Elsevier

Phytomedicine

Volume 68, March 2020, 153146
Phytomedicine

Sweroside promotes osteoblastic differentiation and mineralization via interaction of membrane estrogen receptor-α and GPR30 mediated p38 signalling pathway on MC3T3-E1 cells

https://doi.org/10.1016/j.phymed.2019.153146Get rights and content

Abstract

Background

Dipsaci Radix has been clinically used for thousands of years in China for strengthening muscles and bones. Sweroside is the major active iridoid glycoside isolated from Dipsaci Radix. It has been reported that sweroside can promote alkaline phosphatase (ALP) activity in both the human osteosarcoma cell line MG-63 and rat osteoblasts. However, the underlying mechanism involved in these osteoblastic processes is poorly understood.

Purpose

This study aimed to characterize the bone protective effects of sweroside and to investigate the signaling pathway that is involved in its actions in MC3T3-E1 cells.

Methods

Cell proliferation, differentiation and mineralization were evaluated by the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay, ALP test and Alizarin Red S staining, respectively. The concentration of sweroside in intracellular and extracellular fluids was determined by ultra-performance liquid chromatography coupled to triple quadrupole xevo-mass spectrometry (UPLC/TQ-XS-MS). Proteins associated with the osteoblastic signaling pathway were analysed by western blot and immunofluorescence methods.

Results

Sweroside did not obviously affect the proliferation but significantly promoted the ALP activity and mineralization of MC3T3-E1 cells. The maximal absorption amount 0.465 ng/ml (1.3 × 10−9 M) of sweroside was extremely lower than the tested concentration of 358.340 ng/ml (10−6 M), indicating an extremely low absorption rate by MC3T3-E1 cells. Moreover, the ALP activity, the protein expression of ER-α and G protein-coupled receptor 30 (GPR30) induced by sweroside were markedly blocked by both the ER antagonist ICI 182780 and the GPR30 antagonist G15. In addition, sweroside also activated the phosphorylation of p38 kinase (p-p38), while the phosphorylation effects together with ALP and mineralization activities were completely blocked by a p38 antagonist, SB203580. Additionally, the phosphorylation of p38 induced by sweroside were markedly blocked by both the ER antagonist ICI 182780 and the GPR30 antagonist G15.

Conclusions

The present study indicated that sweroside, as a potential agent in treatment of osteoporosis, might exert beneficial effects on MC3T3-E1 cells by interaction with the membrane estrogen receptor-α and GPR30 that then activates the p38 signaling pathway. This is the first study to report the specific mechanism of the effects of sweroside on osteoblastic differentiation and mineralization of MC3T3-E1 cells.

Introduction

Osteoporosis is a systemic bone disease caused by a disorder of bone reconstruction, namely, an obvious imbalance between osteoclastic bone resorption and osteoblastic bone formation (Nakamura and Udagawa, 2011). It has become a major public health problem with the increase in the aging population around the world (Becker et al., 2010). The current anti-osteoporosis drugs mainly include bone resorption inhibitors (Hadji, 2012; Kishimoto and Maehara, 2015), bone formation stimulators (Silva et al., 2018; Hodsman, 2005) and mineralization drugs (Bolland et al., 2015). However, different side effects, such as hypocalcaemia and secondary hyperparathyroidism (Papapetrou, 2009), photosensitivity and urticaria (Musette et al., 2011), osteonecrosis (Voss et al., 2018), heart disease and breast cancer (Bowring and Francis, 2011) have been reported to be associated with their long-term use. Traditional Chinese medicine has been widely used in the clinic to treat bone diseases for thousands of years in China, and it may be an alternative safe therapeutic approach to the prevention and treatment of osteoporosis. Dipsaci Radix is a routinely used Chinese herbal medicine with the effect of strengthening muscles and bones (Liu et al., 2009). As a major ingredient in Dipsaci Radix, sweroside possesses many pharmacological and biological activities, such as promoting osteoblastogenesis (Sun et al., 2013) and anti-inflammatory (Wang et al., 2019), anti-melanogenesis (Jeong et al., 2015), anti-leukaemia (Han et al., 2017) and anti-allergic activities (Oku et al., 2011). Importantly, recent studies have reported sweroside could improve trabecular thickness, bone mineral density and trabecular number in ovariectomized mice (Ding et al., 2019), and it could increase alkaline phosphatase (ALP) activity in human MG-63 cells and rat osteoblasts (Sun et al., 2013). In addition, some mixtures containing sweroside significantly promoted the proliferation of rat osteoblasts (Sun et al., 2008). However, the underlying mechanism of the anabolic effects of sweroside on bone is far from clear.

Estrogen plays a key role in bone remodeling. The decreased estrogen level after menopause leads to a rapid loss of bone mineral density and an increase in fracture risk. Estrogen receptor-alpha (ER-α) and estrogen receptor-beta (ER-β) are classical estrogen receptors (ERs) located in the cell nuclei and cell membrane (Bord et al., 2001; Pappas et al., 1995) and distributed widely in bone (Hou et al., 2006), uterus and ovary (Monje and Boland, 2001), neurohypophysis (Takahashi and Kawashima, 2009), prostate (Leav et al., 2001), adipose tissue (Bluher, 2013), skin (Bakry et al., 2014), and so on. Many studies have shown that ER-α is crucial for mediating the estrogenic effects in both trabecular and cortical bone (Borjesson et al., 2011; Borjesson et al., 2013). Smith et al. reported that a point mutation in ER-α caused unfused growth osteoporosis (Smith et al., 1994; Hamilton et al., 2017; Melville et al., 2014). In addition, Wang et al. demonstrated that E2 promotion of osteogenesis is involved in the activation of ER-α by upregulation of OPG, BMP2, TGF-β2, Runx2, and IGF-1, and downregulation of RANKL, while ER-β showed opposite actions to ER-α (Wang et al., 2016). Therefore, the role of ER-α in bone metabolism has become the major focus of recent research (Khosla, 2013; Windahl et al., 2013; Kondoh et al., 2014).

The direct activation of ERs stimulates a series of physiological functions, such as promoting the proliferation and differentiation of osteoblasts (Cheskis et al., 2007), regulating the apoptosis of human breast cancer cells (Won et al., 2014) and modulating the expansion and fibrosis of adipose tissue (Davis et al., 2013). Previous studies revealed that blocking the activation of ERs resulted in inhibition of the transduction of some signaling pathways, such as the mitogen-activated protein kinase (MAPK) (Wu et al., 2017) and phosphatidylinositol 3-kinase (PI3K) (Tsai et al., 2001) signaling pathways, which means ERs can act as upstream modulators in these signaling pathways. The mammalian family of MAPKs includes p38 kinase (p38), c-Jun N terminal kinase (JNK) and extracellular signal-regulated kinase (ERK). They convey signals from the cell surface to the nucleus and play key roles in stimulating osteogenic differentiation (Soundharrajan et al., 2018), inhibiting the proliferation of human gastric cancer AGS cells (Teng et al., 2016), and inducing human nasopharyngeal carcinoma cell apoptosis (Hsieh et al., 2014). Each of the MAPK pathways (ERK, JNK, p38) exerts distinctly biological effects; for instance, the p38 signaling pathway plays a crucial role in ALP activity, while the ERK signaling pathway is involved in cell proliferation (Suzuki et al., 1999). Therefore, it is important to clarify the cascades of ERs to MAPKs when studying the mechanism of bone protective drugs.

G protein-coupled receptor 30 (GPR30), also called G protein-coupled estrogen receptor 1, is a membrane estrogen receptor with seven transmembrane domains (Carmeci et al., 1997; Thomas et al., 2005), but it has also been detected in the cytoplasm of osteoblasts and osteoclasts by an immunodetection method (Heino et al., 2008). Recently, studies have proven that GPR30 exerts important regulatory effects in ovariectomized rats (Kang et al., 2015), osteoblasts (Lin et al., 2019), and osteoclasts (Masuhara et al., 2016). For example, G1, a specific agonist of GPR30, can increase bone mineral content and density, and promote osteoblastic proliferation and differentiation, which indicates that GPR30 might modulate bone remodeling (Kang et al., 2015). Importantly, GPR30 is co-expressed with ER-α and ER-β in some specific organs or tissues of humans and animals such as brain, ovary and bone (Levin, 2008; Hadjimarkou and Vasudevan, 2018; Levin, 2009). This phenomenon indicates the existence of functional cross-talk or interactions between classical ERs and GPR30.

In the current study, we aimed to investigate the osteoblastic proliferation, differentiation and mineralization effects of sweroside on MC3T3-E1 cells and to explore the underlying mechanism of its actions. The results showed that sweroside promoted osteoblastic differentiation and mineralization through activating the p38 signaling pathway and upregulating the expression of ER-α and GPR30 in MC3T3-E1 cells. Most importantly, this study revealed for the first time that sweroside exerted its effects by modulating the membrane receptors of ER-α and GPR30 via their joint effect in activating the p38 signaling pathway. Taken together, our results clearly demonstrated that sweroside promotes osteoblastic differentiation and mineralization via interaction with the membrane estrogen receptor-α and GPR30 mediated p38 signaling pathway in MC3T3-E1 cells. This is the first study to report the specific mechanism of sweroside on osteoblastic differentiation and mineralization of MC3T3-E1 cells, and further supports the application of sweroside as an agent in preventing and treating osteoporosis.

Section snippets

Materials

Sweroside was purchased from Ronghe (Shanghai, China) at 98% purity and was dissolved in ethanol. The reagents used are listed as follows: α-modified minimum essential medium (α-MEM, Gibco, USA), fetal bovine serum (FBS, Gibco, USA), penicillin 100 U/ml and steptomycin 100 μg/ml (P/S, Invitrogen, USA), ICI 182780 (ICI, Tocris, USA, ≥99% purity), MPP (Tocris, USA, ≥98% purity), PHTPP (Tocris, USA, ≥ 99% purity), G15 (Apexbio, USA, 98.00% purity), SB203580 (MCE, USA, 99.92% purity), 17β-estradiol

Sweroside promotes alkaline phosphatase activity without influencing the proliferation of MC3T3-E1 cells

The influence of sweroside on the proliferation of MC3T3E1 cells was determined by an MTS assay. As shown in Fig. 2A, sweroside did not influence the proliferation of MC3T3-E1 cells at all tested concentrations from 5 × 10−7 M to 10−5 M, while the positive control drug 17β-estradiol at 10−8 M significantly stimulated MC3T3-E1 cell proliferation.

Alkaline phosphatase (ALP) activity is a common marker of osteoblastic differentiation during the early stage (Pagani et al., 2005). As shown in Fig. 2

Discussion

In the present study, we found that sweroside at 10−6 M markedly improved ALP activity, bone mineralized nodule formation, and phosphorylation of p38, as well as upregulated the protein levels of ER-α and GPR30, while all of these effects could be abolished by co-treatment with ICI 182780 or G15. Combined with the trace amount of sweroside absorbed into cells, we concluded that sweroside might promote osteoblastic differentiation and mineralization via interaction with the membrane estrogen

Declaration of Competing Interest

The authors declare that they have no conflict of interests.

Acknowledgments

This work was supported by the State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica (SIMM1803KF-17), the Shenzhen Basic Research Program (JCYJ20170818104152702), the National Natural Science Foundation of China (81903616), and by the Project of the Science Foundation for Distinguished Young Scholars of Guangdong Province (grant no. 2014A030306043).

CRediT authorship contribution statement

Qing-chang Wu: Formal analysis, Methodology, Writing - original draft. Xi-yang Tang: Formal analysis, Methodology. Zi-qin Dai: Formal analysis, Methodology. Yi Dai: Methodology, Writing - review & editing. Hui-hui Xiao: Methodology, Writing - review & editing. Xin-sheng Yao: Methodology, Writing - review & editing.

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      Among them, m-TOR, HIF-1, HIF-1, Estrogen receptor, and c-AMP signaling pathway were the top five osteogenic-related pathways (Fig. 6B). Indeed, some studies showed that sweroside could promote osteogenic activity via up-regulation of the mTORc1/PS6 signaling pathway in BMSC (Ding et al., 2019) and stimulation of ER-α and GPR30 mediated p38 signaling pathway in MC3T3-E1 cells (Wu et al., 2020). Other study indicated that psoralen could stimulate the proliferation of osteoblasts through NF-kappa B-MAPK signaling pathway (Li et al., 2017).

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