The Mechanism Of Treponema Pallidum Tp0751 Promoting Apoptosis Of Cerebrovascular Endothelial Cells And Regulating Tight Junctions

Objective: Neurosyphilis is a fearful complication caused by the invasion of Treponema pallidum Subsp.pallidum into the host central nervous system.It can occur at all stages of the course of syphilis.At present,the molecular mechanism of Treponema pallidum breaking the blood-brain barrier has not been fully elucidated.Tp0751 was the first adhesive protein identified in Treponema pallidum,which can promote the transmission of Treponema pallidum in the host by interacting with the host extracellular matrix(e.g: laminin,fibrinogen,fibronectin).Taking cerebrovascular endothelial cells as the research object,this study preliminarily investigated the effect of Treponema pallidum recombinant protein Tp0751 on the tight junction protein of cerebrovascular endothelial cells and the permeability of blood-brain barrier,in order to lay a solid foundation for further clarifying the pathogenesis of neurosyphilis.Method: 1.BEnd3 cells were used to construct a monolayer blood-brain barrier model in vitro.The TEER of blood-brain barrier model was measured by a transendothelial cell resistance meter after the stimulation of recombinant protein TP0751.The damage of tight junction structure was observed by transmission electron microscope(TEM).Western blotting assay was used to measure the expression of tight junction proteins ZO-1,Occludin and Claudin-5.2.After the recombinant protein Tp0751 stimulated bEnd3 cells,Hoechst 33258 staining was used to observe the apoptotic morphology of cells,and flow cytometry was used to detect the apoptosis rate of bEnd3 cells.3.After the recombinant protein Tp0751 stimulated bEnd3 cells,the expression of caspase 3,cleaved caspase 3,casepase 8,cleaved caspase8,Caspase 9 and cleaved caspase 9 at the translation level were analysed by Western blotting,the enzyme activity detection kit was responsible for detecting the enzyme activities of Caspase 3,Caspase 8 and Caspase9.4.After the bEnd3 cells were pretreated with apoptosis pathway inhibitor,the apoptosis rate of bEnd3 cells was detected by flow cytometry,and the expression of tight junction proteins at the translation level was detected by Western blotting.5.After the stimulation of bEnd3 cells by recombinant protein Tp0751,the expression of pro-inflammatory cytokines TNF-α,IL-1β and IL-6 at the transcription and translation levels were detected by q RT-PCR and ELISA,respectively.6.After msi RNAIL-6 pretreatment of bEnd3 cells,the expression of IL-6 was detected by q RT-PCR and ELISA,and the expression of tight junction proteins was detected by Western blotting.7.MAPKs and NF-κB specific inhibitors pretreatment bEnd3 cell,the expression of tight junction proteins were detected by q RT-PCR and Western blotting,while the expression of IL-6 was detected by q RT-PCR and ELISA.8.Phosphorylation levels of ERK1/2,JNK,P38 and IκBα were detected by Western blotting.Result: 1.The TEER value of blood-brain barrier model was decreased but was not significant,and no obvious damage to tight junction structure was observed under transmission electron microscopy after stimulated by Tp0751.Western blotting detection showed changes in the expression of tight junction proteins(ZO-1,Occludin and Claudin-5).2.After Tp0751 was co-incubated with bEnd3 cells,the apoptosis of bEnd3 cells was induced in a concentration and time dependent manner.The optimal concentration and time were 10 μg/ml and 18 h within the range of working concentration and working time.3.After Tp0751 was applied to bEnd3 cells,the protein expression levels of cleaved caspase 3 and cleaved caspase 8 were significantly up-regulated,and the enzyme activities of caspase 3 and Caspase 8 were significantly increased.4.Treatment of bEnd3 cells with inhibitors Z-VAD-FMK,Z-DEVD-FMK and Z-IETD-FMK for 30 minutes in advance not only reduced the apoptosis rate of bEnd3 cells,but also up-regulated the expression level of ZO-1 protein and down-regulated the expression level of Claudin-5 protein.5.After Tp0751 stimulated bEnd3 cells,the cells were induced to secrete IL-6 in a concentration and time dependent manner.After msi RNAIL-6 pretreatment cells,the secretion level of IL-6 was decreased,the protein expression levels of ZO-1 was increased,and the protein expression level of Claudin-5 was down-regulated.6.The expression of ZO-1 at the transcriptional level was upregulated by inhibitors BAY117082 and SP600125,while the expression of Occludin at the transcriptional level was upregulated by SB203580.BAY117082 could inhibit the expression of Claudin-5 at the transcriptional level.BAY117082,SB203580 and PD98059 could compensate for the changes of the tight junction protein ZO-1 at the translation level,the use of the inhibitor SB203580 could reverse the change in occludin at the translation level,and the inhibitors BAY117082 and PD98059 could partially restore the changes in the tight junction protein claudin-5 at the translation level.After incubation of Tp0751 with bEnd3 cells for 1 hour,phosphorylation levels of IκBα,ERK and P38 were significantly increased,while JNK phosphorylation levels were not significantly changed.SB203580,SP600125 and BAY117082 could inhibit IL-6 secretion induced by Tp0751 in bEnd3 cells.Conclusion: 1.Tp0751 is responsible for the change of tight junction proteins expression by inducing exogenous apoptosis in bEnd3 cells.2.Tp0751 induced bEnd3 cells to secrete IL-6,and IL-6 was involved in the expression changes of Tp0751 on tight junction proteins.