Centella Asiatica inhibits TNFα-induced adhesion molecule expression in endothelial cells of umbilical cords from gestational diabetic women.

Background and aims: Diabetes mellitus is associated with inflammatory endothelial activation and increased vascular leukocyte adhesion molecules expression, both playing a prominent role in the development of vascular complications. Centella Asiatica (CA) has shown anti-inflammatory properties in a variety of experimental models: however, its actions on vascular adhesion molecule synthesis and exposure have not yet been tested. Thus, we evaluated the effect of CA on TNFα-stimulated adhesion molecule expression in cultured endothelial cells obtained from umbilical cords of gestational diabetic and control women. Materials and methods: Human Umbilical Vein Endothelial Cells (HUVEC) obtained at delivery form umbilical cords of 10 healthy women (C) and 10 women with gestational diabetes (GD) were stimulated with TNFα (1 ng/mL) after a 48 hours pre-incubation with CA (25 µg/mL) or with buffer. After 12 and 16 hours, vascular cell adhesion molecules (VCAM-1), intercellular cell adhesion molecules (ICAM-1) and E-Selectin protein levels (Western Blot) and their surface expression (biparametric flow cytometry analysis) were assessed. The functional consequences of C- and GD-HUVEC treatment with CA on VCAM-1 membrane exposure were also evaluated by human monocytoid cell (U937 line) adhesion assay. Results: After a 12 hours TNFα stimulation, VCAM-1, ICAM-1 and E-Selectin protein levels were significantly higher in GD- as compared in C-HUVEC (p<0.05, Western Blot analysis). Preincubation with CA significantly decreased the effects of 12 hours TNFα-stimulation on VCAM-1 protein levels in GD-HUVEC (25-30%), while no effect was observed on C-HUVEC. Flow cytometry analysis demonstrated that, following CA pre incubation, the percentage of cells positive for surface VCAM-1 and ICAM-1 expression was modestly but significantly lower both in C- and GD-HUVEC after 12 and 16 hours TNFα stimulation. In addition, as compared to cells not pre-exposed to CA, both VCAM-1 and ICAM-1 MFI ratio (Mean Fluorescence Intensity) was lower in both CA-preincubated C- and GD-HUVEC after 12 (MFI R for VCAM-1: 0.85 and 0.65, respectively; MFI R for ICAM-1: 0.80 and 0.70, respectively) and 16 hours (MFI R for VCAM-1: 0.85 and 0.65, respectively; MFI R for ICAM-1: 0.88 and 0.90, respectively) TNFα-stimulation. We also examined the functional consequences of C- and GD-HUVEC treatment with CA in terms of U937 cell adhesion to cells. In agreement with data on TNFα-increased VCAM-1 expression, treatment of C- and GD-HUVEC with CA for 16 hours produced a significant decrease in U937 cell adhesion (p<0.001). Conclusion: In conclusion, our in vitro study demonstrates a role for Centella Asiatica in mitigating the potentially dangerous effects on endothelium of chronic exposure to hyperglycemia in vivo.