1Universität Osnabrück Institut für Gesundheitsforschung und Bildung Osnabrück, Deutschland
Background: Arthrocen (Ar) is a compound containing unsaponifiables made from fruits and seeds of avocado and soybean oil (ASUs). In the US, ASUs are classified as dietary supplements. ASUs contain phytosterols, lipophilic vitamins, and terpenoids and are commonly used to treat osteoarthritis symptoms. Recent data implicate that ASUs may exert anti-inflammatory effects on acetic acid-mediated colon inflammation through suppressing the NF-κB pathway in a rat model. As dietary supplements such as omega-3 fatty acids have been found to lead to reductions in residual cardiovascular risk we followed the hypothesis that Ar can reduce TNF-α-induced expression of adhesion molecules up-regulated in the pathogenesis of vascular dysfunction. Therefore, we analyzed Ar-mediated effects on adhesion molecule gene expression in HUVEC.
Methods: Ar was bought from Pharmin USA. Contents of one capsule was weighed and dissolved in ethanol (stock concentration of 2500 µg/mL). HUVEC were pre-treated with Ar concentrations of 2.7, 8.3 or 25 µg/mL for 30 minutes before stimulation with TNF-α (10 ng/mL). Viability of HUVEC was determined by the PrestoBlueTM assay. Total RNA was isolated and cDNA was synthesized by reverse transcription. Expression of the following genes was quantified on a CFX96 Real Time System, BIO-RAD: ICAM1, VCAM1, SELE encoding E-selectin, OLR1 encoding LOX-1, and NOS3 encoding eNOS. We used 18S rRNA as reference gene. In addition, we analysed NOS3 expression as a marker for maintaining vascular function.
Results: Ar treatment for 4 and 16 hours had no relevant effects on cell viability. TNF-α-treatment for 16 hours significantly up-regulated ICAM-1, VCAM1, SELE and OLR1 expression (see Table 1). NOS3 expression was reduced significantly by TNF-α-treatment. For ICAM-1, VCAM1, SELE we found that Ar failed to reduce the TNF-α-induced up-regulation with Ar at a concentration of 8,3 µg/mL showing at least a five-fold-higher expression compared to HUVEC treated with TNF-α only. Interestingly, OLR1 expression was up-regulated in a dose-dependent manner in the presence of both TNF-α and Ar. However, Ar at concentrations of 2,7 and 25 µg/mL failed to restore TNF-α-induced down-regulation of NOS3 expression back to normal levels. At a concentration of 8,3 µg/mL, Ar lead to a 1.5-fold higher expression in the presence of TNF-α compared to cells treated with TNF-α only.
Conclusions: Cell viability was not significantly affected by Ar. However, Ar failed to reverse TNF-α-induced up-regulation of adhesion molecule gene expression and further increased adhesion molecule gene expression in a dose-dependent-manner (OLR1). Ar at a concentration of 8.3 µg/mL seemed to enhance the inflammatory reaction induced by TNF-α. Further experiments will be needed to elucidate if Ar exerts potential beneficial effects on vascular function.
| Gene | Control | TNF-α |
TNF-α+ Ar (2.7 µg/mL) |
TNF-α+ Ar (8.3 µg/m) | TNF-α+ Ar (25 µg/mL) | Ethanol |
| ICAM1 (n=4) | 0.00±0.00* | 1.00 | 0.93±0.49 | 8.88±7.70 | 1.29±0.11 | 0.01±0.00 |
| VCAM1 (n=4 | 0.00±0.00* | 1.00 | 0.83±0.47 | 6.10±5.23 | 0.82±0.13 | 0.00±0.00 |
| SELE (n=4) | 0.00±0.00* | 1.00 | 0.61±0.31 | 5.53±4.50 | 1.27±0.34 | 0.00±0.00 |
| OLR1 (n=3) | 0.34±0.19† | 1.00 | 1.52±1.14 | 6.34±3.14† | 18.83±6.25 | 0.80±0.31 |
| NOS3 (n=3) | 9.60±6.81† | 1.00 | 1.00±0.37 | 1.53±0.87 | 0.99±0.59 | 2.56±0.81† |
Table 1. Fold-changes of gene expression ± SEM in TNF-α-treated HUVEC normalized to 18S rRNA. * p<0.05 vs TNF-α-treated HUVEC (rank sum test); † p<0.05 vs TNF-α-treated HUVEC (t-test).