Heme Oxygenase-1Induced By A Mycoplasmal Lipopeptide Negativly Regulates COX-2Expression In Monocytes

Objective Excessive inflammatory response caused by Mycoplasma infection isan important factor of inducing tissue damage. Once Mycoplasma infection, immunesystem of host may have the appropriate self-defense mechanism against infection.This study was performed to study Heme oxygenase (HO)-1production and itspotential mechanisms induced by macrophage-activating lipopeptide-2(MALP-2) inTHP-1cells and the potential effect of HO-1on MALP-2-induced COX-2expression.Methods The human monocyte cell line THP-1was incubated with variousconcentrations (0,0.01,0.1,1.0or5.0ng/mL) of MALP-2for16h, or5.0ng/mLMALP-2for different time points (0h,4h,8h,12h,16h or24h). The HO-1mRNAand protein expression induced by MALP-2was detected by Realtime-PCR techniqueand Western blot, respectively. HO-1enzymic activity was detected by colorimetrictechnique. THP-1cells were pretreated with different concentrations of actinomycinD (Act.D) and cycloheximide (CHX) prior to5ng/mL MALP-2stimulation, theexpression of HO-1was determined by Western blot. In order to investigate MAPKssignaling pathways involved in MALP-2induced HO-1production, THP-1cells werepreincubated with different concentrations of PD98059, SP600125, SB203580for30min, then5ng/mL MALP-2was added into the medium for another16h, the HO-1level was detected by Western blot. To investigate whether MALP-2-induced HO-1expression was modulated by Nrf2pathway, THP-1cells were incubated with5ng/mL MALP-2for different time points, NF-E2-related factor2(Nrf2) protein wasdetected by Western blot. THP-1cells (1×106/well) were transfected with a finalconcentration of100nM of Nrf2siRNA and HO-1siRNA, Nrf2, HO-1andcyclooxygenase (COX)-2proteins were assessed by Western blot respectively. Results(1)0,0.01,0.1,1.0,5.0ng/ml of MALP-2induced significantly increase in HO-1mRNA and protein expression. In addition, HO-1enzymatic activity stimulated withdifferent concentrations of MALP-2in THP-1cells were (0.424±0.022) nmol/mg/h,(0.563±0.133) nmol/mg/h,(1.058±0.189) nmol/mg/h,(1.121±0.148) nmol/mg/h,(1.299±0.186) nmol/mg/h, respectively.(2) THP-1cells were pretreated with30μM MAPKs specific inhibitors SB203580,PD98059and SP600125prior to5.0ng/ml MALP-2stimulation. The level of HO-1protein expression was inhibited by38%,44%, and56%, respectively.(3)5ng/mL MALP-2induced Nrf2translocation, and transient transfection withNrf2siRNA, the HO-1protein expression was reduced by40%.(4)5ng/mL MALP-2induced COX-2protein expression was increased in atime-dependent manner, and transient transfection of THP-1cells with HO-1siRNAresulted in significant knockdown of HO-1protein expression, and permittedsignificant induction of COX-2(2.6fold).Conclusions（1）MAPKs and Nrf2signaling pathways involved in MALP-2induced HO-1production.（2）HO-1may ameliorate inflammation by downregulating COX-2overexpression.