The Lipid-associated Membrane Proteins Derived From Mycoplasma Pneumoniae LAMPs Activate Nrf2and Effect The Expression Of HO-1and NQO1

Objectives A series of inflammatory responses caused by Mycoplasmapneumoniae largely depend on the lipid-associated membrane proteins (LAMPs). Thisstudy was to investigate the activation of nuclear transcription factor2(NF-E2-related factor2, Nrf2) and the expression of heme oxygenase1(Heme oxygenase, HO-1) and quinone oxidoreductase (NADPH: quinine oxidoreductase, NQO1) inducedby the lipid-associated membrane proteins derived from Mycoplasma pneumoniaeactivate in the human monocyte cell line THP-1cells.Methods Mp M129strains were cultivated to logarithmic phase in SP4liquidmedium. Then the precipitate was collected by centrifugation. LAMPs were extractedand BCA kit was performed to determinate the concentration. THP-1cells werecultured in vitro and stimulated with increasing concentrations of LAMPs(1.0,2.5,5.0,7.5, and10.0ug/mL) for12h, PBS and LPS were used respectively for negative andpositive control. The cytotoxicity of LAMPs and the expression levels of HO-1andNQO1were respectively examined by CCK-8kit and Western blot.THP-1cells weretreated with5.0μg/mL LAMPs for0,4,8,12,16,20or24h.The expression levels ofHO-1and NQO1were determined by Western blot. HO-1enzymic activity wasexamined by colorimetric technique.THP-1cells were incubated with5.0μg/mLLAMPs and the expression levels of Nrf2protein at nucleus and cytoplasm weredetected by Western blot and IFA respectively.THP-1cells were cultured with5.0μg/mL LAMPs for3,6,9h after being transfected with100nM Nrf2siRNA for30hours, then the expression of HO-1and NQO1mRNA were determined by qRT-PCRrespectively.Results1. The concentration of LAMPs derived from Mycoplasma pneumoniae was1.029mg/mL. 2. The cytotoxicity results showed that the OD450nm light absorption valuesbetween different concentrations of LAMPs and PBS groups weren’t significantdifferent (P＞0.05).3. HO-1and NQO1protein could be induced in THP-1cells after beingstimulated by LAMPs. The expression level of NQO1protein was increased in adose-dependent manner. When the concentrations of LAMPs ranged from0~10μg/mL,the expression of HO-1was increased at low dose of LAMPs, and declined at highdose of LAMPs. The optimal concentration of LAMPs for HO-1expression was5.0μg/mL.4. The expression of HO-1and NQO1protein was increased in a time-dependentmanner in0~12h, and then declined. And the expression levels of both proteins have apeak at12h after stimulated by LAMPs.5. The activities of HO-1enzyme was（1.278±0.171）nmol/mg/h in THP-1cellsafter being treated with5.0μg/mL LAMPs for12h. The enzymatic activities wereobviously increased.6. With5.0μg/mL LAMPs treated THP-1cells, the expression level of cytoplasmNrf2protein ireduced gradually while nuclear Nrf2protein increased. IFA resultsshowed that the Nrf2protein translated from plasma to nucleus.7. After treated with Nrf2siRNA, the mRNA expression levels of Nrf2and HO-1were significant declined, but NQO1didn’t have an obvious change.Conclusions1. Mp LAMPs could increase the protein expression levels of HO-1and NQO1in human mononuclear cell line THP-1cells.2. Nrf2could regulate the expression of HO-1in THP-1cells with Mp LAMPs.But the regulation of Nrf2on the expression of NQO1is not obviously.