Pharmacological inhibition of protein S-palmitoylation suppresses osteoclastogenesis and ameliorates ovariectomy-induced bone loss

Piperlongumine, a Piper longum-derived amide alkaloid, protects mice from ovariectomy-induced osteoporosis by inhibiting osteoclastogenesis via suppression of p38 and JNK signaling

“…Bone marrow-derived macrophages (BMMs) were prepared from osteoporotic mice as previously described with minor modifications. 29 Briefly, bone marrow cells were isolated from hindlimbs of 14- to 16-week-old C57BL/6 mice that were bilaterally ovariectomized at 8 weeks of age, and cultured with α-MEM (Minimum Essential Medium) growth medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin (P/S). After 24 h of culture, the suspended cells were collected and then continued to grow in fresh medium containing 15 ng ml −1 M-CSF.…”

“…The effect of PLM on cell viability was evaluated by CCK-8 assays as previously described. 29 Specifically, BMMs were inoculated into three sets of 96-well plates at a density of 5 × 10 3 per well and cultured overnight in α-MEM medium supplemented with 10% FBS, 1% P/S, and 15 ng ml −1 M-CSF. On the second day, cells in each set were treated with 0, 0.25, 0.5, 1, 2, 4, or 8 μM of PLM with 6 wells for each concentration.…”

“…To assess the impact of PLM on F-actin ring formation, phalloidin staining was performed as previously reported. 29 Briefly, BMMs were seeded onto glass coverslips and then subjected to osteoclast differentiation in the absence or presence of indicated concentrations of PLM. After osteoclast induction, cells were immobilized with 4% paraformaldehyde in phosphate buffered saline (PBS), and subsequently penetrated with Triton X-100 (1 : 1000 diluted in PBS).…”

“…30,31 For osteogenic differentiation, BMSCs were seeded in 24- or 6-well tissue culture plates at a density of 4 × 10 4 cm −2 , and then were induced with an osteogenic cocktail as described before. 29 To test the effect of PLM on osteogenic differentiation of BMSCs, different concentrations of PLM dissolved in DMSO were added to osteogenic medium (OM) and replaced every 2 days. After 6 days of osteogenic induction, activity of alkaline phosphatase (ALP), a marker of early-stage osteoblasts, in BMSCs cultured in 24-well plates was examined by ALP staining and ALP activity assay using BCIP/NBT Alkaline Phosphatase Color Development Kit (C3206, Beyotime Biotechnology, Shanghai, China) and Alkaline Phosphatase Assay Kit (P0321, Beyotime Biotechnology, Shanghai, China), respectively, according to the kit's instruction.…”

“…To investigate the effect of PLM on protein expression and pathway activation in BMMs in response to osteoclastogenic induction, BMMs were subjected to osteoclastogenic induction and chemical treatment as previously described, 29 except that 1 μM PLM was used. At each endpoint, total proteins were isolated from cultured cells using a radioimmunoprecipitation assay (RIPA) buffer supplemented with protease and phosphatase inhibitors and protein concentration of each sample was determined using a BCA Protein Assay Kit (#P0012, Beyotime Biotechnology, Shanghai, China).…”