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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 To investigate the effects of mycoplasma macrophage activating lipopeptide-2 (MALP-2) on the expression of HO-1 and NAD (P) H: quinone oxidoreductase-1 (NQO1 ) Expression in order to clarify the self-defense mechanism of the body against inflammatory injury caused by 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 medium was added. In the experimental group, different concentrations of MALP-2 were added for 5 ~ 120 min or 12 h according to different experimental purposes. The expression of HO-1, NQO1 and Akt phosphorylation were detected by Western blot. Meanwhile, cells were treated with PI3K inhibitor LY294002 to confirm that PI3K is involved in HO-1 expression. The DNA binding activity and nuclear translocation of NF-E2-related factor 2 (Nrf2) were detected by immunocytochemistry and nuclear translocation. Nrf2 was silenced by specific siRNA. Western blot was used to detect the effect of HO-1 and Effect of NQO1 expression. Results Western blot results showed that MALP-2 induced HO-1 and NQO1 expression in THP-1 cells in a dose-dependent manner. In addition, MALP-2 can activate PI3K, and PI3K inhibitor can inhibit the expression of HO-1 and NQO1; MALP-2 can enhance the DNA binding activity and nuclear translocation of Nrf2, The PI3K inhibitor treatment, Nrf2 DNA binding activity and nuclear translocation level is further reduced. RNA interference with Nrf2, HO-1 and NQO1 expression was significantly reduced. Conclusions MALP-2 can induce the expression of HO-1 and NQO1 in THP-1 cells, which may be regulated by PI3K / Nrf2.