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

Mycoplasmal macrophage-activating lipopeptide-2 induces hemeoxygenase-1expression via Btk/PI3K/Nrf2 pathway in human monocytes

目的观察支原体巨噬细胞活化脂肽2(macrophage-activating lipopeptide-2,MALP-2)诱导人单核细胞系THP-1表达血红素氧合酶-1(hemeoxygenase,HO-1)的分子机制。方法体外培养THP-1细胞,用5 ng/ml的MALP-2刺激12 h。Real-time PCR检测HO-1 mRNA的表达,Western blot检测其蛋白水平及Akt磷酸化;THP-1细胞与不同浓度的Bruton’酪氨酸激酶(Bruton’s tyrosine kinase,Btk)抑制剂LFM-A13作用1 h,随后用MALP-2刺激12 h,Western blot观察其对HO-1表达以及Akt磷酸化的影响;分别采用EMSA和免疫荧光检测核转录因子Nrf2的DNA结合活性和核转位,并观察PI3K抑制剂LY294002对Nrf2的激活和DNA结合活性的改变情况;siRNA干扰Nrf2表达,以证实其在HO-1表达中的作用;最后,采用LFM-A13和血红素处理THP-1细胞,MTT法检测细胞的存活率。结果 MALP-2处理可显著诱导THP-1细胞表达HO-1蛋白和mRNA,LFM-A13处理后,HO-1表达水平随其浓度的增高而降低;同时,LFM-A13也能抑制MALP-2诱导PI3K的激活(Akt磷酸化)。MALP-2能促进Nrf2的核转位并增强其DNA结合活性,而PI3K抑制剂LY294002处理后可抑制Nrf2的激活;RNA干扰Nrf2表达后,HO-1表达显著降低;此外,LFM-A13也可降低血红素处理后THP-1细胞的存活率。而采用HO-1激动剂CoPP诱导后,THP-1细胞的存活率有所增高。结论 MALP-2通过Btk/PI3k/Nrf2诱导THP-1细胞表达HO-1,从而发挥对细胞的保护作用。

To investigate the molecular mechanism involved in hemoxygenase-1（HO-1） expression of THP cells induced by a macrophage-activating lipopeptide-2（MALP-2）, THP-1 cells were cultured in vitro and stimulated by 5 ng/ml of MALP-2 for 12 h. Western blot and real-time PCR indicated that MALP-2 could induce THP-1 cells to express HO-1 protein and mRNA, which was significantly abrogated by pretreatment of different concentration of a Bruton＇s tyrosine kinase（Btk） inhibitor LFM-A13. Western blot also showed that MALP-2 could activate PI3K（Akt phosphorylation）. While immunofluorescence and EMSA demonstrated that MALP-2 could induce Nrf2 translocation to the nucleus and increase Nrf2 DNA-binding activity. LFM-A13 treatment significantly inhibited MALP-2-induced PI3K activation. In addition, Nrf2 nuclear translocation and DNA-binding activity were also inhibited by pretreatment of THP-1 cells with a PI3K inhibitor, LY294002. Silencing of Nrf2 expression significantly inhibited MALP-2-induced HO-1 expression. Furthermore, heme treatment reduced the viability of THP-1 cells, as compared with the control cells. By contrast, LFA-A13-treated cells were more sensitive to hemedependent toxicity, and CoPP, a agonist of HO-1, could reverse the decrease of viability of THP-1 cells induced by heme. These results indicate that MALP-2 can induce THP-1 cells expression of HO-1 through Btk/PI3K/Nrf2pathways, and then protect the cells from pathological damage.