Recombinant Treponema Pallidum Protein Tp0768 Enhances The Chemotactic Effect Of HUVECs On Neutrophils Through TLR2/ER Stress Pathway

Purpose:In this study,human umbilical vein endothelial cells(HUVECs)were used as target cells,and the effects of recombinant protein Tp0768 of Treponema pallidum on HUVECs’ chemotaxis of neutrophils were discussed,and the roles of signal molecules such as TLR2 and ER stress in HUVECs’ chemokines secretion were determined,which provided a new possibility for the mechanism of Tp0768 promoting vascular inflammation and laid a foundation for further revealing the pathogenic mechanism of Treponema pallidum.Methods:1.Construction of prokaryotic vector,expression and purification of Treponema pallidum Tp0768 recombinant protein.2.After the HUVECs were stimulated by Tp 0768,RT-PCR was used to detect the mRNA expression levels of CXCL1,CXCL2 and CXCL8 in HUVECs;ELISA was used to detect the protein expression levels of CXCL1,CXCL2 and CXCL8 in supernatant;the supernatants after co-culture were taken for neutrophil transwell experiment to detect the ability of neutrophil chemotaxis.3.After the HUVECs were stimulated by Tp 0768,RT-PCR was used to detect the mRNA expression levels of TLR1,TLR2,TLR4 and MyD88;WB was used to detect the protein expression levels of TLR2,PERK and IRE1;transmission electron microscope observes the morphology and structure of ER of HUVECs.4.After using TLR2 inhibitor,RT-qPCR was used to detect the mRNA expression levels of CXCL1,CXCL2,CXCL8 and MyD88;ELISA was used to detect the protein expression levels of CXCL1,CXCL2 and CXCL8;WB was used to detect the protein expression levels of PERK,IRE1 and BIP;neutrophil transwell experiment was used to detect the ability of neutrophil chemotaxis.5.After using MyD88 inhibitor,RT-PCR was used to detect the mRNA levels of CXCL1,CXCL2 and CXCL8;CCK8 was used to detect the activity level of HUVECs.6.After using inhibitors of PERK and IRE1 respectively,RT-PCR was used to detect the mRNA expression levels of CXCL1,CXCL2 and CXCL8;ELISA was used to detect the protein expression levels of CXCL1,CXCL2 and CXCL8;Neutrophil transwell experiment was used to detect the change of chemotactic ability of neutrophils.Results:1.After constructing prokaryotic expression vector and expressing and purifying,Tp0768 protein with concentration of 1.1mg/m L and purity of above 90% was obtained.2.After Tp0768 stimulated HUVECs,the expression levels of CXCL1,CXCL2 and CXCL8 mRNA and protein were improved in a dose-dependent manner,and the chemotaxis of neutrophils was promoted.3.After Tp0768 stimulated HUVECs,the mRNA expression level of TLR2 increased,but there was no correlation between the mRNA expression levels of TLR1、 TLR4 and the concentration of Tp0768.After TLR2 inhibitor was used,the mRNA and protein expression levels of CXCL1,CXCL2 and CXCL8 decreased significantly,and the chemotaxis of neutrophils also showed the same trend.4.After Tp0768 stimulated HUVECs,the mRNA expression level of MyD88 increased to some extent,but the correlation with the concentration of Tp0768 was not very obviously.The expression level of MyD88 mRNA could not be reduced by using TLR2 inhibitor,at the same time,after pretreatment of endothelial cells with MyD88 inhibitor,the mRNA of CXCL1,CXCL2 and CXCL8 did not decrease with the increase of inhibitor concentration,although their expression decreased at20μM,CCK8 results showed that the activity of endothelial cells was seriously affected at 20 μM.5.Tp0768 can cause morphological changes in the ER of endothelial cells,the lumen of ER becomes larger and vacuoles are formed,the rough ER is obviously increased,and the protein expression of ER stress-related signaling pathways PERK and IRE1 is increased.After pretreatment of endothelial cells with inhibitors of PERK and IRE1 respectively,the mRNA and protein expression levels of CXCL1,CXCL2 and CXCL8 are obviously decreased,and the chemotaxis of neutrophils is also obviously inhibited.6.After pretreatment with TLR2 inhibitor,the protein expression levels of ER stress-related signal molecules PERK,IRE1 and BIP were obviously inhibited.Conclusions:1.Tp0768,the recombinant protein of Treponema pallidum,upregulates the neutrophil chemokines CXCL1,CXCL2 and CXCL8 secreted by HUVECs,thus promoting the chemotaxis of neutrophils.2.Tp0768 upregulates HUVECs’ chemotaxis of neutrophils,which is regulated by TLR2/ER stress,and MyD88 signal molecule may not be involved in this process.