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. 2020 Apr;24(7):4105-4117.
doi: 10.1111/jcmm.15064. Epub 2020 Mar 3.

Osthole-loaded N-octyl-O-sulfonyl chitosan micelles (NSC-OST) inhibits RANKL-induced osteoclastogenesis and prevents ovariectomy-induced bone loss in rats

Lining Wang  1 , Suyang Zheng  1 , Guicheng Huang  1   2 , Jie Sun  1 , Yalan Pan  1   3 , Yuhao Si  1 , Pengcheng Tu  1 , Guihua Xu  3 , Yong Ma  1   2 , Yang Guo  1
Affiliations

Osthole-loaded N-octyl-O-sulfonyl chitosan micelles (NSC-OST) inhibits RANKL-induced osteoclastogenesis and prevents ovariectomy-induced bone loss in rats

Lining Wang et al. J Cell Mol Med. 2020 Apr.

Abstract

Osthole (OST), a derivative of Fructus Cnidii, has been proved to have potential anti-osteoporosis effects in our recent studies. However, its pharmacological effects are limited in the human body because of poor solubility and bioavailability. Under the guidance of the classical theory of Chinese medicine, Osthole-loaded N-octyl-O-sulfonyl chitosan micelles (NSC-OST), which has not previously been reported in the literature, was synthesized in order to overcome the defects and obtain better efficacy. In this study, we found that NSC-OST inhibited on the formation and resorption activity of osteoclasts through using a bone marrow macrophage (BMM)-derived osteoclast culture system in vitro, rather than affecting the viability of cells. We also found that NSC-OST inhibited osteoclast formation, hydroxyapatite resorption and RANKL-induced osteoclast marker protein expression. In terms of mechanism, NSC-OST suppressed the NFATc1 transcriptional activity and the activation of NF-κB signalling pathway. In vivo, ovariectomized (OVX) rat models were established for further research. We found that NSC-OST can attenuate bone loss in OVX rats through inhibiting osteoclastogenesis. Consistent with our hypothesis, NSC-OST is more effective than OST in parts of the results. Taken together, our findings suggest that NSC-OST can suppress RANKL-induced osteoclastogenesis and prevents ovariectomy-induced bone loss in rats and could be considered a safe and more effective anti-osteoporosis drug than OST.

Keywords: NFATc1; NSC-OST; homology of medicine and food; mutual promotion; osteoclasts; osteoporosis.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Effect of NSC‐OST on the body and uterus weight in OVX rats. (A) Bodyweight was measured once a week during the period of the experiment in the sham group, OVX group and OVX rats with oral administration of NIL at 1 mg/kg/wk or OST at 10 mg/kg/d or NSC‐OST at 100 mg/kg/d. (B) After killing the rats, uterus was isolated and weighted. The uterine index is the ratio of uterine weight to bodyweight. Dates are the Mean ± SEM (n = 8). #P < .05 vs Sham group, *P < .05 vs OVX group
Figure 2
Figure 2
Effect of NSC‐OST on the bone metabolism biochemical markers and biomechanical parameters in OVX rats. (A) Bone gla protein (BGP); (B) tartrate‐resistant acid phosphatase (TRAP); (C) bone alkaline phosphatase (B‐ALP); (D) β‐isomerized C‐terminal telopeptides (β‐CTX) of type I collagen; (E) maximum load; (F) maximum deflection; (G) stiffness in each group was determined. Dates are the Mean ± SEM (n = 8). #P < .05 vs Sham group, ##P < .01 vs Sham group, *P < .05 vs OVX group, **P < .01 vs OVX group
Figure 3
Figure 3
Effect of NSC‐OST on bone mass in OVX rats. (A) Representative 2D images of the vertical plane of the bone microstructure of the distal femur from the micro‐CT scans. (B) Micro‐CT 3D morphometry reconstruction of distal femur. (C) Bone mineral density (BMD); (D) bone volume/tissue volume (BV/TV); (E) trabecular number (Tb. N); (F) trabecular thickness (Tb. Th); (G) trabecular separation (Tb. Sp). Dates are the Mean ± SEM (n = 5). #P < .05 vs Sham group, ##P < .01 vs Sham group, *P < .05 vs OVX group, **P < .01 vs OVX group, &P < .05 vs OST group, &&P < .01 vs OST group
Figure 4
Figure 4
Effect of NSC‐OST on the histopathological changes of femurs in OVX rats. (A) Morphologies of cortical and trabecular bone tissue of proximal femur were examined by H&E staining. Original magnification was 40×. (B) Effect of OST on the formation of osteoclasts in vivo was examined by TRAP staining. Original magnification was 200×. (C) Osteoclast surface/bone surface (OC.s/BS) and (D) Osteoclast number/bone surface (OC.N/BS) ratios were analysed by Image Pro Plus 6.0 software. Dates are the Mean ± SEM (n = 3). #P < .05 vs Sham group, ##P < .01 vs Sham group, *P < .05 vs OVX group, **P < .01 vs OVX group, &P < .05 vs OST group, &&P < .01 vs OST group
Figure 5
Figure 5
Effect of NSC‐OST on the expression of osteoclast‐specific markers in OVX rats. (A) The expression of NFATc1, c‐Fos and CTSK with tibia platform from rats in each group was detected by IHC staining. Original magnification was 100×. Semi‐quantitative analysis was performed for measuring the mean optical density of (B) NFATc1, (C) c‐Fos and (D) CTSK by Image Pro Plus 6.0 software. The osteoclast‐specific gene expressions of (E) NFATc1, (F) CTSK, (G) MMP‐9 and (H) TRAP with tibia platform from rats in each group were measured and analysed by the 2−ΔΔCt method. Dates are the Mean ± SEM (n = 3). #P < .05 vs Sham group, ##P < .01 vs Sham group, *P < .05 vs OVX group, **P < .01 vs OVX group
Figure 6
Figure 6
Effect of NSC‐OST on the osteoclastogenesis in vitro. (A) Representative TRAP staining images of RANKL‐induced osteoclast formation in different groups for 5 d, Original magnification was 40×. (B) Representative images of resorbing area in hydroxyapatite‐coated plates. Original magnification was 40×. (C) Quantification of TRAP‐positive multinucleated cells, five microscopic fields of view were randomly selected at a magnification of 40×, and the cells with ≥3 nuclei were enumerated. (D) Quantification of relative bone absorption area was analysis by Image Pro Plus 6.0 software in different groups. (E) Viability of BMMs cells treated by NSC‐OST with CCK‐8 method. Dates are the Mean ± SEM (n = 3), **P < .01
Figure 7
Figure 7
Effect of NSC‐OST on the NFATc1 transcriptional activity, osteoclast‐related protein expression and the activation NF‐κB pathway. (A) The stably transfected RAW264.7 cells were pre‐treated with OST or NSC‐OST and CsA for half an hour, then administrated with 100 ng/mL RANKL for 24 h. The results show that NSC‐OST suppresses the transcriptional activity of NFATc1 by the luciferase reporter gene assay. (B) BMMs were pre‐treated with OST or NSC‐OST for 1 h then offered 25 ng/mL M‐CSF and 100 ng/mL RANKL stimulation for 24 h. Afterwards, whole cell proteins were extracted, and the protein expressions of related proteins were measured by Western blotting analysis, including NFATc1, c‐Fos, CTSK and TRAP. In addition, proteins of NF‐κB signalling pathway from (C) cytoplasm and (D) nucleus were extracted and measured by Western blotting analysis separately. (E‐H) NFATc1‐related protein expression were analysed to those in control. (I‐L) NF‐κB‐related protein expression of IκB‐α, p‐iκB and NF‐κB p65 were analysed to those in control. Dates are the Mean ± SEM (n = 3). #P < .05 vs Sham group, ##P < .01 vs Sham group, *P < .05 vs CON group, **P < .01 vs CON group, &P < .05 vs OST group, &&P < .01 vs OST group
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References

    1. Langdahl B, Ferrari S, Dempster DW. Bone modeling and remodeling: potential as therapeutic targets for the treatment of osteoporosis. Ther Adv Musculoskelet Dis. 2016;8(6):225‐235. - PMC - PubMed
    1. Lagari VS, Levis S. Phytoestrogens in the prevention of postmenopausal bone loss. J Clin Densitom. 2013;16(4):445‐449. - PubMed
    1. D'Amelio P, Grimaldi A, Di Bella S, et al. Estrogen deficiency increases osteoclastogenesis up‐regulating T cells activity: a key mechanism in osteoporosis. Bone. 2008;43(1):92‐100. - PubMed
    1. Bar‐Shavit Z. The osteoclast: a multinucleated, hematopoietic‐origin, bone‐resorbing osteoimmune cell. J Cell Biochem. 2007;102(5):1130‐1139. - PubMed
    1. Boyle WJ, Simonet WS, Lacey DL. Osteoclast differentiation and activation. Nature. 2003;423(6937):337‐342. - PubMed

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