Treponema Pallidum Lipoprotein Tpn32 Induces The Upregulation Of HUVECs MCP-1 And ICAM-1 To Promote The Migration And Adhesion Of THP-1 Cells

Objective: To explore the mechanism of MCP-1 and ICAM-1upregulation in human umbilical vein endothelial cells(HUVECs)induced by lipoprotein Tpn32 in Treponema pallidum to promote the migration and adhesion of human myeloid leukemia mononuclear cells(THP-1)and provide scientific basis for prevention and treatment of syphilis infection.Methods: The prokaryotic expression vector p ET28a(+)/Tpn32 was transformed into E.coli BL21(DE3),The expressed product was induced by IPTG and purified by Ni-NTA column,then determined by BCA method.Polymyxins B was used to remove endotoxin,the endotoxin was determined by limulus lysate reagent to obtain a high purity r Tpn32.HUVECs were stimulated by r Tpn32,the m RNA transcription and protein expression levels of MCP-1 and ICAM-1 were detected by RT-PCR and ELISA method.The phosphorylation of related protein kinases were detected by Western blotting.Transwell and adhesion assay was used to detect the migration and adhesion between HUVECs and THP-1 cells.Anti-MCP-1 and anti-ICAM-1 neutralizing antibodies were used to verify the migration and adhesion activities of MCP-1 and ICAM-1 on THP-1cells.After pretreatment with ERK1/2,JNK and P38 specific inhibitors,the m RNA transcription and protein expression levels of MCP-1 and ICAM-1,the phosphorylation level of related signaling molecules in the pathway and the adhesion rate and migration rate of HUVECs to THP-1cells were detected.Results: SDS-PAGE showed that the recombinant plasmid p ET28a(+)/Tpn32 was successfully transferred into E.coli BL21,which was induced by IPTG and purified by Ni-NTA column,a high purity single target band at a relative molecular weight of 35 k Da was obtained.Western blotting indicated that the recombinant protein was Tpn32.The concentration of r Tpn32 protein was 114 μg/m L and the endotoxin was0.0982 EU/m L(< 0.1 EU/m L)after treatment with polymyxins B.When HUVECs were stimulated by r Tpn32 for different stimulation time(0～48h)and concentrations(0～800 ng/m L),the m RNA transcription and protein expression levels of MCP-1 and ICAM-1 were increased,the chemotaxis and adhesion ability of THP-1 cells to HUVECs were also promoted.However,the migration and adhesion of THP-1 cells to HUVECs were significantly suppressed by anti-MCP-1 and anti-ICAM-1 neutralizing antibodies.After pretreatment of HUVECs with ERK1/2 and P38 specific inhibitors,the m RNA transcription and protein expression levels of MCP-1 and ICAM-1,as well as the adhesion and migration rate between HUVECs and THP-1 cells were significantly inhibited.There was significant difference between treatment group and control group(P<0.05).However,JNK inhibitors exhibited poor inhibitory effects on m RNA transcription and protein expression levels of MCP-1 and ICAM-1,the differences were not statistically significant(P>0.05).Conclusion: Treponema pallidum membrane lipoprotein Tpn32 induces the upregulation of MCP-1 and ICAM-1 by HUVECs,and promotes the migration and adhesion of THP-1 cells to HUVECs,which is related to the ERK/P38 MAPK signaling pathway.