Essential Oils from Mediterranean Plants Inhibit In Vitro Monocyte Adhesion to Endothelial Cells from Umbilical Cords of Females with Gestational Diabetes Mellitus

Abstract

Essential oils (EOs) are mixtures of volatile compounds belonging to several chemical classes derived from aromatic plants using different distillation techniques. Recent studies suggest that the consumption of Mediterranean plants, such as anise and laurel, contributes to improving the lipid and glycemic profile of patients with diabetes mellitus (DM). Hence, the aim of the present study was to investigate the potential anti-inflammatory effect of anise and laurel EOs (AEO and LEO) on endothelial cells isolated from the umbilical cord vein of females with gestational diabetes mellitus (GDM-HUVEC), which is a suitable in vitro model to reproduce the pro-inflammatory phenotype of a diabetic endothelium. For this purpose, the Gas Chromatographic/Mass Spectrometric (GC-MS) chemical profiles of AEO and LEO were first analyzed. Thus, GDM-HUVEC and related controls (C-HUVEC) were pre-treated for 24 h with AEO and LEO at 0.025% v/v, a concentration chosen among others (cell viability by MTT assay), and then stimulated with TNF-α (1 ng/mL). From the GC-MS analysis, trans-anethole (88.5%) and 1,8-cineole (53.9%) resulted as the major components of AEO and LEO, respectively. The results in C- and GDM-HUVEC showed that the treatment with both EOs significantly reduced: (i) the adhesion of the U937 monocyte to HUVEC; (ii) vascular adhesion molecule-1 (VCAM-1) protein and gene expression; (iii) Nuclear Factor-kappa B (NF-κB) p65 nuclear translocation. Taken together, these data suggest the anti-inflammatory efficacy of AEO and LEO in our in vitro model and lay the groundwork for further preclinical and clinical studies to study their potential use as supplements to mitigate vascular endothelial dysfunction associated with DM.

Keywords: HUVEC; NF-κB p65; VCAM-1; anise; anti-inflammatory; diabetes; endothelial dysfunction; essential oils; inflammation; laurel.

Figure 1

C- and GDM-HUVEC cell viability after 24 h and 48 h of treatment with (a) AEO and (b) LEO at different concentrations (0.025, 0.05, and 0.1% v/v) or with medium alone (BASAL). Results are shown as mean ± Standard Error of the Mean (SEM). All experiments were conducted in technical and biological triplicate using at least 3 different primary culture strains of C- and GDM-HUVEC.

Figure 2

Effects of AEO on U937 monocyte adhesion to C- and GDM-HUVEC. Cells were serum-starved and incubated for 16h with TNF-α (1 ng/mL) following 24 h of pre-incubation with AEO (0.025% v/v) or with medium alone (BASAL). Quantitative data express the number of U937 cells adhering within a high-power field (3.5 mm²). Each measurement is expressed as mean ± SEM of adhering cells from 3 independent experiments, consisting of 8 counts per condition. (a) **** p < 0.0001 TNF-α vs. BASAL; * p < 0.05 TNF-α vs. TNF-α + AEO. (b) **** p < 0.0001 TNF-α vs BASAL; **** p < 0.0001 TNF-α vs. TNF-α+AEO. (c) Representative images of U937 cell adhesion to C- and GDM-HUVEC. Scale bar: 100 µm.

Figure 3

Effects of LEO on U937 monocyte adhesion to C- and GDM-HUVEC. Cells were serum-starved and incubated for 16h with TNF-α (1 ng/mL) following 24 h of pre-incubation with LEO (0.025% v/v) or with medium alone (BASAL). Quantitative data express the number of U937 cells adhering within a high-power field (3.5 mm²). Each measurement is expressed as mean ± SEM of adhering cells from 3 independent experiments, consisting of 8 counts per condition. (a,b) **** p < 0.0001 TNF-α vs. BASAL; * p < 0.05 TNF-α vs. TNF-α+LEO. (c) Representative images of U937 cell adhesion to C- and GDM-HUVEC. Scale bar: 100 µm.

Figure 4

Effects of (a,b) AEO and (c,d) LEO on VCAM-1 membrane exposure levels in C- and GDM-HUVEC. Cells were serum-starved and incubated for 16h with TNF-α (1 ng/mL), following 24 h of pre-incubation with AEO or LEO (0.025% v/v) or with medium alone (BASAL). Results are presented as Mean Fluorescence Intensity (MFI) Ratio and shown as mean ± Standard Deviation (SD). All experiments were conducted in technical and biological triplicate using at least 3 different primary culture strains of C- and GDM-HUVEC. (a–d) *** p < 0.001 TNF-α vs. BASAL; * p < 0.05 TNF-α vs AEO+TNF-α or LEO+TNF-α.

Figure 5

Effects of (a,b) AEO and (c,d) LEO on VCAM-1 mRNA expression in C- and GDM-HUVEC. Cells were serum-starved and incubated for 16 h with TNF-α (1 ng/mL), following 24 h of pre-incubation with AEO or LEO (0.025% v/v) or with medium alone (BASAL). Results of RT-qPCR analysis are shown as individual values and mean ± SD of at least 3 independent experiments. (a,c) * p < 0.05 TNF-α vs. AEO+TNF-α or LEO+TNF-α; (b,d) ** p < 0.01 TNF-α vs. AEO+TNF-α or LEO+TNF-α.

Figure 6

Effects of AEO on NF-κB p65 nuclear translocation in C- and GDM-HUVEC. Graphs and representative immunofluorescence images showed NF-κB p65 nuclear translocation in C- and GDM-HUVEC by immunofluorescence staining. Cells have been marked with NF-κB p65 primary antibody and Alexa Fluor 488 anti-rabbit secondary antibody, and nuclei were stained with 40, 6-diamidino-2-phenylindole (DAPI) following 24 h of treatment with AEO (0.025%). Data, calculated as the ratio of the MFI of NF-κB p65 (green) on the MFI of nuclei (blue), were obtained by analyzing at least 3 different fields for each image with ImageJ software (NIH, USA ImageJ software, public domain available at: http://rsb.info.nih.gov/nih-image/). Results are expressed as mean ± SEM. (a,b) * p < 0.05 TNF-α vs. AEO+TNF-α; (b) * p < 0.05 TNF-α vs. BASAL. Scale bar: 100 µm.

Figure 7

Effects of LEO on NF-κB p65 nuclear translocation in C- and GDM-HUVEC. Graphs and representative immunofluorescence images show NF-κB p65 nuclear translocation in C- and GDM-HUVEC by immunofluorescence staining. Cells have been marked with NF-κB p65 primary antibody and Alexa Fluor 488 anti-rabbit secondary antibody, and nuclei were stained with DAPI, following 24 h of treatment with LEO (0.025%). Data, calculated as the ratio of the MFI of NF-κB p65 (green) on the MFI of nuclei (blue), were obtained by analyzing at least 3 different fields for each image with ImageJ software (NIH, ImageJ software). (a,b) ** p < 0.01 TNF-α vs. LEO+TNF-α; (b) * p < 0.05 BASAL vs. TNF-α or LEO. Scale bar: 100 µm.