Experimental Study Of Cannabinoid Receptor 2 Inhibitor AM630 On Macrophages Stimulated By Titanium Particles

Objective:To explore the effects of cannabinoid receptor 2 inhibitor AM630 on macrophage-like RAW264.7 cells stimulated by titanium particles and provide a new target for the prevention and treatment of aseptic loosening.Methods: The growth inhibitory effect of cannabinoid receptor 2 inhibitor AM630 on macrophage-like RAW264.7 cells was observed by using MTT assay.The RAW264.7 cells were stimulated by titanium particles with or without the addition of AM630,the levels of tumor necrosis factor (TNF-α) and interleukin-1β(IL-1β) of the supernatants were measured using an enzyme-linked immunosorbent assay(ELISA). Morphological changes of cells were observed under optical microscopy, cells apoptosis was analyzed by flow cytometry and caspase-3 cleaved staining. Transformation of RAW264.7 cells to osteoclasts was measured by TRAP Staining using a commercial kit. The extracellular regulated kinase (ERK) pathway was determined by western blot.Results: Cannabinoid receptor 2 inhibitor AM630 (0-200 nm ) did not affect RAW264.7 cell viability, which was assessed by MTT assay.The RAW264.7 cells proliferated rapidly. After RAW264.7 cells was incubated with titanium particles at dosages of 0.1,0.5 and 1mg/ml for 24h, cells are transformed to a macrophage-like morphology, and titanium particles are phagocytosed. the levels of TNF-αin culture supernatants of RAW264.7 cells stimulated by titanium particles showed a dose- dependent increase,and were respectively, significantly higher than those of the control group(p<0.05). the levels of IL-1βin culture supernatants of cells induced by titanium particles at dosages of 0.5 and 1mg/ml were significantly higher than those of the control group.While the RAW264.7 cels were stimulated by titanium particles with the addition of AM630(100nm) for 24h, the levels of cytokines were significantly decreased.The results of caspase-3 cleaved staining demostrated that the caspase-3-positive cells were observed in both titanium particles group and AM630 treatment group,and typical morphological changes could be observed by using normal microscope in both groups, including nuclear chromatin condensation, fragmentation, cell shrinkage, and formation of apoptosis bodies. Typical subdiploid peak before phase G0/G1 was detected by flow cytometry in the titanium particles group,the cell cycle was blocked at"S"phase. The "S" phase ratio(0.579±0.0105) was obviously higher than the ratio of control group(0.423±0.0079;t=23.74,p<0.01), and the cells were decreased at phase G2/M.In the AM630 treatment group, subdiploid peak before phase G0/G1 was also detected,but the "S" phase ratio(0.436±0.0116) was similar with the ratio of control group(t=1.88,p=0.1086>0.05). In our study, mature osteoclast were obtained from RAW264.7 cells stimulated with RANKL using TRAP staining,and treatment with RANKL markedly induced the phosphorylation of ERK.AM630 could significantly inhibit the formation of TRAP-positive cells and the increase in ERK phosphorylation.Conclusion: (1) Macrophages(RAW264.7 cells) stimulated by titanium particles released TNF-αand IL-1βin a dose-dependent mode.The apoptosis in cells could be induced by titanium particles,and the proliferation of cells was inhibited. Transformation of macrophages stimulated with RANKL to osteoclasts was possible. (2) The cannabinoid receptor 2 inhibitor AM630 coule decrease the release of cytokines and induce the macrophages apoptosis,and inhibit transformation of macrophages to osteoclasts by blocking the activations of ERK pathway. Therefore, cannabinoid receptor 2 selective inhibitor represent a suitable therapeutic candidate for the prevention and treatment of aseptic loosening.