mycoplasma pneumoniae,Mp)脂质相关膜蛋白(lipid-associated membrane protein,LAMPs)对人单核细胞表达血红素氧合酶-1(heme oxygenase-1,HO-1)的影响,并探讨可能的调控机制。方法 体外培养THP-1细胞,用不同浓度的LAMPs作用后,分别采用realtime-PCR和Western blot检测HO-1 mRNA和蛋白的表达。同时采用不同浓度的放线菌素D(ActD)和放线菌酮(CHX)预处理细胞,观察其对HO-1表达的影响,以证实HO-1的表达是否通过转录和翻译水平;提取LAMPs作用前后的核蛋白,凝胶迁移率实验检测Nrf2的核转位、Western blot检测Akt的磷酸化情况;同时采用PI3K抑制剂LY294002处理细胞,观察其对LAMPs诱导Nrf2核转位及HO-1表达的影响;最后采用 siRNA干扰Nrf2表达,以证实Nrf2是否参与调控HO-1表达。结果 (1)0~5 μg/mL LAMPs能以剂量依赖性方式诱导THP-1表达HO-1 mRNA和蛋白;(2)5 μg/mL LAMPs能诱导THP-1细胞Akt磷酸化,并能增强其DNA结合活性;PI3K抑制剂LY294002处理后,可明显抑制LAMPs诱导的HO-1表达和Nrf2核转位;(3)干扰Nrf2表达后,可明显下调LAMPs诱导THP-1细胞表达HO-1。结论 LAMPs可诱导THP-1细胞表达HO-1,其机制可能与PI3K/Nrf2通路有关。

Mycoplasma pneumoniae lipid-associated membrane proteins inducing Heme oxygenase-1 expression via PI3K/Nrf2 in THP-1 cell

The aim of this study is to investigate the mechanisms of Mycoplasma pneumonia-derived lipid-associated membrane proteins (LAMPs) inducing heme oxygenase-1 expression in human mononuclear cell line THP-1 cells. THP-1 cells were cultured in vitro, and treated by different concentration of LAMPs; the mRNA and protein level of HO-1 were detected by real time-PCR and western blot, respectively. Meanwhile, actinomycin D and cycloheximide were administered to pretreat THP-1 cell, subsequently challenged by 5.0 μg/mL LAMPs for 16 hrs, and the expression of HO-1 was detected by western blot. To determine the phosphorylation of Akt and the DNA-binding activity of Nrf2, western blot and electrophoretic mobility shift assay were used to detect the nucleoprotein and cytoplasm protein extracted from the THP-1 cell administered by LAMPs. The PI3K inhibitor LY294002 was also administered to investigate the effects of LAMPs on the activity of Nrf2. At last, siRNA was used to confirm the involvement of Nrf2 in regulation of HO-1 expression. Results showed that (Ⅰ) 0-5 μg/mL LAMPs induced HO-1 mRNA and protein dose-dependent expression in THP-1 cells; (Ⅱ)5 μg/mL LAMPs could induce Akt phosphorylation in THP-1 cells, and raise the DNA-binding activity of Nrf2, and PI3K inhibitor LY294002 could significantly inhibit its activity; (Ⅲ) Silencing of Nrf2 by siRNA could downregulate LAMPs induced HO-1 expression in THP-1 cell. In conclusion, LAMPs could induce HO-1 expression in THP-1 cell via PI3K/Nrf2 pathways.