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Drynaria fortunei J. Sm. improves the bone mass of ovariectomized rats through osteocalcin-involved endochondral ossification
Journal of Ethnopharmacology 158 (2014) 94–101
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Drynaria fortunei J. Sm. improves the bone mass of ovariectomized rats through osteocalcin-involved endochondral ossification Yong-Eng Lee a,b, Hwa-Chang Liu c, Yi-Ling Lin b, Shing-Hwa Liu d, Rong-Sen Yang c, Ruei-Ming Chen b,e,f,n a
Department of Orthopedic Surgery, Shin Kong Wu Ho-Su Memorial Hospital, Taipei, Taiwan Cell Physiology and Molecular Image Research Center, Taipei Medical University's Wan-Fang Hospital, Taipei, Taiwan c Department of Orthopedic Surgery, National Taiwan University Hospital, Taipei, Taiwan d Institute of Toxicology, College of Medicine, National Taiwan University, Taipei, Taiwan e Graduate Institute of Medical Sciences, Taipei Medical University, Taipei, Taiwan f Anesthetics Toxicology Research Center, Taipei Medical University Hospital, Taipei, Taiwan b
art ic l e i nf o
a b s t r a c t
Article history: Received 1 May 2014 Received in revised form 1 September 2014 Accepted 13 October 2014 Available online 23 October 2014
Aim of this study: Our previous study showed that Drynaria fortunei J. Sm. (Kunze), a traditional Chinese medical herb, can promote osteoblast differentiation and maturation. This study was further aimed to confirm the traditional effects of Kunze on the bone mass of ovariectomized rats. Materials and methods: Female Wistar rats were given an ovariectomy and then administered the water extract of Kunze (WEK). Systemic and tissue toxicities of WEK were assessed. A biomechanical test, bone mineral contents, and bone histomorphometry were analyzed to determine the effects of the WEK on the bone mass. Levels of osteocalcin (OCN) in bone tissues were determined by immunohistochemistry and immunoblotting. The effects of naringin, one of the bioactive compounds of the WEK, on the bone mass were evaluated. Results: A bilateral ovariectomy in rats caused a time-dependent decrease in levels of serum 17βestradiol. Exposure of ovariectomized rats to the WEK at 0.5 and 1 g/kg body weight/day for 1, 2, 3, and 6 months did not induce systemic or tissue toxicities. Biomechanical testing and a bone mineral content analysis showed that the ovariectomy decreased the bone torsion force and bone ash in time-dependent manners. In comparison, after exposure to the WEK, the ovariectomy-induced reductions in the bone torsion force and bone ash were significantly alleviated. In parallel, results of a bone histomorphometric assay further revealed that the ovariectomy caused significant diminution in the production of prehypertrophic chondrocytes and trabecular bone but enhanced hypertrophic chondrocyte numbers in the growth plate. However, exposure to the WEK lowered ovariectomy-induced changes in these cellular events. As to the mechanism, the WEK increased OCN biosynthesis in bone tissues of ovariectomized rats. Administration of naringin to ovariectomized rats caused significant amelioration of the bone strength, bone mineral contents, and trabecular bone amounts. Conclusions: This study shows that the WEK can translationally promote the bone mass in ovariectomized rats through stimulating OCN-involved endochondral ossification. & 2014 Elsevier Ireland Ltd. All rights reserved.
Keywords: Drynaria fortunei J. Sm. Bone mass Ovariectomy Osteocalcin Endochondral ossification
1. Introduction Osteoporosis, a progressive bone disease, is characterized by a decrease in the bone mass and density which can induce bones to
Abbreviations: ALT, aminotransferase; AST, aspartate aminotransferase; BUN, bilirubin; CRE, creatinine; LDH, lactate dehydrogenase; OCN, osteocalcin; OPN, osteopontin; UA, uric acid; WEK, water extracts of Kunze n Corresponding author at: Graduate Institute of Medical Sciences, College of Medicine, Taipei Medical University, 250 Wu-Xing St., Taipei, 110, Taipei, Taiwan. Tel.: þ 886 2 27361661x3222; fax: þ886 2 86621119. E-mail address: [email protected] (R.-M. Chen). http://dx.doi.org/10.1016/j.jep.2014.10.016 0378-8741/& 2014 Elsevier Ireland Ltd. All rights reserved.
weaken and be more likely to break (Adams, 2012). In the clinic, osteoporosis is also called as a silent disease because it proceeds without pain or symptoms until bone fracture occurs (Bradbury et al., 2012). Bone fractures typically linked to osteoporosis are very dangerous and can cause patients to suffer permanent disabilities or even death (Edwards et al., 2012). A variety of risk factors contribute to the causes of osteoporosis, including low levels of sex hormones, inadequate calcium intake, heredity, chronic diseases, lifestyle habits, medication use, and aging (Rubin et al., 2013). Among these, sex hormones are reported to play the most crucial roles in regulating bone remodeling and maintaining bone mass. However, post-menopausal hormone
Y.-E. Lee et al. / Journal of Ethnopharmacology 158 (2014) 94–101
therapy for osteoporosis may increase the risk of coronary heart disease, stroke, and breast cancer (Rozenberg et al., 2013). Hence, discovering hormone analogs such as phytoestrogen would be helpful in developing de novo therapeutic strategies for osteoporosis. The bone structure is maintained through a dynamic balance between osteoblast-mediated bone formation and osteoclastmediated bone resorption (Seeman and Delmas, 2006). In skeletal development and bone healing, bone is formed through two essential processes, namely endochondral ossification and intramembranous ossification (Shore and Kaplan, 2010). In endochondral ossification, bone is formed with hyaline cartilage as the model (Hojo et al., 2010). In comparison, cartilage is not present in the course of intramembranous ossification (Shore and Kaplan, 2010). Miscellaneous stages are involved in endochondral ossification, including collar formation, cavity formation, vascular invasion, elongation, and epiphyseal ossification (Hojo et al., 2010). During the process of endochondral ossification, chondrocytes differentiated from stem cells first proliferate and then differentiate into hypertrophic chondrocytes (Mackie et al., 2011). Lastly, hypertrophic chondrocytes are gradually replaced by bone, leading to an increase in trabecular bone. As a result, osteoblasts participate in endochondral ossification by colonizing regions of the cartilage (Aubin, 1998; Medici and Olsen, 2012). A complicated network of differentiation proteins is involved in regulating osteoblast-mediated osteogenesis (Stein et al., 1996; Vandenput and Ohlsson, 2009). The process of endochondral bone development needs to be evaluated to discover innovative biomaterials that can prevent or treat osteoporosis. Drynaria fortunei J. Sm. (Korean name; Kunze) is a variety of the traditional Chinese herb, Gusuibu, which is frequently used by Chinese people to prevent or treat bone-related diseases. Long et al. (2005) reported that the flavonoid fractions of Kunze can prevent nephrotoxicity and promote regeneration of kidney primary epithelial tubular cells. Previous studies further showed that the components of Kunze can stimulate proliferation of osteoblast-like UMR106 cells and induce bone formation in mice (Wong and Rabie, 2006; Wang et al., 2008). Our previous study showed that the water extract of Kunze (WEK) can protect rat calvarial osteoblasts from hydrogen peroxide-induced insults (Liu et al., 2001). Recently, we further showed the effects of the WEK on promoting osteoblast maturation by inducing differentiation-related gene expression and protecting against oxidative/nitrosative stress-induced apoptotic insults (Huang et al., 2010; Hsu et al., 2011). Osteocalcin (OCN) is an early osteoblast biosignature that participates in controlling osteoblast function and bone extracellular matrix mineralization (van Leeuwen et al., 2001). A previous study reported that 11 flavonoids were extracted from Kunze (Wang et al., 2008). In our lab, we demonstrated that naringin, one of these flavonoids, can induce OCN expression during osteoblast differentiation (Huang et al., 2010). Thus, this study was further designed to corroborate the translational effects of Kunze and naringin on improving bone mass using ovariectomized rats as our experimental model.
95
2.2. Animals Female Wistar rats (200–250 g) were purchased from the Animal Center of the College of Medicine, National Taiwan University (Taipei, Taiwan). Before the experiments began, animals were allowed to acclimatize for 1 week in their animal quarters with air conditioning and an automatically controlled photoperiod of 12 h of light daily. All experimental procedures were performed according to the National Institutes of Health Guidelines for the Use of Laboratory Animals and approved by the Institutional Animal Care and Use Committee of Taipei Medical University (Taipei, Taiwan). Rats were randomly divided into 4 groups: a sham control, an ovariectomy, an ovariectomy with the WEK at 0.5 g/kg body weight, and an ovariectomy with the WEK at 1 g/kg body weight. Animals were allowed free access to rodent laboratory chow (Purina Mills, St. Louis, MO, USA). 2.3. Surgical procedures and drug treatment Rats were anesthetized using ketamine (100 mg/kg) and xylazine hydrochloride (10 mg/kg) intramuscularly. A bilateral ovariectomy was conducted following a previous method (Brennan et al., 2009). A mid-ventral incision was made, and the bilateral ovaries and ovarian fat were removed. The ovaries were isolated by ligation of the most proximal portion of the oviduct before removal. In the sham-operated group, animals were subjected to the same procedure, but the ovaries were not removed. Animals were kept warm during the procedure and recovery. After surgery, rats were administered different doses of WEK by oral gavage for various time intervals. 2.4. Measurements of serum clinical parameters Systemic toxicity of the WEK was assayed by measuring serum clinical parameters as described previously (Chen et al., 1998). Briefly, animals were sacrificed after drug treatment, and blood samples were collected for assessment of serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), lactate dehydrogenase (LDH), bilirubin (BUN), creatinine (CRE), and uric acid (UA). 2.5. Measurement of serum 17β-estradiol After drug treatment, rats were sacrificed, and blood samples were collected. Serum fractions were prepared for analysis of 17β-estradiol. Levels of serum 17β-estradiol were assayed following the instructions of the Elecsys-Estradiol II enzyme-linked immunosorbent assay (Roche Diagnostics, Mannheim, Germany). 2.6. Hematoxylin and eosin (HE) staining
2. Materials and methods
After drug treatment, animals were sacrificed. Livers and kidneys were removed and collected. Samples were fixed using phosphatebuffered 4% paraformaldehyde and processed for routine paraffin embedding. All tissue samples were sliced into 5-μm transverse sections and stained with HE. Specimens were observed and photographed using a light microscope.
2.1. Preparation of the WEK
2.7. Biomechanical testing
Kunze (D. fortunei) was kindly provided by the Brion Research Institute, Sun Ten Group (Taipei, Taiwan). The herb was identified by institutional experts, and its chemical and physical characteristics were routinely analyzed (Huang et al., 2010). The WEK was prepared as previously described (Liu et al., 2001). The extract was stored at room temperature and protected from light and moisture as described previously (Hsu et al., 2011). Naringin was purchased from Sigma (St. Louis, MO, USA).
After drug treatment, animals were sacrificed, and femur tissues were removed and collected. Following collection of muscle and connective tissues, the femurs were examined by peripheral quantitative computed tomography using a standardized cantileverbending technique as described previously (Wang et al., 2000). Briefly, distal condyles of the femurs were loaded in the anteroposterior direction at a constant rate of 1.0 mm/min until failure in a universal testing device (Avalon Technologies, Rochester, MI, USA).
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The force-displacement curves recorded were used to measure the ultimate bending force to failure (N) and bending stiffness (N/mm).
3. Results 3.1. An ovariectomy decreased levels of serum estradiol
After drug treatment, rats were sacrificed. The right tibias of rats were collected, and the muscle and connective tissues were removed. Then, the tibias were dehydrated at 100 1C for 14 h and weighed. The dehydrated bones were then calcinated at 600 1C for another 16 h. Dry bone ashes were weighed. The ratio of dry bone ash to the dehydrated tibia was calculated.
2.9. Bone histomorphometry Proximal tibias were dehydrated in a graded series of ethanol and xylene and embedded undecalcified in modified methyl methacrylate following a previously described method (Iwaniec et al., 2008). Specimens were longitudinally cut into 5-μm-thick sections. Specimens were stained for assessment of bone area and cell-based measurements. The derived architectural index of trabecular bone number (mm-1) in a standard sampling site 0.5 mm distal to the growth plate was measured using the OsteoMeasure System (OsteoMetrics, Atlanta, GA, USA).
Fifteen days after Wistar rats had undergone an ovariectomy, a significant 40% decrease in levels of serum 17β-estradiol was detected (Fig. 1). One, 2, 3, and 6 months after the ovariectomy, amounts of serum 17β-estradiol were significantly diminished by 48%, 69%, 73%, and 82%, respectively (Fig. 1). 3.2. Effects of the WEK on the body weight, liver weight, and kidney weight One, 2, 3, and 6 months after the ovariectomy, Wistar rats exhibited significant increases in body weight (Table 1). Compared to ovariectomy-treated groups, the WEK at a low dose (0.5 g/kg body weight/day) and a high dose (1 g/kg body weight/day) did not change the body weights of experimental animals. Treatment of animals with an ovariectomy and the WEK at low and high doses for 1, 2, 3, and 6 months did not affect the liver weight of rats 200
150 Estradiol, pmol/L
2.8. Quantification of bone mineral contents
2.10. Immunohistochemical (IHC) analysis of OCN IHC was carried out as described previously (Chang et al., 2014). After drug treatment, animals were sacrificed, and the tibias were removed, collected, and sliced. Bone tissues were fixed with a fixing reagent (acetone: methanol, 1: 1) at 20 1C for 10 min and incubated with 0.2% Triton X-100 at room temperature for 15 min. Immunodetection of OCN in bone tissues was carried out using a polyclonal antibody against rat OCN (Santa Cruz Biotechnology, Santa Cruz, CA, USA) by incubation at 4 1C overnight. After washing, slices were reacted with the second antibody at room temperature for 1 h. Staining was visualized with 3,30 ,3taminobenzidine. Specimens were observed and photographed using a light microscope.
2.11. Immunoblot analysis of OCN After drug treatment, rats were sacrificed, and the femurs were removed and homogenized in lysis buffer (50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EGTA, 1% NP-40 supplemented with 1 mM PMSF, 1 μM aprotinin, 1 μM leupeptin, 1 mM Na2VO4, and 1 mM NaF). Protein concentrations were quantified using a bicinchonic acid protein assay kit (Pierce, Rockford, IL, USA). Proteins (50 μg/well) were subjected to sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE), and transferred to nitrocellulose membranes as described previously (Liao et al., 2014). Membranes were blocked with 5% nonfat milk at 37 1C for 1 h. OCN protein was immunodetected using a polyclonal antibody. β-Actin was immunodetected using a mouse monoclonal antibody against mouse β-actin (Sigma) as an internal control. Intensities of the immunoreactive protein bands were determined using an UVIDOCMW vers. 99.03 digital imaging system (UVtec, Cambridge, UK).
2.12. Statistical analyses The statistical significance of differences between each group was evaluated using a one-way analysis of variance (ANOVA) with the Duncan's multiple-range test. Differences were considered statistically significant at P values ofo0.05.
*
100
* *
50
0
0
15
*
30 60 Time, days
90
* 180
Fig. 1. Effects of an ovariectomy on changes in serum 17β-estradiol. Female Wistar rats were given a bilateral ovariectomy. Then 15, 30, 60, 90, and 180 days after surgery, animals were sacrificed, and blood samples were collected. Levels of serum 17β-estradiol were quantified using an ELISA. Each value represents the mean 7SEM for n¼ 9. n Indicates that the value significantly differed from the respective control, p o0.05. Table 1 Effects of the water extract of Kunze (WEK) on the body weight (BW), liver weight (LW), and kidney weight (KW) of ovariectomized (OVX) rats.
1 Month BW LW/BW KW/BW 2 Months BW LW/BW KW/BW 3 Months BW LW/BW KW/BW 6 Months BW LW/BW KW/BW
Control
OVX
WEK (0.5 g)
WEK (1.0 g)
230 711 3.9 70.3 0.8 70.1
263 7 13n 4.17 0.2 0.7 7 0.1
245 721 4.2 70.3 0.8 70.1
2317 22 3.3 7 0.7 0.7 7 0.1
247 712 4.2 70.1 0.7 70.1
289 7 25n 3.8 7 0.1 0.7 7 0.1
288 710n 3.6 70.1 0.6 70.1
293 7 14n 3.7 7 0.2 0.6 7 0.1
260 710 3.7 70.4 0.7 70.0
3177 20n 3.4 7 0.4 0.7 7 0.1
291 721n 3.2 70.4 0.6 70.1
3247 26n 3.17 0.2 0.6 7 0.1
266 79 3.6 70.3 0.7 70.0
309 7 28n 3.4 7 0.3 0.6 7 0.1
305 79n 3.1 70.3 0.7 70.1
328 7 11n 3.2 7 0.1 0.6 7 0.1
Female Wistar rats were anesthetized and given a bilateral ovariectomy (OVX). After surgery, rats were administered the water extract of Kunze (WEK) at 0.5 and 1 g/kg BW per day by oral gavage for 1, 2, 3, and 6 months. Animals were sacrificed and the BW, LW, and KW were measured. Each value represents the mean 7 SEM for n¼ 9. n Indicates that the value significantly differed from the respective control, po 0.05.
Y.-E. Lee et al. / Journal of Ethnopharmacology 158 (2014) 94–101
Table 2 Effects of the water extract of Kunze (WEK) on serum factors of ovariectomized (OVX) rats.
LDH, U/L AST, U/L ALT, U/L BUN, mg/dl CRE, mg/dl UA, mg/dl
Control
OVX
WEK(0.5 g)
WEK (1 g)
919 7102 121 711 39 77 19 72 0.6 70.1 1.7 70.3
885 7 126 1407 16 327 10 177 2 0.7 7 0.1 1.4 7 0.1
904 7240 146 726 48 716 18 71 0.6 70.1 1.2 70.2
832 7 129 1497 37 417 11 177 2 0.7 7 0.1 1.4 7 0.1
Female Wistar rats were anesthetized and given a bilateral ovariectomy (OVX). After surgery, rats were administered the WEK at 0.5 and 1 g/kg body weight per day by oral gavage for 6 months. Animals were sacrificed, and serum samples were collected for analyses of clinical parameters. Each value represents the mean 7SEM for n ¼9.
Control
OVX
3.3. Systemic and tissue toxicities of the WEK Six months after the ovariectomy, serum LDH of rats had not changed (Table 2). Separately, the ovariectomy did not affect serum indices of AST and ALT that indicate liver injury or those of BUN, CRE, and UA that indicate kidney injury. In comparison, exposure to low and high doses of the WEK did not change serum indices of LDH, AST, ALT, BUN, CRE, or UA (Table 2). Between the animals treated with a low and a high dose of WEK, there was no difference in these serum indices. Tissue toxicities of the WEK to the liver and kidney were confirmed using histological analyses (Fig. 2). Compared to control animals, 6 months after an ovariectomy, rats did not exhibit tissue damages to the liver or kidney (Fig. 2A and B). In ovariectomized rats, administration of the WEK at 0.5 and 1 g/kg body weight/day for 6 months did not induce hepatotoxicity or nephrotoxicity (Fig. 2A and B).
120
Bone torsion, % of control
(Table 1). In parallel, the kidney weight of animals subjected to and ovariectomy and various doses of the WEK for 1, 2, 3, and 6 months was not influenced (Table 1). There was no statistical difference in the body weight, liver weight, and kidney weight between the animals treated with WEK at 0.5 and 1 g /kg body weight/day.
97
100 80 60 40 20 0
Control
OVX
140
WEK-1.0
*
80 60 40
0
OVX
#
100
20
Control
Contr ol Control
OVX
140
WEK (0.5)
Bone torsion, % of control
WEK-1.0
100 80
WEK (1.0)
#
120
WEK-0.5
WEK (1.0)
#
120
Bone torsion, % of control
WEK -0.5
WEK (0.5)
#
GSB0.5
*
60 40 20 0
Fig. 2. Toxicities of the water extract of Kunze (WEK) to the liver and kidneys. Female Wistar rats were given a bilateral ovariectomy (OVX). After surgery, animals were treated by oral gavage with WEK at 0.5 and 1 g/kg body weight/day for 60 days. Following drug treatment, the animals were sacrificed, and the livers (A) and kidneys (B) were removed for hematoxylin and eosin staining analyses. Specimens were observed and photographed using a light microscope. 100 .
Control
OVX
WEK (0.5)
WEK (1.0)
Fig. 3. Effects of the water extract of Kunze (WEK) on the biomechanical strength of rat femurs. Female Wistar rats were given a bilateral ovariectomy (OVX). After surgery, animals were treated with the WEK at 0.5 and 1 g/kg body weight/day by oral gavage for 1 (A), 3 (B), and 6 (C) months. Bone torsion was assayed using a standardized cantilever-bending technique. Each value represents the mean 7 SEM for n ¼6. n and # Indicate that values significantly (po 0.05) differed from the control and OVX-treated groups, respectively.
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3.4. The WEK improves the biomechanical strength of ovariectomized rat femurs Biomechanical assays revealed that an ovariectomy and administration of the WEK for 1 month did not affect the bone torsion force of rat femurs (Fig. 3A). However, 3 and 6 months after the ovariectomy, the torsion force of rat femurs had significantly decreased by 24% and 28%, respectively (Fig. 3B and C). Interestingly, administration of 0.5 and 1 g of the WEK/kg body weight per day for 3 and 6 months caused significant alleviation of the ovariectomy-induced reduction in femur strength (Fig. 3B and C). However, our data indicated that there was no difference in the bone torsion force between the animals treated with various doses of WEK.
3.5. The WEK increases bone mineral contents of ovariectomized rats One month after the ovariectomy, the bone ash weights of rat tibias were significantly reduced by 12% (Fig. 4A). When exposed to the WEK at 0.5 and 1 g/kg body weight/day for 1 month, the ovariectomyinduced reduction in the bone mineral content was alleviated (Fig. 4A). The bone ash weights of rat tibias had decreased by 15% and 25% at 3 and 6 months after the ovariectomy, respectively (Fig. 4B and C). In comparison, the WEK at both low and high doses lessened the ovariectomy-induced loss of bone mineral contents. In the 3-monthadministered time interval, the bone ash weights were less in a high dose of WEK-treated rats than those in a low dose of WEK-treated animals (Fig. 4B). However, there was no significant difference in the bone ash weights between the animals treated with these two doses of WEK for 1 and 6 months (Fig. 4A–C). 3.6. The WEK convalesces ovariectomy-induced alterations in bone histomorphometry
120
# #
Bone ash, % of control
100
*
80
Six months after the ovariectomy, the trabecular bone index and numbers of prehypertrophic chondrocytes had decreased, but hypertrophic chondrocytes in the growth plate had increased (Fig. 5A). The WEK at a low dose (0.5 g/kg body weight) enhanced proliferating
60 40
Control
OVX
WEK-0.5
WEK-1.0
20 0
Control
OVX
WEK (0.5)
#
120
#†
100
*
80 60 40 20 0
Contr ol Control
OVX
WEK (0.5)
#
120
WEK (1.0)
#
Bone ash, % of control
100 80
GSB0.5
*
60 40
6 Trabe ecular bone, numb ber/mm
Bone ash, % of control
WEK (1.0)
#
#
4
* 2
20
0 0
Control
OVX
WEK (0.5)
WEK (1.0)
Fig. 4. Effects of the water extract of Kunze (WEK) on bone mineral contents of rat tibias. Female Wistar rats were given a bilateral ovariectomy (OVX). After surgery, animals were treated with the WEK at 0.5 and 1 g/kg body weight/day by oral gavage for 1 (A), 3 (B), and 6 (C) months. Changes in bone mineral content were analyzed by measuring the ratio of dry bone ash over the dehydrated tibia. Each value represents the mean 7SEM for n¼ 6. n, #, and † Indicate that the values significantly (p o 0.05) differed from the control, OVX-, and WEK (0.5 g/kg body weight/day)-treated groups, respectively.
Control
OVX
WEK (0.5)
WEK (1.0)
Fig. 5. Effects of the water extract of Kunze (WEK) on bone histomorphometry of rat tibias. Female Wistar rats were given a bilateral ovariectomy (OVX). After surgery, animals were treated with the WEK at 0.5 and 1 g/kg body weight/day by oral gavage for 6 months. Proximal tibias were removed for assessment of bone area and cell-based measurements: a, trabecular bone; b, hypertrophic chondrocytes; c, prehypertrophic chondrocytes; d, proliferating chondrocytes (A). The index of trabecular bone was measured using the OsteoMeasure System (B). Each value represents the mean 7SEM for n¼ 6. n and # Indicate that values significantly (p o 0.05) differed from the control and OVX-treated groups, respectively.
Y.-E. Lee et al. / Journal of Ethnopharmacology 158 (2014) 94–101
99
chondrocytes but reduced the numbers of hypertrophic chondrocytes. In comparison, administration of the WEK at a high dose (1 g/kg body weight/day) for 6 months to ovariectomized rats significantly diminished the number of hypertrophic chondrocytes (Fig. 5A). The WEK at a high dose also enlarged the trabecular bone thickness and connections (Fig. 5A). An index of trabecular bone was quantified and analyzed (Fig. 5B). An ovariectomy caused a significant 52% decrease in the trabecular bone index. After exposure to the WEK at 0.5 and 1 g/kg body weight for 6 months, the ovariectomy-induced drop in the trabecular bone index was eased by 61% and 74%, respectively (Fig. 5B). Between the animals treated with WEK at 0.5 and 1 g/kg body weight/day, there was no statistical difference in the trabecular bone index.
weight/day for 6 months, OCN expression was obviously enhanced. An immunoblot analysis further showed that the WEK augmented levels of OCN in the bone of ovariectomized rats (Fig. 6B, top panel, lanes 3 and 4). Amounts of β-actin were immunodetected as an internal control (bottom panel). The immunoreacted protein bands were quantified and analyzed (Fig. 6C). Administration of the WEK at 0.5 and 1 g/kg body weight/day to ovariectomized rats raised levels of OCN in the bone by 80% and 91%, respectively. However, there was no significant difference in the OCN levels between the animals treated with various doses of WEK.
3.7. The WEK increases OCN biosynthesis in ovariectomized rat bone
The effects of naringin, an active compound of WEK, on the bone mass of ovariectomized rats were further analyzed (Table 3). Exposure of ovariectomized rats to naringin alone did not change the bone torsion, bone ash, or trabecular bone index. However, an ovariectomy caused 41%, 19%, and 50% decreases in the bone torsion, bone ash, and trabecular bone index, respectively (Table 3). After exposure to naringin for 6 months, the ovariectomy-induced alterations in bone torsion, bone ash, and trabecular bone index were significantly ameliorated (Table 3).
Effects of the WEK on OCN expression in the bone of ovariectomized rats was analyzed using IHC and immunoblotting (Fig. 6). Subjecting rats to an ovariectomy slightly decreased OCN levels in femurs (Fig. 6A). After exposure to 0.5 and 1 g of the WEK/body
Control
OVX
3.8. Naringin increased the bone strength, bone mineral contents, and trabecular bone index in ovariectomized rats
4. Discussion
WEK-0.5
1
WEK-1.0
2
3
4 OCN
β C
OVX
WEK-0.5
WEK-1
OC CN, arbitrary unit x 100
120
*
90
*
This study proves the translational effects of Kunze on improving the bone mass of ovariectomized rats. An ovariectomy is a useful model which can accelerate osteoporosis in experimental animals (Brennan et al., 2009). In the present study, levels of serum 17β-estradiol time-dependently dropped after the ovariectomy. In parallel, the ovariectomy altered the bone strength, bone mineral contents, and bone microarchitecture. Hence, subjecting female rats to a bilateral ovariectomy successfully decreased serum estrogen synthesis and consequently induced osteoporosis in this study. Our results demonstrated enhancement of the bone mass in ovariectomized rats after exposure to Kunze. A deficiency in the sex hormones usually leads to an imbalance of bone remodeling, leading to loss of bone mass (Rozenberg et al., 2013). Thus, post-menopausal hormone therapy was developed to treat osteoporotic patients. However, hormone replacement therapy may increase the risks of breast cancer and other diseases (Rozenberg et al., 2013). Phytoestrogens, a class of bioactive compounds exerting various estrogenic effects, were shown to increase bone formation and repress bone resorption (Ming et al., 2013). There are more than 11 flavonoids found in the WEK (Wang et al., 2008). The data presented in this study reveal that Table 3 Effects of naringin on the biomechanical strength, bone mineral contents, and trabecular bone index of ovariectomized (OVX) rat femurs.
60
Naringin, mg/kg
Bone torsion (% of control)
Bone ash (% of control)
Trabecular bone (number/mm)
Control OVX Naringin NaringinþOVX
100 597 10n 1017 13 877 10#
100 817 4n 987 6 947 6#
67 1 37 1n 77 2 57 1#
30
0
Control
OVX
WEK (0.5)
WEK (1.0)
Fig. 6. Effects of the water extract of Kunze (WEK) on osteocalcin OCN biosynthesis. Female Wistar rats were given a bilateral ovariectomy (OVX). After surgery, animals were treated with the WEK at 0.5 and 1 g/kg body weight/day by oral gavage for 6 months. Immunohistochemical analysis of OCN in bone tissues was carried out (A). Levels of OCN were further determined using an immunoblot analysis (B, top panel). Amounts of β-actin were immunodetected as the internal control (bottom panel). The immunoreactive protein bands were quantified and statistically analyzed (C). Each value represents the mean 7 SEM for n¼6. n and # Indicate that values significantly (p o 0.05) differed from the control and OVXtreated groups, respectively.
Female Wistar rats were anesthetized and given a bilateral ovariectomy (OVX). After surgery, rats were administered the water extract of Kunze (WEK) at 0.5 and 1 g/kg body weight per day by oral gavage for 6 months. Animals were sacrificed, and bone samples were collected for analyses of bone torsion, bone ash, and the trabecular bone index. Each value represents the mean7 SEM for n¼ 6. n Indicate that values significantly differed from the respective control and OVX-treated groups, po 0.05. # Indicate that values significantly differed from the respective control and OVX-treated groups, po 0.05.
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the WEK did not induce systemic or tissue toxicities to animals. Therefore, Kunze may have therapeutic potential at maintaining bone health in the clinic. Kunze may promote the bone mass of ovariectomized rats by alleviating osteoblast insults. Oxidative stress is directly linked to estrogen decline and osteoporosis occurrence (Manolagas, 2010). Osteoblasts also mediate bone formation in bone remodeling but are easily insulted by oxidative stress (Ducy et al., 2000; Chen et al., 2010). Our previous studies reported that Kunze could protect osteoblasts from oxidative- and nitrosative stress-induced apoptotic injury (Liu et al., 2001; Huang et al., 2010). Moreover, those studies further validated the effects of Kunze at improving osteoblast differentiation and maturation through inducing certain osteoblast differentiation-related genes, including insulin-like growth factor-1, matrix maturation-related bone morphogenetic protein (BMP)-2 and BMP-6, alkaline phosphatase, ostepontin, and OCN (Huang et al., 2010; Hsu et al., 2011). In this study, we provide supplementary in vivo data to illustrate the effects of Kunze on the bone mass of the ovariectomized rats. Therefore, two possible reasons explaining the Kunze-induced promotion of bone mass in ovariectomized rats are that it protects osteoblasts against oxidative stress-triggered apoptosis while promoting osteoblast maturation. Kunze expands the bone mass of ovariectomized rats possibly via endochondral ossification. Endochondral ossification is a process that uses hyaline cartilage as a model to form new bone (Hojo et al., 2010). Chondrocytes were reported to play an important role in the progression of endochondral ossification. At first, chondrocytes multiply, and proliferated chondrocytes subsequently differentiate into hypertrophic chondrocytes (Mackie et al., 2011). This study found that administration of the WEK lessened ovariectomy-induced suppression of chondrocyte proliferation and construction of hypertrophic chondrocytes. When exposed to Kunze, the ovariectomy-induced reduction in the trabecular bone index also significantly recovered in ovariectomized rats. In the final stage of endochondral ossification, hypertrophic chondrocytes are replaced by osteoblasts to form new bone (Hojo et al., 2010; Mackie et al., 2011). As a result, the Kunze-induced promotion of bone mass in ovariectomized rats may occur mainly via an endochondral ossification pathway. In addition, bone fractures are a chief risk to patients suffering osteoporosis (Edwards et al., 2012). In bone fractures, endochondral ossification is reported to be a key process during bone healing (Shore and Kaplan, 2010). Accordingly, Kunze may be helpful in the clinical treatment of osteoporosis and osteoporosis-induced bone fractures. OCN contributes to Kunze-induced improvement of bone mass. Our previous study used neonatal rat calvarial osteoblasts as the experimental model to show the role of OCN in Kunze-triggered osteoblast differentiation and maturation (Huang et al., 2010). The present study further demonstrates that the WEK stimulated OCN biosynthesis in ovariectomized rats. OCN is an early biomarker for bone ECM mineralization and bone formation (van Leeuwen et al., 2001). A previous study showed that downregulation of OCN expression led to repression of osteoblast differentiation (Lin et al., 2010). Okamura et al. (2011) stated that upregulation of OCN expression simultaneously enhanced bone formation. When OCN synthesis is abrogated by silencing expression of the Cbfa1 gene, the endochondral ossification process is concurrently inhibited (Sun et al., 2012). Therefore, the Kunze-provoked alleviation of bone mass in ovariectomized rats may be via OCN-involved endochondral ossification. Kunze enhances bone strength and bone mineral contents in ovariectomized rats. The decline in the bone torsion force is positively correlated to bone loss (Beaupied et al., 2007). A previous study reported that icaritin, one of the epimedium-derived flavonoids, remarkably restored bone biomechanical properties in ovariectomized rats (Peng et al., 2013). This study further demonstrated that after exposure to the WEK, the ovariectomy-induced lessening in the bone torsion force was significantly alleviated. This finding indicates the
beneficial property of Kunze on the bone mass due to enhancement of bone strength. In parallel, the amount of bone ash in ovariectomized rats was simultaneously increased following administration of the WEK. In female Sprague–Dawley rats, an increase in bone mineral contents concurrently improved the bone biomechanical strength (Khan et al., 2013). Accordingly, the Kunze-induced augmentation of bone mineral contents contributes to the enrichment of bone strength. Based on the present results, Kunze can assuage ovariectomy-induced decreases in bone mineral contents and bone strength and subsequently improve the bone mass of ovariectomized rats. Naringin is one of the active components present in the WEK and can enhance bone development in ovariectomized rats. Previous studies showed that naringin is a component of the WEK (Wang et al., 2008; Wong et al., 2013). Our previous study reported the osteogenic effects of naringin on inducting OCN gene expression (Huang et al., 2010). The present study demonstrated the favorable translational effects of naringin on intensifying the bone mineral contents and bone potency in ovariectomized rats. In parallel, after exposure to naringin, the trabecular bone index in ovariectomized rats was also augmented. Such enhancement indicates the promoting effects of naringin of stimulating the formation of new bone. A previous study also showed that naringin can provoke osteogenesis by enlarging the proliferation and differentiation of bone marrow stromal cells (Li et al., 2013). Wong et al. (2013) reported that naringin possesses the potential to inhibit osteoclastogenesis. Therefore, naringin may be one of the active components in the WEK that overcomes ovariectomy-induced bone loss. In summary, this study has shown that the WEK did not induce systemic or tissue toxicity in rats. Meanwhile, Kunze alleviated ovariectomy-induced reductions in bone mechanics and bone mineral contents. Analyses of bone histomorphometry further showed that ovariectomy-triggered alterations in proliferating chondrocytes, hypertrophic chondrocytes, and trabecular bone significantly recovered following Kunze administration. In parallel, Kunze increased OCN synthesis in bone tissues of ovariectomized rats. Naringin, one of the active components found in the WEK, lessened ovariectomy-induced decreases in bone torsion, bone ash, and the trabecular bone index. Therefore, this study has translationally confirmed that Kunze can promote the bone mass of ovariectomized rats through an OCNinvolved endochondral ossification mechanism. Our in vitro and in vivo data suggest the potential application of Kunze for osteoporosis therapy. There are certain study limitations in this study. For example, there was no significant difference in most of our assays between the animals treated with various doses of WEK. However, the present results cannot explain why in the 3-month-administered time interval, the bone ash weights of a high dose of WEK-treated rats were less than those of a low dose of WEK-treated animals. To confirm and elucidate such a difference, more experiments, including quantification of bone calcium, magnesium, and phosphate, will be conducted in our upcoming mechanism study.
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Journal of Ethnopharmacology journal homepage: www.elsevier.com/locate/jep
Drynaria fortunei J. Sm. improves the bone mass of ovariectomized rats through osteocalcin-involved endochondral ossification Yong-Eng Lee a,b, Hwa-Chang Liu c, Yi-Ling Lin b, Shing-Hwa Liu d, Rong-Sen Yang c, Ruei-Ming Chen b,e,f,n a
Department of Orthopedic Surgery, Shin Kong Wu Ho-Su Memorial Hospital, Taipei, Taiwan Cell Physiology and Molecular Image Research Center, Taipei Medical University's Wan-Fang Hospital, Taipei, Taiwan c Department of Orthopedic Surgery, National Taiwan University Hospital, Taipei, Taiwan d Institute of Toxicology, College of Medicine, National Taiwan University, Taipei, Taiwan e Graduate Institute of Medical Sciences, Taipei Medical University, Taipei, Taiwan f Anesthetics Toxicology Research Center, Taipei Medical University Hospital, Taipei, Taiwan b
art ic l e i nf o
a b s t r a c t
Article history: Received 1 May 2014 Received in revised form 1 September 2014 Accepted 13 October 2014 Available online 23 October 2014
Aim of this study: Our previous study showed that Drynaria fortunei J. Sm. (Kunze), a traditional Chinese medical herb, can promote osteoblast differentiation and maturation. This study was further aimed to confirm the traditional effects of Kunze on the bone mass of ovariectomized rats. Materials and methods: Female Wistar rats were given an ovariectomy and then administered the water extract of Kunze (WEK). Systemic and tissue toxicities of WEK were assessed. A biomechanical test, bone mineral contents, and bone histomorphometry were analyzed to determine the effects of the WEK on the bone mass. Levels of osteocalcin (OCN) in bone tissues were determined by immunohistochemistry and immunoblotting. The effects of naringin, one of the bioactive compounds of the WEK, on the bone mass were evaluated. Results: A bilateral ovariectomy in rats caused a time-dependent decrease in levels of serum 17βestradiol. Exposure of ovariectomized rats to the WEK at 0.5 and 1 g/kg body weight/day for 1, 2, 3, and 6 months did not induce systemic or tissue toxicities. Biomechanical testing and a bone mineral content analysis showed that the ovariectomy decreased the bone torsion force and bone ash in time-dependent manners. In comparison, after exposure to the WEK, the ovariectomy-induced reductions in the bone torsion force and bone ash were significantly alleviated. In parallel, results of a bone histomorphometric assay further revealed that the ovariectomy caused significant diminution in the production of prehypertrophic chondrocytes and trabecular bone but enhanced hypertrophic chondrocyte numbers in the growth plate. However, exposure to the WEK lowered ovariectomy-induced changes in these cellular events. As to the mechanism, the WEK increased OCN biosynthesis in bone tissues of ovariectomized rats. Administration of naringin to ovariectomized rats caused significant amelioration of the bone strength, bone mineral contents, and trabecular bone amounts. Conclusions: This study shows that the WEK can translationally promote the bone mass in ovariectomized rats through stimulating OCN-involved endochondral ossification. & 2014 Elsevier Ireland Ltd. All rights reserved.
Keywords: Drynaria fortunei J. Sm. Bone mass Ovariectomy Osteocalcin Endochondral ossification
1. Introduction Osteoporosis, a progressive bone disease, is characterized by a decrease in the bone mass and density which can induce bones to
Abbreviations: ALT, aminotransferase; AST, aspartate aminotransferase; BUN, bilirubin; CRE, creatinine; LDH, lactate dehydrogenase; OCN, osteocalcin; OPN, osteopontin; UA, uric acid; WEK, water extracts of Kunze n Corresponding author at: Graduate Institute of Medical Sciences, College of Medicine, Taipei Medical University, 250 Wu-Xing St., Taipei, 110, Taipei, Taiwan. Tel.: þ 886 2 27361661x3222; fax: þ886 2 86621119. E-mail address: [email protected] (R.-M. Chen). http://dx.doi.org/10.1016/j.jep.2014.10.016 0378-8741/& 2014 Elsevier Ireland Ltd. All rights reserved.
weaken and be more likely to break (Adams, 2012). In the clinic, osteoporosis is also called as a silent disease because it proceeds without pain or symptoms until bone fracture occurs (Bradbury et al., 2012). Bone fractures typically linked to osteoporosis are very dangerous and can cause patients to suffer permanent disabilities or even death (Edwards et al., 2012). A variety of risk factors contribute to the causes of osteoporosis, including low levels of sex hormones, inadequate calcium intake, heredity, chronic diseases, lifestyle habits, medication use, and aging (Rubin et al., 2013). Among these, sex hormones are reported to play the most crucial roles in regulating bone remodeling and maintaining bone mass. However, post-menopausal hormone
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therapy for osteoporosis may increase the risk of coronary heart disease, stroke, and breast cancer (Rozenberg et al., 2013). Hence, discovering hormone analogs such as phytoestrogen would be helpful in developing de novo therapeutic strategies for osteoporosis. The bone structure is maintained through a dynamic balance between osteoblast-mediated bone formation and osteoclastmediated bone resorption (Seeman and Delmas, 2006). In skeletal development and bone healing, bone is formed through two essential processes, namely endochondral ossification and intramembranous ossification (Shore and Kaplan, 2010). In endochondral ossification, bone is formed with hyaline cartilage as the model (Hojo et al., 2010). In comparison, cartilage is not present in the course of intramembranous ossification (Shore and Kaplan, 2010). Miscellaneous stages are involved in endochondral ossification, including collar formation, cavity formation, vascular invasion, elongation, and epiphyseal ossification (Hojo et al., 2010). During the process of endochondral ossification, chondrocytes differentiated from stem cells first proliferate and then differentiate into hypertrophic chondrocytes (Mackie et al., 2011). Lastly, hypertrophic chondrocytes are gradually replaced by bone, leading to an increase in trabecular bone. As a result, osteoblasts participate in endochondral ossification by colonizing regions of the cartilage (Aubin, 1998; Medici and Olsen, 2012). A complicated network of differentiation proteins is involved in regulating osteoblast-mediated osteogenesis (Stein et al., 1996; Vandenput and Ohlsson, 2009). The process of endochondral bone development needs to be evaluated to discover innovative biomaterials that can prevent or treat osteoporosis. Drynaria fortunei J. Sm. (Korean name; Kunze) is a variety of the traditional Chinese herb, Gusuibu, which is frequently used by Chinese people to prevent or treat bone-related diseases. Long et al. (2005) reported that the flavonoid fractions of Kunze can prevent nephrotoxicity and promote regeneration of kidney primary epithelial tubular cells. Previous studies further showed that the components of Kunze can stimulate proliferation of osteoblast-like UMR106 cells and induce bone formation in mice (Wong and Rabie, 2006; Wang et al., 2008). Our previous study showed that the water extract of Kunze (WEK) can protect rat calvarial osteoblasts from hydrogen peroxide-induced insults (Liu et al., 2001). Recently, we further showed the effects of the WEK on promoting osteoblast maturation by inducing differentiation-related gene expression and protecting against oxidative/nitrosative stress-induced apoptotic insults (Huang et al., 2010; Hsu et al., 2011). Osteocalcin (OCN) is an early osteoblast biosignature that participates in controlling osteoblast function and bone extracellular matrix mineralization (van Leeuwen et al., 2001). A previous study reported that 11 flavonoids were extracted from Kunze (Wang et al., 2008). In our lab, we demonstrated that naringin, one of these flavonoids, can induce OCN expression during osteoblast differentiation (Huang et al., 2010). Thus, this study was further designed to corroborate the translational effects of Kunze and naringin on improving bone mass using ovariectomized rats as our experimental model.
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2.2. Animals Female Wistar rats (200–250 g) were purchased from the Animal Center of the College of Medicine, National Taiwan University (Taipei, Taiwan). Before the experiments began, animals were allowed to acclimatize for 1 week in their animal quarters with air conditioning and an automatically controlled photoperiod of 12 h of light daily. All experimental procedures were performed according to the National Institutes of Health Guidelines for the Use of Laboratory Animals and approved by the Institutional Animal Care and Use Committee of Taipei Medical University (Taipei, Taiwan). Rats were randomly divided into 4 groups: a sham control, an ovariectomy, an ovariectomy with the WEK at 0.5 g/kg body weight, and an ovariectomy with the WEK at 1 g/kg body weight. Animals were allowed free access to rodent laboratory chow (Purina Mills, St. Louis, MO, USA). 2.3. Surgical procedures and drug treatment Rats were anesthetized using ketamine (100 mg/kg) and xylazine hydrochloride (10 mg/kg) intramuscularly. A bilateral ovariectomy was conducted following a previous method (Brennan et al., 2009). A mid-ventral incision was made, and the bilateral ovaries and ovarian fat were removed. The ovaries were isolated by ligation of the most proximal portion of the oviduct before removal. In the sham-operated group, animals were subjected to the same procedure, but the ovaries were not removed. Animals were kept warm during the procedure and recovery. After surgery, rats were administered different doses of WEK by oral gavage for various time intervals. 2.4. Measurements of serum clinical parameters Systemic toxicity of the WEK was assayed by measuring serum clinical parameters as described previously (Chen et al., 1998). Briefly, animals were sacrificed after drug treatment, and blood samples were collected for assessment of serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), lactate dehydrogenase (LDH), bilirubin (BUN), creatinine (CRE), and uric acid (UA). 2.5. Measurement of serum 17β-estradiol After drug treatment, rats were sacrificed, and blood samples were collected. Serum fractions were prepared for analysis of 17β-estradiol. Levels of serum 17β-estradiol were assayed following the instructions of the Elecsys-Estradiol II enzyme-linked immunosorbent assay (Roche Diagnostics, Mannheim, Germany). 2.6. Hematoxylin and eosin (HE) staining
2. Materials and methods
After drug treatment, animals were sacrificed. Livers and kidneys were removed and collected. Samples were fixed using phosphatebuffered 4% paraformaldehyde and processed for routine paraffin embedding. All tissue samples were sliced into 5-μm transverse sections and stained with HE. Specimens were observed and photographed using a light microscope.
2.1. Preparation of the WEK
2.7. Biomechanical testing
Kunze (D. fortunei) was kindly provided by the Brion Research Institute, Sun Ten Group (Taipei, Taiwan). The herb was identified by institutional experts, and its chemical and physical characteristics were routinely analyzed (Huang et al., 2010). The WEK was prepared as previously described (Liu et al., 2001). The extract was stored at room temperature and protected from light and moisture as described previously (Hsu et al., 2011). Naringin was purchased from Sigma (St. Louis, MO, USA).
After drug treatment, animals were sacrificed, and femur tissues were removed and collected. Following collection of muscle and connective tissues, the femurs were examined by peripheral quantitative computed tomography using a standardized cantileverbending technique as described previously (Wang et al., 2000). Briefly, distal condyles of the femurs were loaded in the anteroposterior direction at a constant rate of 1.0 mm/min until failure in a universal testing device (Avalon Technologies, Rochester, MI, USA).
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The force-displacement curves recorded were used to measure the ultimate bending force to failure (N) and bending stiffness (N/mm).
3. Results 3.1. An ovariectomy decreased levels of serum estradiol
After drug treatment, rats were sacrificed. The right tibias of rats were collected, and the muscle and connective tissues were removed. Then, the tibias were dehydrated at 100 1C for 14 h and weighed. The dehydrated bones were then calcinated at 600 1C for another 16 h. Dry bone ashes were weighed. The ratio of dry bone ash to the dehydrated tibia was calculated.
2.9. Bone histomorphometry Proximal tibias were dehydrated in a graded series of ethanol and xylene and embedded undecalcified in modified methyl methacrylate following a previously described method (Iwaniec et al., 2008). Specimens were longitudinally cut into 5-μm-thick sections. Specimens were stained for assessment of bone area and cell-based measurements. The derived architectural index of trabecular bone number (mm-1) in a standard sampling site 0.5 mm distal to the growth plate was measured using the OsteoMeasure System (OsteoMetrics, Atlanta, GA, USA).
Fifteen days after Wistar rats had undergone an ovariectomy, a significant 40% decrease in levels of serum 17β-estradiol was detected (Fig. 1). One, 2, 3, and 6 months after the ovariectomy, amounts of serum 17β-estradiol were significantly diminished by 48%, 69%, 73%, and 82%, respectively (Fig. 1). 3.2. Effects of the WEK on the body weight, liver weight, and kidney weight One, 2, 3, and 6 months after the ovariectomy, Wistar rats exhibited significant increases in body weight (Table 1). Compared to ovariectomy-treated groups, the WEK at a low dose (0.5 g/kg body weight/day) and a high dose (1 g/kg body weight/day) did not change the body weights of experimental animals. Treatment of animals with an ovariectomy and the WEK at low and high doses for 1, 2, 3, and 6 months did not affect the liver weight of rats 200
150 Estradiol, pmol/L
2.8. Quantification of bone mineral contents
2.10. Immunohistochemical (IHC) analysis of OCN IHC was carried out as described previously (Chang et al., 2014). After drug treatment, animals were sacrificed, and the tibias were removed, collected, and sliced. Bone tissues were fixed with a fixing reagent (acetone: methanol, 1: 1) at 20 1C for 10 min and incubated with 0.2% Triton X-100 at room temperature for 15 min. Immunodetection of OCN in bone tissues was carried out using a polyclonal antibody against rat OCN (Santa Cruz Biotechnology, Santa Cruz, CA, USA) by incubation at 4 1C overnight. After washing, slices were reacted with the second antibody at room temperature for 1 h. Staining was visualized with 3,30 ,3taminobenzidine. Specimens were observed and photographed using a light microscope.
2.11. Immunoblot analysis of OCN After drug treatment, rats were sacrificed, and the femurs were removed and homogenized in lysis buffer (50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EGTA, 1% NP-40 supplemented with 1 mM PMSF, 1 μM aprotinin, 1 μM leupeptin, 1 mM Na2VO4, and 1 mM NaF). Protein concentrations were quantified using a bicinchonic acid protein assay kit (Pierce, Rockford, IL, USA). Proteins (50 μg/well) were subjected to sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE), and transferred to nitrocellulose membranes as described previously (Liao et al., 2014). Membranes were blocked with 5% nonfat milk at 37 1C for 1 h. OCN protein was immunodetected using a polyclonal antibody. β-Actin was immunodetected using a mouse monoclonal antibody against mouse β-actin (Sigma) as an internal control. Intensities of the immunoreactive protein bands were determined using an UVIDOCMW vers. 99.03 digital imaging system (UVtec, Cambridge, UK).
2.12. Statistical analyses The statistical significance of differences between each group was evaluated using a one-way analysis of variance (ANOVA) with the Duncan's multiple-range test. Differences were considered statistically significant at P values ofo0.05.
*
100
* *
50
0
0
15
*
30 60 Time, days
90
* 180
Fig. 1. Effects of an ovariectomy on changes in serum 17β-estradiol. Female Wistar rats were given a bilateral ovariectomy. Then 15, 30, 60, 90, and 180 days after surgery, animals were sacrificed, and blood samples were collected. Levels of serum 17β-estradiol were quantified using an ELISA. Each value represents the mean 7SEM for n¼ 9. n Indicates that the value significantly differed from the respective control, p o0.05. Table 1 Effects of the water extract of Kunze (WEK) on the body weight (BW), liver weight (LW), and kidney weight (KW) of ovariectomized (OVX) rats.
1 Month BW LW/BW KW/BW 2 Months BW LW/BW KW/BW 3 Months BW LW/BW KW/BW 6 Months BW LW/BW KW/BW
Control
OVX
WEK (0.5 g)
WEK (1.0 g)
230 711 3.9 70.3 0.8 70.1
263 7 13n 4.17 0.2 0.7 7 0.1
245 721 4.2 70.3 0.8 70.1
2317 22 3.3 7 0.7 0.7 7 0.1
247 712 4.2 70.1 0.7 70.1
289 7 25n 3.8 7 0.1 0.7 7 0.1
288 710n 3.6 70.1 0.6 70.1
293 7 14n 3.7 7 0.2 0.6 7 0.1
260 710 3.7 70.4 0.7 70.0
3177 20n 3.4 7 0.4 0.7 7 0.1
291 721n 3.2 70.4 0.6 70.1
3247 26n 3.17 0.2 0.6 7 0.1
266 79 3.6 70.3 0.7 70.0
309 7 28n 3.4 7 0.3 0.6 7 0.1
305 79n 3.1 70.3 0.7 70.1
328 7 11n 3.2 7 0.1 0.6 7 0.1
Female Wistar rats were anesthetized and given a bilateral ovariectomy (OVX). After surgery, rats were administered the water extract of Kunze (WEK) at 0.5 and 1 g/kg BW per day by oral gavage for 1, 2, 3, and 6 months. Animals were sacrificed and the BW, LW, and KW were measured. Each value represents the mean 7 SEM for n¼ 9. n Indicates that the value significantly differed from the respective control, po 0.05.
Y.-E. Lee et al. / Journal of Ethnopharmacology 158 (2014) 94–101
Table 2 Effects of the water extract of Kunze (WEK) on serum factors of ovariectomized (OVX) rats.
LDH, U/L AST, U/L ALT, U/L BUN, mg/dl CRE, mg/dl UA, mg/dl
Control
OVX
WEK(0.5 g)
WEK (1 g)
919 7102 121 711 39 77 19 72 0.6 70.1 1.7 70.3
885 7 126 1407 16 327 10 177 2 0.7 7 0.1 1.4 7 0.1
904 7240 146 726 48 716 18 71 0.6 70.1 1.2 70.2
832 7 129 1497 37 417 11 177 2 0.7 7 0.1 1.4 7 0.1
Female Wistar rats were anesthetized and given a bilateral ovariectomy (OVX). After surgery, rats were administered the WEK at 0.5 and 1 g/kg body weight per day by oral gavage for 6 months. Animals were sacrificed, and serum samples were collected for analyses of clinical parameters. Each value represents the mean 7SEM for n ¼9.
Control
OVX
3.3. Systemic and tissue toxicities of the WEK Six months after the ovariectomy, serum LDH of rats had not changed (Table 2). Separately, the ovariectomy did not affect serum indices of AST and ALT that indicate liver injury or those of BUN, CRE, and UA that indicate kidney injury. In comparison, exposure to low and high doses of the WEK did not change serum indices of LDH, AST, ALT, BUN, CRE, or UA (Table 2). Between the animals treated with a low and a high dose of WEK, there was no difference in these serum indices. Tissue toxicities of the WEK to the liver and kidney were confirmed using histological analyses (Fig. 2). Compared to control animals, 6 months after an ovariectomy, rats did not exhibit tissue damages to the liver or kidney (Fig. 2A and B). In ovariectomized rats, administration of the WEK at 0.5 and 1 g/kg body weight/day for 6 months did not induce hepatotoxicity or nephrotoxicity (Fig. 2A and B).
120
Bone torsion, % of control
(Table 1). In parallel, the kidney weight of animals subjected to and ovariectomy and various doses of the WEK for 1, 2, 3, and 6 months was not influenced (Table 1). There was no statistical difference in the body weight, liver weight, and kidney weight between the animals treated with WEK at 0.5 and 1 g /kg body weight/day.
97
100 80 60 40 20 0
Control
OVX
140
WEK-1.0
*
80 60 40
0
OVX
#
100
20
Control
Contr ol Control
OVX
140
WEK (0.5)
Bone torsion, % of control
WEK-1.0
100 80
WEK (1.0)
#
120
WEK-0.5
WEK (1.0)
#
120
Bone torsion, % of control
WEK -0.5
WEK (0.5)
#
GSB0.5
*
60 40 20 0
Fig. 2. Toxicities of the water extract of Kunze (WEK) to the liver and kidneys. Female Wistar rats were given a bilateral ovariectomy (OVX). After surgery, animals were treated by oral gavage with WEK at 0.5 and 1 g/kg body weight/day for 60 days. Following drug treatment, the animals were sacrificed, and the livers (A) and kidneys (B) were removed for hematoxylin and eosin staining analyses. Specimens were observed and photographed using a light microscope. 100 .
Control
OVX
WEK (0.5)
WEK (1.0)
Fig. 3. Effects of the water extract of Kunze (WEK) on the biomechanical strength of rat femurs. Female Wistar rats were given a bilateral ovariectomy (OVX). After surgery, animals were treated with the WEK at 0.5 and 1 g/kg body weight/day by oral gavage for 1 (A), 3 (B), and 6 (C) months. Bone torsion was assayed using a standardized cantilever-bending technique. Each value represents the mean 7 SEM for n ¼6. n and # Indicate that values significantly (po 0.05) differed from the control and OVX-treated groups, respectively.
98
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3.4. The WEK improves the biomechanical strength of ovariectomized rat femurs Biomechanical assays revealed that an ovariectomy and administration of the WEK for 1 month did not affect the bone torsion force of rat femurs (Fig. 3A). However, 3 and 6 months after the ovariectomy, the torsion force of rat femurs had significantly decreased by 24% and 28%, respectively (Fig. 3B and C). Interestingly, administration of 0.5 and 1 g of the WEK/kg body weight per day for 3 and 6 months caused significant alleviation of the ovariectomy-induced reduction in femur strength (Fig. 3B and C). However, our data indicated that there was no difference in the bone torsion force between the animals treated with various doses of WEK.
3.5. The WEK increases bone mineral contents of ovariectomized rats One month after the ovariectomy, the bone ash weights of rat tibias were significantly reduced by 12% (Fig. 4A). When exposed to the WEK at 0.5 and 1 g/kg body weight/day for 1 month, the ovariectomyinduced reduction in the bone mineral content was alleviated (Fig. 4A). The bone ash weights of rat tibias had decreased by 15% and 25% at 3 and 6 months after the ovariectomy, respectively (Fig. 4B and C). In comparison, the WEK at both low and high doses lessened the ovariectomy-induced loss of bone mineral contents. In the 3-monthadministered time interval, the bone ash weights were less in a high dose of WEK-treated rats than those in a low dose of WEK-treated animals (Fig. 4B). However, there was no significant difference in the bone ash weights between the animals treated with these two doses of WEK for 1 and 6 months (Fig. 4A–C). 3.6. The WEK convalesces ovariectomy-induced alterations in bone histomorphometry
120
# #
Bone ash, % of control
100
*
80
Six months after the ovariectomy, the trabecular bone index and numbers of prehypertrophic chondrocytes had decreased, but hypertrophic chondrocytes in the growth plate had increased (Fig. 5A). The WEK at a low dose (0.5 g/kg body weight) enhanced proliferating
60 40
Control
OVX
WEK-0.5
WEK-1.0
20 0
Control
OVX
WEK (0.5)
#
120
#†
100
*
80 60 40 20 0
Contr ol Control
OVX
WEK (0.5)
#
120
WEK (1.0)
#
Bone ash, % of control
100 80
GSB0.5
*
60 40
6 Trabe ecular bone, numb ber/mm
Bone ash, % of control
WEK (1.0)
#
#
4
* 2
20
0 0
Control
OVX
WEK (0.5)
WEK (1.0)
Fig. 4. Effects of the water extract of Kunze (WEK) on bone mineral contents of rat tibias. Female Wistar rats were given a bilateral ovariectomy (OVX). After surgery, animals were treated with the WEK at 0.5 and 1 g/kg body weight/day by oral gavage for 1 (A), 3 (B), and 6 (C) months. Changes in bone mineral content were analyzed by measuring the ratio of dry bone ash over the dehydrated tibia. Each value represents the mean 7SEM for n¼ 6. n, #, and † Indicate that the values significantly (p o 0.05) differed from the control, OVX-, and WEK (0.5 g/kg body weight/day)-treated groups, respectively.
Control
OVX
WEK (0.5)
WEK (1.0)
Fig. 5. Effects of the water extract of Kunze (WEK) on bone histomorphometry of rat tibias. Female Wistar rats were given a bilateral ovariectomy (OVX). After surgery, animals were treated with the WEK at 0.5 and 1 g/kg body weight/day by oral gavage for 6 months. Proximal tibias were removed for assessment of bone area and cell-based measurements: a, trabecular bone; b, hypertrophic chondrocytes; c, prehypertrophic chondrocytes; d, proliferating chondrocytes (A). The index of trabecular bone was measured using the OsteoMeasure System (B). Each value represents the mean 7SEM for n¼ 6. n and # Indicate that values significantly (p o 0.05) differed from the control and OVX-treated groups, respectively.
Y.-E. Lee et al. / Journal of Ethnopharmacology 158 (2014) 94–101
99
chondrocytes but reduced the numbers of hypertrophic chondrocytes. In comparison, administration of the WEK at a high dose (1 g/kg body weight/day) for 6 months to ovariectomized rats significantly diminished the number of hypertrophic chondrocytes (Fig. 5A). The WEK at a high dose also enlarged the trabecular bone thickness and connections (Fig. 5A). An index of trabecular bone was quantified and analyzed (Fig. 5B). An ovariectomy caused a significant 52% decrease in the trabecular bone index. After exposure to the WEK at 0.5 and 1 g/kg body weight for 6 months, the ovariectomy-induced drop in the trabecular bone index was eased by 61% and 74%, respectively (Fig. 5B). Between the animals treated with WEK at 0.5 and 1 g/kg body weight/day, there was no statistical difference in the trabecular bone index.
weight/day for 6 months, OCN expression was obviously enhanced. An immunoblot analysis further showed that the WEK augmented levels of OCN in the bone of ovariectomized rats (Fig. 6B, top panel, lanes 3 and 4). Amounts of β-actin were immunodetected as an internal control (bottom panel). The immunoreacted protein bands were quantified and analyzed (Fig. 6C). Administration of the WEK at 0.5 and 1 g/kg body weight/day to ovariectomized rats raised levels of OCN in the bone by 80% and 91%, respectively. However, there was no significant difference in the OCN levels between the animals treated with various doses of WEK.
3.7. The WEK increases OCN biosynthesis in ovariectomized rat bone
The effects of naringin, an active compound of WEK, on the bone mass of ovariectomized rats were further analyzed (Table 3). Exposure of ovariectomized rats to naringin alone did not change the bone torsion, bone ash, or trabecular bone index. However, an ovariectomy caused 41%, 19%, and 50% decreases in the bone torsion, bone ash, and trabecular bone index, respectively (Table 3). After exposure to naringin for 6 months, the ovariectomy-induced alterations in bone torsion, bone ash, and trabecular bone index were significantly ameliorated (Table 3).
Effects of the WEK on OCN expression in the bone of ovariectomized rats was analyzed using IHC and immunoblotting (Fig. 6). Subjecting rats to an ovariectomy slightly decreased OCN levels in femurs (Fig. 6A). After exposure to 0.5 and 1 g of the WEK/body
Control
OVX
3.8. Naringin increased the bone strength, bone mineral contents, and trabecular bone index in ovariectomized rats
4. Discussion
WEK-0.5
1
WEK-1.0
2
3
4 OCN
β C
OVX
WEK-0.5
WEK-1
OC CN, arbitrary unit x 100
120
*
90
*
This study proves the translational effects of Kunze on improving the bone mass of ovariectomized rats. An ovariectomy is a useful model which can accelerate osteoporosis in experimental animals (Brennan et al., 2009). In the present study, levels of serum 17β-estradiol time-dependently dropped after the ovariectomy. In parallel, the ovariectomy altered the bone strength, bone mineral contents, and bone microarchitecture. Hence, subjecting female rats to a bilateral ovariectomy successfully decreased serum estrogen synthesis and consequently induced osteoporosis in this study. Our results demonstrated enhancement of the bone mass in ovariectomized rats after exposure to Kunze. A deficiency in the sex hormones usually leads to an imbalance of bone remodeling, leading to loss of bone mass (Rozenberg et al., 2013). Thus, post-menopausal hormone therapy was developed to treat osteoporotic patients. However, hormone replacement therapy may increase the risks of breast cancer and other diseases (Rozenberg et al., 2013). Phytoestrogens, a class of bioactive compounds exerting various estrogenic effects, were shown to increase bone formation and repress bone resorption (Ming et al., 2013). There are more than 11 flavonoids found in the WEK (Wang et al., 2008). The data presented in this study reveal that Table 3 Effects of naringin on the biomechanical strength, bone mineral contents, and trabecular bone index of ovariectomized (OVX) rat femurs.
60
Naringin, mg/kg
Bone torsion (% of control)
Bone ash (% of control)
Trabecular bone (number/mm)
Control OVX Naringin NaringinþOVX
100 597 10n 1017 13 877 10#
100 817 4n 987 6 947 6#
67 1 37 1n 77 2 57 1#
30
0
Control
OVX
WEK (0.5)
WEK (1.0)
Fig. 6. Effects of the water extract of Kunze (WEK) on osteocalcin OCN biosynthesis. Female Wistar rats were given a bilateral ovariectomy (OVX). After surgery, animals were treated with the WEK at 0.5 and 1 g/kg body weight/day by oral gavage for 6 months. Immunohistochemical analysis of OCN in bone tissues was carried out (A). Levels of OCN were further determined using an immunoblot analysis (B, top panel). Amounts of β-actin were immunodetected as the internal control (bottom panel). The immunoreactive protein bands were quantified and statistically analyzed (C). Each value represents the mean 7 SEM for n¼6. n and # Indicate that values significantly (p o 0.05) differed from the control and OVXtreated groups, respectively.
Female Wistar rats were anesthetized and given a bilateral ovariectomy (OVX). After surgery, rats were administered the water extract of Kunze (WEK) at 0.5 and 1 g/kg body weight per day by oral gavage for 6 months. Animals were sacrificed, and bone samples were collected for analyses of bone torsion, bone ash, and the trabecular bone index. Each value represents the mean7 SEM for n¼ 6. n Indicate that values significantly differed from the respective control and OVX-treated groups, po 0.05. # Indicate that values significantly differed from the respective control and OVX-treated groups, po 0.05.
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the WEK did not induce systemic or tissue toxicities to animals. Therefore, Kunze may have therapeutic potential at maintaining bone health in the clinic. Kunze may promote the bone mass of ovariectomized rats by alleviating osteoblast insults. Oxidative stress is directly linked to estrogen decline and osteoporosis occurrence (Manolagas, 2010). Osteoblasts also mediate bone formation in bone remodeling but are easily insulted by oxidative stress (Ducy et al., 2000; Chen et al., 2010). Our previous studies reported that Kunze could protect osteoblasts from oxidative- and nitrosative stress-induced apoptotic injury (Liu et al., 2001; Huang et al., 2010). Moreover, those studies further validated the effects of Kunze at improving osteoblast differentiation and maturation through inducing certain osteoblast differentiation-related genes, including insulin-like growth factor-1, matrix maturation-related bone morphogenetic protein (BMP)-2 and BMP-6, alkaline phosphatase, ostepontin, and OCN (Huang et al., 2010; Hsu et al., 2011). In this study, we provide supplementary in vivo data to illustrate the effects of Kunze on the bone mass of the ovariectomized rats. Therefore, two possible reasons explaining the Kunze-induced promotion of bone mass in ovariectomized rats are that it protects osteoblasts against oxidative stress-triggered apoptosis while promoting osteoblast maturation. Kunze expands the bone mass of ovariectomized rats possibly via endochondral ossification. Endochondral ossification is a process that uses hyaline cartilage as a model to form new bone (Hojo et al., 2010). Chondrocytes were reported to play an important role in the progression of endochondral ossification. At first, chondrocytes multiply, and proliferated chondrocytes subsequently differentiate into hypertrophic chondrocytes (Mackie et al., 2011). This study found that administration of the WEK lessened ovariectomy-induced suppression of chondrocyte proliferation and construction of hypertrophic chondrocytes. When exposed to Kunze, the ovariectomy-induced reduction in the trabecular bone index also significantly recovered in ovariectomized rats. In the final stage of endochondral ossification, hypertrophic chondrocytes are replaced by osteoblasts to form new bone (Hojo et al., 2010; Mackie et al., 2011). As a result, the Kunze-induced promotion of bone mass in ovariectomized rats may occur mainly via an endochondral ossification pathway. In addition, bone fractures are a chief risk to patients suffering osteoporosis (Edwards et al., 2012). In bone fractures, endochondral ossification is reported to be a key process during bone healing (Shore and Kaplan, 2010). Accordingly, Kunze may be helpful in the clinical treatment of osteoporosis and osteoporosis-induced bone fractures. OCN contributes to Kunze-induced improvement of bone mass. Our previous study used neonatal rat calvarial osteoblasts as the experimental model to show the role of OCN in Kunze-triggered osteoblast differentiation and maturation (Huang et al., 2010). The present study further demonstrates that the WEK stimulated OCN biosynthesis in ovariectomized rats. OCN is an early biomarker for bone ECM mineralization and bone formation (van Leeuwen et al., 2001). A previous study showed that downregulation of OCN expression led to repression of osteoblast differentiation (Lin et al., 2010). Okamura et al. (2011) stated that upregulation of OCN expression simultaneously enhanced bone formation. When OCN synthesis is abrogated by silencing expression of the Cbfa1 gene, the endochondral ossification process is concurrently inhibited (Sun et al., 2012). Therefore, the Kunze-provoked alleviation of bone mass in ovariectomized rats may be via OCN-involved endochondral ossification. Kunze enhances bone strength and bone mineral contents in ovariectomized rats. The decline in the bone torsion force is positively correlated to bone loss (Beaupied et al., 2007). A previous study reported that icaritin, one of the epimedium-derived flavonoids, remarkably restored bone biomechanical properties in ovariectomized rats (Peng et al., 2013). This study further demonstrated that after exposure to the WEK, the ovariectomy-induced lessening in the bone torsion force was significantly alleviated. This finding indicates the
beneficial property of Kunze on the bone mass due to enhancement of bone strength. In parallel, the amount of bone ash in ovariectomized rats was simultaneously increased following administration of the WEK. In female Sprague–Dawley rats, an increase in bone mineral contents concurrently improved the bone biomechanical strength (Khan et al., 2013). Accordingly, the Kunze-induced augmentation of bone mineral contents contributes to the enrichment of bone strength. Based on the present results, Kunze can assuage ovariectomy-induced decreases in bone mineral contents and bone strength and subsequently improve the bone mass of ovariectomized rats. Naringin is one of the active components present in the WEK and can enhance bone development in ovariectomized rats. Previous studies showed that naringin is a component of the WEK (Wang et al., 2008; Wong et al., 2013). Our previous study reported the osteogenic effects of naringin on inducting OCN gene expression (Huang et al., 2010). The present study demonstrated the favorable translational effects of naringin on intensifying the bone mineral contents and bone potency in ovariectomized rats. In parallel, after exposure to naringin, the trabecular bone index in ovariectomized rats was also augmented. Such enhancement indicates the promoting effects of naringin of stimulating the formation of new bone. A previous study also showed that naringin can provoke osteogenesis by enlarging the proliferation and differentiation of bone marrow stromal cells (Li et al., 2013). Wong et al. (2013) reported that naringin possesses the potential to inhibit osteoclastogenesis. Therefore, naringin may be one of the active components in the WEK that overcomes ovariectomy-induced bone loss. In summary, this study has shown that the WEK did not induce systemic or tissue toxicity in rats. Meanwhile, Kunze alleviated ovariectomy-induced reductions in bone mechanics and bone mineral contents. Analyses of bone histomorphometry further showed that ovariectomy-triggered alterations in proliferating chondrocytes, hypertrophic chondrocytes, and trabecular bone significantly recovered following Kunze administration. In parallel, Kunze increased OCN synthesis in bone tissues of ovariectomized rats. Naringin, one of the active components found in the WEK, lessened ovariectomy-induced decreases in bone torsion, bone ash, and the trabecular bone index. Therefore, this study has translationally confirmed that Kunze can promote the bone mass of ovariectomized rats through an OCNinvolved endochondral ossification mechanism. Our in vitro and in vivo data suggest the potential application of Kunze for osteoporosis therapy. There are certain study limitations in this study. For example, there was no significant difference in most of our assays between the animals treated with various doses of WEK. However, the present results cannot explain why in the 3-month-administered time interval, the bone ash weights of a high dose of WEK-treated rats were less than those of a low dose of WEK-treated animals. To confirm and elucidate such a difference, more experiments, including quantification of bone calcium, magnesium, and phosphate, will be conducted in our upcoming mechanism study.
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