MgPa Of Mycoplasma Genitalium Induce Secreation Of Inflammatory Factors In SV-HUC-1 Cells Via CypA-CD147-ERK-pathway

Background: Mycoplasma genitalium was firstly isolated from urethral secretions of 2 patients with non-gonococcal uresitis(NGU)by Tully in 1981.M.genitalium is the most minimal prokaryotic cell microorganism that has no cell wall and can replicate by itself.And M.genitalium's membrane protein is anchored to the cell membrane through acyl anchors and is the basis that mediates M.genitalium adhesion to the surface of the host cells.MgPa(M.genitalium protein of adhesion,MgPa)is the most abundant membrane protein with a molecular weight of140 KDa.M.genitalium may attach to or invade cells by MgPa,and thus causing disease to host.The previous studies of our group showed that Cyclophilin A(CypA)is a receptor protein of MgPa on human urethral epithelial(SV-HUC-1).But the specific pathogenic mechanism which based on the interaction of MgPa and CypA has not been reported.The purpose of this study is to verify the effect of MgPa on the host cell after interacting with CypA,and to illuminate the possible signal pathway,which providing new ideas and targets for the prevention and treatment of M.genitalium.Objective: To study whether MgPa can induces SV-HUC-1 to promote eCypA secretion;Further study the interaction of eCypA-CD147,and then verify whether ERK1/2-NF-?B pathway can induce secretory of inflammatory cytokines to clarify the possible pathogenic mechanism ofM.genitalium.Methods:(1)Western blot(WB)and enzyme linked immunosorbent assay(ELISA)were performed to detect the expression of CypA protein in cells stimulated by MgPa;(2)WB and indirect immunofluorescence were used to verify the expression of CypA and CD147 induced by MgPa at different times;(3)The Indirect immunofluorescence and immune co-positioning tests showed that CypA and CD147 could be interacted in SV-HUC-1 under MgPa induction;(4)The expression of CypA and CD147 were detected using WB before and after the CypA-siRNA or CD147-siRNA was transfected;(5)RT-qPCR and ELISA were performed to detected the expression of cytokines after MgPa treatment at different times;(6)Western blot(WB)was used to test the influence of eCypA to the phosphorylation of ERK,p38,JNK,I?B?;(7)Indirect immunofluorescence and WB were used to detect p-65 nuclear translocation;(8)ELISA and RT-qPCR were performed to verify the influence of NF-? B inhibitors BAY 11-7082,ERK inhibitors PD 98059,JNK inhibitors SP600125 and p38 inhibitors SB202190 to the expression of inflammatory factors.Results:(1)After different concentrations of MgPa stimulating cells,the amount of CypA secretion was gradually increased and peaks at 20 ?g/ mL which is the optimal stimulus concentration;(2)The results of WB and ELISA verified that MgPa could induce cell expression and secretion of CypA in SV-HUC-1 cell and CypA was gradually increased with the prolong of time and peaked at 32 h,and then decreased;(3)The results of indirect immunofluorescence showed that the expression increased with the increasing stimulus time,and the highest expression was in the stimulation for 24h;(4)The results of indirect immunofluorescence assay and immuno-co-precipitation showed that CypA-CD147 could interact inSV-HUC-1 cells;(5)The results of WB showed that CypA-siRNA and CD147-siRNA can interfer the expression of CypA and CD147;(6)RT-qPCR and ELISA results demonstrated that different concentration of MgPa can induce the expression of IL-1?,IL-6,TNF-? and MMP-9 in protein and mRNA levels;(7)CypA-siRNA and CD147-siRNA significantly inhibit the expression of IL-1?,IL-6,TNF-? and MMP-9;(8)The levels of phosphorylation of I? B? and ERK were increased significantly in SV-HUC-1 cells treated with MgPa;(9)The results of WB and indirect immunofluorescence assay showed that MgPa can induce p65 nuclear translocation in SV-HUC-1 cells.(10)The expression of protein and mRNA levels of inflammatory factor(IL-1?,IL-6,TNF-?,MMP-9)could be significantly inhibited by NF-?B and ERK inhibitors.Conclusions: The MgPa of M.genitalium can induce SV-HUC-1 cells to secrete eCypA and thus produce inflammatory cytokines IL-1?,IL-6,TNF-? and MMP-9 via the CypA-CD147-ERK-NF-?B signaling pathway.