Nuclear Factor Nrf2 Negatively Regulated Inflammatory Substances Productioninduced By Lipid-Associated Membrane Proteins Derivedfrom Mycoplasma Pneumoniaein Human Monocyte Cell Line THP-1 Cells

[Objective]Lipid-associated membrane proteins(LAMPs) played a key role in the pathogenesis of severe inflammation response induced by Mycoplasma pneumoniae(M.pneumoniae).Whereas it is still unknown how host cells regulate inflammatory responses and protect against dysfunctional inflammation during LAMPs infection. This study aims to explore the role of nuclear factor E2-related factor 2(Nrf2) in the inflammation process induced by LAMPs derived from M.pneumoniae through investigating the effect of Nrf2 on the expression of inflammatory factors,proteins and mediators in human monocyte cell line THP-1 cells,and to put forward new theoretical basis for further research on the pathogenic mechanism of M.pneumoniae.[Methods]M.pneumoniae M129 strains were cultivated and the precipitate was collected by centrifugation to extracted LAMPs.BCA was performed for LAMPs concentration determination.The lentiviral p LKO.1-Nrf2 sh RNA vectorwas constructed and transiently transfected into HEK 293 T along with packaging plasmids(ps PAX2, p MD2G) to generate lentivirus, then lentivirus were performed to infect THP-1 cells and puromycin was performed for positive infected cells screening,after that,q RT-PCR and western blot was executed to detect Nrf2 m RNA and protein expression,respectively.5μg/m L or 10μg/m L M.pneumoniae LAMPs were utilized to stimulate Nrf2 sh RNA lentivirus infected cells, control lentivirus infected cells or non-infected cells respectively,PBS stimulation as a negative control, lipopolysaccharide stimulation as a positive control,afterwards,prostaglandin E2(PGE2) production and cytokines production of interleukine 6(IL-6), interleukine 8(IL-8) were tested by ELISA,intracellular nitric oxide(NO) production was measured by Griess, cyclooxygenase 2(COX-2) and inducible nitric oxide synthase(i NOS) protein expression were determined by western blot,ROS production was detected by fluorescent probe DCFH-DA.7.5μg/m L LAMPs was intended to stimulate THP-1 cells which pre-incubated with ROS scavenger N-acetylcysteine(NAC), HO-1 specific agonist cobalt protoporphyrin(Co PP) or specific inhibitor zinc protoporphyrin(Zn PP), ultimately DCFH-DA was performed for the ROS production detection. [Results](1)The sequencing results showed that we correctly constructed the lentivirus p LKO.1-Nrf2 sh RNA. Compared with the control lentiviral infected cells as well as non-infected cells,the expression level of Nrf2m RNA was inhibited by 64.73% and 62.98%, respectively, and the Nrf2 protein expression was repressed by 44.43% and 40.48%, respectively, in the Nrf2 sh RNA lentivirus infected cells; The expression level of Nrf2 m RNA and protein in the Nrf2 sh RNA infected cells after M.pneumoniae LAMPs stimulation was suppressed by 66.12% and 52.12%,severally;(2)M.pneumoniae LAMPs stimulation significantly enhanced the expression of IL-6, IL-8, PGE2, COX-2, i NOS and the production of NO and ROS in THP-1 cells;In addition, M.pneumoniae LAMPs was applied to stimulate the THP-1 cells knocked down Nrf2 expression, the production of IL-6, IL-8 and PGE2 was 150.09pg/m L, 196.18pg/m L and 123.88pg/m L respectively,which were remarkably greater than that in the control cells(the production of IL-6, IL-8 and PGE2 was 75.03pg/m L, 90.09pg/m L and 66.42pg/m L severally);Comparison of the control cells,the expression level of COX-2 and i NOS protein separately enlarged by 20.37% and 26.65%,synchronally the NO and ROS production respectively augmented by 73.17% and 19.57% after M.pneumoniae LAMPs stimulation in the THP-1 cells knocked down Nrf2 expression;(3) THP-1 cells were pretreated with NAC, HO-1 specific agonist Co PP and specific inhibitor Zn PP,respectivly,prior to M.pneumoniae LAMPs stimulation.Compared with the untreated cells,the ROS production reduced by 8.28%, simultaneously the expression level of HO-1 m RNAmultiplied by 12.76 times in the NAC pretreated cells;Furthermore, contrasting of the untreated cells,M.pneumoniae LAMPs induced ROS production decreased by 7.64% in the Co PP pretreated cells and increased by 15.28% in the Zn PP pretreated cells.[Conclusion]Nrf2 negative regulate the expression of IL-6, IL-8, COX-2, i NOS, PGE2 and the production of NO and ROS induced by M.pneumoniae LAMPs in the THP-1 cells.