APOPTOSIS IS NOT THE MAJOR DEATH MECHANISM INDUCED BY RESEVERATROL AND OTHER AGENTS ON OSTEOARTHRITIS AND RHEUMATOID ARTHRITIS SYNOVIAL FIBROBLASTS

Abstract

In animal experiments antioxidants like Resveratrol, Quercetin-dihydrate and Selen-L-Methionine cause a growth rate decrease in synovial tissue and furthermore an inhibition of pro-infiammatory factors. We investigated the effect of these antioxidants on synovial fibroblasts of Osteoarthritis (OA) patients compared to Rheumatoid Arthritis (RA) patients.

Random biopsies of synovial membrane were obtained aseptically from joints of OA and RA patients. After in vitro expansion cells were cultivated until passage three, seeded in 96 well microtiterplates and treated with 0μM, 50μM, 100μM and 200μM of Resveratrol, Quercetin-dihydrate and Selen-L-Methionin. After 24 and 48 hours incubation cell proliferation assays and apoptosis FACS analysis were performed. Additionally woundhealing assays and photographic documentation of resettlement of synovial fibroblasts was accomplished.

The results of cell proliferation assays showed a highly significant reduction as well in OA and RA cells. In OA synovial fibroblasts 200μM of Resveratrol evoked a decrease of 72,3 ±1,7% (***), 200 μM of Quercetin-dihydrate induced a reduction of 16,11 ±3% (***). 200μM of Selen-L-Methionine evoked a decrease of 27,3 ±3,8% (***). In RA cultures 200 μM of Resveratrol evoked a decrease of 77,7 ±1,8% (***), 200μM of Quercetin-dehydrate induced a reduction of 20,38 ±15,3%(**), 200μM of Seleno-L-Methionine evoked a decrease of 23,3 ±4,8%(***)(n=20). The results of photographic documentation correlated with cell experiments. Analysis with untreated and treated OA and RA synovial fibroblasts for their content of apoptotic and necrotic cells by Annexin/7AAD staining displayed only few apoptotic cells. Caspase 3, a key mediator of apoptosis, was not activated in resveratrol-treated OA and RA synovial fibroblasts.

Resveratrol, Quercetin-dihydrate and Selen-L-Methionine showed a significant growth rate decrease in OA and RA synovial fibroblasts. In OA and RA the pharmacologic treatment with these antioxidants may be a therapeutic approach. Different apoptosis assays represented only few apoptotic cells. We therefore conclude that apoptosis is not the major pathway in resveratrol-treated synovial fibroblasts.