Dose-Dependent Cannabidiol-Induced Elevation of Intracellular Calcium and Apoptosis in Human Articular Chondrocytes

Abstract

Cannabidiol (CBD) is the most abundant non-psychoactive compound of Cannabis sativa extracts. Cannabinoids have been shown to exhibit anti-inflammatory, analgesic, antioxidant, neuroprotective, and anti-tumorigenic effects. In the present study, we investigated the effects of CBD on human articular chondrocytes. Cell viability was determined by Resazurin assays. Apoptosis was analyzed by annexin-V/7-actinomycin D (7-AAD) staining followed by flow cytometry. Caspase 3/7 activity was measured with caspase assays. Intracellular Ca2+ ([Ca2+]i) was monitored by time-lapse fluorescence imaging. The perforated whole-cell patch-clamp technique was used for measuring the cell membrane potential. Erk1/2 phosphorylation was assessed by western blot analysis. The chondrocyte cell line C28/I2 and primary chondrocytes showed a reduced viability after treatment with concentrations of CBD greater than 4 µM. This apoptotic effect was accompanied by an increase of caspase 3/7 activity and an increase in the early apoptotic cell population. CBD elevated [Ca2+]i, which was accompanied by depolarization of the cell membrane potential. The increase of [Ca2+]i was abrogated, when Ca2+ was omitted from the bath solution, indicating an influx of extracellular Ca2+. The cannabinoid receptor 1 (CB1) antagonist AM251 inhibited the Ca2+ influx triggered by CBD. Preincubation with AM251 reduced the toxic effects of CBD. By looking for mediators of the apoptotic CBD effect downstream of the CB1 receptor, enhanced Erk1/2 phosphorylation could be detected after CBD treatment. However, this Erk1/2 activation proved to be unaffected by CB1 receptor blockage. The present study demonstrates that CBD promotes apoptosis and [Ca2+]i elevation in human articular chondrocytes via a CB1-receptor-mediated mechanism. © 2019 The Authors. Journal of Orthopaedic Research® published by Wiley Periodicals, Inc. on behalf of Orthopaedic Research Society J Orthop Res 37:2540–2549, 2019.