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Sclareol inhibits RANKL-induced osteoclastogenesis and promotes osteoblastogenesis through promoting CCN1 expression via repressing the MAPK pathway

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Abstract

Osteoclasts are crucial cellular components of bone and are the cause of various bone problems like osteoporosis. Various biological activities such as anti-tumorous, anti-inflammatory, antibacterial, and immunomodulatory function are influenced by Sclareol, as a natural diterpene compound. However, studies on the effect and mechanism of Sclareol on osteoporosis are rare. In the current research, the influence of Sclareol on osteoclastogenesis and osteoblastogenesis was targeted to be discovered in ovariectomy (OVX)-induced animal models and in vitro. The expression levels of osteoclast-related genes such as c-Fos, NFATc1, and CTSK were detected by RT-qPCR and western blotting to understand the inhibition of Sclareol on the creation of osteoclast. The influence of Sclareol on osteoblastogenesis and the expression of osteoblastogenic markers were also examined. Sclareol inhibited the osteoclastogenesis caused by receptor activator of nuclear factor-κB ligand (RANKL) which promoted osteoblastogenesis through upregulating the expression of cysteine-rich protein 61 (CYR61/CCN1), which is a matricellular protein of the CCN family. The p-ERK and p-P38 protein expression levels were considerably downregulated by Sclareol. Furthermore, CCN1 overexpression partially mimicked the inhibitory effect of Sclareol, while the opposite results were obtained after CCN1 silencing. Additionally, Sclareol protected against loss of bones in an osteoporosis mouse model generated by OVX. The acquired results indicated that Sclareol represses RANKL-induced osteoclastogenesis and promotes osteoblastogenesis via promoting the expression of CCN1 by constraining the mitogen-activated protein kinase (MAPK) pathway. Our findings proposed that for the avoidance and treatment of osteoclast-linked disorders, Sclareol is a potentially effective drug.

Graphical abstract

A proposed model for mediated regulation of osteoclastogenesis and osteoblastogenesis by Sclareol. The basic model of the process by which Sclareol prevents osteoclastogenesis and promotes osteoblastogenesis. Sclareol may increase the expression of CCN1 through inhibiting the MAPK pathway, thereby inhibiting osteoclast differentiation and attenuating bone resorption. Sclareol represses the expression of c-Fos, which stimulates the formation of osteoclast. In contrast, Sclareol promotes osteoblast differentiation by upregulating Runx2 expression, thereby improving the formation of bones. Consequently, Sclareol protects against loss of bones by regulating the stability of bone makeover via inhibition of bone formation and stimulation of bone resorption.

Graphical Headlights

1. Sclareol represses RANKL-induced osteoclastogenesis.

2. Sclareol promotes osteoblast differentiation.

3. Sclareol inhibits the MAPK pathway through induction of CCN1.

4. Sclareol protects against bone loss by regulating the balance of bone remodeling via inhibition of bone formation and stimulation of bone resorption.

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All data generated or analyzed during this study are included in this article.

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Funding

This work was supported by A Project of Scientific Research Fund of the Zhejiang Provincial Education Department (188310-542126/015). No benefits in any form have been or will be received from a commercial party related directly or indirectly to the subject of this manuscript.

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  1. Xiang Li and Yuxin Wang contributed equally to this work.

Authors and Affiliations

  1. Department of Orthopaedic Surgery, The First Affiliated Hospital, Zhejiang University School of Medicine, No. 79 Qingchun Road, Hangzhou, 310003, People’s Republic of China

    Xiang Li, Yuxin Wang, Shengji Zhou & Fengchao Zhao

  2. Department of Surgery, The Second Affiliated Hospital of Zhejiang University School of Medicine, Hangzhou, 310009, People’s Republic of China

    Liangping Li

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  1. Xiang Li
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Contributions

Fengchao Zhao and Xiang Li contributed to the study conception and design. Material preparation, data collection, and analysis were performed by Xiang Li and Yuxin Wang. The first draft of the manuscript was written by Xiang Li and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

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Correspondence to Fengchao Zhao.

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All animal studies were carried out in conformity to the National Institutes of Health (NIH) guidelines and protocols for laboratory animal use and care. The study was approved by the Animal Experimental Ethical Committee of the First Affiliated Hospital College of Medicine, Zhejiang University.

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Supplementary information

Supplementary Figure 1.

Sclareol promotes BMSCs differentiation into OB and the expression of the osteoblast-specific genes via modulating CCN1 expression. (A) Effects of Sclareol on the viability of BMSCs by CCK8 assays at 48h and 96 h. (B) ALP and ARS expression in BMSCs after their treatment with various concentrations of Sclareol for seven days. Alizarin Red S staining for 21 days was used to determine the mineralized extracellular matrix. (C) The ALP-positive cells number and the OD values were obtained for mineralized matrix solutions after their treatment with Sclareol. (D) Expression levels of the CCN1, OPN, OCN, and Runx2 in BMSCs treated-with Sclareol were detected using RT-qPCR. (E) The expression levels of osteoblast-specific genes (Runx2) and CCN1 in BMSCs treated with indicated concentrations of Sclareol. (F) The CCN1 expression in BMSCs after the treatment with Sclareol. (G) Immunofluorescence was used to detect CCN1 expression in BMSCs. All experiments were carried out in triplicate. The results were presented as mean ± SEM. *P < 0.05 and **P < 0.01 in comparison to control group. (PNG 79122 kb)

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Supplementary Figure 2.

CCN1 silencing promotes osteoblastogenesis and the expression of osteoblast-specific gene expression in BMSCs. (A) The transfection efficiency of the CCN1 vector and siCCN1 were detected by western blot. (B) The CCN1 expression in BMSCs following transfection with CCN1 siRNA or CCN1 vector and Sclareol treatment. ALP-positive cells number and OD values following transfection with CCN1 siRNA (C) and CCN1 vector (D). The untreated cells served as a control in this study. (E-G) The expression of osteoblast-specific genes in BMSCs after the transfection with CCN1 siRNA or CCN1 vector was detected using western blot and RT-qPCR. (H) Treatment of BMSCs with or without 0.5 μM of Sclareol for the designated times. The protein expression levels of AKT, p-AKT, p-P38, and P38 were evaluated. (I) The effects of MAPK pathway inhibitor, SB203580, on the protein expression of CCN1 induced by Sclareol in BMSCs. (J, K) The effect of MAPK pathway inhibitor, SB203580, on the ALP and ARS staining induced by Sclareol in BMSCs. All experiments were carried out in triplicate. The results were presented as mean ± SEM. *P < 0.05 and **P < 0.01 in comparison to control group. (PNG 3147 kb)

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Li, X., Wang, Y., Li, L. et al. Sclareol inhibits RANKL-induced osteoclastogenesis and promotes osteoblastogenesis through promoting CCN1 expression via repressing the MAPK pathway. Cell Biol Toxicol 37, 849–871 (2021). https://doi.org/10.1007/s10565-020-09578-6

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