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, 37 (12), 2540-2549

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

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

Martina Winklmayr et al. J Orthop Res.

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.

Keywords: CBD; Ca2+; apoptosis; calcium; cannabidiol; chondrocyte; osteoarthritis; viability.

Figures

Figure 1
Figure 1
Cannabidiol (CBD) reduces viability and induces apoptosis in human chondrocytes. Relative viability of C28/I2 cells (A) or human primary chondrocytes (B) treated with 3–30 µM CBD for 2 and 24 h. Results are given as % of untreated controls (UTC). Mean ± standard deviation (SD) of three independent 7‐hydroxy‐3H‐phenoxazin‐3‐one‐10‐oxide sodium salt (Resazurin) experiments. (C) Flow cytometry analysis of annexin‐V/7‐actinomycin D (7‐AAD) staining in C28/I2 cells treated ± CBD. (D) Caspase 3/7 activity (% of UTC) of C28/I2 cells treated with rising concentrations of CBD. Mean ± SD of three independent experiments. (E) Microscopy images of C28/I2 cells untreated, starved, or starved and treated with 10 or 30 µM CBD for 2 h.
Figure 2
Figure 2
Cannabidiol (CBD) induces [Ca2+]i elevation and a depolarization of the chondrocyte cell membrane potential (V mem). Time‐lapse fluorescence imaging of [Ca2+]i (A–D) and whole‐cell patch‐clamp time course of V mem and mean V mem (E, F). (A) Time course of a single [Ca2+]i measurement using Fura‐2/AM. C28/I2 cells were perfused with 10 µM histamine (His) as a positive control, subsequently washed with bath solution containing the CBD solvent, ethanol (Ve = Vehicle) and then treated with 100 µM CBD, followed by another washing step and histamine application. Gray lines represent recordings of single cells, the black line represents the average of the single‐cell recordings. Boxes above indicate the perfusion conditions. (B) Pseudo‐color image of C28/I2 cells stained with Fura‐2/AM. A shift toward yellow and red indicates accumulation of [Ca2+]i. (C) Mean ± standard deviation (SD) of maxima of 340/380 ratios of C28/I2 cells treated with rising concentrations of CBD (10 µM: three independent experiments, 73 single cells; 30 µM: two independent experiments, 85 single cells; 100 µM: five independent experiments; 93 single cells). (D) Mean ± SD of maxima of 340/380 ratios of primary human chondrocytes treated with histamine as positive control followed by rising concentrations of CBD (10 µM: one experiment, six single cells; 30 µM: two independent experiments, 26 single cells). (E) Time course of V mem of a single experiment. Each dot represents the mean V mem over 15 s. (F) Mean ± SD V mem in the absence (Ctrl) and the presence of 30 µM CBD. [Color figure can be viewed at wileyonlinelibrary.com]
Figure 3
Figure 3
Absence of extracellular Ca2+ abolishes the cannabidiol (CBD)‐induced [Ca2+]i elevation and improves cell viability after CBD treatment. Time‐lapse fluorescence imaging of cytosolic Ca2+ of C28/I2 and primary chondrocytes with CBD treatment with and without extracellular Ca2+ (A–C) and relative viability and caspase 3/7 activity tested in C28/I2 cells with and without Ca2+ chelating compounds (D–F). (A) Time course of a single [Ca2+]i measurement. C28/I2 cells were perfused with 10 µM histamine (His) as positive control, subsequently vehicle and CBD without Ca2+ were applied, followed by CBD and histamine in the presence of Ca2+. (B, C) Mean ± standard deviation (SD) of maxima of 340/380 ratios of C28/I2 cells (B; four independent experiments; 83 single cells) and primary chondrocytes (C; two independent experiments; 55 single cells) treated with 100 µM CBD in the absence or presence of extracellular Ca2+. (D, E) Relative cell viability of C28/I2 cells treated with rising concentrations of CBD with or without 2 mM ethylenediaminetetraacetic acid (EDTA) (d) or 4 mM 1,2‐bis(o‐aminophenoxy)ethane‐N,N,N′,N′‐tetraacetic acid (BAPTA) (E). (F) Relative caspase 3/7 activity in C28/I2 cells treated with rising concentrations of CBD in the absence or presence of 4 mM BAPTA. (D–F) Means ± SD of three independent experiments.
Figure 4
Figure 4
Mean ± standard deviation (SD) of maxima of 340/380 ratios of three independent [Ca2+]i measurements on C28/I2 cells treated with 30 µM CBD in the absence or presence of 2 mM CdCl2 (A; three independent experiments; 51 single cells) or 20 µM Nifedipine (B; three independent experiments; 79 single cells).
Figure 5
Figure 5
The cannabinoid receptor 1 (CB1) receptor blocker AM251 partially antagonizes cannabidiol (CBD)‐induced effects. (A) Mean ± standard deviation (SD) of maxima of 340/380 ratios of three independent experiments on C28/I2 cells treated with 100 µM CBD in the absence or presence of 16 µM CB1 receptor antagonist AM251 or CB2 receptor antagonist AM630. (B) Relative viability of C28/I2 cells treated with 30 µM CBD and rising concentrations of AM251. (C) Relative caspase 3/7 activity in C28/I2 cells treated with rising concentrations of CBD with or without AM251. Means ± SD of three independent experiments.
Figure 6
Figure 6
Western blot analysis of Erk1/2 phosphorylation in C28/I2 cells treated with cannabidiol (CBD) (15 µM, 3 h; six independent experiments) in the presence or absence of AM251 (16 µM, 2 h pre‐treatment, 3 h co‐treatment; three independent experiments). (A) Mean ± SD of normalized densitometry values of 6 (CBD) or 3 (CBD + AM251) independent western blot experiments for CBD. (B) and (C) Representative western blots for β‐actin, total Erk1/2, and phospho Erk1/2 under control conditions (Ctrl), in the presence of CBD and after co‐treatment with CBD and AM251.

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