Cistanche Deserticola Polysaccharide Can Attenuate Osteoclastogenesis And Bone Resorptiontion

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Dezhi Song, et al

Osteoporosis is a metabolic disease characterized by osteopenia and bone microstructural deterioration. Osteoclasts are the primary effector cells that degrade the bone matrix and their abnormal function leads to the development of osteoporosis. Reactive oxygen species (ROS) accumulation during cellular metabolism promotes osteoclast proliferation and differentiation, therefore, playing an important role in osteoporosis. Cistanche deserticola polysaccharide (CDP) possesses antitumor, anti‐inflammatory, and antioxidant activity. However, the impact of CDPon osteoclasts is unclear. In this study, tartrate‐resistant acid phosphatase staining, immunofluorescence, reverse transcription‐polymerase chain reaction, and western blot analysis were utilized to demonstrate that CDP inhibited osteoclastogenesis and hydroxyapatite resorption. In addition, CDP also inhibited the expression of osteoclast marker genes including Ctsk, Mmp9, and Acp5, and had no effect on receptor activator of nuclear factor κB (RANK) expression. Mechanistic analyses revealed that CDP increases the expression of antioxidant enzymes to attenuateRANKL‐mediated ROS production in osteoclasts and inhibits nuclear factor of activated T cells and mitogen‐activated protein kinase activation. These results suggest that CDP may represent a candidate drug for the treatment of osteoporosis caused by excessive osteoclast activity.

KEYWORDS: bone resorption, Cistanche deserticola polysaccharide, MAPK, osteoclast, reactive oxygen species

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1. INTRODUCTION

The balance between bone formation, mediated by osteoblasts, and bone resorption, mediated by osteoclasts, plays a vital role in maintaining bone metabolic homeostasis (Manolagas, 2000; Zhu et al., 2018). When bone resorption exceeds bone formation, osteoporosis occurs, which is characterized by reduced bone mass and bone microstructural damage (Ikeda, 2008). Osteoporosis is a common disease in elderly individuals and postmenopausal women and its pathogenesis has not been fully elucidated (Cooper & Melton,1992). Estrogen deficiency is a primary cause of osteoporosis(Manolagas, O'Brien, & Almeida, 2013). In addition, reactive oxygen species (ROS) can be induced by receptor activator of nuclear factorκB ligand (RANKL) and are associated with osteoclast formation(Yip et al., 2005), and thus may contribute to the development of osteoporosis (Manolagas, 2010). Some studies have found thatNrf2‐antioxidant deficiency increases ROS levels and promotes rank‐induced osteoclast differentiation (Hyeon, Lee, Yang, &Jeong, 2013). Therefore, reducing ROS production during osteoclast differentiation should be evaluated as a therapeutic strategy for the treatment of osteoporosis.

Osteoclasts are derived from the monocyte or macrophage hematopoietic lineage and are the only multinucleated cells that perform bone resorption (Teitelbaum, 2000). Therefore, research involving osteoclast formation is of great significance in developing effective treatments for bone metabolism diseases (Lorenzo,2017). Macrophage colony-stimulating factor (M‐CSF) and RANKL, produced by osteoblasts and activated T cells, are important cytokines that regulate osteoclastogenesis (Kim & Kim, 2016; Teitelbaum & Ross, 2003). RANKL induces the expression of the nuclear factor of activated T cells (NFATc1), which is a critical transcription factor active during osteoclast formation (Ishidaet al., 2002). Activated NFATc1 promotes the expression of osteoclast marker genes such as tartrate‐resistant acid phosphatase (TRAcP) and cathepsin K (CTSK) that regulate osteoclastogenesis and osteoclast function (Balkan et al., 2009; Crottiet al., 2008). These results suggest that CDP might be used to treat osteoporosis caused by excessive osteoclastic bone resorption.

2 | MATERIALS AND METHODS

2.1 | Materials

CDP (purity > 98%) was purchased from Solarbio (Beijing, China) and prepared at a stock concentration of 1 mM in phosphate-buffered saline(PBS). Antibodies are specific for c‐Fos, CTSK, GSR, TRX1, NOS2, TRAF6, RANK, NFATc1, ERK, JNK, p38, phosphorylated (p)‐ERK,p‐p38, p‐JNK, and β‐actin were obtained from Santa Cruz Biotechnology(San Jose, CA). Antibodies to V‐ATPase d2 were produced as previously described (H. Feng et al., 2009). The 3‐(4,5‐dimethylthiazol‐2‐yl)‐5‐(3‐carboxymethoxyphenyl)‐2‐(4‐sulfophenyl)‐2H‐tetrazolium (MTS) and luciferase assay system were obtained from Promega (Sydney, Australia). Recombinant M‐CSF was purchased from R&D Systems(Minneapolis, MN). Recombinant GST‐rRANKL protein was expressed and purified as previously described (Xu et al., 2000).

2.2 | Cell culture

RAW264.7 cells (mouse macrophage cells) were obtained from American Type Culture Collection (ATCC, Manassas, VA) and cultured in α‐modified minimal essential medium (Thermo FisherScientific, Scoresby, Australia) supplemented with 10% fetal bovine serum, 2 mM of L‐glutamine, 100 U/ml of penicillin, and 100 μg/ml of streptomycin (complete medium). Bone marrow-derived monocytes(BMMs) were isolated from 6‐week‐old C57BL/6J mice, which were euthanized according to procedures approved by the Animal ethics committee of the University of Western Australia (RA/3/100/1244). Long bones were dissected free of soft tissues and the bone marrow was flushed from the femur and tibia, which was then cultured in a complete medium in the presence of M‐CSF (50 ng/ml).

2.3 | Osteoclastogenesis assay

BMMs were plated into 96‐well culture plates at a density of 6 × 103cells per well and treated with a complete medium containing M‐CSF(50 ng/ml) and GST‐rRANKL (100 ng/ml), in the presence or absence of varying concentrations of CDP. The cell culture medium was changed every 2 days. After 5 days, cells were fixed with 4%paraformaldehyde for 10 min, washed three times with PBS, and then stained for TRAcP‐enzymatic activity using the leukocyte acid phosphatase staining kit (Sigma‐Aldrich, Sydney, Australia), following the manufacturer’s procedures. TRAcP‐positive multinucleated cells(>three nuclei) were identified as osteoclasts.

2.4 | Cytotoxicity assays

BMMs were seeded into 96‐well plates at 6 × 103 cells per well and left overnight to adhere. The following day, the cells were incubated with varying concentrations of CDP. After a further 48 hr, MTS solution (20 µl/well) was added and incubated with cells for 2 hr. The absorbance at 490 nm was determined with a microplate reader (MultiscanSpectrum; Thermo Labsystems, Chantilly, VA).

2.5 | Immunofluorescent staining

BMMs were seeded at a density of 6 × 103 cells per well in the presence of M‐CSF (50 ng/ml) overnight. Cells were then stimulated with M‐CSFand GST‐rRANKL (100 ng/ml) until mature osteoclasts formed. The osteoclasts were then treated with varying concentrations of CDP for 48hr before fixing with 4% paraformaldehyde, permeabilizing with 0.1%Triton X‐100–PBS, and blocking with 3% bovine serum albumin in PBS. Prepared cells were incubated with rhodamine‐conjugated phalloidin for45 min in the dark to stain for F‐actin. Cells were then washed with PBS, nuclei counterstained with 4′,6‐diamidino‐2‐phenylindole (DAPI), and mounted with coverslips for confocal microscopy.

2.6 | Hydroxyapatite resorption assay

To measure osteoclast activity BMMs (1 × 105 cells per well)cultured on six‐well collagen‐coated plates (BD Biocoat; ThermoFisher Scientific) were stimulated with GST‐rRANKL (100 ng/ml)and M‐CSF (50 ng/ml) until mature osteoclasts were generated(Zhou et al., 2016). Cells were then gently detached from the plate using cell dissociation solution (Sigma‐Aldrich) and equal numbers of mature osteoclasts were seeded onto individual wells hydroxyapatite‐coated 96‐well plates (Corning Osteoassay, Corning, NY). Mature osteoclasts were incubated in a medium containing‐rRANKL and M‐CSF with or without CDP at the indicated concentrations. After 48 hr, half of the wells were immunohistochemically stained for TRAcP activity, as described above, to assess the number of multinucleated cells per well. The remaining wells were bleached for 10 min to remove cells and allow measurement of the resorbed areas. Resorbed areas were photographed under standard light microscopy and the ImageJ software (National Institutes of Health, Bethesda, MD) was used to quantify the percentage area of hydroxyapatite surface resorbed by the osteoclasts.

2.7 | Luciferase reporter assays

To investigate NFATc1 transcriptional activation, RAW264.7 cells were stably transfected with an NFATc1‐responsive luciferase reporter construct (Cheng et al., 2018; van der Kraan et al., 2013). Transfected cells were cultured in 48‐well plates at a density of1.5 × 105 cells per well and pretreated with various concentrations of CDP for 1 hr. Following pretreatment, cells were stimulated with GST‐rRANKL (100 ng/ml) for 24 hr, and luciferase activity was measured using the luciferase reporter assay system according to the manufacturer’s protocol (Promega).

2.8 | Quantitative reverse transcription‐polymerase chain reaction (RT‐PCR) analysis

Total RNA was isolated from cells using Trizol reagent according to the manufacturer's protocol (Thermo Fisher Scientific). Complementary DNA was synthesized using Moloney murine leukemia virus reverse transcriptase with 1 μg of RNA template and oligo‐dT primers. Polymerase chain reaction amplification of specific sequences was performed using the following program: 94°C for 5 min, followed by 30 cycles of 94°C for40 s, 60°C for 40 s, and 72°C for 40 s, and a final extension step of 5 min at 72°C. The detailed information on specific primers is shown in Table 1. Relative messenger RNA levels were calculated by normalization to the expression of the housekeeping gene Hmbs.

2.9 | Western blot analysis

BMMs were cultured in a complete medium with M‐CSF in six‐well plates and stimulated with GST‐rRANKL (100 ng/ml) for the stated times. Cells were lysed in radioimmunoprecipitation lysis buffer and proteins were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to poly(vinylidene fluoride) membranes (GE Healthcare, Silverwater, Australia). The membranes were blocked in 5% skim milk for1 hr and then probed with various specific primary antibodies with gentle shaking overnight at 4°C. Membranes were washed and subsequently incubated with horseradish peroxidase-conjugated secondary antibodies. Antibody reactivity was then detected with enhanced chemiluminescence reagent (Amersham Pharmacia Biotech, Piscataway, NJ) and visualized with an Image‐quant LAS 4000 (GE Healthcare).

2.10 | Intracellular ROS detection

Intracellular ROS levels were detected using a 2′,7′‐dichlorofluorescein diacetate cellular ROS detection assay kit (Abcam, Melbourne, Australia). BMMs (5 × 103 cells per well) were cultured in 96‐well plates and treated with RANKL (100 ng/ml), M‐CSF (50 ng/ml), and CDP for 72 hr. Intracellular ROS levels were measured using2′,7′‐dichlorofluorescein diacetate, which oxidizes into fluorescent CSF in the presence of ROS. Cells were washed in Hank’s buffer and incubated in the dark for 30 min with 10 µM of DCFH‐DA. Images were obtained using confocal microscopy.

2.11 | Statistical analyses

All data are representative of at least three experiments performed in triplicate unless otherwise indicated. Data are expressed as mean ± SD. One‐way analysis of variance followed by Student–Newman–Keulspost hoc tests were used to determine the significance of differences between results, with p < 0.05 regarded as significant.

3 | RESULTS

3.1 | CDP inhibits RANKL‐induced osteoclastogenesis and osteoclast fusion

To determine if CDP can suppress RANKL‐induced osteoclast formation, we first performed an osteoclastogenesis assay using mouse BMMs (Liu et al., 2013; Song et al., 2016). BMMs were treated with RANKL and M‐CSF for 5 days with increasing concentrations of CDP. CDP reduced the number of TRAcP‐positive multinucleated cells when the concentration of CDPreached 5 µM or higher (Figure 1a,b). To evaluate CDP toxicity and confirm that these results were not a reflection of cell death or number, we performed an MTS assay. BMMs were treated withRANKL and M‐CSF for 48 hr with varying dosages of CDP. CDP had no effect on BMM proliferation at the concentration of 15 µMor less (Figure 1c).

To test the effects of CDP on osteoclast fusion, osteoclasts were induced with RANKL and M‐CSF treatment, with or without varying doses of CDP. Osteoclasts were stained with rhodamine‐phalloidin and DAPI to assess the number of nuclei per osteoclast (Figure 2a). Both osteoclast number and the average number of nuclei per osteoclast decreased following CDP treatment (5–10 µM; Figure 2b,c). Therefore, CDP had inhibitory effects on RANKL‐induced osteoclastogenesis and osteoclast fusion in a dose‐dependent manner.

3.2 | CDP attenuates RANKL‐induced osteoclastic hydroxyapatite resorption activity

A hydroxyapatite resorption assay was performed to detect the effect of CDP on osteoclast function (Figure 3a). After a 24‐hr incubation, the number of osteoclasts per well did not change while the hydroxyapatite resorption area was significantly decreased by treatment with 5 and 10 µM CDP when compared to control groups(Figure 3b,c). These results demonstrate that CDP has strong inhibitory effects on both osteoclast formation and osteoclast resorption activity without any cytotoxic effects.

3.3 | CDP inhibits osteoclast marker gene expression

To further investigate the inhibitory effects of CDP on osteoclastogenesis and osteoclastic bone resorption, BMMs were treated withRANKL and M‐CSF for 5 days with varying concentrations of CDP.RT‐PCR was then performed to detect the expression of osteoclast marker genes. The expression of Nfatc1, a crucial transcription factor during osteoclastogenesis, was inhibited by CDP in a dose‐dependent manner (Figure 4a). In addition, CDP (5 and 10 µM) downregulated the expression of bone resorption–related genes, including Mmp9, Ctsk, and Acp5 (Figure 4bd).

3.4 | CDP suppresses NFATc1 activity and downstream protein expression

To examine the effect of CDP on RANKL‐induced NFATc1 activity, a luciferase reporter assay was performed. Treatment with CDP, at concentrations of 5 µM and higher, significantly inhibited RANKL‐induced NFATc1 activity (Figure 5a). In addition, western blot analysis showed that CDP significantly suppressed protein expression of NFATc1 and c‐Fos in BMMs treated with RANKL and M‐CSF for 3 and 5 days(Figure 5b). Moreover, the expression of osteoclast function-related proteins, such as V‐ATPase‐d2 and CTSK, was downregulated in the presence of CDP compared to control groups. However, CDP had no effect on RANK expression (Figure 5b).

3.5 | CDP promotes the expression of antioxidant enzymes to scavenge ROS production duringRANKL‐induced osteoclastogenesis

To explore the underlying mechanism of CDP‐dependent osteoclastogenesis inhibition, BMMs were treated with RANKL (100 ng/ml)and M‐CSF (50 ng/ml) together with PBS or CDP for 72 hr. We examined the effect of CDP on intracellular ROS production stimulated by RANKL. Intracellular ROS levels were increased byRANKL treatment, which was attenuated by CDP (5 and 10 µM; Figure 6a). Both the number of ROS‐positive cells and the intensity ofROS staining were decreased by CDP treatment in a dose‐dependentmanner (Figure 6b,c). Western blot analysis showed that CDPpromoted thioredoxin (TRX1) and glutathione reductase (GSR)expression while suppressing the expression of inducible nitric oxide synthase (NOS2) in BMMs which were treated with RANKL and‐CSF for 3 days (Figure 6d).

To further explore if CDP suppressed osteoclast differentiation by reducing ROS production, we then treated BMMs with peroxide (10 µM) to imitate the high ROS status in cells. BMMs were induced by RANKL and M‐CSF for 3 days and the results of western blot analysis and RT‐PCR showed that peroxide promoted NFATc1and c‐Fos expression compared with the control groups. Consistent with the results in Figure 5, the expression of NFATc1 and c‐Fos was suppressed by CDP while peroxide rescued the inhibitory effect of CDP (Figure 6e,f). These data indicated that CDP repressed the expression of NFATc1 and c‐Fos by scavenging ROS production.

3.6 | CDP represses MAPK pathways duringRANKL‐induced osteoclastogenesis

Next, we investigated the effect of CDP treatment on RANKL‐mediatedTRAF6 expression and MAPK pathway activation. Following incubation in serum‐free medium for 2 hr, BMMs were stimulated with RANKL, with or without CDP, for 60 min. Stimulation with CDP (10 µM) had no effect on TRAF6 expression and attenuated phosphorylation of JNK2 andERK1/2 at 10 and 20 min (Figure 7). Additionally, p38 phosphorylation was significantly inhibited by CDP treatment of 60 min compared to control groups (Figure 7). These data reveal that CDP suppressesRANKL‐induced MAPK signaling pathways, consistent with its inhibitory effect on osteoclast formation and activity.

4 | DISCUSSION

Cistanche, known as “desert ginseng,” has attracted much attention recently for its ability to modulate immunity and act protectively during aging and oxidative stress (Jia et al., 2012; Snytnikovaet al., 2012). Phenylpropanoid‐substituted diglycosides, the major active components of Cistanche, have been shown to inhibit activity in macrophages (Ahn, Chae, Chin, & Kim, 2017). In addition, a Cistanche extract reduced oxidative stress in reperfused myocardium following ischemia and played a significant role in the inhibition of apoptotic pathways leading to cardioprotection(Yu, Li, & Cao, 2016). As an important component of Cistanche, CDP has a variety of pharmacological functions. Our current study found that CDP repressed RANKL‐activated osteoclast differentiation and activation by attenuating ROS production as well as NFATand MAPK activation.

As an acid phosphatase, TRAcP exists in a variety of cells and is abundant in osteoclasts and alveolar macrophages (Snipes, Lam, Dodd, Gray, & Cohen, 1986). TRAcP is a characteristic enzyme of osteoclasts and its expression is closely related to osteoclast function, regarded as an indicator of osteoclast activity and bone resorption (Minkin, 1982). In our study, CDP inhibited the number of TRAcP‐positive cells, indicating that RANKL‐induced osteoclastogenesis was blocked by CDP. Degradation of bone matrix by osteoclasts depends on cathepsin K (CTSK) and MPs (Gruber, 2015). Here, CDP significantly downregulated theexpression of osteoclast functional genes such as Mmp9, Ctsk, and Acp5.

Osteoclast differentiation and function are regulated by multiple signaling pathways (Boyle, Simonet, & Lacey, 2003). After binding to RANK, RANKL recruits adapter protein TRAF6 to activate the expression of NFATc1, which is an important transcription factor for osteoclast formation, affecting osteoclast‐specific gene expression, including TRAcP and CTSK (X. Feng,2005; Takayanagi et al., 2002). In this study, we found that CDPhad no effect on RANK and TRAF6 expression in osteoclasts. However, CDP inhibited RANKL‐induced NFATc1 activation during osteoclastogenesis of BMMs. Besides, it was demonstrated that ROS, produced by mitochondria in the process of delivering electrons, promoted the proliferation and differentiation of osteoclasts and regulated bone matrix degradation (Ha et al., 2004). A recent study found that RANKL induced Bach1 nuclear import and attenuated Nrf2‐mediated antioxidant enzyme production, thereby augmenting intracellular ROS expression and osteoclastogenesis in mice (Kanzaki et al., 2017). In addition, increased intracellular ROS generation by homocysteine enhanced osteoclast formation and activity (Koh et al., 2006). Our study found that CDP reduced ROS accumulation in osteoclasts by inhibiting NOS2 expression and promoting the expression of antioxidant enzymes, such as TRX1 and glutathione reductase. When we treated BMMs with peroxide to enhance intracellularROS accumulation the results that increased ROS could improveNFATc1 expression revealed that ROS was upstream of NFATc1.We also found that peroxide rescued the inhibitory effect of CDPon NFATc1 expression. So, our data suggest that CDP suppressesROS accumulation to inhibit NFATc1 expression, then repress osteoclast formation and function.

ERK, JNK, and p38 belong to the MAPK family, which is also involved in the regulation of osteoclast differentiation (Seger & Krebs, 1995).RANKL activates the MAPK pathway by increasing the phosphorylation of ERK, JNK, and p38 (Mizukami et al., 2002). We demonstrated that CDP repressed the RANKL‐mediated phosphorylation of key proteins in the MAPK pathway, thereby contributing to the inhibitory effect of CDPon the expression of osteoclast marker genes. To our knowledge, this is the first study showing the inhibitory effects of CDP on ROS production, NFAT, and MAPK activation, representing novel mechanisms of action of CDP in vitro.

In summary, we demonstrated that CDP attenuates osteoclastogenesis and hydroxyapatite resorption, and the expression of osteoclast marker genes including Ctsk, Mmp9, and Acp5. CDP was able to suppress RANKL‐mediated ROS production, as well as NFATand MAPK activation (Figure 8). Collectively, our results suggest that CDP may represent a candidate drug for the treatment of osteoclast-related conditions accompanied by ROS overproduction.

ACKNOWLEDGMENTS

This study was supported by the Nature Science Foundation of China(81572164), the National Key Technology Research and Development Program of China (2017YFC1103300), the Natural ScienceFoundation of Guangxi Province (2016GXNSFAA380295), and the University Science and Technology Research Project of GuangxiProvince (KY2015YB054). It is also supported in part by GuangxiScientific Research and Technology Development Plan Project(GKG13349003, 1598013‐15), Western Australia Medical & HealthResearch Infrastructure Fund, Arthritis Australia Foundation, the TheUniversity of Western Australia (UWA) Research CollaborationAwards, and the Australian Health and Medical Research Council(NHMRC, Nos. 1107828 and 1027932).

CONFLICTS OF INTEREST

The authors declare that there are no conflicts of interest.

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