Abstract:

Zhang Ruili, Wang Qianqiu Department of Dermatology, Wuxi No.2 People′s Hospital, Wuxi 214002, Jiangsu, China （Zhang R）; Department of Sexually Transmitted Disease Clinical Management, Institute of Dermatology, Chinese Academy of Medical Sciences and Peking Union Medical College, Nanjing 210042, China （Wang QQ） Corresponding author: Wang Qianqiu, Email: doctorwqq@163.com 【Abstract】 Objective To investigate mechanisms underlying the regulation of the permeability of vascular endothelial cells by the Treponema pallidum membrane protein Tpp47. Methods Human umbilical vascular endothelial cell （HUVEC） monolayers were established as a model, and were directly cultured with the presence of recombinant Tpp47 protein （rTpp47-treated group）, or boiled and inactivated rTpp47 （negative control group）. Some HUVEC monolayers, which were pretreated with the RhoA/ROCK signal pathway inhibitor Y-27632 for 30 minutes and then cultured with the presence of rTpp47, served as the pretreatment group. After 1- and 4-hour additional culture, enzyme-linked immunosorbent assay （ELISA） was performed to estimate the permeability of these cell monolayers to horseradish peroxidase （HRP）. After 12 hours of culture, rhodamine-phalloidin was used to stain cytoskeletal proteins, and confocal laser scanning microscopy was performed to observe the arrangement of the cytoskeletal protein F-actin. Western-blot analysis was conducted to measure the expressions of RhoA in HUVECs treated with rTpp47 or inactivated rTpp47. Results The supernatant level of HRP （expressed as the absorbance value at 450 nm） was significantly higher in the rTpp47-treated group than in the negative control group （0.81 ± 0.10 vs. 0.39 ± 0.09, P < 0.05）, but no significant difference was observed between the pretreatment group （0.51 ± 0.10） and rTpp47-treated group or negative control group （both P > 0.05） after 1-hour culture. Similarly, the rTpp47-treated group showed significantly increased levels of HRP compared with the pretreatment group and negative control group （2.31 ± 0.14 vs. 1.21 ± 0.12 and 0.73 ± 0.12, both P < 0.05）, while there was no significant difference between the pretreatment group and negative control group after 4-hour culture. The expression of RhoA in HUVECs treated with rTpp47 was significantly higher than that in those treated with inactivated rTpp47. Confocal laser scanning microscopy showed that rTpp47 treatment led to the rearrangement of F-actin in HUVECs followed by the formation of stress fibers in cytoplasm, while Y-27632 could partly inhibit the rearrangement of F-actin. Conclusion The recombinant Treponema pallidum membrane protein Tpp47 can regulate the permeability of vascular endothelial cells through the RhoA/ROCK signal pathway.