Sinensetin serves as an AMPK activator to inhibit RANKL-induced osteoclastogenesis via osteoclast cytoskeleton reorganization

Abstract

Osteoporosis is a skeletal condition caused by an excess of osteoclasts, resulting in an imbalance in bone metabolism. Sinensetin (SIN), one of the main ingredients in citrus fruits, provides a variety of pharmacological properties, like antioxidant, but its effects on osteoporosis remains unknown. Herein, we explored at how SIN affected RANKL-induced osteoclastogenesis and ovariectomy (OVX)-induced osteoporotic mice. Our research found that SIN, without compromising cell viability, inhibited RANKL-mediated osteoclastogenesis and the NFATc1 signaling pathway in a concentration-dependent manner. Further, RNA sequencing analysis suggested that the molecular mechanism of SIN inhibitory effect on osteoclasts is related to the cytoskeleton reorganization. The results indicated that SIN prevents the cytoskeleton reorganization of preosteoclasts via the c-Src-mediated PI3K/PAK4/AKT signaling axis. Meanwhile, SIN enhanced the expression of phosphorylation and activity of AMP-activated protein kinase (AMPK) in response to RANKL. Further, SIN targets AMPK to reduce intracellular Reactive oxygen species (ROS) levels, thereby blocking c-Src activation. Finally, we verified that SIN inhibits osteoclast activity, thus preventing OVX-induced bone loss. These findings suggest that SIN serves as an AMPK activator that abrogates RANKL-induced osteoclastogenesis and OVX-induced bone loss via hindering cytoskeleton reorganization.

Keywords: AMPK; Cytoskeleton reorganization; Osteoclast; ROS; Sinensetin.

Fig. 1

SIN inhibits RANKL-induced osteoclastogenesis and function in vitro. a The 2D structure of Sinensetin (SIN). b The cell viability of SIN on BMMs in 96 h was assessed by CCK-8 assay (n = 3). c Representative images of BMMs treated with RANKL (50 ng/mL) and M-CSF (25 ng/mL) in the presence of SIN from 1 μM to 10 μM. d, e TRAcP + osteoclast with nuclei > 3 in each well were quantitatively counted and their area was calculated as a proportion of the entire field of vision. f Representative images of actin belt of osteoclasts in the presence or absence of SIN (5 μM and 10 μM). g Actin belts area was quantitatively measured. h The number of actin belts was counted. i The resorption area on bone slices with or without SIN (5 μM and 10 μM). j The ImageJ program was used to quantify the bone resorption pit area as a percentage of the total bone surface well area. *p < 0.05, **p < 0.01, ***p < 0.001. All data are expressed as mean ± SD

Fig. 2

SIN inhibits RANKL-induced NFATc1 signaling pathway. a The expressed mRNA levels of Acp5, Dcstamp, Ctsk, Nfatc1, Atp6v0d2, and Fos were quantitatively measured by qPCR. b Representative images of NFATc1 signaling activation during osteoclast differentiation in the presence or absence of SIN (5 μM and 10 μM). c NFATc1/DAPI fluorescence levels were assessed and quantified for cells in each field of view. d BMMs were treated with or without SIN (10 μM) on day 1, day 3, day 5 and the proteins including c-Fos, NFATc1, CTSK and ATP6V0D2 were measured using western-blot. e–h Quantitative analysis of c-Fos, NFATc1, CTSK, and ATP6V0D2 normalized to β-actin (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001. All data are expressed as mean ± SD

Fig. 3

SIN inhibition of osteoclast activation is associated with podosomes. a, b and d, e Representative images of TRAcP⁺ osteoclasts under the treatment of SIN (10 μM) on indicated days. c, f The number of TRAcP⁺ osteoclasts nuclei > 3 per well was quantitatively counted. g Representative images of actin belt in the presence or absence of SIN (5 μM and 10 μM) for 72 h. h, i Actin belts area and number were counted. j, l The expressed mRNA levels of RhoA, Cdc42 and Rac1 were quantitatively measured by qPCR. *p < 0.05, **p < 0.01, ***p < 0.001. All data are expressed as mean ± SD

Fig. 4

SIN exerts the anti-osteoclast effect by modulating the cytoskeleton. a RNA sequencing detected the DEGs of the positive group and the SIN group, and the heatmap obtained by cluster analysis. The color bars indicate gene expression levels. b–e DEGs were further analyzed for enrichment in the KEGG pathway and GO analysis including biological processes, cellular component and molecular function. f Heatmap for cluster analysis of specific DEGs, which associated with osteoclast differentiation, osteoclast cytoskeleton and oxidative phosphorylation. g–i The expressed mRNA levels of Pik3r3, Itgav and Vcl were quantitatively measured by qPCR. **p < 0.01, ***p < 0.001. All data are expressed as mean ± SD

Fig. 5

SIN prevents the cytoskeleton reorganization of preosteoclasts. a Schematic diagram of osteoclast differentiation and maturation. b Images of crystalline violet staining for the transwell migration assay were recorded. c Statistical area ratio of invaded cells. d Images of cells at the same scratch location after 0 and 24 h in the presence and absence of SIN. e The number of cells in the same region were counted and quantified the difference in number between 0 and 24 h. f Representative images of western blot showed the expression of c-Src, Integrin β3 and Cortactin standardized to β-actin. g–i Quantitative analysis of c-Src, Integrin β3 and Cortactin normalized to β-actin (n = 3). j Representative images of western blot showed the expression of p-PAK4, p-PI3K and p-AKT individually standardized to β-actin, PI3K and AKT. (K-M) Quantitative analysis of p-PAK4, p-PI3K and p-AKT proteins expression (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001. All data are expressed as mean ± SD

Fig. 6

SIN prevents mitochondria energy supply of preosteoclast. a, c BMMs were treated in the presence or absence of SIN (5 μM and 10 μM) for 48 h, then the intracellular superoxide levels and mitochondrial membrane potential were measured separately by MitoSOX red and JC-1. b, d Fluorescence intensity and scale were quantified by ImageJ. e, f The expressed mRNA levels of Atp5b and Idh3a were quantitatively measured by qPCR. g, h The relative levels of intracellular ATP activity and NAD⁺/NADH were detected after 48 h of treatment in the presence or absence of SIN (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001. All data are expressed as mean ± SD

Fig. 7

SIN reduces intracellular ROS levels by targeting AMPK. a BMMs were treated with or without SIN (5 μM and 10 μM) in 48 h and the proteins including p-AMPK and AMPK were measured using western blot. b Quantitative analysis of p-AMPK normalized to AMPK (n = 3). c–e Molecular docking prediction image of SIN with AMPK. f SRP analyses of SIN interaction with AMPK. g Heatmap for cluster analysis of antioxidant enzyme-related DEGs. h BMMs were treated with or without SIN for 48 h, and then intracellular ROS levels were measured by a DCFH-DA assay. i DCF fluorescence area as a percentage of each field of view was evaluated and quantified by ImageJ. j ARE luciferase activity of RAW 264.7 cells stably transfected with the ARE luciferase in the presence or absence of SIN was measured. k Representative images of western blot showed the expression of proteins including Traf6, Nrf2, Keap1, and HO-1 standardized to β-actin. l Quantitative analysis of Traf6 normalized to β-actin (n = 3). m Quantitative analysis of Nrf2/Keap1 ratio (n = 3). n–p Quantitative analysis of Nrf2, Keap1, and HO-1 normalized to β-actin (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001. All data are expressed as mean ± SD

Fig. 8

SIN protects OVX-induced bone loss in vivo. a Schematic illustration of the establishment of OVX mice model and the experimental design to evaluate SIN’s therapeutic effects. b The micro-CT representative images of tibia structures were captured in different groups (n = 6 per group). c The BV/TV, Tb.N, Tb.Th and Tb.Sp were quantitatively analyzed with micro-CT Skyscan software. d Histological analysis of tibia with H&E staining (n = 6 per group). e, f Quantitative analysis of BV/TV and bone surface area of tibia by H&E staining. *p < 0.05, **p < 0.01, ***p < 0.001. All data are expressed as mean ± SD

Fig. 9

SIN inhibits osteoclastogenesis associated with AMPK activity in vivo. a Histological analysis of tibia with TRAcP staining (n = 6 per group). b, c Quantitative analysis of N.Oc/BS and Oc.S/BS of TRAcP-positive cells in tibia. d Representative images of western blot showed the expression of bone proteins including CTSK and ATP6V0D2 standardized to β-actin. e, f Quantitative analysis of CTSK and ATP6V0D2 normalized to β-actin (n = 3). g, i Histological analysis of tibia with p-PAK4 and p-AMPK staining (n = 6 per group). h, j Quantitative analysis of staining area in tibia bone tissue. *p < 0.05, **p < 0.01, ***p < 0.001. All data are expressed as mean ± SD