RESUMEN
Objective:To explore the effect of Duanteng Yimu decoction （DTYM） on the activation of the human umbilical vein endothelial cell （HUVEC） model and the effect on related activated proteins and vascular endothelial growth factor （VEGF） signaling pathway. Method:After DTYM （200， 400 g·mL<sup>-1</sup>） treatment of HUVEC induced by VEGF and tumor necrosis factor-<italic>α</italic> （TNF-<italic>α</italic>）， cell proliferation， migration， and tubulogenesis were detected by cell counting kit-8 （CCK-8） assay， 5-ethynyl-2'-deoxyuridine （EdU） assay， transwell migration assay， phalloidin staining， and matrix gel card method. The mRNA expression of adhesion factors， including E-selectin， intercellular adhesion molecule-1 （ICAM-1）， and vascular cell adhesion molecule-1 （VCAM-1） was detected by real-time fluorescence quantitative polymerase chain reaction （Real-time PCR）. The expression of von Willebrand factor （VWF）， platelet-endothelial cell adhesion molecule-31 （CD31）， angiogenic factor cysteine-rich-61 （CYR61）， angiopoietin-1 （ANG-1）， VEGF， and VEGF receptor-2 （VEGFR2） was detected by Western blot. Immunofluorescence was used to determine CD31 expression. Result:Compared with the normal group， the model group showed potentiated proliferation， migration， and tubulogenesis of HUVEC （<italic>P</italic><0.05， <italic>P</italic><0.01）， elevated mRNA expression of E-selectin， ICAM-1， and VCAM-1 （<italic>P</italic><0.01）， up-regulated protein expression of VWF， CD31， ANG-1， CYR61， VEGF-<italic>α</italic>， and phospho （p）-VEGFR2 （<italic>P</italic><0.05，<italic> P</italic><0.01）， and increased CD31 immunofluorescence intensity （<italic>P</italic><0.01）. Compared with the model group， the DTYM groups displayed blunted proliferation， migration， and tubulogenesis of HUVEC （<italic>P</italic><0.05，<italic> P</italic><0.01）， decreased mRNA expression of E-selectin， ICAM-1， and VCAM-1 （<italic>P</italic><0.05， <italic>P</italic><0.01）， down-regulated protein expression of VWF， CD31， ANG-1， CYR61， VEGF-<italic>α</italic>， and p-VEGFR2 （<italic>P</italic><0.05， <italic>P</italic><0.01）， and weakened CD31 immunofluorescence intensity （<italic>P</italic><0.01）. Conclusion:DTYM inhibits HUVEC proliferation， migration， adhesion， and tubulogenesis， which is associated with the regulation of CD31， VWF， CYR61， and ANG-1 expression in HUVEC and the VEGF signaling pathway.
RESUMEN
OBJECTIVE@#To investigate the effects of multi-walled carbon nanotubes (MWCNTs) with different length or chemical modification on endothelial cell activation and to explore the role of nucleotide-binding oligomerization domain-like receptor family pyrin domain containing 3 (NLRP3) inflammasome.@*METHODS@#MWCNTs were characterized by dynamic light scattering (DLS) after being suspended in culture medium. The immortalized mouse cerebral microvascular endothelial cell line b.End3 was treated with short MWCNTs (S-MWCNT, 0.5 to 2 μm), long MWCNTs (L-MWCNT, 10 to 30 μm) and the above long MWCNTs functionalized by carboxyl-(L-MWCNT-COOH), amino-(L-MWCNT-NH2) or hydroxyl-(L-MWCNT-OH) modification. Cytotoxicity of MWCNTs in b.End3 cells was determined by cell counting kit-8 (CCK-8) assay and lactate dehydrogenase (LDH) release assay, and non-toxic low dose was selected for subsequent experiments. Effects of all types of MWCNTs on the endothelial activation of b.End3 were determined by the measurement of vascular cell adhesion molecule-1 (VCAM-1) concentration in cell supernatant and adhesion assay of human monocytic cell line THP-1 to b.End3.To further elucidate the mechanism involved, the protein expressions of nucleotide-binding oligomerization domain-like receptor family pyrin domain containing 3(NLRP3) in cells treated with S-MWCNT, L-MWCNT and L-MWCNT-COOH were measured by Western blot.@*RESULTS@#At a higher concentration (125 μg/cm2) and treated for 24 h, all types of MWCNTs significantly inhibited viability of b.End3 cells. At a sub-toxic concentration (6.25 μg/cm2), all types of MWCNTs treated for 12 h significantly induced the activation of b.End3 cells, as evidenced by the elevated VCAM-1 release and THP-1 adhesion. Compared with S-MWCNT, L-MWCNT significantly promoted endothelial cell activation. L-MWCNT and L-MWCNT-COOH activated b.End3 cells to a similar extent. Furthermore, treatment with S-MWCNT, L-MWCNT and L-MWCNT-COOH increased NLRP3 expression in a time-dependent manner at 6.25 μg/cm2. Compared with S-MWCNT, cells treated with L-MWCNT for 4 h and 12 h exhibited significantly increased protein expressions of NLRP3. However, no significant differences were detected in the level of NLRP3 protein in cells treated with L-MWCNT and L-MWCNT-COOH.@*CONCLUSION@#Compared with the surface chemical modification, length changes of MWCNTs exerted more influence on endothelial cell activation, which may be related to the activation of NLRP3 inflammasome. Our study contributes further understanding of the impact of MWCNTs on endothelial cells, which may have implications for the improvement of safety evaluation of MWCNTs.