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Emodin suppresses cadmium-induced osteoporosis by inhibiting osteoclast formation
Environmental Toxicology and Pharmacology 54 (2017) 162–168
Contents lists available at ScienceDirect
Environmental Toxicology and Pharmacology journal homepage: www.elsevier.com/locate/etap
Research Paper
Emodin suppresses cadmium-induced osteoporosis by inhibiting osteoclast formation Xiao Chena,b,c, Shuai Renc, Guoying Zhud, Zhongqiu Wangc, Xiaolin Wene,
MARK
⁎
a
Division of Nephrology, Zhongshan Hospital Fudan University, Shanghai 200032, China Shanghai Key Laboratory of Kidney and Dialysis, Shanghai 200032, China c Department of Radiology, Affiliated Hospital of Nanjing University of Chinese Medicine, Nanjing 210029, China d Institute of Radiation Medicine, Fudan University, Shanghai 200032, China e Zhejiang Provincial Key Laboratory of Geriatrics, Zhejiang Hospital, Hangzhou 310013, China b
A R T I C L E I N F O
A B S T R A C T
Keywords: Cadmium Bone Osteoporosis Osteoclast Emodin
Environmental level of cadmium (Cd) exposure can induce bone loss. Emodin, a naturally compound found in Asian herbal medicines, could influence osteoblast/osteoclast differentiation. However, the effects of emodin on Cd-induced bone damage are not clarified. The aim of this study was to investigate the role of emodin on Cdinduced osteoporosis. Sprague-Dawley male rats were divided into three groups which were given 0 mg/L, 50 mg Cd/L and 50 mg Cd/L plus emodin (50 mg/kg body weight). Bone histological investigation, microCT analysis, metabolic biomarker determination and immunohistochemical staining were performed at the 12th week. The bone mass and bone microstructure index of rats treated with Cd were obviously lower than in control. Cd markedly enhanced the osteoclast formation compared with control. Emodin significantly abolished the Cd-induced bone microstructure damage (p < 0.05), osteoclast formation and increase of tartrate-resistant acid phosphatase 5b level (p < 0.05). Our data further showed that emodin attenuated the Cd-induced inhibition of osteoprotegerin expression and stimulation of receptor activator for nuclear factor-κ B ligand expression. Our data show that emodin suppresses the Cd-induced osteoporosis by inhibiting osteoclast formation.
1. Introduction Cadmium (Cd) is one of the important environmental contaminants. Food and tobacco are the main source of Cd exposure in general population (Järup and Akesson, 2009). Cd exposure can induce serious adverse effects, including renal dysfunction, liver damage, osteoporosis and cardiovascular diseases (WHO/ICPS, 1992). Due to its long biological half-life in the body, low level of Cd exposure also could cause bone loss which may increase the risk of osteoporosis and bone fractures (Wang et al., 2003; Åkesson et al., 2006; Wallin et al., 2016). Cd exposure may inhibit the production of 1, 25(OH)2D, which subsequently diminish the calcium uptake in intestine (Berglund et al., 2000; Brzóska and Moniuszko-Jakoniuk, 2005). In addition, Cd also can directly affect the activatity of osteoblasts and osteoclasts (Wilson et al., 1996; Chen et al., 2009; Brama et al., 2012; Chen et al., 2013), resulting in the imbalance of bone resorption and formation. No proven effective treatments for Cd intoxication have been found (Waalkes, 2003). Many explorations have been performed in the past two decades. Vitamin (C, D and E) and trace elements (Se) can protect against Cd-induced liver and kidney injury (Karabulut-Bulan et al.,
⁎
Corresponding author. E-mail address: [email protected] (X. Wen).
http://dx.doi.org/10.1016/j.etap.2017.07.007 Received 10 April 2017; Received in revised form 8 July 2017; Accepted 16 July 2017 Available online 18 July 2017 1382-6689/ © 2017 Elsevier B.V. All rights reserved.
2008; Kara et al., 2008). In addition, numerous studies have shown that natural products, such as green tea catechins, daidzein, quercetin compounds and curcumin, are also valuable in protecting against Cd toxicity (Kim et al., 2016). However, only a few studies focused on the treatments of Cd-induced osteoporosis. Recently, aronia melanocarpa polyphenols and tannic acid may be valuable in protecting against Cdinduced skeleton damage (Brzóska et al., 2015; Tomaszewska et al., 2016), which suggests that natural products may be also valuable for the treatment of Cd-induced osteoporosis. Emodin, a naturally occurring anthraquinone derivative present in the roots and bark of numerous plants of thegenus Rhamnus, is present with variety of pharmacologic effects (Zhang et al., 2017; Iwanowycz et al., 2016; Song et al., 2017), including anticancer, antioxidant and anti-inflammatory activities. Lee et al. (2008) showed that emodin accelerated osteoblasts differentiation by up-regulating bone morphogenetic protein-2(BMP-2) expression. Kim et al. (2013) further demonstrated that emodin can suppress osteoclastogenesis and stimulate osteoblast formation. However, the effects of emodin on Cd-induced osteoporosis have not been investigated. Our previous studies showed that osteoclast is one of target cells for
Environmental Toxicology and Pharmacology 54 (2017) 162–168
X. Chen et al.
Fig. 1. The structure of emodin (A) and body weight of control and cadmium (Cd) treated rats with or without emodin (B). Body weight was obtained every week. Data are shown as means ± SD (n = 8).
paraffin and sliced for Hematoxylin and Eosin (HE) stain or immunochemistry staining. The right tibia was embedded in methylmethacrylate and sliced for Goldner’s trichrome staining.
low-level of Cd exposure (Chen et al., 2009). We speculated that emodin may protect against bone loss induced by Cd via its inhibitory effects on osteoclast formation. In this study, we observed the role of emodin on Cd-induced bone injury in a rat model.
2.4. Histochemistry and immunohistochemistry
2. Materials and methods
Osteoclast formation in tibia was determined by tartrate resistant acid phosphatase (TRAP) staining. Briefly, the sections were incubated in oven at 52 °C for one hour, dewaxed in dimethylbenzene for three times, then hydrated through a series of descending ethanol solution (100%-40%). TRAP staining was used for the identification of osteoclasts following the manufacturer’s instructions (Sigma 387-A, St. Louis, USA). TRAP positive area was measured by using SimplePCI (Compix Inc., Arizona, USA) imaging software. Immunohistochemistry for osteoprotegerin (OPG) and receptor activator of nuclear factor (NF)-kB ligand (RANKL) was performed as our previous study (Chen et al., 2013). Briefly, sections were dewaxed in dimethylbenzene and rinsed in PBS. Antigen retrieval was done using heat mediated retrieval solution. Then, the sections were incubated in 1% H2O2. Subsequently, sections were incubated with primary antibodies [anti-OPG (1:100 dilution) and anti-RANKL (1:100 dilution)] overnight at 4 °C, followed by 2-h incubation with secondary antibodies (1:1000) at room temperature. Finally, the antigen-antibody complex was visualized by 3,30-diaminobenzidine (DAB).
2.1. Experimental design The animal experiments were approved by the Institutional Animal Care Committee of our institution. Cd was purchased from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China). Emodin (Fig. 1A) was obtained from Sigma-Aldrich Chemical Co., (≥90% purity, St Louis, MO, USA). Twenty four specific pathogen free Sprague Dawley male rats aged 8 weeks old were feed in the animal facilities under standard conditions (21 ± 1 °C and a 12-h light-dark cycle). They were randomly divided into three groups of eight rats (control, Cd-treated, Cd and emodin-treated groups) and acclimated to the laboratory for one week. They freely access to water (Cd ≤ 0.004 mg/L) and rat chow (Cd ≤ 0.2 mg/kg). Cd was administrated via drinking water (50 mg/L) for 12 weeks; emodin was administrated intragastically (50 mg/kg body weight) every other days for 12 weeks. The control groups received normal water containing sodium chloride. The exposure level used in this study is very close to human environmental exposure (Brzóska and Moniuszko-Jakoniuk, 2005). 2.2. MicroCT analysis
2.5. Biomarker determination
Before sacrifice, the rats underwent MicroCT examinations (Ge eXplore, GE healthcare, USA) as our previous study (Tang et al., 2016). Briefly, rats were anaesthetized by 7.0% chloral hydrate (0.5 mL/100 g body weight) and placed on the scanning platform. The scan protocol was as following: tube voltage 80 kV, current 450 μA, a rotation step of 0.5°, field of view 3.2 cm, and spatial resolution with a voxel size of 45 × 45 × 45 μm. The proximal tibia was figured out and reconstructed for further analysis. Then, bone volume fraction (BV/TV), trabecular thickness (Tb.Th), trabecular number (Tb.N) and trabecular separation (Tb.Sp) were analyzed by using Microview 2.5.0 software. The region of interest was 1 mm distally from the growth plate, and extending a further longitudinal distance of 2 mm in the distal direction.
Serum tartrate-resistant acid phosphatase 5b (Tracp5b) was measured using EIA methods (Rat TRAP Assay, IDS, UK) as manufacturer’s protocol. A standard sample (1.8 U/L) was supplied by manufacturer and the obtained result in our laboratory was 1.7 U/L. The intraassay and interassay were both lower than 5%.
2.3. Samples collection and preparation
3. Results
The rats were anesthetized using chloral hydrate (7%, 0.5 mL/ 100 g). Blood was collected from carotid artery and centrifuged for serum isolation. Lumbar spine and tibia were taken off and then removed the soft tissues. The lumbar spine (L5) and tibia were fixed with 4% polyoxymethylene at 4 °C for 3 days. Then, L5 and left tibia were decalcified by 10% EDTA at 4 °C for 1–2 weeks, dehydrated through a series of ascending ethanol solution (40–100%) and embedded in
3.1. Body weight
2.6. Statistical analysis Data are given as mean ± SD or boxplot diagrams. Statistical comparisons between groups were analyzed using non-parametric as Mann-Whitney ‘U’ Test, with p < 0.05 was considered statistically significant.
The body weight of all rats increased during the whole experiment (Fig. 1). Slight decrease was found in rats treated with Cd compared with control, but there were no significant differences. In addition, no differences of body weight was observed in rats treated with both Cd and emodin compared with control. 163
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Fig. 2. Three-dimensional (3D) and two-dimensional (2D) reconstructed images derived by micro-computed tomography in control and cadmium(Cd) treated rats with or without emodin.
and chondral cell (Fig. 7). Cd-treated mice displayed profound decreases in OPG expression and increases in RANKL expression. However, emodin obviously attenuated the increased expression of OPG and decreased expression of RANKL induced by Cd exposure.
3.2. Bone microstructure Bone microstructure in lumbar spine and tibia were evaluated by microCT analysis (Figs. 2 and 3) and histologic examinations (HE and Goldner’s trichrome staining) (Fig. 4). Qualitative analysis from HE and Goldner’s trichrome staining and quantitative data from microCT both showed the Tb.N and conjunction points was obviously decreased and Tb.Sp increased in Cd-treated rats. Emodin treatment protected the mice against bone loss induced by Cd exposure as shown by increased BV/TV and Tb.N and decreased Tb.Sp (p < 0.05 or 0.01).
4. Discussion Cd exposure can cause bone loss, such as osteoporosis and osteomalacia, which increase the risk of bone fractures. Recent several studies have shown that natural product, such as tea, aronia melanocarpa polyphenols and tannic acid, may have protective effects on bone loss evoked by Cd exposure (Brzóska et al., 2015; Tomaszewska et al., 2016, 2017). In the present study, we demonstrated that emodin, a naturally occurring anthraquinone derivative, could also decrease the bone loss induced by Cd exposure. Moreover, we observed that emodin administration inhibited the osteoclast formation enhanced by Cd exposure. Our data showed that emodin might represent an efficient drug for the treatment of Cd-induced osteoporosis. More and more studies have shown that low-level of Cd exposure are associated with bone loss and high risk of bone fractures (Åkesson et al., 2006; Wallin et al., 2016). Brzóska and Moniuszko-Jakoniuk (2005) have reported that the exposure level of 50 mg Cd/L was relative to the human exposure. In our previous study, we reported that the Cd in blood and urine were about 3.3 μg/L and 5 μg/g creatinine in rats received 50 mg Cd/L exposure, respectively (Chen et al., 2013). The exposure levels were close to human exposure via food consumption. Emodin is a natural compound extracted from the roots and bark of numerous plants of thegenus Rhamnus. Emodin was also found to have many biological effects, such as anti-Inflammatory (Zhang et al., 2017), anti-cancer (Iwanowycz et al., 2016), antihyperglycaemic (Abu et al., 2017) and antioxidant (Song et al., 2017). Few studies also investigated the role of emodin on bone. Lee et al. (2008) showed that emodin
3.3. TRAP positive cell in bone tissues Few TRAP positive cells were observed epiphyseal-metaphyseal region of tibia in control (Figs. 5 , 6 A). Cd exposure significantly enhanced osteoclast formation. The TRAP positive area was higher in Cd group compared with that of control (p < 0.01) (Fig. 6A). The TRAP positive area was significantly decreased in rats treated with Cd plus emodin compared with rats treated with Cd alone. 3.4. Metabolic biomarkers Tracp5b is a biomarker reflecting osteoclast number and activity. The Tracp5b level of rats treated with Cd was significantly higher than that in control (Fig. 6B), increasing by approximately 100% (p < 0.05). The level of Tracp5b was significantly decreased in rats treated with both Cd and emodin compared with rats treated with Cd alone (p < 0.05). 3.5. OPG/RANKL expression Weak RANKL and evident OPG expression were observed in control rats as displayed brown immunostaining in cytoplasm of osteogenic cell 164
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Fig. 3. Quantitative analysis of bone microstructure parameters based on the reconstructed images derived by micro-computed tomography in control and cadmium(Cd) treated rats with or without emodin, including bone volume fraction (BV/TV), trabecular number (Tb.N), trabecular thickness (Tb.Th) and trabecular separation (Tb.Sp). Data are given as boxplot diagrams medians (lines in the boxes), 25 and 75% quartiles (boxes) and minimum and maximum ranges (whiskers) (n = 8).
induced bone damage. Our data showed that emodin could diminish the bone loss in cancellous bone caused by Cd exposure, since the bone volume fraction and Tb.N were increased while the Tb.Sp was decreased. Our previous studies have shown that low-level of Cd exposure can stimulate osteoclast formation (Chen et al., 2013). Therefore, subsequently, we observed the effects of emodin on Cd-stimulated
enhanced osteoblast differentiation and mineralization by stimulating BMP-2 expression. Yang et al. (2014) found that emodin could enhance osteoblast differentiation and inhibit adipocyte differentiation from bone marrow stem cells. Furthermore, several studies also reported that emodin inhibited osteoclast formation (Hwang et al., 2014; Kim et al., 2013). Therefore, we aimed to show the effects of emodin on Cd-
Fig. 4. Histologic feature of tibia and lumbar spine using Golder’s trichrome (upper) and hemotoxylin and eosin staining (lower) in control and cadmium (Cd) treated rats with or without emodin treatment.
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Fig. 5. Tartrate-resistant acid phosphatase (TRAP) positive cells in bone tissues in control and cadmium (Cd) treated rats with or without emodin. The osteoclasts were stained in red.
and up-regulate the gene expression related with differentiation. OPG is a decoy receptor which can bind with RANKL and inhibit the activation of RANK. However, the role of emodin on OPG and RANKL has not been investigated. Our results showed that emodin significantly inhibited the Cd-related RANKL overexpression and reversed the Cd-induced OPG down-regulation. These findings suggest that emodin may inhibit the osteoclast formation by suppressing RANKL expression and stimulating OPG expression. The mechanisms of damaging Cd action in the skeleton are also associated with oxidative stress (Brzóska et al., 2011, 2016). Emodin also has the biological activity of antioxidant. Therefore, the effects of emodin on Cd-induced bone loss may be also related with its antioxidative function. A recent study also showed that the osteoprotective activity of aronia melanocarpa polyphenols was related with its function of oxidative defense (Brzóska et al., 2016). There are several limitations in our study. First, the role of emodin
osteoclast formation. Our data showed that emodin inhibited the Cdstimulated osteoclast formation in bone tissues. We speculated that emodin protect against Cd-induced bone loss by inhibiting osteoclast formation. The mechanisms of emodin on osteoclast are not fully clarified. Kim et al. (2013) showed that emodin could inhibit the expression c-fos and NFATc1, which are essential for the osteoclast differentiation. In addition, emodin also inhibit the RANKL induced IKKb activation, resulting in the suppression of NF-ƙB transcriptional activation. Their data suggests that emodin can inhibit the RANKL-associated signal pathways leading to osteoclast formation. Previous studies also displayed that Cd-induced bone loss was associated with RANKL and OPG (Chen et al., 2013; Brzóska and Rogalska, 2013). OPG and RANKL are the two important cellular factors for osteoclast formation and activation (Boyce and Xing, 2008). RANKL can bind with its receptor RANK
Fig. 6. The area of tartrate-resistant acid phosphatase (TRAP) positive cells in bone tissue (A) and TRACP5b level in serum (B). Data are given as boxplot diagrams medians (lines in the boxes), 25 and 75% quartiles (boxes) and minimum and maximum ranges (whiskers).
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Fig. 7. Immunochemical staining shows the osteoprotegerin (OPG) and receptor activator of nuclear factor (NF)-kB ligand (RANKL) expression in bone tissues in control and cadmium (Cd) treated rats with or without emodin. Tomczyk, M., 2015. Protective effect of Aronia melanocarpa polyphenols against cadmium-induced disorders in bone metabolism: a study in a rat model of lifetime human exposure to this heavy metal. Chem. Biol. Interact. 229, 132–146. Brzóska, M.M., Rogalska, J., Roszczenko, A., Galazyn-Sidorczuk, M., Tomczyk, M., 2016. The mechanism of the osteoprotective action of a polyphenol-rich aronia melanocarpa extract during chronic exposure to cadmium is mediated by the oxidative defense system. Planta Med. 82, 621–631. Chen, X., Zhu, G., Gu, S., Jin, T., Shao, C., 2009. Effects of cadmium on osteoblasts and osteoclasts in vitro. Environ. Toxicol. Pharmacol. 28, 232–236. Chen, X., Wang, G., Li, X., Gan, C., Zhu, G., Jin, T., Wang, Z., 2013. Environmental level of cadmium exposure stimulates osteoclasts formation in male rats. Food Chem. Toxicol. 60, 530–535. Hwang, J.K., Noh, E.M., Moon, S.J., Kim, J.M., Kwon, K.B., Park, B.H., You, Y.O., Hwang, B.M., Kim, J.Y., Cheon, Y.H., Kwak, S.C., Baek, J.M., Yoon, K.H., Lee, M.S., Oh, J., 2014. Emodin regulates bone remodeling by inhibiting osteoclastogenesis and stimulating osteoblast formation. J. Bone. Miner. Res. 29, 1541–1553. Iwanowycz, S., Wang, J., Hodge, J., Wang, Y., Yu, F., Fan, D., 2016. Emodin inhibits breast cancer growth by blocking the tumor-promoting feedforward loop between cancer cells and macrophages. Mol. Cancer. Ther. 15, 1931–1942. Järup, L., Akesson, A., 2009. Current status of cadmium as an environmental health problem. Toxicol. Appl. Pharmacol. 238, 201–208. Kara, H., Cevik, A., Konar, V., Dayangac, A., Servi, K., 2008. Effects of selenium with vitamin E and melatonin on cadmium-induced oxidative damage in rat liver and kidneys. Biol. Trace. Elem. Res. 125, 236–244. Karabulut-Bulan, O., Bolkent, S., Yanardag, R., Bilgin-Sokmen, B., 2008. The role of vitamin C, vitamin E, and selenium on cadmium-induced renal toxicity of rats. Drug Chem. Toxicol. 31, 413–426. Kim, H.J., Kim, B.S., Lee, S.J., Kim, J.S., Lee, Y.R., 2013. Emodin suppresses inflammatory responses and joint destruction in collagen-induced arthritic mice. Rheumatology (Oxford) 52, 1583–1591. Kim, M.Y., Shon, W.J., Park, M.N., Lee, Y.S., Shin, D.M., 2016. Protective effect of dietary chitosan on cadmium accumulation in rats. Nutr. Res. Pract. 10, 19–25. Lee, S.U., Shin, H.K., Min, Y.K., Kim, S.H., 2008. Emodin accelerates osteoblast differentiation through phosphatidylinositol 3-kinase activation and bone morphogenetic protein-2 gene expression. Int. Immunopharmacol. 8, 741–747. Song, C., Liu, B., Xie, J., Ge, X., Zhao, Z., Zhang, Y., Zhang, H., Ren, M., Zhou, Q., Miao, L., Xu, P., Lin, Y., 2017. Comparative proteomic analysis of liver antioxidant mechanisms in Megalobrama amblycephala stimulated with dietary emodin. Sci. Rep. 7, 40356. Tang, L., Chen, X., Bao, Y., Xu, W., Lv, Y., Wang, Z., Wen, X., 2016. CT imaging biomarkers of bone damage induced by environmental level of cadmium exposure in male rats. Biol. Trace. Elem. Res. 170, 146–151. Tomaszewska, E., Dobrowolski, P., Winiarska-Mieczan, A., Kwiecień, M., Tomczyk, A., Muszyńsk, I.S., Radzki, R., 2016. Alteration in bone geometric and mechanical properties, histomorphometrical parameters of trabecular bone, articular cartilage, and growth plate in adolescent rats after chronic co-exposure tocadmium and lead in the case of supplementation with green, black, red and white tea. Environ. Toxicol. Pharmacol. 46, 36–44. Tomaszewska, E., Dobrowolski, P., Winiarska-Mieczan, A., Kwiecień, M., Tomczyk, A., Muszyński, S., 2017. The effect of tannic acid on the bone tissue of adult male Wistar rats exposed to cadmium and lead. Exp. Toxicol. Pathol. 69, 131–141. WHO/IPCS, 1992. Environmental Health Criteria Document 134 Cadmium. Geneva, WHO. Waalkes, M.P., 2003. Cadmium carcinogenesis. Mutat. Res. 533, 107–120. Wallin, M., Barregard, L., Sallsten, G., Lundh, T., Karlsson, M.K., Lorentzon, M., Ohlsson,
alone on bone was not investigated because the previous study has confirmed the role of emodin on bone. Second, we only observed the effects of emodin on Cd-induced osteoclast formation. The role of emodin on Cd-related inhibition in bone formation needs further exploration. Finally, single level of Cd and emodin were adopted in our study. In conclusion, our data showed that emodin can protect against Cdinduced bone loss by inhibiting osteoclast formation. Furthermore, our data indicated that the role of emodin on osteoclast may be related with its effects on RANKL and OPG. These results suggest that emodin could be explored as a potential therapeutic agent against Cd-induced osteoporosis. Declaration of interest The authors report no conflicts of interest. Acknowledgement This study was funded by Natural Science Foundation of Jiangsu Province (no. BK20161609). References Åkesson, A., Bjellerup, P., Lundh, T., Lidfeldt, J., Nerbrand, C., Samsioe, G., Skerfving, S., Vahter, M., 2006. Cadmium induced effects on bone in a population-based study of women. Environ. Health. Perspect. 114, 830–834. Abu, Eid. S., Adams, M., Scherer, T., Torres-Gómez, H., Hackl, M.T., Kaplanian, M., Riedl, R., Luge, r A., Fürnsinn, C., 2017. Emodin, a compound with putative antidiabetic potential, deteriorates glucose tolerance in rodents. Eur. J. Pharmacol. 798, 77–84. Berglund, M., Akesson, A., Bjellerup, P., Vahter, M., 2000. Metal-bone interactions. Toxicol. Lett. 112–113, 219–225. Boyce, B.F., Xing, L., 2008. Functions of RANKL/RANK/OPG in bone modeling and remodeling. Arch. Biochem. Biophys. 473, 139–146. Brama, M., Politi, L., Santini, P., Migliaccio, S., Scandurra, R., 2012. Cadmium-induced apoptosis and necrosis in human osteoblasts: role of caspases and mitogen-activated protein kinases pathways. J. Endocrinol. Invest. 35, 198–208. Brzóska, M.M., Moniuszko-Jakoniuk, J., 2005. Bone metabolism of male rats chronically exposed to cadmium. Toxicol. Appl. Pharmacol. 207, 195–211. Brzóska, M.M., Rogalska, J., 2013. Protective effect of zinc supplementation against cadmium-induced oxidative stress and the RANK/RANKL/OPG system imbalance in the bone tissue of rats. Toxicol. Appl. Pharmacol. 272, 208–220. Brzóska, M.M., Rogalska, J., Kupraszewicz, E., 2011. The involvement of oxidative stress in the mechanisms of damaging cadmium action in bone tissue: a study in a rat model of moderate and relatively high human exposure. Toxicol. Appl. Pharmacol. 250, 327–335. Brzóska, M.M., Rogalska, J., Galazyn-Sidorczuk, M., Jurczuk, M., Roszczenko, A.,
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Yang, F., Yuan, P.W., Hao, Y.Q., Lu, Z.M., 2014. Emodin enhances osteogenesis and inhibits adipogenesis.BMC. Complement. Altern. Med. 14, 74. Zhang, W., Lu, X., Wang, W., Ding, Z., Fu, Y., Zhou, X., Zhang, N., Cao, Y., 2017. Inhibitory effects of emodin, thymol, and astragalin on leptospira interrogans-induced inflammatory response in the uterine and endometrium epithelial cells of mice. Inflammation 40, 666–675.
C., Mellström, D., 2016. Low-level cadmium exposure is associated with decreased bone mineral density and increased risk of incident fractures in elderly men: the MrOS Sweden study. J. Bone. Miner. Res. 31, 732–741. Wilson, A.K., Cerny, E.A., Smith, B.D., Wagh, A., Bhattacharyya, M.H., 1996. Effects of cadmium on osteoclast formation and activity in vitro. Toxicol. Appl. Pharmacol. 140, 451–460.
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Contents lists available at ScienceDirect
Environmental Toxicology and Pharmacology journal homepage: www.elsevier.com/locate/etap
Research Paper
Emodin suppresses cadmium-induced osteoporosis by inhibiting osteoclast formation Xiao Chena,b,c, Shuai Renc, Guoying Zhud, Zhongqiu Wangc, Xiaolin Wene,
MARK
⁎
a
Division of Nephrology, Zhongshan Hospital Fudan University, Shanghai 200032, China Shanghai Key Laboratory of Kidney and Dialysis, Shanghai 200032, China c Department of Radiology, Affiliated Hospital of Nanjing University of Chinese Medicine, Nanjing 210029, China d Institute of Radiation Medicine, Fudan University, Shanghai 200032, China e Zhejiang Provincial Key Laboratory of Geriatrics, Zhejiang Hospital, Hangzhou 310013, China b
A R T I C L E I N F O
A B S T R A C T
Keywords: Cadmium Bone Osteoporosis Osteoclast Emodin
Environmental level of cadmium (Cd) exposure can induce bone loss. Emodin, a naturally compound found in Asian herbal medicines, could influence osteoblast/osteoclast differentiation. However, the effects of emodin on Cd-induced bone damage are not clarified. The aim of this study was to investigate the role of emodin on Cdinduced osteoporosis. Sprague-Dawley male rats were divided into three groups which were given 0 mg/L, 50 mg Cd/L and 50 mg Cd/L plus emodin (50 mg/kg body weight). Bone histological investigation, microCT analysis, metabolic biomarker determination and immunohistochemical staining were performed at the 12th week. The bone mass and bone microstructure index of rats treated with Cd were obviously lower than in control. Cd markedly enhanced the osteoclast formation compared with control. Emodin significantly abolished the Cd-induced bone microstructure damage (p < 0.05), osteoclast formation and increase of tartrate-resistant acid phosphatase 5b level (p < 0.05). Our data further showed that emodin attenuated the Cd-induced inhibition of osteoprotegerin expression and stimulation of receptor activator for nuclear factor-κ B ligand expression. Our data show that emodin suppresses the Cd-induced osteoporosis by inhibiting osteoclast formation.
1. Introduction Cadmium (Cd) is one of the important environmental contaminants. Food and tobacco are the main source of Cd exposure in general population (Järup and Akesson, 2009). Cd exposure can induce serious adverse effects, including renal dysfunction, liver damage, osteoporosis and cardiovascular diseases (WHO/ICPS, 1992). Due to its long biological half-life in the body, low level of Cd exposure also could cause bone loss which may increase the risk of osteoporosis and bone fractures (Wang et al., 2003; Åkesson et al., 2006; Wallin et al., 2016). Cd exposure may inhibit the production of 1, 25(OH)2D, which subsequently diminish the calcium uptake in intestine (Berglund et al., 2000; Brzóska and Moniuszko-Jakoniuk, 2005). In addition, Cd also can directly affect the activatity of osteoblasts and osteoclasts (Wilson et al., 1996; Chen et al., 2009; Brama et al., 2012; Chen et al., 2013), resulting in the imbalance of bone resorption and formation. No proven effective treatments for Cd intoxication have been found (Waalkes, 2003). Many explorations have been performed in the past two decades. Vitamin (C, D and E) and trace elements (Se) can protect against Cd-induced liver and kidney injury (Karabulut-Bulan et al.,
⁎
Corresponding author. E-mail address: [email protected] (X. Wen).
http://dx.doi.org/10.1016/j.etap.2017.07.007 Received 10 April 2017; Received in revised form 8 July 2017; Accepted 16 July 2017 Available online 18 July 2017 1382-6689/ © 2017 Elsevier B.V. All rights reserved.
2008; Kara et al., 2008). In addition, numerous studies have shown that natural products, such as green tea catechins, daidzein, quercetin compounds and curcumin, are also valuable in protecting against Cd toxicity (Kim et al., 2016). However, only a few studies focused on the treatments of Cd-induced osteoporosis. Recently, aronia melanocarpa polyphenols and tannic acid may be valuable in protecting against Cdinduced skeleton damage (Brzóska et al., 2015; Tomaszewska et al., 2016), which suggests that natural products may be also valuable for the treatment of Cd-induced osteoporosis. Emodin, a naturally occurring anthraquinone derivative present in the roots and bark of numerous plants of thegenus Rhamnus, is present with variety of pharmacologic effects (Zhang et al., 2017; Iwanowycz et al., 2016; Song et al., 2017), including anticancer, antioxidant and anti-inflammatory activities. Lee et al. (2008) showed that emodin accelerated osteoblasts differentiation by up-regulating bone morphogenetic protein-2(BMP-2) expression. Kim et al. (2013) further demonstrated that emodin can suppress osteoclastogenesis and stimulate osteoblast formation. However, the effects of emodin on Cd-induced osteoporosis have not been investigated. Our previous studies showed that osteoclast is one of target cells for
Environmental Toxicology and Pharmacology 54 (2017) 162–168
X. Chen et al.
Fig. 1. The structure of emodin (A) and body weight of control and cadmium (Cd) treated rats with or without emodin (B). Body weight was obtained every week. Data are shown as means ± SD (n = 8).
paraffin and sliced for Hematoxylin and Eosin (HE) stain or immunochemistry staining. The right tibia was embedded in methylmethacrylate and sliced for Goldner’s trichrome staining.
low-level of Cd exposure (Chen et al., 2009). We speculated that emodin may protect against bone loss induced by Cd via its inhibitory effects on osteoclast formation. In this study, we observed the role of emodin on Cd-induced bone injury in a rat model.
2.4. Histochemistry and immunohistochemistry
2. Materials and methods
Osteoclast formation in tibia was determined by tartrate resistant acid phosphatase (TRAP) staining. Briefly, the sections were incubated in oven at 52 °C for one hour, dewaxed in dimethylbenzene for three times, then hydrated through a series of descending ethanol solution (100%-40%). TRAP staining was used for the identification of osteoclasts following the manufacturer’s instructions (Sigma 387-A, St. Louis, USA). TRAP positive area was measured by using SimplePCI (Compix Inc., Arizona, USA) imaging software. Immunohistochemistry for osteoprotegerin (OPG) and receptor activator of nuclear factor (NF)-kB ligand (RANKL) was performed as our previous study (Chen et al., 2013). Briefly, sections were dewaxed in dimethylbenzene and rinsed in PBS. Antigen retrieval was done using heat mediated retrieval solution. Then, the sections were incubated in 1% H2O2. Subsequently, sections were incubated with primary antibodies [anti-OPG (1:100 dilution) and anti-RANKL (1:100 dilution)] overnight at 4 °C, followed by 2-h incubation with secondary antibodies (1:1000) at room temperature. Finally, the antigen-antibody complex was visualized by 3,30-diaminobenzidine (DAB).
2.1. Experimental design The animal experiments were approved by the Institutional Animal Care Committee of our institution. Cd was purchased from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China). Emodin (Fig. 1A) was obtained from Sigma-Aldrich Chemical Co., (≥90% purity, St Louis, MO, USA). Twenty four specific pathogen free Sprague Dawley male rats aged 8 weeks old were feed in the animal facilities under standard conditions (21 ± 1 °C and a 12-h light-dark cycle). They were randomly divided into three groups of eight rats (control, Cd-treated, Cd and emodin-treated groups) and acclimated to the laboratory for one week. They freely access to water (Cd ≤ 0.004 mg/L) and rat chow (Cd ≤ 0.2 mg/kg). Cd was administrated via drinking water (50 mg/L) for 12 weeks; emodin was administrated intragastically (50 mg/kg body weight) every other days for 12 weeks. The control groups received normal water containing sodium chloride. The exposure level used in this study is very close to human environmental exposure (Brzóska and Moniuszko-Jakoniuk, 2005). 2.2. MicroCT analysis
2.5. Biomarker determination
Before sacrifice, the rats underwent MicroCT examinations (Ge eXplore, GE healthcare, USA) as our previous study (Tang et al., 2016). Briefly, rats were anaesthetized by 7.0% chloral hydrate (0.5 mL/100 g body weight) and placed on the scanning platform. The scan protocol was as following: tube voltage 80 kV, current 450 μA, a rotation step of 0.5°, field of view 3.2 cm, and spatial resolution with a voxel size of 45 × 45 × 45 μm. The proximal tibia was figured out and reconstructed for further analysis. Then, bone volume fraction (BV/TV), trabecular thickness (Tb.Th), trabecular number (Tb.N) and trabecular separation (Tb.Sp) were analyzed by using Microview 2.5.0 software. The region of interest was 1 mm distally from the growth plate, and extending a further longitudinal distance of 2 mm in the distal direction.
Serum tartrate-resistant acid phosphatase 5b (Tracp5b) was measured using EIA methods (Rat TRAP Assay, IDS, UK) as manufacturer’s protocol. A standard sample (1.8 U/L) was supplied by manufacturer and the obtained result in our laboratory was 1.7 U/L. The intraassay and interassay were both lower than 5%.
2.3. Samples collection and preparation
3. Results
The rats were anesthetized using chloral hydrate (7%, 0.5 mL/ 100 g). Blood was collected from carotid artery and centrifuged for serum isolation. Lumbar spine and tibia were taken off and then removed the soft tissues. The lumbar spine (L5) and tibia were fixed with 4% polyoxymethylene at 4 °C for 3 days. Then, L5 and left tibia were decalcified by 10% EDTA at 4 °C for 1–2 weeks, dehydrated through a series of ascending ethanol solution (40–100%) and embedded in
3.1. Body weight
2.6. Statistical analysis Data are given as mean ± SD or boxplot diagrams. Statistical comparisons between groups were analyzed using non-parametric as Mann-Whitney ‘U’ Test, with p < 0.05 was considered statistically significant.
The body weight of all rats increased during the whole experiment (Fig. 1). Slight decrease was found in rats treated with Cd compared with control, but there were no significant differences. In addition, no differences of body weight was observed in rats treated with both Cd and emodin compared with control. 163
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Fig. 2. Three-dimensional (3D) and two-dimensional (2D) reconstructed images derived by micro-computed tomography in control and cadmium(Cd) treated rats with or without emodin.
and chondral cell (Fig. 7). Cd-treated mice displayed profound decreases in OPG expression and increases in RANKL expression. However, emodin obviously attenuated the increased expression of OPG and decreased expression of RANKL induced by Cd exposure.
3.2. Bone microstructure Bone microstructure in lumbar spine and tibia were evaluated by microCT analysis (Figs. 2 and 3) and histologic examinations (HE and Goldner’s trichrome staining) (Fig. 4). Qualitative analysis from HE and Goldner’s trichrome staining and quantitative data from microCT both showed the Tb.N and conjunction points was obviously decreased and Tb.Sp increased in Cd-treated rats. Emodin treatment protected the mice against bone loss induced by Cd exposure as shown by increased BV/TV and Tb.N and decreased Tb.Sp (p < 0.05 or 0.01).
4. Discussion Cd exposure can cause bone loss, such as osteoporosis and osteomalacia, which increase the risk of bone fractures. Recent several studies have shown that natural product, such as tea, aronia melanocarpa polyphenols and tannic acid, may have protective effects on bone loss evoked by Cd exposure (Brzóska et al., 2015; Tomaszewska et al., 2016, 2017). In the present study, we demonstrated that emodin, a naturally occurring anthraquinone derivative, could also decrease the bone loss induced by Cd exposure. Moreover, we observed that emodin administration inhibited the osteoclast formation enhanced by Cd exposure. Our data showed that emodin might represent an efficient drug for the treatment of Cd-induced osteoporosis. More and more studies have shown that low-level of Cd exposure are associated with bone loss and high risk of bone fractures (Åkesson et al., 2006; Wallin et al., 2016). Brzóska and Moniuszko-Jakoniuk (2005) have reported that the exposure level of 50 mg Cd/L was relative to the human exposure. In our previous study, we reported that the Cd in blood and urine were about 3.3 μg/L and 5 μg/g creatinine in rats received 50 mg Cd/L exposure, respectively (Chen et al., 2013). The exposure levels were close to human exposure via food consumption. Emodin is a natural compound extracted from the roots and bark of numerous plants of thegenus Rhamnus. Emodin was also found to have many biological effects, such as anti-Inflammatory (Zhang et al., 2017), anti-cancer (Iwanowycz et al., 2016), antihyperglycaemic (Abu et al., 2017) and antioxidant (Song et al., 2017). Few studies also investigated the role of emodin on bone. Lee et al. (2008) showed that emodin
3.3. TRAP positive cell in bone tissues Few TRAP positive cells were observed epiphyseal-metaphyseal region of tibia in control (Figs. 5 , 6 A). Cd exposure significantly enhanced osteoclast formation. The TRAP positive area was higher in Cd group compared with that of control (p < 0.01) (Fig. 6A). The TRAP positive area was significantly decreased in rats treated with Cd plus emodin compared with rats treated with Cd alone. 3.4. Metabolic biomarkers Tracp5b is a biomarker reflecting osteoclast number and activity. The Tracp5b level of rats treated with Cd was significantly higher than that in control (Fig. 6B), increasing by approximately 100% (p < 0.05). The level of Tracp5b was significantly decreased in rats treated with both Cd and emodin compared with rats treated with Cd alone (p < 0.05). 3.5. OPG/RANKL expression Weak RANKL and evident OPG expression were observed in control rats as displayed brown immunostaining in cytoplasm of osteogenic cell 164
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Fig. 3. Quantitative analysis of bone microstructure parameters based on the reconstructed images derived by micro-computed tomography in control and cadmium(Cd) treated rats with or without emodin, including bone volume fraction (BV/TV), trabecular number (Tb.N), trabecular thickness (Tb.Th) and trabecular separation (Tb.Sp). Data are given as boxplot diagrams medians (lines in the boxes), 25 and 75% quartiles (boxes) and minimum and maximum ranges (whiskers) (n = 8).
induced bone damage. Our data showed that emodin could diminish the bone loss in cancellous bone caused by Cd exposure, since the bone volume fraction and Tb.N were increased while the Tb.Sp was decreased. Our previous studies have shown that low-level of Cd exposure can stimulate osteoclast formation (Chen et al., 2013). Therefore, subsequently, we observed the effects of emodin on Cd-stimulated
enhanced osteoblast differentiation and mineralization by stimulating BMP-2 expression. Yang et al. (2014) found that emodin could enhance osteoblast differentiation and inhibit adipocyte differentiation from bone marrow stem cells. Furthermore, several studies also reported that emodin inhibited osteoclast formation (Hwang et al., 2014; Kim et al., 2013). Therefore, we aimed to show the effects of emodin on Cd-
Fig. 4. Histologic feature of tibia and lumbar spine using Golder’s trichrome (upper) and hemotoxylin and eosin staining (lower) in control and cadmium (Cd) treated rats with or without emodin treatment.
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Fig. 5. Tartrate-resistant acid phosphatase (TRAP) positive cells in bone tissues in control and cadmium (Cd) treated rats with or without emodin. The osteoclasts were stained in red.
and up-regulate the gene expression related with differentiation. OPG is a decoy receptor which can bind with RANKL and inhibit the activation of RANK. However, the role of emodin on OPG and RANKL has not been investigated. Our results showed that emodin significantly inhibited the Cd-related RANKL overexpression and reversed the Cd-induced OPG down-regulation. These findings suggest that emodin may inhibit the osteoclast formation by suppressing RANKL expression and stimulating OPG expression. The mechanisms of damaging Cd action in the skeleton are also associated with oxidative stress (Brzóska et al., 2011, 2016). Emodin also has the biological activity of antioxidant. Therefore, the effects of emodin on Cd-induced bone loss may be also related with its antioxidative function. A recent study also showed that the osteoprotective activity of aronia melanocarpa polyphenols was related with its function of oxidative defense (Brzóska et al., 2016). There are several limitations in our study. First, the role of emodin
osteoclast formation. Our data showed that emodin inhibited the Cdstimulated osteoclast formation in bone tissues. We speculated that emodin protect against Cd-induced bone loss by inhibiting osteoclast formation. The mechanisms of emodin on osteoclast are not fully clarified. Kim et al. (2013) showed that emodin could inhibit the expression c-fos and NFATc1, which are essential for the osteoclast differentiation. In addition, emodin also inhibit the RANKL induced IKKb activation, resulting in the suppression of NF-ƙB transcriptional activation. Their data suggests that emodin can inhibit the RANKL-associated signal pathways leading to osteoclast formation. Previous studies also displayed that Cd-induced bone loss was associated with RANKL and OPG (Chen et al., 2013; Brzóska and Rogalska, 2013). OPG and RANKL are the two important cellular factors for osteoclast formation and activation (Boyce and Xing, 2008). RANKL can bind with its receptor RANK
Fig. 6. The area of tartrate-resistant acid phosphatase (TRAP) positive cells in bone tissue (A) and TRACP5b level in serum (B). Data are given as boxplot diagrams medians (lines in the boxes), 25 and 75% quartiles (boxes) and minimum and maximum ranges (whiskers).
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Fig. 7. Immunochemical staining shows the osteoprotegerin (OPG) and receptor activator of nuclear factor (NF)-kB ligand (RANKL) expression in bone tissues in control and cadmium (Cd) treated rats with or without emodin. Tomczyk, M., 2015. Protective effect of Aronia melanocarpa polyphenols against cadmium-induced disorders in bone metabolism: a study in a rat model of lifetime human exposure to this heavy metal. Chem. Biol. Interact. 229, 132–146. Brzóska, M.M., Rogalska, J., Roszczenko, A., Galazyn-Sidorczuk, M., Tomczyk, M., 2016. The mechanism of the osteoprotective action of a polyphenol-rich aronia melanocarpa extract during chronic exposure to cadmium is mediated by the oxidative defense system. Planta Med. 82, 621–631. Chen, X., Zhu, G., Gu, S., Jin, T., Shao, C., 2009. Effects of cadmium on osteoblasts and osteoclasts in vitro. Environ. Toxicol. Pharmacol. 28, 232–236. Chen, X., Wang, G., Li, X., Gan, C., Zhu, G., Jin, T., Wang, Z., 2013. Environmental level of cadmium exposure stimulates osteoclasts formation in male rats. Food Chem. Toxicol. 60, 530–535. Hwang, J.K., Noh, E.M., Moon, S.J., Kim, J.M., Kwon, K.B., Park, B.H., You, Y.O., Hwang, B.M., Kim, J.Y., Cheon, Y.H., Kwak, S.C., Baek, J.M., Yoon, K.H., Lee, M.S., Oh, J., 2014. Emodin regulates bone remodeling by inhibiting osteoclastogenesis and stimulating osteoblast formation. J. Bone. Miner. Res. 29, 1541–1553. Iwanowycz, S., Wang, J., Hodge, J., Wang, Y., Yu, F., Fan, D., 2016. Emodin inhibits breast cancer growth by blocking the tumor-promoting feedforward loop between cancer cells and macrophages. Mol. Cancer. Ther. 15, 1931–1942. Järup, L., Akesson, A., 2009. Current status of cadmium as an environmental health problem. Toxicol. Appl. Pharmacol. 238, 201–208. Kara, H., Cevik, A., Konar, V., Dayangac, A., Servi, K., 2008. Effects of selenium with vitamin E and melatonin on cadmium-induced oxidative damage in rat liver and kidneys. Biol. Trace. Elem. Res. 125, 236–244. Karabulut-Bulan, O., Bolkent, S., Yanardag, R., Bilgin-Sokmen, B., 2008. The role of vitamin C, vitamin E, and selenium on cadmium-induced renal toxicity of rats. Drug Chem. Toxicol. 31, 413–426. Kim, H.J., Kim, B.S., Lee, S.J., Kim, J.S., Lee, Y.R., 2013. Emodin suppresses inflammatory responses and joint destruction in collagen-induced arthritic mice. Rheumatology (Oxford) 52, 1583–1591. Kim, M.Y., Shon, W.J., Park, M.N., Lee, Y.S., Shin, D.M., 2016. Protective effect of dietary chitosan on cadmium accumulation in rats. Nutr. Res. Pract. 10, 19–25. Lee, S.U., Shin, H.K., Min, Y.K., Kim, S.H., 2008. Emodin accelerates osteoblast differentiation through phosphatidylinositol 3-kinase activation and bone morphogenetic protein-2 gene expression. Int. Immunopharmacol. 8, 741–747. Song, C., Liu, B., Xie, J., Ge, X., Zhao, Z., Zhang, Y., Zhang, H., Ren, M., Zhou, Q., Miao, L., Xu, P., Lin, Y., 2017. Comparative proteomic analysis of liver antioxidant mechanisms in Megalobrama amblycephala stimulated with dietary emodin. Sci. Rep. 7, 40356. Tang, L., Chen, X., Bao, Y., Xu, W., Lv, Y., Wang, Z., Wen, X., 2016. CT imaging biomarkers of bone damage induced by environmental level of cadmium exposure in male rats. Biol. Trace. Elem. Res. 170, 146–151. Tomaszewska, E., Dobrowolski, P., Winiarska-Mieczan, A., Kwiecień, M., Tomczyk, A., Muszyńsk, I.S., Radzki, R., 2016. Alteration in bone geometric and mechanical properties, histomorphometrical parameters of trabecular bone, articular cartilage, and growth plate in adolescent rats after chronic co-exposure tocadmium and lead in the case of supplementation with green, black, red and white tea. Environ. Toxicol. Pharmacol. 46, 36–44. Tomaszewska, E., Dobrowolski, P., Winiarska-Mieczan, A., Kwiecień, M., Tomczyk, A., Muszyński, S., 2017. The effect of tannic acid on the bone tissue of adult male Wistar rats exposed to cadmium and lead. Exp. Toxicol. Pathol. 69, 131–141. WHO/IPCS, 1992. Environmental Health Criteria Document 134 Cadmium. Geneva, WHO. Waalkes, M.P., 2003. Cadmium carcinogenesis. Mutat. Res. 533, 107–120. Wallin, M., Barregard, L., Sallsten, G., Lundh, T., Karlsson, M.K., Lorentzon, M., Ohlsson,
alone on bone was not investigated because the previous study has confirmed the role of emodin on bone. Second, we only observed the effects of emodin on Cd-induced osteoclast formation. The role of emodin on Cd-related inhibition in bone formation needs further exploration. Finally, single level of Cd and emodin were adopted in our study. In conclusion, our data showed that emodin can protect against Cdinduced bone loss by inhibiting osteoclast formation. Furthermore, our data indicated that the role of emodin on osteoclast may be related with its effects on RANKL and OPG. These results suggest that emodin could be explored as a potential therapeutic agent against Cd-induced osteoporosis. Declaration of interest The authors report no conflicts of interest. Acknowledgement This study was funded by Natural Science Foundation of Jiangsu Province (no. BK20161609). References Åkesson, A., Bjellerup, P., Lundh, T., Lidfeldt, J., Nerbrand, C., Samsioe, G., Skerfving, S., Vahter, M., 2006. Cadmium induced effects on bone in a population-based study of women. Environ. Health. Perspect. 114, 830–834. Abu, Eid. S., Adams, M., Scherer, T., Torres-Gómez, H., Hackl, M.T., Kaplanian, M., Riedl, R., Luge, r A., Fürnsinn, C., 2017. Emodin, a compound with putative antidiabetic potential, deteriorates glucose tolerance in rodents. Eur. J. Pharmacol. 798, 77–84. Berglund, M., Akesson, A., Bjellerup, P., Vahter, M., 2000. Metal-bone interactions. Toxicol. Lett. 112–113, 219–225. Boyce, B.F., Xing, L., 2008. Functions of RANKL/RANK/OPG in bone modeling and remodeling. Arch. Biochem. Biophys. 473, 139–146. Brama, M., Politi, L., Santini, P., Migliaccio, S., Scandurra, R., 2012. Cadmium-induced apoptosis and necrosis in human osteoblasts: role of caspases and mitogen-activated protein kinases pathways. J. Endocrinol. Invest. 35, 198–208. Brzóska, M.M., Moniuszko-Jakoniuk, J., 2005. Bone metabolism of male rats chronically exposed to cadmium. Toxicol. Appl. Pharmacol. 207, 195–211. Brzóska, M.M., Rogalska, J., 2013. Protective effect of zinc supplementation against cadmium-induced oxidative stress and the RANK/RANKL/OPG system imbalance in the bone tissue of rats. Toxicol. Appl. Pharmacol. 272, 208–220. Brzóska, M.M., Rogalska, J., Kupraszewicz, E., 2011. The involvement of oxidative stress in the mechanisms of damaging cadmium action in bone tissue: a study in a rat model of moderate and relatively high human exposure. Toxicol. Appl. Pharmacol. 250, 327–335. Brzóska, M.M., Rogalska, J., Galazyn-Sidorczuk, M., Jurczuk, M., Roszczenko, A.,
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