| Background and objective: Disease caused by chlamydia trachomatis(C.trachomatis)are preventable blindness and bacterial sexually-transmitted.At present,the incidence of C.trachomatis is increasing year by year in China.A primary reason for pathophysiology of C.tarchomatis infection is link to inflammatory responses.However,the virulence factors on how C.trachomatis triggers the production of proinflammatory cytokines and the precise mechanism remains unknown.C.trachomatis glycogen synthase(GlgA)was identified as chlamydia secreted protein,which may be associated with C.trachomatis pathogenesis.Here,we examined the host inflammatory responses to GlgA and illustrated the signaling pathway involved in the inflammatory mechanism for knowing the molecular basis of C.trachomatis infection.Methods:1.We Constructed pGeX-6P-1-GlgA plasmid and induced the expression of pGeX-6P-1-GlgA at 20℃ with 0.5 mM IPTG.PurifedGlgA is identified by Western Blot.2.THP-1 cells were incubated with 15 μg/mL GlgA,qRT-PCR and ELISA were used to measure the transcription and protein expression level of IL-6,IL-8,IL-10,IL-1β and TNF-α.3.THP-1 cells were stimulated with 15 μg/mL of GlgA from 0-48 h,and THP-1 cells were treated with 0-15 μg/mL GlgA at different time,qRT-PCR was used to measure the mRNA expression and ElisA was used to detecte the protein expression level of TNF-α,IL-1β,IL-8.4.qRT-PCR and ElisA were used to detecte the response of the proinflammatory cytokines in gene and protein expression,followed by pDeNy-hMyD88 transfected with THP-1 cell.5.THP-1 cells were transfected with specific siRNA of TLR2,TLR4,TLR6,QRT-PCR and ElisA were used to detecte the transcription and protein expression level of TNF-α,IL-1β and IL-8.6.THP-1 cells was treated with 10 μg/mL GlgA for 15,30,60 or120 min to assess the influences on molecules in the p38,ERK and JNK signaling pathway by Western Blot.7.THP-1 cells were pretreated with p38 inhibitor,ERK inhibitor and JNK inhibitor at 30 μM for 30 min,respectively.Then,THP-1 cells were stimulated 10 μg/mL GlgA to measure the transcription and protein expression level of proinflammatory cytokines by qRT-PCR and ELISA.8.THP-1 cells was treated with 10 μg/mL GlgA for 15,30,60 or120 min to assess the influences on molecules in the IкBα signaling pathway by Western Blot.Further more,indirect immunofluorescence was to analysed nucleus translocation of NF-кB p65.Results:1.Purified GlgA protein was identified by Western blot and showed an obvious blot at the size of 25 KDa.2.THP-1 cells were incubated with 15 μg/mL GlgA,qRT-PCR and ELISA results shown that GlgA significantly induced TNF-α,IL-1β,IL-8mRNA expression,and also significantly induced TNF-α,IL-1β,IL-8secretion.3.THP-1 cells were incubated with 0-15 μg/mL GlgA from 0-36 h,the level of TNF-α,IL-1β,IL-8 mRNA was significantly increased in 8 h,while the level of TNF-α,IL-1β,IL-8 expression was significantly increased in 24 h.we observed that GlgA with 10 μg/ml concentration obtained a significant production of proinflammatory cytokines in THP-1cells.4.pDeNy-hMyD88 was transfected with THP-1 cell,then GlgA stimulated THP-1 cells,qRT-PCR and ELISA results showed that the transcription and protein level of cytokines were downregulation.5.Blockade the TLR2 and TLR4 signaling abrogated induction level of IL-8,TNF-α and IL-1β.Nevertheless,transfection of TLR6 siRNA,the expression of proinflammation cytokines did not show downregulation.6.Western Blot results showed that when THP-1 cells was treated with 10 μg/mL GlgA,the phosphorylation levels of ERK and JNK was significant but transient increased at 30 min but not p38.7.THP-1 cells were pretreated with specific inhibitors,p38 inhibitor,ERK inhibitor and JNK inhibitor,respectively.The GlgA-induced proinflammatory cytokines was notably reduced by ERK and JNK inhibitor.However,p38 inhibitor did not cause significantly changes in the mRNA and protein expression of IL-8,TNF-α,IL-1β.8.Western blot results showed that protein level of P-IкBαstimulated with GlgA was obviously degradation by 60 min.Also,immunofluorescence analysis confirmed NF-кB p65 subunit was translocated from cytoplasm into nucleus of GlgA treated THP-1 cells for6 h.Conclusions: GlgA induced IL-8,TNF-α and IL-1β production in THP-1cells via TLR2/ TLR4 and MAPK/NF-кB pathways... |
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