The Recombinant Protein TPF-1 Upregulates The Secretion Of IL-6 And IL-8 And Promotes Endothelial Angiogenesis Through The ERS/PI3K/AKT And ROS/NF-κB Pathways

Aims: Human umbilical vein vascular endothelial cells(HUVEC)were used as the target cells to investigate the role of recombinant syphilis protein TPF-1 and its signaling pathway in promoting angiogenesis to lay a foundation for the study of vascular inflammation and angiogenesis of Treponema pallidum.Methods:1.The vascular formation ability of HUVEC stimulated by different concentrations of TPF-1 was determined by an in vitro angiogenesis assay.The CCK-8 assay was used to detect endothelial cell proliferation and survival stimulated by TPF-1;2.The expression of IL-6,IL-8 and VEGF in HUVEC was quantified by real-time quantitative polymerase chain reaction(RT-QPCR)and enzyme-linked immunosorbent assay(ELISA);3.Changes in the structure of the endoplasmic reticulum were observed by transmission electron microscopy(TEM)and the expressions of ERS and PI3K/Akt related pathway proteins detected by western blotting.TPF-1 stimulated nuclear translocation was assessed by immunofluorescence and changes in the expression of P-65 protein in the NF-κB signaling pathway were determined by western blotting;4.ERS,PI3K/Akt and ROS inhibitors(4-PBA,GSK2656157,STF-083010,LY294002 and NAC)were added to TPF-1-stimulated HUVEC and the expression of related pathway proteins was detected by western blotting.The expression of ROS in endothelial cells was detected by flow cytometry.The CCK-8 assay was used to observe whether the addition of ERS,PI3 K and ROS-related inhibitors would affect HUVEC cell activity after TPF-1 stimulation;5.The Transwell assay and endothelial tubularization assay were used to determine whether the addition of ERS,PI3K/ Akt and ROS-related inhibitors affected the tubularization and migration ability of TPF-1-stimulated HUVEC.Results:1.TPF-1 stimulated HUVEC at concentrations of 1.5,3,6,and 12(μg /m L),respectively.After 18 h of stimulation,it was found that TPF-1 stimulated HUVEC at concentrations of 6.25(μg/m L).The most apparent induction of HUVEC angiogenesis was observed at concentrations of 12.5(μg / m L).Furthermore,CCK-8 results showed that with the increase of TPF-1 protein concentration,the cell activity of HUVEC also increased gradually,which was statistically significant;2.The expression of cytokines IL-6 and IL-8 increased in a concentrationdependent manner after stimulation of HUVEC with different concentrations of TPF-1,while the expression of VEGF did not change significantly;3.The transmission electron microscopy results showed that the endoplasmic reticulum structure of cells in the syphilis protein stimulation group was significantly swollen,and the endoplasmic reticulum structure was broken.Western Blot results also showed that the endoplasmic reticulum stress-related proteins were significantly upregulated after the syphilis protein TPF-1 stimulation in a concentration-dependent way;4.After the addition of ERS and PI3K/ Akt inhibitors(4-PBA,GSK2656157,STF-083010,LY294002),it was observed that ERS and PI3 K related proteins were significantly inhibited,and the ERS pathway could inhibit the expression of downstream PI3K/ Akt pathway-related proteins.The expression of IL-6 and IL-8 were also inhibited;5.Angiogenesis was significantly reduced after the addition of ERS and NAC inhibitors through in vitro tubular experiments;6.CCK-8 results showed that the cell activity of cells treated with ERS,ROS,and PI3 K inhibitors was significantly reduced compared with the cells treated with no inhibitors.However,there was no statistical difference in the activity change of cells treated with PBS;7.Immunofluorescence results showed that nuclear translocation occurred in the cells treated with TPF-1 protein.In addition,western blot results showed that the expression of NF-κB related protein was significantly higher than that in the group without protein stimulation.Conclusion:1.TPF-1 promotes HUVEC to secrete IL-6 and IL-8 to induce angiogenesis.2.TPF-1 stimulated the formation of endothelial cell tubes via the ERS/PI3K/Akt pathway.3.TPF-1 can induce endothelial cell tube formation through the ROS and the NF-κB pathway.