Aconine inhibits RANKL-induced osteoclast differentiation in RAW264.7 cells by suppressing NF-κB and NFATc1 activation and DC-STAMP expression.

AIM: Aconiti Lateralis Radix Preparata is a traditional Chinese medicine used to treat chronic arthritis and is highly effective against rheumatoid arthritis. However, the effects of aconine, a derivative of aconitum alkaloids, on osteoclasts, which can absorb bone, remain unknown. Here, we investigated the effects of aconine on osteoclast differentiation and bone resorption in vitro. METHODS: The viability of mouse leukemic monocyte/macrophage cell line RAW264.7 was measured using CCK-8 assays. Osteoclast differentiation was induced by incubation of RAW264.7 cells in the presence of RANKL, and assessed with TRAP staining assay. Bone resorption was examined with bone resorption pits assay. The expression of relevant genes and proteins was analyzed using RT-PCR and Western blots. The activation of NF-κB and nuclear factor of activated T-cells (NFAT) was examined using stable NF-κB and NFATc1 luciferase reporter gene systems, RT-PCR and Western blot analysis. RESULTS: Aconine (0.125, 0.25 µmol/L) did not affect the viability of RAW264.7 cells, but dose-dependently inhibited RANKL-induced osteoclast formation and bone resorptive activity. Furthermore, aconine dose-dependently inhibited the RANKL-induced activation of NF-κB and NFATc1 in RAW264.7 cells, and subsequently reduced the expression of osteoclast-specific genes (c-Src, ß3-Integrin, cathepsin K and MMP-9) and the expression of dendritic cell-specific transmembrane protein (DC-STAMP), which played an important role in cell-cell fusion. CONCLUSION: These findings suggest that aconine inhibits RANKL-induced osteoclast differentiation in RAW264.7 cells by suppressing the activation of NF-κB and NFATc1 and the expression of the cell-cell fusion molecule DC-STAMP.

Sinomenine induces apoptosis in RAW 264.7 cell-derived osteoclasts in vitro via caspase-3 activation.

AIM: Sinomenine (SIN) is an alkaloid found in the roots and stems of Sinomenium acutum, which has been used to treat rheumatic arthritis in China and Japan. In this study we investigated the effects of SIN on osteoclast survival in vitro and the mechanisms of the actions. METHODS: Mature osteoclasts were differentiated from murine monocyte/macrophage cell line RAW264.7 through incubation in the presence of receptor activator of NF-κB ligand (RANKL, 100 ng/mL) for 4 d. The cell viability was detected using the CCK-8 method. The survival and actin ring construction of the osteoclasts were scored using TRACP staining and phalloidin-FITC staining, respectively. The apoptosis of the osteoclasts was detected by DNA fragmentation and Hoechst 33258 staining, and the cell necrosis was indicated by LDH activity. The activation of caspase-3 in osteoclasts was measured using Western blotting and the caspase-3 activity colorimetric method. RESULTS: SIN (0.25-2 mmol/L) inhibited the viability of mature osteoclasts in dose-dependent and time-dependent manners, but did not affect that of RAW264.7 cells. Consistently, SIN dose-dependently suppressed the survival of mature osteoclasts. The formation of actin ring, a marker associated with actively resorbing osteoclasts, was also impaired by the alkaloid. SIN (0.5 mmol/L) induced the apoptosis of mature osteoclasts, which was significantly attenuated in the presence of the caspase-3 inhibitor Ac-DEVD-CHO. SIN increased the cleavage of caspase-3 in mature osteoclasts in dose-dependent and time-dependent manners. Furthermore, SIN dose-dependently enhanced caspase-3 activity, which was blocked in the presence of Ac-DEVD-CHO. CONCLUSION: Sinomenine inhibits osteoclast survival in vitro through caspase-3-mediated apoptosis, thus it is a potential agent for treating excessive bone resorption diseases.

[Inhibitory effects of HIV-1 gp41 fusion peptide on CD3 antibody activated regulatory T cells].

AIM: To investigate whether the HIV-1 gp41 fusion peptide (FP) could affect the regulatory T cell (Treg) function activated by CD3 antibody. METHODS: Murine CD4(+);CD25(+); Treg and CD4(+);CD25(-); effector T cells (Teff) were isolated from mice spleens by the immune magnetic beads. The splenocytes were treated with mitomycin C to obtain antigen presenting cells (APCs). CCK-8 assay and CFSE loading were employed to evaluate the effects of FP on the Treg inhibitory function via measuring Teff proliferation activated by CD3 antibody. In addition, IL-10 secretion of activated Treg was detected by ELISA. The distribution of FP and TCR on Treg surface was observed by laser scanning confocal microscope. RESULTS: Through the CCK-8 assay and flow cytometry, we found that Teff cells have significant proliferation stimulated by the CD3 antibodies. When Treg and Teff were co-cultured, the proliferation of Teff was significantly inhibited by Treg. When added with 25 µg/mL FP, the proliferation of Treg+Teff group and Teff group was not significantly affected, but when 5 µg/mL FP was added, the proliferation rate of Treg+Teff group was significantly lower than that of Teff group. IL-10 secretion was low when Treg were not activated, but it significantly increased by CD3 antibodies stimulation. When the concentration of FP was 25 µg/mL, IL-10 level significantly decreased, but 5 µg/mL FP did not significantly influence IL-10 secretion. Through the laser scanning confocal microscope, we found that T cell receptors (TCR) of non-activated Treg showed uniform distribution on the cell surface, and that FP and TCR had no common distribution. When Treg were stimulated by CD3 antibodies, the activated TCR formed half crescent, and FP and the activated TCR had common distribution on cell surface. CONCLUSION: Treg inhibitory function is significantly inhibited by 25 µg/mL FP in vitro, but 5 µg/mL FP does not affect it. This may be due to the suppression of IL-10 secretion and the influence of the signaling cross-talk between APC and TCR.