The Novel Antioxidant Compound JSH-23 Prevents Osteolysis by Scavenging ROS During Both Osteoclastogenesis and Osteoblastogenesis

Abstract

Inflammatory osteolysis is a pathological skeletal disease associated with not only the production of inflammatory cytokines but also local oxidative status. Excessive reactive oxygen species (ROS) promote bone resorption by osteoclasts and induce the apoptosis of osteoblasts. In consideration of the lack of effective preventive or treatments options against osteolysis, the exploitation of novel pharmacological compounds/agents is critically required. In our study, we found that a novel antioxidant compound, JSH-23, plays a role in restoring bone homeostasis by scavenging intracellular ROS during both osteoclastogenesis and osteoblastogenesis. Mechanically, JSH-23 suppressed RANKL-induced osteoclastogenesis, bone resorption and the expression of specific genes (including NFATc1, c-Fos, TRAP, CTSK and DC-STAMP) via inhibition of the NF-κB signaling pathway. Meanwhile, JSH-23 suppressed RANKL-induced ROS generation via the TRAF6/Rac1/NOX1 pathway and the enhanced expression of Nrf2/HO-1. In addition, JSH-23 attenuated H₂O₂-induced apoptosis and mineralization reduction in osteoblasts by reducing ROS production and enhancing Nrf2/HO-1 expression. Our in vivo results further revealed that JSH-23 exerts its protective effects on bone mass through its antioxidant activity. In conclusion, our results show that the application of JSH-23 might be a novel and plausible strategy for the treatment of osteolysis-related disease.

Keywords: HO-1 (heme oxygenase-1); JSH-23 (PubChem CID: 16760588); Nrf2; ROS–reactive oxygen species; osteoblast (OB); osteoclast (OC).

FIGURE 1

JSH-23 suppresses RANKL-induced osteoclastogenesis and bone resorption without cytotoxicity. (A) Chemical structure of JSH-23. (B) Effects of JSH-23 on BMMs viability as measured by CCK-8 assay. (C) BMMs were stimulated with M-CSF and RANKL in the presence of different concentrations of JSH-23 (10, 20, and 40 μM). TRAP staining showed that JSH-23 inhibited osteoclastogenesis dose-dependently. (D) Quantification of TRAP-positive cell numbers and area per well. (E) Cells were fixed and stained for F-actin. (F) Representative fluorescence images showing that recombinant JSH-23 treatment significantly decreased the size of F-actin ring structures. (G) Representative SEM and enlarged images of bone resorption pits. (H) The bone resorption area was measured relative to that in the control group. The values are shown as the means ± SDs, n = 3; *p < 0.05, **p < 0.01.

FIGURE 2

Osteoclast-specific gene expression is inhibited by JSH-23 during osteoclastogenesis. (A) BMMs were cultured to generate mature osteoclasts for 5 days with different doses of JSH-23 (10, 20, and 40 μM). (B) BMMs were stimulated with M-CSF and RANKL and treated with 40 μM JSH-23 at 1, 3, and 5 days. The mRNA expression levels of NFATc1, c-Fos, TRAP, CTSK, and DC-STAMP were measured by qPCR. (C) The protein expression of NfATc1 and c-Fos was analyzed by western blotting (days 1, 3, and 5) and (D) quantitatively analyzed. (E) Immunofluorescence analysis of BMMs stimulated with RANKL for 3 days to detect the expression of NFATc1. (F) Quantitative analysis of cells with NFATc1 nuclear translocation using ImageJ. The values are shown as the means ± SDs, n = 3; *p < 0.05, **p < 0.01.

FIGURE 3

JSH-23 suppresses osteoclastogenesis via inhibition of the NF-κB signaling pathway. (A) BMMs were pretreated with JSH-23 (40 μM) for 4 h and thereafter exposed to RANKL (100 ng/ml) for the indicated times (0, 5, 10, 20, 30 and 60 min). The activation of the NF-κB, MAPK and PI3K/Akt signaling pathways was examined by western blotting analysis and (B) quantified accordingly. (C) BMMs were pretreated with JSH-23 for 4 h and then stimulated with RANKL for 30 min. The representative immunofluorescence images show that recombinant JSH-23 treatment inhibited p65 nuclear transportation. (D) Quantitative analysis of cells with p65 nuclear translocation using ImageJ. The values are shown as the means ± SDs, n = 3; ns, no significance; *p < 0.05, **p < 0.01.

FIGURE 4

JSH-23 reduces RANKL-induced ROS production during osteoclastogenesis by downregulating the TRAF6/Rac1/NOX1 signaling pathway and enhancing the expression of Nrf2/HO-1. (A) Representative images of RANKL-induced ROS generation in BMMs with or without JSH-23 treatment at different concentrations. (B) Quantification of the average DCF intensity per well. (C) BMMs were stimulated with RANKL in the absence or presence of JSH-23 (20 and 40 μM) for 48 h, and then the ROS production pathway was analyzed by western blotting. (D) BMMs were cultured in α-MEM containing M-CSF and RANKL in the presence of JSH-23 (40 μM) at the indicated times (1, 3, 5 days). The expression of Nrf2 was analyzed by western blotting. (E) Western blotting images of the effects of JSH-23 on antioxidant enzyme, HO-1, catalase, NQO1, and GSR expression. (F–H) Quantitative analysis of the western blotting results. (I) Representative immunofluorescence images showing Nrf2 translocation and (J) HO-1 expression. (K–L), BMMs were transfected with siRNA against Nrf2 or (M, N) HO-1 for 48 h. The silencing efficiency was evaluated by western blotting. (O) Transfected BMMs were treated with RANKL and JSH-23 (40 μM) for 5 days. TRAP staining was measured, and (P) the osteoclasts were counted. The values are shown as the means ± SDs, n = 3; *p < 0.05, **p < 0.01.

FIGURE 5

Inhibitory effects of JSH-23 on H₂O₂-induced apoptosis in MC3T3-E1 cells. (A) MC3T3-E1 cells were treated with various concentrations of H₂O₂ (0–800 μM) for 1, 2, 4 and 8 h, and cell viability was analyzed by CCK-8 assay. (B) Cells were treated with 0, 200, 400 and 800 μM H₂O₂ for 4 h, and apoptosis was determined by flow cytometry followed by FITC-Annexin V and PI double staining. (C) The percentage of apoptotic cells in each well was quantified. (D–E) The protein levels of cleaved Caspase-9, cleaved Caspase-3, Bcl-2 and Bax were detected by western blotting. (F) MC3T3-E1 cells were pretreated with JSH-23 (40 μM) for 24 h and then treated with H₂O₂ (400 μM) for 4 h. Apoptosis was measured by flow cytometry followed by FITC-Annexin V and PI double staining. (G) The percentage of apoptosis was determined. (H) Cells were treated for 4 h with H₂O₂ in the presence or absence of JSH-23. Western blotting analysis was performed using the indicated antibodies. (I) Quantitative analysis of the western blotting results. The values are shown as the means ± SDs, n = 3; *p < 0.05, **p < 0.01.

FIGURE 6

JSH-23 alleviates H₂O₂-induced inhibition of osteoblast differentiation in MC3T3-E1 cells. (A) Cells were pretreated with JSH-23 for 24 h before induction of differentiation until day 7 or 21 and then exposed to H₂O₂ (400 μM) for 4 h. ALP or AR staining was performed. (B) Quantitative analysis of ALP activity and AR staining in cells based on absorbance. (C) The expression of the osteoblast-specific genes (Runx2, OCN, BMP2 and OSX) was analyzed by qPCR. (D) The protein expression levels of Runx2, OCN and BMP2 were measured using western blotting. (E) Quantitative analysis of the western blotting results. The values are shown as the means ± SDs, n = 3; *p < 0.05, **p < 0.01.

FIGURE 7

JSH-23 scavenges intracellular ROS through increased activation of the Nrf2/HO-1 pathway in MC3T3-E1 cells. (A) MC3T3-E1 cells were pretreated with JSH-23 (20 and 40 μM) for 24 h before stimulation with H₂O₂ (400 μM), and ROS generation was observed using fluorescence microscopy. (B) The fluorescence intensity of ROS was measured with ImageJ software. (C) Nrf2 and HO-1 expression was determined by western blotting analysis. (D) Quantitative analysis of the western blotting results. (E) MC3T3-E1 cells were pretreated with JSH-23 (20 and 40 μM) for 24 h before stimulation with H₂O₂ (400 μM). The representative immunofluorescence images show Nrf2 translocation and (F) HO-1 expression. (G–H) MC3T3-E1 cells were transfected with siRNA against Nrf2 or (I, J) HO-1 for 48 h. The transfection efficiency was confirmed by western blotting. (K) The protective effects of JSH-23 on the expression of the bone markers (Runx2, OCN and BMP2) in H₂O₂-treated MC3T3-E1 cells were greatly weakened by Nrf2 or HO-1 knockdown. (L) Quantitative analysis of the western blotting results. The values are shown as the means ± SDs, n = 3; **p < 0.01.

FIGURE 8

JSH-23 prevents LPS-induced osteolysis in vivo. (A) Micro-CT scanning and 3D reconstruction of whole calvaria from the sham group (PBS), vehicle group (LPS), low group (LPS with 1 mg/kg JSH-23) and high group (LPS with 3 mg/kg JSH-23). (B) Quantitative analysis of the bone volume/total volume (BV/TV) ratio and porosity percentage. (C) Representative images of calvaria stained with H&E and TRAP from each group. (D) The total TRAP-positive cell number and the OC.S/BS percentage were quantified. (E) Immunofluorescence analysis of the expression of Runx2 and OCN in calvarial tissue sections from each group. (F) Quantitative analysis of the relative fluorescence intensity of Runx2 and OCN in each experimental group. The values are shown as the means ± SDs, n = 6; **p < 0.01.

FIGURE 9

JSH-23 reduces ROS production and enhances the expression of Nrf2/HO-1 in vivo. (A) Representative images of bone sections showing DHE fluorescence in different groups. (B) Quantification of the DHE fluorescence intensity of each group. (C) Proteins were isolated from bone tissues, and Nrf2/HO-1 expression was investigated using western blotting. (D) Quantitative analysis of the western blotting results. (E) Immunofluorescence double staining was performed to detect the expression of Nrf2 and HO-1. (F) Quantitative analysis of Nrf2 and HO-1 immunofluorescence intensity. The values are shown as the means ± SDs, n = 6; **p < 0.01.

FIGURE 10

Schematic diagram of the possible mechanism by which JSH-23 elevates bone mass by scavenging ROS production and activating Nrf2/HO-1 signaling.