支原体巨噬细胞活化脂肽-2经PI3K/Nrf2诱导单核细胞表达HO-1和NQO1

Macrophage-activating Lipopeptide-2 Induces the Expression of HO-1 and NQO1 via PI3K /Nrf2 Pathways in Monocytes

目的 观察支原体巨噬细胞活化脂肽-2（MALP-2）对THP-1单核细胞血红素氧合酶-1（HO-1）和NAD（P）H：醌氧化还原酶-1（NQO1）表达的影响,以明确机体抵抗支原体感染所致炎性损伤的自我防御机制.方法 体外培养THP-1细胞并分为对照组和实验组.其中对照组加入等体积培养基,实验组根据不同的实验目的 加入不同浓度的MALP-2作用5～120 min或12 h,Western blot 检测HO-1和NQO1表达以及Akt磷酸化水平.同时,采用PI3K抑制剂LY294002处理细胞,以证实PI3K参与HO-1表达.提取细胞核蛋白,凝胶迁移率实验和免疫荧光观察NF-E2相关因子2（Nrf2）的DNA结合活性和核转位情况,并采用特异性siRNA沉默Nrf2后,Western blot观察其对HO-1和NQO1表达的影响.结果 Western blot结果显示,MALP-2能诱导THP-1细胞表达HO-1和NQO1蛋白,且呈一定的剂量依赖性.此外,MALP-2能激活PI3K,且PI3K抑制剂能抑制HO-1和NQO1的表达;凝胶迁移率实验和激光共聚焦结果显示,MALP-2能增强Nrf2的DNA结合活性及核转位,而PI3K抑制剂处理后,Nrf2的DNA结合活性以及核转位水平进一步降低.RNA干扰Nrf2后,HO-1和NQO1表达显著降低.结论 MALP-2能诱导THP-1细胞表达HO-1和NQO1,其机制可能受PI3K/Nrf2调控.

Objective This study aimed to determine the role of macrophage-activating lipopeptide-2 （MALP-2） on the expression of hemoxygenase-1 （ HO-1 ） and NAD（P） H： quinone oxidoreductase 1 （ NQO1 ） ,and then investigate its regulatory mechanism to better understand the self-protecting mechanism upon mycoplasma infection. Methods THP-1 cells were cultured in vitro and divided into control group and experimental group. In the control group, equal volume of cul- ture medium was added. For the experimental group, different concentrations of MALP-2 were added for 5 ~ 120 min or 12 h,and the expression of HO-1 and NQO1, phosphorylation of Akt were detected by Western blot. Also, PI3K inhibitor LY294002 was used to investigate the role of PI3K in HO-1 expression, lmmunofluorescence and electrophoretic mobility shift assay were applied to examine the nuclear translocation and DNA-binding activity of NF-E2-related factor 2 （Nrf2）. Silencing of Nrf2 was achieved by RNA interference and expression of HO-1 and NQO1 were detected by Western blot. Results MALP-2 induced THP-1 cells production of HO-1 and NQO1 in a dose-dependent manner, as demonstrated by Western blot. In addition,MALP-2 could also activate PI3K,and LY294002 inhibited HO-1 and NQO1 expression. MALP-2 could also induce the translocation of Nrf2 to the nucleus, and increase its DNA-binding activity;but this activity could be inhibited by PI3K inhibitor. Silencing of Nrf2 expression significantly inhibited HO-1 and NQO1 expression. Conclusion MALP-2 can induce THP-1 cells expression of HO-1 and NQO1 through PI3K/Nrf2 pathways.