CB2 regulates oxidative stress and osteoclastogenesis through NOX1-dependent signaling pathway in titanium particle-induced osteolysis

Abstract

Periprosthetic osteolysis (PPO) induced by wear particles at the interface between the prosthesis and bone is a crucial issue of periprosthetic bone loss and implant failure. After wear and tear, granular material accumulates around the joint prosthesis, causing a chronic inflammatory response, progressive osteoclast activation and eventual loosening of the prosthesis. Although many studies have been conducted to address bone loss after joint replacement surgeries, they have not fully addressed these issues. Focusing on osteoclast activation induced by particles has important theoretical implications. Cannabinoid type II receptor (CB2) is a seven-transmembrane receptor that is predominantly distributed in the human immune system and has been revealed to be highly expressed in bone-associated cells. Previous studies have shown that modulation of CB2 has a positive effect on bone metabolism. However, the exact mechanism has not yet been elucidated. In our experiments, we found that NOX1-mediated ROS accumulation was involved in titanium particle-stimulated osteoclast differentiation. Furthermore, we confirmed that CB2 blockade alleviated titanium particle-stimulated osteoclast activation by inhibiting the NOX1-mediated oxidative stress pathway. In animal experiments, downregulation of CB2 alleviated the occurrence of titanium particle-induced cranial osteolysis by inhibiting osteoclasts and scavenging intracellular ROS. Collectively, our results suggest that CB2 blockade may be an attractive and promising therapeutic scheme for particle-stimulated osteoclast differentiation and preventing PPO.

Fig. 1. Ti particles promoted the production of ROS and downregulated antioxidant proteins in macrophages.

A Electron micrographs of the purchased Ti particles. B Size analysis of Ti particles. C The whole process of Ti particles being phagocytosed into RAW264.7 cells under electron microscopy. D Intracellular ROS immunofluorescence staining and ROS(+) RAW264.7 cell counts after intervention of RAW264.7 cells with different concentrations of Ti particles. Scale bars, 100 µm. Statistical significance was determined by a one-way ANOVA with Tukey’s multiple-comparison test. E Intracellular ROS levels were detected by flow cytometry. F The expression and quantitative analysis of NOX1 and NOX2 were detected by western blotting after treatment with RANKL and Ti particles. G qRT-PCR analysis of the mRNA expression levels of NOX1 and NOX2. H qRT-PCR analysis of the mRNA expression levels of Nrf2, NQO1, HO-1, and SOD2. The data were represented as mean ± SD of three independent experiments. Statistical significance was determined by Student’s t test. *P < 0.05, **P < 0.01.

Fig. 2. Ti particles promoted the activation of osteoclasts and the formation of superoxide in mitochondria.

A The expression and quantitative analysis of MMP9, NFATc1, and CTSK were detected by western blotting after treatment with RANKL and Ti particles. B qRT-PCR analysis of the mRNA expression levels of CTSK, Atp6v0d2, DC-STAMP, and MMP9. C TRAcP staining and quantitative analysis of the number of positive osteoclasts and the percentage of osteoclasts in the area. Scale bars, 100 µm. D The expression of superoxide in osteoclasts was detected by MitoSOX immunofluorescence staining. Scale bars, 100 µm. E The microstructure under TEM after intervention with Ti particles. Scale bars, 2 µm. The data were represented as mean ± SD of three independent experiments. Statistical significance was determined by a one-way ANOVA with Tukey’s multiple-comparison test. *P < 0.05, **P < 0.01.

Fig. 3. Inhibition of CB2 alleviated Ti particles-stimulated osteoclast activation.

A Immunofluorescence assay was used to detect the expression and fluorescence quantification of CB2 under the intervention of Ti particles. Statistical significance was determined by Student’s t test. B Western blot assay and relative quantification of CB2 protein after intervention with Ti particles and RANKL. C RT-PCR was used to detect the effect of siRNA intervention on CB2 expression in RAW264.7. D Representative TRAcP staining was used to detect the effects of Cnr2-457 and Cnr2-689 on osteoclast differentiation and quantitative analysis of the number and area of osteoclasts per well. Scale bars, 200 µm. E Western blot revealed the effects of Cnr2-457 and Cnr2-689 on the expression of MMP9, NFATc1, and CTSK during osteoclast differentiation stimulated by Ti particles. F RT-PCR revealed the effects of Cnr2-457 and Cnr2-689 on osteoclast-related genes during osteoclast differentiation stimulated by Ti particles. G, H Immunofluorescence staining revealed the effects of Cnr2-457 and Cnr2-689 on the expression of ACP5 and CTSK. The data were represented as mean ± SD of three independent experiments. Statistical significance was determined by a one-way ANOVA with Tukey’s multiple-comparison test. *P < 0.05, **P < 0.01.

Fig. 4. Inhibition of CB2 alleviated osteoclast activation through NOX1-mediated oxidative stress signaling.

A RT-PCR analysis of the mRNA expression levels of NOX1, Nrf2, SOD2, HO-1, and NOX2. B RT-PCR was used to detect the inhibitory effect of ML171 intervention on NOX1 expression in RAW264.7. C RT-PCR analysis of the mRNA expression levels of CTSK, NFATc1, Atp6v0d2, and MMP9. D, G Representative TRAcP staining and quantitative analysis were used to detect the effects of Cnr2-689 and ML171 on osteoclast differentiation. E, H Immunofluorescence staining revealed the effects of Cnr2-689 and ML171 on the expression of NFATc1. F, I SEM images of the bone resorption area. J, L Immunofluorescence staining and quantitative analysis of MitoTracker Red. K, M Immunofluorescence staining and quantitative analysis of ROS. The data were represented as mean ± SD of three independent experiments. Statistical significance was determined by a one-way ANOVA with Tukey’s multiple-comparison test. *P < 0.05, **P < 0.01.

Fig. 5. Inhibition of CB2 alleviated cranial bone loss in osteolytic mice.

A Schematic diagram of animal modeling intervention. B Representative 3D and 2D reconstruction images of micro-CT. C–F Related bone parameters. n = 7 per group. The data were represented as mean ± SD. Statistical significance was determined by a one-way ANOVA with Tukey’s multiple-comparison test. *P < 0.05, **P < 0.01.

Fig. 6. Inhibition of CB2 inhibited the number of osteoclasts and ROS levels in osteolytic mice.

A H&E staining of skulls, Scale bar = 100 µm. B, E TRAcP staining of the skull and quantitative analysis of osteoclasts in each group of mice. Scale bar = 100 µm. C, F ROS staining and quantitative analysis of immunofluorescence staining in each group of mice, Scale bar = 100 µm. D, G, H Immunohistochemical staining and quantitative analysis of NOX1 and NFATc1 in decalcified bone sections. n = 7 per group. The data were represented as mean ± SD. Statistical significance was determined by a one-way ANOVA with Tukey’s multiple-comparison test. *P < 0.05, **P < 0.01.