http://informahealthcare.com/phb ISSN 1388-0209 print/ISSN 1744-5116 online Editor-in-Chief: John M. Pezzuto Pharm Biol, 2015; 53(1): 110–116 ! 2015 Informa Healthcare USA, Inc. DOI: 10.3109/13880209.2014.911920
ORIGINAL ARTICLE
Treatment of Radix Dipsaci extract prevents long bone loss induced by modeled microgravity in hindlimb unloading rats Yinbo Niu1, Chenrui Li1, Yalei Pan1, Yuhua Li2,3, Xianghe Kong4, Shuo Wang1, YuanKun Zhai1, Xianglong Wu1, Wutu Fan1, and Qibing Mei1,2 1
Key Laboratory for Space Bioscience and Biotechnology, School of Life Sciences, Northwestern Polytechnical University, Xi’an, PR China, Department of Pharmacology, School of Pharmacy, Fourth Military Medical University, Xi’an, PR China, 3Department of Pharmacy, No. 422 Hospital of PLA, Zhanjiang, PR China, and 4Training and Teaching Research Office, Graduate School of Chang’an University, Xi’an, PR China
2
Abstract
Keywords
Context: Radix Dipsaci is a kidney tonifying herbal medicine with a long history of safe use for treatment of bone fractures and joint diseases in China. Previous studies have shown that Radix Dipsaci extract (RDE) could prevent bone loss in ovariectomized rats. Objective: This study investigates the effect of RDE against bone loss induced by simulated microgravity. Materials and methods: A hindlimb unloading rat model was established to determine the effect of RDE on bone mineral density and bone microarchitecture. Twenty-four male Sprague– Dawley rats were divided into four groups (n ¼ 6 per group): control (CON), hindlimb unloading with vehicle (HLU), hindlimb unloading treated with alendronate (HLU-ALN, 2.0 mg/kg/d), and hindlimb unloading treated with RDE (HLU-RDE, 500 mg/kg/d). RDE or ALN was administrated orally for 4 weeks. Results: Treatment with RDE had a positive effect on mechanical strength, BMD, BMC, bone turnover markers, and the changes in urinary calcium and phosphorus excretion. MicroCT analysis showed that RDE significantly prevented the reduction of the bone volume fraction, connectivity density, trabecular number, thickness, tissue mineral density, and tissue mineral content as well as improved the trabecular separation and structure model index. Discussion and conclusion: RDE was demonstrated to prevent the loss of bone mass induced by HLU treatment, which suggests the potential application of RDE in the treatment of microgravity-induced bone loss.
Bone density, bone strength, Dipsacus asperoides
Introduction A marked and potentially debilitating reduction in bone mass is the major skeletal problem after prolonged space flight and weightlessness (Collet et al., 1997; Smith et al., 2012). In the most severe forms of microgravity-induced bone loss, there is an approximately 2% decrease in bone mineral density in only 1 month, which roughly approximates that of a postmenopausal woman in 1 year (Riggs et al., 1998). Microgravity changes the metabolic environment of bone leading to site-specific alterations in bone remodeling. Bone formation is decreased and bone resorption is increased, which results in significant bone loss with a consequent increase in fracture risk (Smith et al., 2002). Over the past few years, several countermeasures have been examined to prevent bone loss during exposure to microgravity, including pharmacological therapy with bisphosphonates and calcitonin, and several active and passive exercise regimes, but
Correspondence: Qibing Mei, School of Life Sciences, Northwestern Polytechnical University, Xi’an 710072, PR China. Tel: +86 29 84779212. Fax: +86 29 84779212. E-mail: [email protected]
History Received 19 November 2013 Revised 10 January 2014 Accepted 1 April 2014 Published online 22 September 2014
positive outcomes have been limited (Czarnik & Vernikos, 1999; Derendorf, 1994). To date, the offered modalities do not show much success in preventing or alleviating bone loss in astronauts and cosmonauts (Nagaraja & Risin, 2013). The pathology remains a key concern, and the development of effective countermeasures is still a major task. Natural products have been shown to be excellent and reliable sources for the prevention of bone loss (Habold et al., 2011; Siu et al., 2013; Zhang et al., 2009, 2011). Radix Dipsaci, the dried root of Dipsacus asperoides C. Y. Cheng and T. M. Ai (Dipsacaceae), has long been used as an antiosteoporosis tonic and anti-aging agent in China. It has also been used for the treatment of lower back pain, traumatic hematoma, and bone fractures (Hung et al., 2006). Recent studies confirmed that Radix Dipsaci extract (RDE) could increase bone density and alter bone histomorphology in mice and rats (Liu et al., 2009; Wong et al., 2007). However, there is no direct evidence supporting RDE inhibitory effect on bone loss induced by microgravity. Thus, the current study aimed to determine the osteoprotective effect of RDE on biological indices of bone metabolism, including biochemical, bone mineral density (BMD), bone mineral content (BMC), biomechanical, and
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bone microparameters in the hindlimb unloading (HLU) rats (Hindlimb unloading rat model is extensively utilized as a simulated mechanical inactivity model under microgravity environment) (Morey-Holton & Globus, 1998).
Materials and methods Identification and preparation of Radix Dipsaci extract Radix Dipsaci was purchased from a local Chinese medicine store in 2011, and was identified morphologically, histologically, and chemically by Guo Yaowu, a pharmacist in laboratory for the control of drugs of Shaanxi Province. A voucher specimen including the identification and classification of the plant material was preserved in the Research Center of Clinic Pharmacology and Pharmacy of the Fourth Military Medical University (Xi’an, China). In brief, the crude plant was identified by using the thin-layer chromatography method. During the procedure, dipsacus saponin VI was used as a standard substance, and methanol as a developing solvent. Radix Dipsaci extract was prepared using the protocol for ethyl alcohol extraction (Liu et al., 2009). Briefly, the raw herbs (10 kg) were crushed into small pieces and extracted in 50 L of 60% ethanol for 2 h by heating and refluxing method for three times. The extracts were then collected, filtered, decompression concentrated, and lyophilized. At last, the powder with an extraction yield of 20% was obtained. Overview of study design Male Sprague–Dawley rats aged 12 weeks (Experimental Animal Center of the Fourth Military Medical University, Xi’an, China) were housed in a facility maintained at 22 C and with a 12/12 h light/dark cycle. During the experimental period, the rats were maintained on standard rodent chow (Animal Center of the Key Laboratory for Space Bioscience and Biotechnology, Xi’an, China) that contained 0.9% calcium and 0.7% phosphate, and filtered water available ad libitum. After 1 week of acclimatization to the keeping environment, rats were randomized into four groups: (n ¼ 6 per group): control (CON), hindlimb unloading with vehicle (HLU), hindlimb unloading treated with alendronate (HLUALN, 2.0 mg/kg/d) (Mosekilde et al., 2000; Qi et al., 2012), and hindlimb unloading treated with RDE (HLU-RDE, 500 mg/kg/d) (Liu et al., 2009). Animals were assigned to groups by total body bone mineral density and body mass in a manner to minimize differences between groups at baseline. For the CON group, rats were allowed to move freely with their four limbs (without hindlimb unloading). In groups HLU, HLU-ALN, and HLU-RDE, the tails of the rats were suspended so that their hindlimbs were unloaded according to the recommendations of Morey-Holton and Globus (2002). RDE or ALN was administrated orally for 4 weeks. Rats in both CON and HLU groups received equal volume of distilled water vehicle for four weeks. All tail-suspended rats were adjusted weekly to ensure that the hindlimb paws could not touch the ground. All animals were treated in compliance with the Guidance for Care and Use of Laboratory Animals with the approval of the Institutional Ethics Committee of the Fourth Military Medical University.
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Tissue collection and sample preparation A blood sample was collected by abdominal aorta puncture from each anesthetized rat at the end of the laparotomy and was centrifuged at 2000 rpm for 20 min to get the serum. Urine was collected over 24 h from fasted, individually housed rats in metabolic cages. Urine and serum samples were then stored at 80 C for biochemical determinations. Bone mineral content (BMC) and bone mineral density (BMD) were measured before rats euthanized by dual-energy X-ray absorptiometry (DXA). Femurs were harvested and cleaned off soft tissue. The right femur was prepared for biomechanical testing by wrapping in saline-soaked gauze and freezing at 20 C. The left femur was prepared for microCT in 10% neutral buffered formalin at 4 C for 48 h, and then transferred to 70% ethanol at 4 C. Assay for serum and urine chemistry Urine calcium (U-Ca), urine phosphorus (U-P), and creatinine (Cr) concentrations were measured by an automatic biochemical analyzer (Cobas Integra 400 plus, Roche Diagnostics, Basel, Switzerland) using the original kits from Roche Diagnostics (Indianapolis, IN). Serum calcium (S-Ca) and phosphorus (S-P) concentrations were analyzed by the same method used for the urine samples. Serum bone alkaline phosphatase (BALP), tartrate-resistant acid phosphatase (TRAP), osteocalcin (OC), urinary deoxypyridinoline (DPD), C-terminal crosslinked telopeptides of collagen type I (CTx), and N-terminal crosslinked telopeptides of collagen type I (NTx) concentrations were assessed using rat ELISA kits (Beijing Sino-uk Institute of Biological Technology, Beijing, China). Urinary excretion of Ca, P, and DPD was expressed as the ratio to Cr concentration (Ca/Cr; P/Cr; DPD/Cr). BMD and BMC analysis The BMC and BMD were measured by DXA (Lunar Prodigy Advance DXA, GE healthcare, Madison, WI) using the small laboratory animal scan mode (Pastoureau et al., 1995). The BMC and BMD of the whole femur were calculated automatically by the purpose-designed software (enCOREÔ 2006, GE Healthcare, Madison, WI). Biomechanical testing Bones were mechanically tested to failure via three-point bending using an MTS MiniBionix 858 testing apparatus (MTS Systems, Eden Prairie, MN). Each specimen was placed on two supports spaced 20 mm apart, and a load was applied to the bone midway between the supports at a deformation rate of 2 mm/min until the occurrence of fracture. The central loading point was displaced, and the load and displacement were recorded until the specimen was broken. All force and displacement data were collected for later evaluation, which were performed after measurement of the inner and outer sizes (i.e., width and height) of the femur at the point of fracture. The calculation of the biomechanical parameters was based on the formulas described previously (Brodt et al., 1999; Turner & Burr, 1993).
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MicroCT analysis The distal metaphysis of each femur was scanned using a desktop eXplore Locus SP Pre-Clinical Specimen microCT (GE Healthcare, Madison, WI). Three-dimensional (3D) image data were acquired with a voxel size of 12 lm in all spatial directions with microCT Evaluation Program (V5.0A) (Laib et al., 2000). Trabecular bone was separated from cortical bone by free drawing regions of interests with the Micro-View program (GE Healthcare, Madison, WI) and a multiple IntelÕ processor-based microCT workstation provided with the scanner. The regions of interest, which were located 1.5 mm from the metaphyseal line below, were chosen for the data analysis. Morphologic measurements were performed, and the following 3D parameters were obtained for the trabecular bone: (1) relative bone volume (BV/TV; %), (2) connectivity density (Conn.D; 1/mm3), (3) trabecular number (Tb.N; 1/mm), (4) trabecular separation (Tb.Sp; mm), (5) trabecular thickness (Tb.Th; mm), (6) structure model index (SMI), (7) tissue mineral density (TMD; mg/cm3), and (8) tissue mineral content (TMC; mg) (Bouxsein et al., 2010). Statistical analyses Data were analyzed by a one-way analysis of variance, and followed by Tukey’s multiple comparison test as a post-test to compare the group means if overall p50.05. Results were presented as mean ± SEM. Probability (p value) of 50.05 was considered to be statistically significant.
Figure 1. Effects of treatment with RDE or alendronate on (A) the bone mineral density (BMD) and (B) the bone mineral content (BMC) in the femurs of rats. Values are mean ± SEM, n ¼ 6. *p50.05, **p50.01 versus HLU; ##p50.01 versus CON.
Results Bone mineral content and density The BMD of the HLU group was significantly lower than that of the CON group (p50.01). Four weeks of treatment with RDE (500 mg/kg/d) and ALN significantly increased the BMD compared with the HLU vehicle group (p50.01) (Figure 1A). Similarly, the decrease in the BMC level induced by unloading was prevented by treatment with both RDE and ALN (p50.05 or p50.01) (Figure 1B). MicroCT analysis of trabecular bone Three-dimensional images of the distal femoral metaphysis with differences in trabecular microarchitecture among the four groups are presented in Figure 2. Analysis of the properties of trabecular bone revealed that HLU rats had significantly lower trabecular BV/TV, Conn.D, Tb.N, Tb.Th, TMD, and TMC (p50.01), as compared with the CON rats. In contrast, SMI and Tb.Sp in the HLU distal femoral were significantly increased as compared with the CON group (p50.01). Treatment of HLU rats with RDE and ALN could significantly reverse the changes in these parameters induced by hindlimb unloading (p50.01 or p50.05), and maintain the microarchitecture of trabecular bone in the distal femoral metaphysis (Figure 3). Biomechanical testing of femur To determine if RDE treatment could improve bone strength, three-point bending test of femur was performed. Femoral maximum stress, Young’s modulus, and maximum load were
significantly lower in HLU as compared with the CON (p50.01). Compared with the HLU vehicle group, 4 weeks of treatment with both ALN and RDE significantly increased the maximum stress, Young’s modulus, and maximum load (p50.01 or p50.05). Hindlimb unloading rats had no significant effect on bending stiffness and energy of the femur with/without the supplementation of RDE and ALN (Figure 4). Biochemical markers of bone metabolism Biochemical parameters in serum and urine for all groups studied are shown in Table 1. U-Ca/Cr and U-P/Cr levels were significantly increased in response to hindlimb unloading (p50.01). Nevertheless, treatments with both RDE and ALN prevented the unloading-induced increase in U-Ca/Cr and U-P/Cr levels (p50.01). In contrast, although the HLU groups had increased S-Ca and S-P excretion, the differences were not statistically significant as compared with those in the CON groups (p40.05). HLU-RDE and HLU-ALN groups had decreased S-Ca and S-P excretion; with the differences being not significant as compared with those in the HLU groups as well (p40.05). Urinary DPD/Cr, NTx, and CTx levels were significantly higher in the HLU group in compared with those in the CON group (p50.01). Both RDE and ALN treatments significantly suppressed the unloading-induced increase in the urinary DPD/Cr, NTx, and CTx levels (p50.05 or p50.01). Similarly, in comparison with the CON group, the serum TRAP level increased in the HLU group (p50.01).
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Figure 2. Representative sample from each group: 3D architecture of trabecular bone within the distal femoral metaphyseal region. Treatment of HLU rats with RDE or alendronate could significantly maintain the microarchitecture of trabecular bone in the distal femoral metaphysis.
The increase in the serum TRAP level induced by hindlimb unloading was prevented by the treatment with both RDE and ALN (p50.05). Serum BALP and OC levels decreased in the HLU group (p50.05 or p50.01), but they were restored to significantly higher levels in RDE treatment groups than those in the HLU group (p50.05 or p50.01). ALN treatments restored the BALP to a significantly higher level than in the HLU group (p50.05) as well, but there was no difference in the level of serum OC between the ALN group and the HLU group.
Discussion The strength and integrity of our bones depend on a delicate balance between bone resorption (by osteoclasts) and bone formation (by osteoblasts). This delicate balancing act becomes tipped in favor of osteoclasts so that bone resorption exceeds bone formation, rendering bones brittle, and prone to fracture (Rodan & Martin, 2000). The balance of bone resorption and formation is regulated not only by hormones and cytokines, but also by mechanical stimuli. Reduced mechanical loading of bones during space flight in microgravity environments has been shown to cause rapid bone loss (Kim et al., 2003; Vico et al., 1998). The prevention and treatment of microgravity-induced bone loss have drawn considerable attention. Radix Dipsaci has long been used as an analgesic, antiosteoporosis, and anti-aging agent in China for the therapy of low back pain, traumatic hematoma, and bone fractures
(Hung et al., 2006; Peng et al., 2010). The primary objective of this study was to provide direct evidence supporting the inhibitory effect of RDE on bone loss of rats exposed to hindlimb unloading. Alendronate was included in the study as a reference drug for the effect of bone modeling and remodeling. As expected, 4 week hindlimb unloading resulted in a significant decrease in the femoral BMD. Treatment with RDE led to a beneficial effect in the setting of unloading, as evidenced by increases in BMD, trabecular microarchitecture, and femoral strength values in the HLU-RDE group, which were higher than those in the HLU group. Furthermore, treatment with RDE not only led to a decrease in serum and urinary bone resorption markers (TRAP, NTx, CTx, and DPD) but also resulted in an increase in serum bone formation markers (BALP and OC). In addition, an increase in urinary Ca and P as well as a decrease in Ca and P absorption efficiency might contribute to the reduction of BMD (Gaumet et al., 1997). RDE could prevent the HLU-induced increase in urinary Ca and P. These effects of RDE and ALN treatment were consistent with the maintenance of bone mass by inhibiting bone remodeling in both groups. Although such inhibition would generally be considered as beneficial, biomechanical competence of bone may be compromised if bone remodeling is inhibited excessively (Ferretti et al., 1990). The three-point bending test was carried out to investigate the effects of RDE on the biomechanical properties of HLU rat femurs. We computed maximum load, stiffness, and energy as extrinsic property
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Figure 3. Effects of treatment with RDE or alendronate on microCT 3D parameters of trabecular bone in the distal femur of rats. Values are mean ± SEM, n ¼ 6. *p50.05, **p50.01.
indices, as well as Young’s modulus and maximum stress as intrinsic property indices. Results showed that daily treatment with RDE effectively promoted the intrinsic (Young’s modulus and maximum stress) and extrinsic properties (maximum load) of the femur. The preservation of trabecular bone architecture significantly promotes bone strength, and may be more important to the decrease of fracture risk than the improvement in bone mineral density (Mu¨ller et al., 2004). Our data revealed that RDE treatment could restore the BV/TV, Tb.N,
Tb.Th, TMD, TMC, and Conn.D, and decrease Tb.Sp parameters. SMI distinguishes rods from plates of bony trabeculae. SMI, 0 and 3, represents bone that consists purely of plate or rod-like structures, respectively. The results suggested a moderate but significant evolution of trabecular structure return from rods to mixed plates and rods forms by RDE treatment. Although far from conclusive, our observations suggest that RDE appears to beneficially affect bone metabolism based on the indicators of both bone formation and resorption.
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Figure 4. Effects of treatment with RDE or alendronate on bone biomechanical parameters in the femoral diaphysis of rats. Values are mean ± SEM, n ¼ 6. *p50.05, **p50.01. Table 1. Effects of treatment with RDE or alendronate on biochemical parameters in serum and urine of HLU rats. Parameters
CON
HLU
HLU-ALN
HLU-RDE
Urine
U-Ca/Cr (mM/mM) U-P/Cr (mM/mM) DPD/Cr (nM/mM) NTx (nM) CTx (mg/L)
0.74 ± 0.19 5.60 ± 0.16 177.45 ± 28.59 11.20 ± 1.26 4.13 ± 0.30
2.67 ± 0.42## 7.60 ± 0.59## 323.94 ± 27.07## 16.74 ± 0.87## 6.12 ± 1.26##
0.79 ± 0.08** 6.41 ± 0.42* 196.24 ± 18.14** 13.20 ± 0.80* 4.36 ± 0.45**
1.35 ± 0.18** 5.81 ± 0.31* 217.17 ± 19.94* 13.29 ± 0.91* 4.95 ± 0.52*
Serum
S-Ca ( mM) S-P (mM) Trap (ng/L) BALP (mg/L) OC (ng/L)
2.37 ± 0.07 1.69 ± 0.03 1.66 ± 0.19 270.41 ± 14.83 0.227 ± 0.008
2.48 ± 0.09 1.77 ± 0.02 2.87 ± 0.28## 217.17 ± 10.95# 0.178 ± 0.012##
2.36 ± 0.08 1.69 ± 0.06 2.37 ± 0.18 289.58 ± 17.49** 0.210 ± 0.008
2.37 ± 0.08 1.75 ± 0.02 1.84 ± 0.22* 300.29 ± 21.91** 0.224 ± 0.011*
Values are means ± SEM, n ¼ 6. *p50.05, **p50.01 versus HLU; #p50.05, ##p50.01 versus CON.
Several limitations of this study merit mention. The molecular mechanisms behind the results, the regulation of related proteins, the role of target genes, and signal pathways need further study. In addition, the experimental drug used in the present study was a traditional Chinese medicine complex preparation. Although the results demonstrated that the RDE compound drug treatment had a preventive effect on bone loss induced by microgravity, it has yet to be investigated which ingredient in the complex plays the key role and how it acts on the bone cells. Despite these limitations, this study yielded novel information about the ability of RDE to induce bone formation and reduce bone resorption in the situation of reduced mechanical loading. In conclusion, treatment with RDE induces a protective skeletal response in an established rodent model of microgravity-induced bone loss, such that unloaded animals treated with RDE had bone mineral density, microarchitecture, and mechanical strength values at or above the normally loaded HLU rats. Moreover, our data demonstrated that the boneprotective effects of RDE might be due to its direct influence
on bone formation and inhibition of osteoclast formation. The results suggested the therapeutic effect of RDE as an alternative supplement to be applied in the prevention and treatment of bone loss induced by simulated microgravity.
Declaration of interest The authors declare no conflict of interest. This work was financially supported by the National Natural Science Foundation of China (Grant no. 81202457) and the China Postdoctoral Science Foundation (Grant nos. 2012T50822 and 2011M501482).
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ORIGINAL ARTICLE
Treatment of Radix Dipsaci extract prevents long bone loss induced by modeled microgravity in hindlimb unloading rats Yinbo Niu1, Chenrui Li1, Yalei Pan1, Yuhua Li2,3, Xianghe Kong4, Shuo Wang1, YuanKun Zhai1, Xianglong Wu1, Wutu Fan1, and Qibing Mei1,2 1
Key Laboratory for Space Bioscience and Biotechnology, School of Life Sciences, Northwestern Polytechnical University, Xi’an, PR China, Department of Pharmacology, School of Pharmacy, Fourth Military Medical University, Xi’an, PR China, 3Department of Pharmacy, No. 422 Hospital of PLA, Zhanjiang, PR China, and 4Training and Teaching Research Office, Graduate School of Chang’an University, Xi’an, PR China
2
Abstract
Keywords
Context: Radix Dipsaci is a kidney tonifying herbal medicine with a long history of safe use for treatment of bone fractures and joint diseases in China. Previous studies have shown that Radix Dipsaci extract (RDE) could prevent bone loss in ovariectomized rats. Objective: This study investigates the effect of RDE against bone loss induced by simulated microgravity. Materials and methods: A hindlimb unloading rat model was established to determine the effect of RDE on bone mineral density and bone microarchitecture. Twenty-four male Sprague– Dawley rats were divided into four groups (n ¼ 6 per group): control (CON), hindlimb unloading with vehicle (HLU), hindlimb unloading treated with alendronate (HLU-ALN, 2.0 mg/kg/d), and hindlimb unloading treated with RDE (HLU-RDE, 500 mg/kg/d). RDE or ALN was administrated orally for 4 weeks. Results: Treatment with RDE had a positive effect on mechanical strength, BMD, BMC, bone turnover markers, and the changes in urinary calcium and phosphorus excretion. MicroCT analysis showed that RDE significantly prevented the reduction of the bone volume fraction, connectivity density, trabecular number, thickness, tissue mineral density, and tissue mineral content as well as improved the trabecular separation and structure model index. Discussion and conclusion: RDE was demonstrated to prevent the loss of bone mass induced by HLU treatment, which suggests the potential application of RDE in the treatment of microgravity-induced bone loss.
Bone density, bone strength, Dipsacus asperoides
Introduction A marked and potentially debilitating reduction in bone mass is the major skeletal problem after prolonged space flight and weightlessness (Collet et al., 1997; Smith et al., 2012). In the most severe forms of microgravity-induced bone loss, there is an approximately 2% decrease in bone mineral density in only 1 month, which roughly approximates that of a postmenopausal woman in 1 year (Riggs et al., 1998). Microgravity changes the metabolic environment of bone leading to site-specific alterations in bone remodeling. Bone formation is decreased and bone resorption is increased, which results in significant bone loss with a consequent increase in fracture risk (Smith et al., 2002). Over the past few years, several countermeasures have been examined to prevent bone loss during exposure to microgravity, including pharmacological therapy with bisphosphonates and calcitonin, and several active and passive exercise regimes, but
Correspondence: Qibing Mei, School of Life Sciences, Northwestern Polytechnical University, Xi’an 710072, PR China. Tel: +86 29 84779212. Fax: +86 29 84779212. E-mail: [email protected]
History Received 19 November 2013 Revised 10 January 2014 Accepted 1 April 2014 Published online 22 September 2014
positive outcomes have been limited (Czarnik & Vernikos, 1999; Derendorf, 1994). To date, the offered modalities do not show much success in preventing or alleviating bone loss in astronauts and cosmonauts (Nagaraja & Risin, 2013). The pathology remains a key concern, and the development of effective countermeasures is still a major task. Natural products have been shown to be excellent and reliable sources for the prevention of bone loss (Habold et al., 2011; Siu et al., 2013; Zhang et al., 2009, 2011). Radix Dipsaci, the dried root of Dipsacus asperoides C. Y. Cheng and T. M. Ai (Dipsacaceae), has long been used as an antiosteoporosis tonic and anti-aging agent in China. It has also been used for the treatment of lower back pain, traumatic hematoma, and bone fractures (Hung et al., 2006). Recent studies confirmed that Radix Dipsaci extract (RDE) could increase bone density and alter bone histomorphology in mice and rats (Liu et al., 2009; Wong et al., 2007). However, there is no direct evidence supporting RDE inhibitory effect on bone loss induced by microgravity. Thus, the current study aimed to determine the osteoprotective effect of RDE on biological indices of bone metabolism, including biochemical, bone mineral density (BMD), bone mineral content (BMC), biomechanical, and
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bone microparameters in the hindlimb unloading (HLU) rats (Hindlimb unloading rat model is extensively utilized as a simulated mechanical inactivity model under microgravity environment) (Morey-Holton & Globus, 1998).
Materials and methods Identification and preparation of Radix Dipsaci extract Radix Dipsaci was purchased from a local Chinese medicine store in 2011, and was identified morphologically, histologically, and chemically by Guo Yaowu, a pharmacist in laboratory for the control of drugs of Shaanxi Province. A voucher specimen including the identification and classification of the plant material was preserved in the Research Center of Clinic Pharmacology and Pharmacy of the Fourth Military Medical University (Xi’an, China). In brief, the crude plant was identified by using the thin-layer chromatography method. During the procedure, dipsacus saponin VI was used as a standard substance, and methanol as a developing solvent. Radix Dipsaci extract was prepared using the protocol for ethyl alcohol extraction (Liu et al., 2009). Briefly, the raw herbs (10 kg) were crushed into small pieces and extracted in 50 L of 60% ethanol for 2 h by heating and refluxing method for three times. The extracts were then collected, filtered, decompression concentrated, and lyophilized. At last, the powder with an extraction yield of 20% was obtained. Overview of study design Male Sprague–Dawley rats aged 12 weeks (Experimental Animal Center of the Fourth Military Medical University, Xi’an, China) were housed in a facility maintained at 22 C and with a 12/12 h light/dark cycle. During the experimental period, the rats were maintained on standard rodent chow (Animal Center of the Key Laboratory for Space Bioscience and Biotechnology, Xi’an, China) that contained 0.9% calcium and 0.7% phosphate, and filtered water available ad libitum. After 1 week of acclimatization to the keeping environment, rats were randomized into four groups: (n ¼ 6 per group): control (CON), hindlimb unloading with vehicle (HLU), hindlimb unloading treated with alendronate (HLUALN, 2.0 mg/kg/d) (Mosekilde et al., 2000; Qi et al., 2012), and hindlimb unloading treated with RDE (HLU-RDE, 500 mg/kg/d) (Liu et al., 2009). Animals were assigned to groups by total body bone mineral density and body mass in a manner to minimize differences between groups at baseline. For the CON group, rats were allowed to move freely with their four limbs (without hindlimb unloading). In groups HLU, HLU-ALN, and HLU-RDE, the tails of the rats were suspended so that their hindlimbs were unloaded according to the recommendations of Morey-Holton and Globus (2002). RDE or ALN was administrated orally for 4 weeks. Rats in both CON and HLU groups received equal volume of distilled water vehicle for four weeks. All tail-suspended rats were adjusted weekly to ensure that the hindlimb paws could not touch the ground. All animals were treated in compliance with the Guidance for Care and Use of Laboratory Animals with the approval of the Institutional Ethics Committee of the Fourth Military Medical University.
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Tissue collection and sample preparation A blood sample was collected by abdominal aorta puncture from each anesthetized rat at the end of the laparotomy and was centrifuged at 2000 rpm for 20 min to get the serum. Urine was collected over 24 h from fasted, individually housed rats in metabolic cages. Urine and serum samples were then stored at 80 C for biochemical determinations. Bone mineral content (BMC) and bone mineral density (BMD) were measured before rats euthanized by dual-energy X-ray absorptiometry (DXA). Femurs were harvested and cleaned off soft tissue. The right femur was prepared for biomechanical testing by wrapping in saline-soaked gauze and freezing at 20 C. The left femur was prepared for microCT in 10% neutral buffered formalin at 4 C for 48 h, and then transferred to 70% ethanol at 4 C. Assay for serum and urine chemistry Urine calcium (U-Ca), urine phosphorus (U-P), and creatinine (Cr) concentrations were measured by an automatic biochemical analyzer (Cobas Integra 400 plus, Roche Diagnostics, Basel, Switzerland) using the original kits from Roche Diagnostics (Indianapolis, IN). Serum calcium (S-Ca) and phosphorus (S-P) concentrations were analyzed by the same method used for the urine samples. Serum bone alkaline phosphatase (BALP), tartrate-resistant acid phosphatase (TRAP), osteocalcin (OC), urinary deoxypyridinoline (DPD), C-terminal crosslinked telopeptides of collagen type I (CTx), and N-terminal crosslinked telopeptides of collagen type I (NTx) concentrations were assessed using rat ELISA kits (Beijing Sino-uk Institute of Biological Technology, Beijing, China). Urinary excretion of Ca, P, and DPD was expressed as the ratio to Cr concentration (Ca/Cr; P/Cr; DPD/Cr). BMD and BMC analysis The BMC and BMD were measured by DXA (Lunar Prodigy Advance DXA, GE healthcare, Madison, WI) using the small laboratory animal scan mode (Pastoureau et al., 1995). The BMC and BMD of the whole femur were calculated automatically by the purpose-designed software (enCOREÔ 2006, GE Healthcare, Madison, WI). Biomechanical testing Bones were mechanically tested to failure via three-point bending using an MTS MiniBionix 858 testing apparatus (MTS Systems, Eden Prairie, MN). Each specimen was placed on two supports spaced 20 mm apart, and a load was applied to the bone midway between the supports at a deformation rate of 2 mm/min until the occurrence of fracture. The central loading point was displaced, and the load and displacement were recorded until the specimen was broken. All force and displacement data were collected for later evaluation, which were performed after measurement of the inner and outer sizes (i.e., width and height) of the femur at the point of fracture. The calculation of the biomechanical parameters was based on the formulas described previously (Brodt et al., 1999; Turner & Burr, 1993).
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MicroCT analysis The distal metaphysis of each femur was scanned using a desktop eXplore Locus SP Pre-Clinical Specimen microCT (GE Healthcare, Madison, WI). Three-dimensional (3D) image data were acquired with a voxel size of 12 lm in all spatial directions with microCT Evaluation Program (V5.0A) (Laib et al., 2000). Trabecular bone was separated from cortical bone by free drawing regions of interests with the Micro-View program (GE Healthcare, Madison, WI) and a multiple IntelÕ processor-based microCT workstation provided with the scanner. The regions of interest, which were located 1.5 mm from the metaphyseal line below, were chosen for the data analysis. Morphologic measurements were performed, and the following 3D parameters were obtained for the trabecular bone: (1) relative bone volume (BV/TV; %), (2) connectivity density (Conn.D; 1/mm3), (3) trabecular number (Tb.N; 1/mm), (4) trabecular separation (Tb.Sp; mm), (5) trabecular thickness (Tb.Th; mm), (6) structure model index (SMI), (7) tissue mineral density (TMD; mg/cm3), and (8) tissue mineral content (TMC; mg) (Bouxsein et al., 2010). Statistical analyses Data were analyzed by a one-way analysis of variance, and followed by Tukey’s multiple comparison test as a post-test to compare the group means if overall p50.05. Results were presented as mean ± SEM. Probability (p value) of 50.05 was considered to be statistically significant.
Figure 1. Effects of treatment with RDE or alendronate on (A) the bone mineral density (BMD) and (B) the bone mineral content (BMC) in the femurs of rats. Values are mean ± SEM, n ¼ 6. *p50.05, **p50.01 versus HLU; ##p50.01 versus CON.
Results Bone mineral content and density The BMD of the HLU group was significantly lower than that of the CON group (p50.01). Four weeks of treatment with RDE (500 mg/kg/d) and ALN significantly increased the BMD compared with the HLU vehicle group (p50.01) (Figure 1A). Similarly, the decrease in the BMC level induced by unloading was prevented by treatment with both RDE and ALN (p50.05 or p50.01) (Figure 1B). MicroCT analysis of trabecular bone Three-dimensional images of the distal femoral metaphysis with differences in trabecular microarchitecture among the four groups are presented in Figure 2. Analysis of the properties of trabecular bone revealed that HLU rats had significantly lower trabecular BV/TV, Conn.D, Tb.N, Tb.Th, TMD, and TMC (p50.01), as compared with the CON rats. In contrast, SMI and Tb.Sp in the HLU distal femoral were significantly increased as compared with the CON group (p50.01). Treatment of HLU rats with RDE and ALN could significantly reverse the changes in these parameters induced by hindlimb unloading (p50.01 or p50.05), and maintain the microarchitecture of trabecular bone in the distal femoral metaphysis (Figure 3). Biomechanical testing of femur To determine if RDE treatment could improve bone strength, three-point bending test of femur was performed. Femoral maximum stress, Young’s modulus, and maximum load were
significantly lower in HLU as compared with the CON (p50.01). Compared with the HLU vehicle group, 4 weeks of treatment with both ALN and RDE significantly increased the maximum stress, Young’s modulus, and maximum load (p50.01 or p50.05). Hindlimb unloading rats had no significant effect on bending stiffness and energy of the femur with/without the supplementation of RDE and ALN (Figure 4). Biochemical markers of bone metabolism Biochemical parameters in serum and urine for all groups studied are shown in Table 1. U-Ca/Cr and U-P/Cr levels were significantly increased in response to hindlimb unloading (p50.01). Nevertheless, treatments with both RDE and ALN prevented the unloading-induced increase in U-Ca/Cr and U-P/Cr levels (p50.01). In contrast, although the HLU groups had increased S-Ca and S-P excretion, the differences were not statistically significant as compared with those in the CON groups (p40.05). HLU-RDE and HLU-ALN groups had decreased S-Ca and S-P excretion; with the differences being not significant as compared with those in the HLU groups as well (p40.05). Urinary DPD/Cr, NTx, and CTx levels were significantly higher in the HLU group in compared with those in the CON group (p50.01). Both RDE and ALN treatments significantly suppressed the unloading-induced increase in the urinary DPD/Cr, NTx, and CTx levels (p50.05 or p50.01). Similarly, in comparison with the CON group, the serum TRAP level increased in the HLU group (p50.01).
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Figure 2. Representative sample from each group: 3D architecture of trabecular bone within the distal femoral metaphyseal region. Treatment of HLU rats with RDE or alendronate could significantly maintain the microarchitecture of trabecular bone in the distal femoral metaphysis.
The increase in the serum TRAP level induced by hindlimb unloading was prevented by the treatment with both RDE and ALN (p50.05). Serum BALP and OC levels decreased in the HLU group (p50.05 or p50.01), but they were restored to significantly higher levels in RDE treatment groups than those in the HLU group (p50.05 or p50.01). ALN treatments restored the BALP to a significantly higher level than in the HLU group (p50.05) as well, but there was no difference in the level of serum OC between the ALN group and the HLU group.
Discussion The strength and integrity of our bones depend on a delicate balance between bone resorption (by osteoclasts) and bone formation (by osteoblasts). This delicate balancing act becomes tipped in favor of osteoclasts so that bone resorption exceeds bone formation, rendering bones brittle, and prone to fracture (Rodan & Martin, 2000). The balance of bone resorption and formation is regulated not only by hormones and cytokines, but also by mechanical stimuli. Reduced mechanical loading of bones during space flight in microgravity environments has been shown to cause rapid bone loss (Kim et al., 2003; Vico et al., 1998). The prevention and treatment of microgravity-induced bone loss have drawn considerable attention. Radix Dipsaci has long been used as an analgesic, antiosteoporosis, and anti-aging agent in China for the therapy of low back pain, traumatic hematoma, and bone fractures
(Hung et al., 2006; Peng et al., 2010). The primary objective of this study was to provide direct evidence supporting the inhibitory effect of RDE on bone loss of rats exposed to hindlimb unloading. Alendronate was included in the study as a reference drug for the effect of bone modeling and remodeling. As expected, 4 week hindlimb unloading resulted in a significant decrease in the femoral BMD. Treatment with RDE led to a beneficial effect in the setting of unloading, as evidenced by increases in BMD, trabecular microarchitecture, and femoral strength values in the HLU-RDE group, which were higher than those in the HLU group. Furthermore, treatment with RDE not only led to a decrease in serum and urinary bone resorption markers (TRAP, NTx, CTx, and DPD) but also resulted in an increase in serum bone formation markers (BALP and OC). In addition, an increase in urinary Ca and P as well as a decrease in Ca and P absorption efficiency might contribute to the reduction of BMD (Gaumet et al., 1997). RDE could prevent the HLU-induced increase in urinary Ca and P. These effects of RDE and ALN treatment were consistent with the maintenance of bone mass by inhibiting bone remodeling in both groups. Although such inhibition would generally be considered as beneficial, biomechanical competence of bone may be compromised if bone remodeling is inhibited excessively (Ferretti et al., 1990). The three-point bending test was carried out to investigate the effects of RDE on the biomechanical properties of HLU rat femurs. We computed maximum load, stiffness, and energy as extrinsic property
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Figure 3. Effects of treatment with RDE or alendronate on microCT 3D parameters of trabecular bone in the distal femur of rats. Values are mean ± SEM, n ¼ 6. *p50.05, **p50.01.
indices, as well as Young’s modulus and maximum stress as intrinsic property indices. Results showed that daily treatment with RDE effectively promoted the intrinsic (Young’s modulus and maximum stress) and extrinsic properties (maximum load) of the femur. The preservation of trabecular bone architecture significantly promotes bone strength, and may be more important to the decrease of fracture risk than the improvement in bone mineral density (Mu¨ller et al., 2004). Our data revealed that RDE treatment could restore the BV/TV, Tb.N,
Tb.Th, TMD, TMC, and Conn.D, and decrease Tb.Sp parameters. SMI distinguishes rods from plates of bony trabeculae. SMI, 0 and 3, represents bone that consists purely of plate or rod-like structures, respectively. The results suggested a moderate but significant evolution of trabecular structure return from rods to mixed plates and rods forms by RDE treatment. Although far from conclusive, our observations suggest that RDE appears to beneficially affect bone metabolism based on the indicators of both bone formation and resorption.
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Figure 4. Effects of treatment with RDE or alendronate on bone biomechanical parameters in the femoral diaphysis of rats. Values are mean ± SEM, n ¼ 6. *p50.05, **p50.01. Table 1. Effects of treatment with RDE or alendronate on biochemical parameters in serum and urine of HLU rats. Parameters
CON
HLU
HLU-ALN
HLU-RDE
Urine
U-Ca/Cr (mM/mM) U-P/Cr (mM/mM) DPD/Cr (nM/mM) NTx (nM) CTx (mg/L)
0.74 ± 0.19 5.60 ± 0.16 177.45 ± 28.59 11.20 ± 1.26 4.13 ± 0.30
2.67 ± 0.42## 7.60 ± 0.59## 323.94 ± 27.07## 16.74 ± 0.87## 6.12 ± 1.26##
0.79 ± 0.08** 6.41 ± 0.42* 196.24 ± 18.14** 13.20 ± 0.80* 4.36 ± 0.45**
1.35 ± 0.18** 5.81 ± 0.31* 217.17 ± 19.94* 13.29 ± 0.91* 4.95 ± 0.52*
Serum
S-Ca ( mM) S-P (mM) Trap (ng/L) BALP (mg/L) OC (ng/L)
2.37 ± 0.07 1.69 ± 0.03 1.66 ± 0.19 270.41 ± 14.83 0.227 ± 0.008
2.48 ± 0.09 1.77 ± 0.02 2.87 ± 0.28## 217.17 ± 10.95# 0.178 ± 0.012##
2.36 ± 0.08 1.69 ± 0.06 2.37 ± 0.18 289.58 ± 17.49** 0.210 ± 0.008
2.37 ± 0.08 1.75 ± 0.02 1.84 ± 0.22* 300.29 ± 21.91** 0.224 ± 0.011*
Values are means ± SEM, n ¼ 6. *p50.05, **p50.01 versus HLU; #p50.05, ##p50.01 versus CON.
Several limitations of this study merit mention. The molecular mechanisms behind the results, the regulation of related proteins, the role of target genes, and signal pathways need further study. In addition, the experimental drug used in the present study was a traditional Chinese medicine complex preparation. Although the results demonstrated that the RDE compound drug treatment had a preventive effect on bone loss induced by microgravity, it has yet to be investigated which ingredient in the complex plays the key role and how it acts on the bone cells. Despite these limitations, this study yielded novel information about the ability of RDE to induce bone formation and reduce bone resorption in the situation of reduced mechanical loading. In conclusion, treatment with RDE induces a protective skeletal response in an established rodent model of microgravity-induced bone loss, such that unloaded animals treated with RDE had bone mineral density, microarchitecture, and mechanical strength values at or above the normally loaded HLU rats. Moreover, our data demonstrated that the boneprotective effects of RDE might be due to its direct influence
on bone formation and inhibition of osteoclast formation. The results suggested the therapeutic effect of RDE as an alternative supplement to be applied in the prevention and treatment of bone loss induced by simulated microgravity.
Declaration of interest The authors declare no conflict of interest. This work was financially supported by the National Natural Science Foundation of China (Grant no. 81202457) and the China Postdoctoral Science Foundation (Grant nos. 2012T50822 and 2011M501482).
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