| Background and ObjectiveSyphilis is a common sexually transmitted disease.The painless ulcers(also known as chancres)at the skin where genital infection occurs in patients with primary syphilis can heal themselves in a few weeks,followed by a period of occultation and secondary infection.The exact mechanism behind this is not yet clear.In essence,chancres self-healing is the healing of skin wounds,a process that requires the participation of a variety of cells.Among them,fibroblasts and microvascular endothelial cells in the skin play a key role,specifically secreting fibronectin(FN),an extracellular matrix component that is crucial for wound repair.Treponema pallidum membrane protein Tp0136 is one of the few proteins found to be able to attach to the body fibronectin,and its transcriptional level is increased in the healing stage of infected rabbit chancres,indicating that it could participate in the process of Treponema pallidum infection.Up to date,it has not been reported whether Tp0136 protein adhering to the fibronectin on the cell surface affects the healing of skin wounds through fibroblasts and microvascular endothelial cells.In this study,human skin fibroblasts and human skin microvascular endothelial cells(HMEC-1)were used as the research subjects to investigate the effects of Tp0136 protein on the migration of human skin cells and vascular permeability changes.The following studies were conducted:(1)To explore the effect of Tp0136 protein on the migration of human skin fibroblasts and its mechanism;(2)To investigate the effect of Tp0136 protein on the permeability of microvascular endothelial barrier and its mechanism.Through the above-mentioned studies,it would lay a foundation for clarifying the self-healing effect of Tp0136 on syphilis chancres and for further research on the self-healing mechanism of chancres.Methods1.To investigate the effect of Tp0136 protein on fibroblast migration and its mechanism:(1)The effect of TP0136 on fibroblast migration was detected by cell scratch andTranswell assay.(2)The expression levels of migration-related cytokines were measured by ELISA and RT-PCR.(3)The effect of MCP-1(monocyte chemokine protein-1)neutralizing antibody and CCR2(CC chemokine receptor 2)inhibitor on the migration process of fibrocells induced by Tp0136 protein was analyzed.(4)Western blotting and migration pathway inhibitors were used to analyze the activation of relevant signaling pathways.(5)TLR4 inhibitors and PCR were used to analyze the role of TLR4 in the process of fiber cell migration induced by Tp0136 protein.2.To explore the influence of Tp0136 protein on the permeability of microvascular endothelial barrier and its mechanism:(1)A microvascular endothelial barrier permeability detection system was constructed using microvascular endothelial HMEC-1 cells to evaluate the effect of Tp0136 protein on barrier permeability.(2)The transcripts of HMEC-1 cells treated with Tp0136 at 0 and 6 h were analyzed by RNA sequencing,and the potential factors affecting the upregulation of microvascular endothelial barrier permeability were screened.(3)Using CCK8,cell scratches,Transwell experiment,tubule formation,budding test,ELISA,immunofluorescence,Western blotting,cytokine microarray and other analyses-to investigate the impact of Tie2-PI3K-AKT-FoxO1-IL-6 pathway on angiogenesis of microvascular endothelial cells,and its role in the process of increase vascular permeability barrier.(4)The effects of Tie2-PI3K-Akt-PTBP1/SRSF1 pathway on the morphological abnormalities of microvascular endothelial cells and its role in the up-regulation of vascular barrier permeability were analyzed by RNA variable splicing detection,PCR,RNA-IP,gene knockout and overexpression,Co-IP and Western blotting.(5)The transcripts of HMEC-1 cells treated with Tp0136 at 6 and 12 h were analyzed by RNA sequencing,and the otential factors affecting the down-regulation of microvascular endothelial barrier permeability were screened.(6)Western blotting,immunofluorescence,ELISA and PCR were used to analyze the role of Tie2-PI3K-Akt-FoxO1/PTBP1/SRSF1 pathway in the down-regulation of vascular barrier permeability.Results1.Tp0136 protein promoted fibroblast migration in a concentration-dependent and time-dependent manner(p<0.05).During the process of migration,the migration-related cytokine MCP-1 was significantly increased(p<0.005),and both the neutralizing antibody of MCP-1 and the inhibitor of CCR2(RS102895)could effectively inhibit the migration of fibroblasts(p<0.005).Tp0136 activated ERK/JNK/PI3K/NF-κB pathway,and its inhibitors,includingU0126(ERK),SP600125(JNK),LY294002(PI3K)and Bay11-7082(NF-κB)could effectively inhibit the secretion of MCP-1 and migration of fibroblasts(p<0.05).TLR4 inhibitor TAK242 also effectively inhibited MCP-1 secretion,fibroblast migration and activation of ERK/JNK/PI3K/NF-κB pathway.2.Tp0136 promoted the upregulation of micro vascular endothelial barrier permeability in a concentration-dependent manner.We found that the endothelial barrier permeability of HMEC-1 cells increased significantly after treatment with 10μg/mL Tp0136 for 0-6 h(p<0.01),and reached the peak at 6 h.With the passage of treatment time,the permeability of the endothelial barrier decreased gradually from 6 to 12 hours(p<0.01).The 0-6 h transcriptome changes of Tp0136 in HMEC-1 cells were analyzed,and the upregulated genes were mainly on cell division and proliferation,angiogenesis,and RNA-binding proteins associated with alterable splicing.We found that during 0-6 hours,Tp0136 protein promoted angiogenesis in microvascular endothelial cells and increased endothelial barrier permeability by down-regulating the phosphorylation level of Tie2-PI3K-AKT-FoxO1 pathway.Meanwhile,Tp0136 activated the FN variable shear mechanism by down-regulating the phosphorylation level of Tie2-PI3K-Akt-PTBP1/SRSF1 pathway,promoted the morphology abnormality of microvascular endothelial cells,and increased the permeability of the endothelial barrier.On the contrary,we analyzed the transcriptome changes of Tp0136 protein in HMEC-1 cells during 6-12 h.It was found that the pathways regulated by Tp0136 at 6-12 h was reversed as well as the genes and pathways during 0-6 h.Tp0136 protein up-regulated the phosphorylation level of Tie2-PI3K-Akt-FoxO1 and Tie2-PI3K-Akt-PTBP1/SRSF1 pathway,and decreased the endothelial barrier permeability.Conclusions1.Tp0136 activates the ERK/JNK/PI3K/NF-κB pathway through the receptor TLR4 on the fibroblast membrane to promote the secretion of MCP-1,and MCP-1 binds to the receptor CCR2 on the cell membrane to promote cell migration.2.The first 6 hours in the introduction of Tp0136 on HMEC-1 cells(0-6 hours),it down-regulated the phosphorylation level of Tie2-PI3K-Akt signaling pathway,regulated FoxO1-IL-6 and PTBP1/SRSF1 pathway,activated the variable shear of FN gene,promoted the morphology abnormality of microvascular endothelial cells and mediated the increase of permeability of microvascular endothelial barrier;On the contrary,Tp0136 regulates FoxO1 and PTBP1/SRSF1 pathways by upregulating the phosphorylation level of Tie2-PI3K-Akt pathway in the last 6 h(6-12 h)after the action of HMEC-1 cells,promoting the morphological restoration of microvascular endothelial cells and mediating the decrease of permeability of the microvascular endothelial barrier.Tp0136 protein plays a critical role in promoting fibroblast migration and affecting changes in microvascular endothelial barrier permeability,which is essential for promoting skin healing.An in-depth study on the self-healing effect of Tp0136 on chancroid syphilis would lay a foundation for further research on the self-healing mechanism of chancres. |
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