Cannabinoid receptor CB2 is involved in tetrahydrocannabinol-induced anti-inflammation against lipopolysaccharide in MG-63 cells

Abstract

Cannabinoid Δ9-tetrahydrocannabinol (THC) is effective in treating osteoarthritis (OA), and the mechanism, however, is still elusive. Activation of cannabinoid receptor CB2 reduces inflammation; whether the activation CB2 is involved in THC-induced therapeutic action for OA is still unknown. Cofilin-1 is a cytoskeleton protein, participating in the inflammation of OA. In this study, MG-63 cells, an osteosarcoma cell-line, were exposed to lipopolysaccharide (LPS) to mimic the inflammation of OA. We hypothesized that the activation of CB2 is involved in THC-induced anti-inflammation in the MG-63 cells exposed to LPS, and the anti-inflammation is mediated by cofilin-1. We found that THC suppressed the release of proinflammatory factors, including tumor necrosis factor α (TNF-α), interleukin- (IL-) 1β, IL-6, and IL-8, decreased nuclear factor-κB (NF-κB) expression, and inhibited the upregulation of cofilin-1 protein in the LPS-stimulated MG-63 cells. However, administration of CB2 receptor antagonist or the CB2-siRNA, not CB1 antagonist AM251, partially abolished the THC-induced anti-inflammatory effects above. In addition, overexpression of cofilin-1 significantly reversed the THC-induced anti-inflammatory effects in MG-63 cells. These results suggested that CB2 is involved in the THC-induced anti-inflammation in LPS-stimulated MG-63 cells, and the anti-inflammation may be mediated by cofilin-1.

Figure 1

Experimental protocol diagram. (a) The MG-63 cells were assigned into five groups, including control, LPS, THC + LPS, AM630 + THC + LPS, and AM630 groups. After the treatments for 24 h, immunocytochemistry and western blot were taken to investigate the expression of CB2 receptor. (b) The MG-63 cells were assigned into seven groups as shown in this figure. After the treatments, ELISA was used to evaluate the inflammatory factors concentrations in the supernatants. (c) The MG-63 cells were assigned into five groups, including control, LPS, THC + LPS, CB2-siRNA + THC + LPS, and SC-siRNA + THC + AM630 groups. After the treatments, western blot was taken to investigate the expression of CB2 receptor and ELISA was used to assess the inflammatory factors release. (d) The cells were divided into control, LPS, THC + LPS, doxycycline (DOX) + THC + LPS, and DOX groups, western blot was taken to investigate the expression of cofilin-1 protein, and ELISA was taken to evaluate the concentrations of proinflammatory factors in the supernatants.

Figure 2

THC decreased LPS-induced IL-6 release from MG-63 cells dose-dependently. (a) MG-63 cells were treated with 0, 1, 10, and 100 ng/mL LPS for 24 h. (b) The MG-63 cells were treated with different concentrations of THC plus 10 ng/mL LPS for 24 h. IL-6 release was determined by ELISA kits. Results are means ± SD (n = 8).  ^*: P < 0.05.

Figure 3

THC upregulated CB2 receptor expression. Immunofluorescent staining (a) and western blot (b) were used to determine the CB2 protein expression. The cells were treated with or without 10 ng/mL LPS for 24 h in the presence or absence of 5 μM THC or 10 μM AM630 (CB2 receptor antagonist). The CB2 receptor expression (red) was observed by a confocal microscope, and stronger signal indicated higher expression of CB2 protein. Nuclei were counter-stained with DAPI (blue). Bar = 10 μm. Results are means ± SD (n = 6).  ^*: P < 0.05; NS: no significance.

Figure 4

THC decreased the release of TNF-α, IL-1β, IL-6, and IL-8 from LPS-stimulated MG-63 cells. TNF-α (a), IL-1β (b), IL-6 (c), and IL-8 (d) release were determined by ELISA kits. THC (5 μM) decreased 10 ng/mL LPS-induced release of TNF-α, IL-1β, IL-6, and IL-8, and CB2 antagonist AM630 (10 μM) reversed these effects. Results are means ± SD (n = 6).  ^*: P < 0.05; NS: no significance.

Figure 5

THC reduced NF-κB expression via CB2 receptor in LPS-stimulated MG-63 cells. NF-κB expression was assessed by western blot. THC reduced LPS-induced upregulation of NF-κB expression, and CB2 antagonist AM630 (a), not CB1 antagonist AM251 (b), partially abolished this effect. Results are means ± SD, (n = 6).  ^*: P < 0.05; NS: no significance.

Figure 6

CB2-siRNA reversed THC-induced decrease of inflammatory factors release. MG-63 cells were assigned into five groups, control (no LPS, THC, or siRNA): cultured in drug-free medium; LPS: cells exposed to 10 ng/mL LPS for 24 h; THC + LPS: cells exposed to 5 μM THC plus 10 ng/mL LPS for 24 h; CB2-siRNA + THC + LPS: cells incubated with CB2-siRNA for 24 h and then exposed to 5 μM THC plus 10 ng/mL LPS for 24 h; scrambled siRNA (SC-siRNA) + THC + LPS: cells incubated with SC-siRNA for 24 h and then exposed to 5 μM THC plus 10 ng/mL LPS for 24 h. (a) CB2-siRNA significantly downregulated the expression of CB2 receptor, assessed by western blot (n = 3). (b) CB2-siRNA significantly reversed THC-induced effect on NF-κB expression (n = 6). (c)–(f) CB2-siRNA significantly reversed THC-induced effects on the release of TNF-α, IL-1β, IL-6, and IL-8 (n = 6). Results are means ± SD.  ^*: P < 0.05; NS: no significance.

Figure 7

The upregulation of cofilin-1 inhibited the THC-induced anti-inflammation. (a) CB2 antagonist reversed the THC-induced effect on cofilin-1 expression. (b) Doxycycline induced an overexpression of cofilin-1 in the MG-63 cells. (c) Doxycycline abolished THC-induced reduction of IL-6 release. (d) Doxycycline abolished the THC-induced reduction of TNF-α release. The cells were treated with different drugs for 24 h (LPS: 10 ng/mL; THC: 5 μM; AM630: 10 μM; doxycycline: 0.1 μg/mL), western blot was performed to evaluate the cofilin-1 expression, and ELISA was used to test the concentrations of IL-6 and TNF-α in the supernatants. Results are means ± SD (n = 6).  ^*: P < 0.05.