Icaritin inhibits osteoclast differentiation and reduces bone loss by targeting ESR1 to inhibit miR503/RANK pathway

Abstract

Background: Postmenopausal osteoporosis (PMOP) is a prevalent metabolic disorder characterized by pathogenic mechanisms associated with the dysfunction of osteoclasts (OC) and osteoblasts (OB). Icaritin (ICT) is a flavonoid derived from icariin and epimedium, which is a natural product, and has demonstrated promising anti-osteoporosis properties. Nevertheless, the targets and mechanisms of ICT in osteoclast differentiation and PMOP remain unclear.

Methods: we developed a bilateral ovariectomy-induced osteoporosis model in animals and receptor activator of nuclear factor kappa-B ligand (RANKL) induced RAW264.7 to differentiate into osteoclasts with or without MPP dihydrochloride (MPP) and antagomir-503-5p. Micro-CT, tartrate-resistant acid phosphatase (TRAP) staining, enzyme-linked immunosorbent assay (ELISA), Western blot and qRT-PCR were used to detect bone resorption function, bone metabolism parameters, osteoclast differentiation rate and the expression of related genes, as well as the expression of ESR1, miR-503 and RANK. Molecular docking, cell thermal shift assay (CETSA) and drug affinity responsive target stability (DARTs) experiments were used to confirmed that ESR1 is the direct target of ICT, and binding site of ICT with ESR1.

Results: ICT significantly inhibited OC differentiation and the expression of related genes (Trap, Mmp9, and Nfatc1), reduced bone loss, and improved osteoporosis and bone trabecular structure, and inhibited the levels of TRAP and RANKL in the serum and increase the level of osteoprotegerin (OPG). ICT significantly enhanced the expression of ESR1, ESR2 and miR-503, while inhibiting RANK expression, and ESR1 is the direct target of ICT, and Asparagine at 455 is the direct binding site of ICT with ESR1. Moreover, blocking ESR1 significantly reduced the regulatory effect of ICT on OC differentiation and related gens expression by MPP, especially the expression of miR-503 and RANK, as well as weakened the regulatory effect of ICT on inhibiting bone loss. Antagomir-503-5p significantly reduced the regulatory effect of ICT on OC differentiation, as well as the expression of genes related to OC differentiation.

Conclusion: Taken together, our study confirmed that ESR1 is the direct target of ICT, and Asparagine at 455 is the direct binding site of ICT, and ICT inhibits OC differentiation and reduces bone loss by targeting ESR1 to upregulate miR503 level and weaken miR503/RANK pathway.

Keywords: MiR-503-5p; estrogen receptor; icaritin; osteoclast; osteoporosis.

ESR1 is the direct target of ICT, and Asparagine at 455 is the direct binding site of ICT. ICT inhibit osteoclast differentiation and reduce bone loss by targeting ESR1 to upregulate miR-503 level and weaken miR-503/RANK pathway.

FIGURE 1

Stereological parameter of trabecular bone in the mouse femur after ICT treatment analyzed by micro-CT. (A) Chemical structure of ICT, (B) Region of interest (ROI) and longitudinal section, (C) Bone volume (BV), (D) Bone volume/total volume (BV/TV), (E) Trabecular thickness (Tb.Th), (F) Trabecular bone connection density (Conn.D), (G) Bone mineral content (BMC), (H) Tissue mineral content (TMC), (I) Structure model index (SMI), (J–L) The level of TRAP, RANKL and OPG in serum after ICT treatment. Data are expressed as the mean ± SD (n = 7), *P < 0.05, **P < 0.01, ***P < 0.001 vs. OVX group, scale column: 1 mm.

FIGURE 2

ICT significantly inhibited osteoclast differentiation and promoted the expression of ESR1 and ESR2. (A) ICT effect on the cytotoxicity of RAW 264.7 cells (n = 5). (B) Inhibition of OC differentiation by ICT was detected using TRAP staining (n = 5). (C) The effect of ICT on the expression of genes related to OC differentiation was assessed using RT-qPCR (n = 4). (D) MMP9 expression was detected following ICT treatment using western blot analysis (n = 4). (E) The effect of ICT on the expression levels of Esr1 and Esr2 was investigated using RTq-PCR (n = 4). (F) The effects of ICT on the protein levels of ESR1 and ESR2 were examined using western blot analysis (n = 4). *P < 0.05, **P < 0.01, ***P < 0.001 vs. the RANKL group, scale column:50 μm.

FIGURE 3

ESR1 is the direct targets of ICT. (A) Auto docking ICT and ESR1 (PDB:1ERE). (B) Auto docking ICT and ESR2 (5TOA). (C) The CETSA experiment evaluated the interaction of ICT with ESR1 and ESR2 (n = 3). (D) The DARTs experiment evaluated the interaction of ICT with ESR1 and ESR2 (n = 3). (E) pcDNA-ESR1-WT and pcDNA-ESR1-Mu were transfected into RAW264.7 cells (n = 3). (F,G) DARTs and CETSA experiments confirmed the interaction of ICT with ESR1-WT or ESR1-Mu (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001 vs. the DMSO group or pcDNA-ESR1-Mu group.

FIGURE 4

ICT inhibits OC differentiation by the ESR signaling pathway. (A) MPP blocks the proliferative effect of ICT on MCF-7 cells (n = 5). (B) The cytotoxicity of MPP on RAW264.7 and MCF-7 cells were detected by the CCK-8 assay (n = 5). (C) TRAP staining following ESR1 antagonism by MPP (n = 3). (D) TRAP-positive cells following antagonism of ESR1 by MPP. (E–G) Effect of ICT on the expression levels of genes related to OC differentiation following antagonism of ESR1 by MPP as detected by RT-qPCR (n = 3). (H) MMP9 expression was detected by western blot analysis following antagonism of ESR1 by MPP (n = 3). ^* P < 0.05, ^** P < 0.01, ^*** P < 0.001 vs. the RANKL group, ^# P < 0.05, ^## P < 0.01, ^### P < 0.001 vs. the ICT (5 μM) and ICT (10 μM) group, scale column:50 μm.

FIGURE 5

ICT inhibits OC differentiation via the miR-503-5p/RANK signaling pathway. (A) ICT upregulates miR-503 expression in OC differentiation (n = 3). (B,C) ICT inhibits RANK mRNA and protein expression in OC (n = 3). (D) MPP blocks ICT on miR-503-5p expression (n = 3). (E,F) MPP antagonizes the effect of ICT on the mRNA and protein expressions of RANK. (G,H) Antagomir-503-5p antagonizes the effect of ICT on OC differentiation (n = 3). (I–K) Effect of ICT on the expression levels of genes related to OC differentiation following inhibition of miR-503-5p by antagomiR-503-5p as demonstrated by RT-qPCR (n = 3). (L,M) The expression levels of MMP9 and RANK were detected by western blot analysis following inhibition of miR-503-5p by antagomir-503-5p (n = 3). (N) RANK expression was detected by RT-qPCR (n = 3). ^* P < 0.05, ^** P < 0.01, ^*** P < 0.001 vs. the model group; ^# P < 0.05, ^## P < 0.01 vs. ICT + antagomir-503 group, scale column:50 μm.

FIGURE 6

Stereological parameter of trabecular bone in the mouse femur after ICT and MPP co-treatment analyzed by micro-CT. (A) Region of interest (ROI) and longitudinal section (n = 6), (B) Bone volume (BV) (n = 6), (C) Bone volume/total volume (BV/TV) (n = 6), (D) Trabecular thickness (Tb.Th) (n = 6), (E) Trabecular bone connection density (Conn.D) (n = 6), (F) Bone mineral content (BMC) (n = 6), (G) Tissue mineral content (TMC) (n = 6), (H) Structure model index (SMI) (n = 6), (I–K) The level of TRAP, RANKL and OPG in serum after ICT and MPP co-treatment (n = 6), *P < 0.05, **P < 0.01, ***P < 0.001 vs. OVX group, ^# P < 0.05, ^## P < 0.01, ^### P < 0.001 vs. the ICT (30 mg/kg/d) group, scale column:5 mm. OVX, ovariectomize; ICT, icaritin; MPP, MPP dihydrochloride. TRAP, tartrate-resistant acid phosphatase; OPG, osteoporogeterin; MMP9, Matrix metalloproteinase 9.

FIGURE 7

Icaritin suppresses osteoclast differentiation and anti-osteoporosis by targeting the estrogen receptor to modulate the miR-503-5p/RANK axis. ICT: Icaritin; ESR1: estrogen receptor α.