Du-zhong (Eucommia ulmoides) prevents disuse-induced osteoporosis in hind limb suspension rats.

The American Journal of Chinese Medicine, Vol. 42, No. 1, 143–155 © 2014 World Scientific Publishing Company Institute for Advanced Research in Asian Science and Medicine DOI: 10.1142/S0192415X14500104

Am. J. Chin. Med. 2014.42:143-155. Downloaded from www.worldscientific.com by CHINESE UNIVERSITY OF HONG KONG on 12/28/14. For personal use only.

Du-Zhong (Eucommia ulmoides) Prevents Disuse-Induced Osteoporosis in Hind Limb Suspension Rats Yalei Pan,*,† Yinbo Niu,*,† Chenrui Li,*,† Yuankun Zhai,*,† Rong Zhang,‡ Xin Guo§ and Qibing Mei*,†,‡ †Key

*School of Life Science Laboratory for Space Biosciences and Biotechnology Northwestern Polytechnical University Xi’an 710072, China

‡

Department of Pharmacology School of Pharmacy Fourth Military Medical University Xi’an 710032, China

§Department

of Microbiology and Molecular Cell Biology Eastern Virginia Medical School Norfolk, VA 23507, USA

Abstract: Du-Zhong has a long history of being used in traditional Chinese formulas to treat bone related diseases. The objective of the present study is to systematically investigate the effects of Du-Zhong cortex extract (DZCE) on disuse-induced osteoporosis. Rats were randomly divided into four groups, and three groups were treated with hind limb suspension (HLS). Control and HLS group received deionized distilled water, while the other two groups received alendronate (2.0 mg/kg/day) and DZCE (300 mg/kg/day) respectively by intragastric gavage for six weeks (two weeks prior to and during the four weeks of HLS). Dualenergy X-ray absorptiometry, assay of biochemical markers, and three-point bending test were employed to determine the effect of various treatments on bone mass, turnover, and strength. The trabecular bone microarchitecture was assessed by microCT analysis. DZCE could effectively prevent the bone loss induced by HLS, which was indicated by decreased levels of bone turnover markers as well as the changes in urinary calcium and phosphorus. The DZCE treatment also enhanced the biomechanical strength of bone and prevented the deterioration of trabecular bone microarchitecture. DZCE administration was able to prevent

Correspondence to: Dr. Qibing Mei, School of Life Science, Northwestern Polytechnical University, 127 West Youyi Road, Xi’an 710072, China. Tel: (þ86) 29-8846-0543, Fax: (þ86) 29-8846-0543, E-mail: qbmei@nwpu. edu.cn

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Y. PAN et al. disuse-induced osteoporosis by regulating the bone metabolism, suggesting that DZCE could be used as an alternative therapy for the prevention of disuse-induced osteoporosis. Keywords: Hind Limb Suspension; Du-Zhong Cortex Extract; Disuse-Induced Osteoporosis; Mechanical Testing; Biochemical Assay; MicroCT Analysis.


Introduction The removal of routine mechanical loading would induce deleterious consequences on bone integrity. In humans, the loss of bone density occurs very rapidly in conditions of weightlessness and immobilization, and the incidence of disuse-induced osteoporosis is also relatively high in such conditions (Zerwekh et al., 1998; Vico et al., 2000). Prolonged exposure to a microgravity environment leads to bone loss at an alarming rate of approximately 0.5–2% per month in the load-bearing bones (Collet et al., 1997; Lang et al., 2004). In patients after scoliosis surgery, prolonged bed rest generates a spinal bone loss rate of 2% per week (Hansson et al., 1975). In addition, bed rest of patients with low backache due to protrusion of a lumbar intervertebral disk resulted in a spinal bone loss rate of 1% per week (Krølner and Toft, 1983). Due to the high cost and unavailability of experiments during a space mission, hind limb suspended (HLS) rats, a ground based animal model, has been well established and used to mimic the effects of prolonged bed rest and microgravity on bones, in which the chronic weightless bearing and reduction in hindlimb movement could be reproduced (Turner et al., 2001; Giangregorio and Blimkie, 2002; Morey-Holton and Globus, 2002). The dried bark of Eucommia ulmoides Oliv., also called Du-Zhong or Tu-Chung, is a medicinal herb and has been used in traditional medicine in China, Japan, and Korea with various pharmacological effects. Du-Zhong has been listed in Chinese Pharmacopoeia with the effects of reinforcing the muscles and lungs, lowering blood pressure, preventing miscarriages, improving the tone of liver and kidneys, and increasing longevity (Li et al., 1983, 2013; Huang, 1993; Chinese Pharmacopoeia Commission, 2010; Kim et al., 2012). It has been confirmed in a mice model that Du-Zhong had neuroprotective ability and ameliorated hyperglycemia and hyperlipidemia (Park et al., 2006; Kwon et al., 2011). In the theories of traditional Chinese medicine (TCM), the kidney is responsible for the nourishment of bone. It is recorded in the famous Chinese ancient herbal medicine books “Ben Cao Gang Mu” and “Shen Nong Ben Cao Jing” that Du-Zhong is a kidney-tonifying herb, and has the efficacy of strong bones. Du-Zhong is among the third most frequently used components in 260 traditional Chinese formulas to treat osteoporosis (Xu, 2009). A number of in vitro and in vivo studies demonstrated that Du-Zhong had the osteoprotective effects. Serum containing Du-Zhong can induce differentiation of bone mesenchymal stem cells to osteoblast (Zeng et al., 2009), and the crude water extract of Du-Zhong promoted the osteoblast-like cells UMR106 proliferation (Wang et al., 2000). More importantly, Du-Zhong prevented ovariectomy-induced osteoporosis in vivo (Zhang et al., 2009, 2012). However, there is no investigation of the beneficial effect of Du-Zhong

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to prevent disuse-induced osteoporosis yet. Therefore, the aim of the present study is to evaluate the anti-osteoporotic effect of Du-Zhong cortex extract (DZCE) in HLS rat model. Materials and Methods


Preparation of Du-Zhong Cortex Extract (DZCE) Dried Du-Zhong (Eucommia ulmoides Oliv.) cortex was purchased from a local herbal drug store in Xi’an, China, and was identified morphologically, histologically, and chemically according to the criteria of Chinese Pharmacopoeia. DZCE was prepared according to the protocol for ethyl alcohol extraction by Tiancheng Drugs and Bio-engineering Co. (Xi’an, China). In brief, dry Du-Zhong cortex (10 kg) was homogenized to a fine powder, then extracted in 100 L of 60% ethanol for 4 h by heating and refluxing method three times. The extracts were filtered through Whatman No. 2 filter paper, then concentrated by rotary evaporator and spray-dried by a spray dryer, resulting in 1.05 kg of DZCE. DZCE was identified using high performance liquid chromatography (HPLC) method with use of pinoresinol diglucoside as standard substance. The mobile phase was methanol: H2 O ¼ 25 : 75, stationary phase was Octadecylsilane bonded silica, and DZCE was detected by UV-Vis detector in 277 nm. The detected result showed that pinoresinol diglucoside was 1.86% of DZCE. Animals and Treatments Thirty-two 3-month-old Sprague–Dawley specific germfree male rats (SIPPR-BK Experimental Animal Ltd., China; body weight, 296 10 g) were housed in a facility maintained at 22 C and with a 12 h light/dark cycle. During the experimental period, the rats were maintained on standard rodent chow (Animal Center of the Fourth Military Medical University, Xi’an, China) that contained 0.9% calcium and 0.7% phosphate, and filtered water available ad libitum. All animal experimental procedures were approved by the institutional animal care and use committee. The rats were randomly divided into four groups, eight were non-HLS treated as a control. The other three groups were treated by HLS, in which rats were fixed with a tail harness which raised their hind limbs 4 cm off the cage floor by a 30 head-down angle (Morey-Holton and Globus, 2002) continuously for four weeks. Tail suspension was performed on rat anesthetized with sodium pentobarbital. The rats in the control and one of the HLS groups were administered deionized distilled water. The other two HLS groups received alendronate (Aln, 2.0 mg/kg/day) and DZCE (300 mg/kg/day), respectively. According to the Human Rat Equivalent Dose Conversion Principle (Huang et al., 2004), the experimental doses of DZCE and alendronate were equivalent to the corresponding clinical prescription dose for a 60-kg human subject. DZCE or alendronate was administrated orally through a custommade stomach tube for six weeks, two weeks before HLS and four weeks after HLS. Body weight of the rats was recorded weekly. Before the last day of treatment, urine was collected over 24 h after the rats were fasted for 12 h in metabolic cages. Blood samples were collected by cardiac puncture on the day of sacrifice and serum were

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separated by centrifugation at 2000 rpm for 20 min. Urine and serum samples were then stored at 20 C until further analysis. The right femur was prepared for imaging and biomechanical testing by wrapping it 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 hours, and then transferred to 70% ethanol at 4 C. All animals were treated in compliance with the Guide for Care and Use of Laboratory Animals with the approval of the Institutional Ethics Committee of the Fourth Military Medical University.


Bone Mineral Density (BMD) Analysis In vivo bone mineral density (BMD) of the whole right femur was measured by DXA (Lunar Prodigy Advance DXA, GE healthcare, Madison, WI, USA) using the small laboratory animal scan mode (Pastoureau et al., 1995). Rats were anesthetized with sodium pentobarbital introduced intraperitoneally and positioned prone on the DXA scanning table. The BMD of the whole femur was calculated automatically by purpose-designed software (enCORETM 2006, GE Healthcare, Madison, WI, USA). Biochemistry Assay for Serum and Urine Serum calcium (S-Ca) and phosphorus (S-P) concentrations were measured by an automatic biochemical analyzer (Cobas Integra 400 plus, Roche Diagnostics, Basel, Switzerland) using the original kits from Roche Diagnostics (IN, USA). Urine calcium (U-Ca), Urine phosphorus (U-P) and creatinine (Cr) concentrations were analyzed by the same method used for the serum samples. Serum bone alkaline phosphatase (BALP), tartrateresistant 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 measured using rat ELISA kits (Beijing sino-uk institute of Biological Technology, China). Urinary excretion of Ca and P were both expressed as the ratio to Cr concentration (Ca/Cr; P/Cr). Mechanical Testing Prior to mechanical testing, the right femurs were slowly thawed and held at room temperature on the day of test, the length of the femurs (distance from intermalleolar to intercondylar region) was measured with a micrometer and the middle of the diaphysis was determined. The intact femur then was placed in the material testing machine on two supports separated by a distance of 20 mm and load was applied to the middle of the diaphysis, thus creating a threepoint bending test. The biomechanical quality of the left femoral diaphysis was determined using 858 Mini Bionix material testing machine (MTS, Eden Prairie, Minnesota, USA) at a speed of 2 mm/minute. The central loading point was displaced, and the load and displacement were recorded until the specimen was broken. From the load–deformation curve, maximum load (ultimate strength, Fmax), stiffness (slope of the linear part of the curve representing elastic deformation), energy absorption


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(area under the curve, Wabs), maximum stress (Fmax/cross-sectional area, max) and Young’s modulus (maximum slope of the stress-strain curve, E) were obtained.


MicroCT Analysis The distal metaphysis of each femur was scanned using a desktop explore Locus SP PreClinical Specimen microCT (GE Healthcare, Madison, WI, USA). Three-dimensional (3D) image data were acquired with a voxel size of 12 m in all spatial directions with the 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, USA) and a multiple Intel® processor-based microCT workstation provided with the scanner. The regions of interest, which were located 1.5 mm from the metaphyseal line and the 100 continuous slices below, were chosen for 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), (3) trabecular number (Tb.N), (4) trabecular thickness (Tb.Th), (5) structure model index (SMI). Statistical Analysis All data are presented as mean S.D. A one-way ANOVA with LSD post hoc test was used to detect significant differences between groups. A probability level of less than 0.05 was considered significant. Statistical analysis was performed using SPSS 16.0 software. Results Body Weights The body weights of rats in all groups had similar increase rate during the first two weeks, however the body weight gradually decreased, except for the control group, from the third week when the animals had been treated with HLS (Fig. 1). In the sixth week, the body

Figure 1. Body weight changes in the four groups of rats. The HLS, Aln and DZCE group treated with hind limb suspension since the 3rd week. Values are meansS.D., n ¼ 8, *p < 0:05, **p < 0:01 vs. HLS.


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Figure 2. Bone mineral density (BMD) in femurs of rats. Values are means S.D., n ¼ 8, **p < 0:01 vs. HLS, #p < 0:05 vs. control.

weight of control group was significantly higher than that of the HLS group ( p < 0:01), whereas the body weight was not significantly different between the Aln and DZCE groups. BMD Evaluation The results of BMD evaluation are shown in Fig. 2. In the sixth week, the BMD of the HLS group was significantly lower than that of the control group ( p < 0:01). Compared with the HLS group, the BMDs in Aln and DZCE-treatment groups were increased by 121% and 132% respectively ( p < 0:01). Biochemical Assay Serum and urinary chemistries under various treatments are shown in Table 1. HLS significantly increased urinary Ca ( p < 0:01) and P ( p < 0:05) levels compared with the control group. While the concentration of serum Ca and P was not significantly changed among all groups, both urinary Ca and inorganic P concentrations in Aln and DZCE groups Table 1. Effects of DZCE on the Levels of Biochemical Parameters in Serum and Urine of HLS Rats Parameters U-Ca/Cr(mmol/mmol) U-P/Cr(mmol/mmol) S-Ca(mmol/L) S-P(mmol/L) BALP(g/L) Trap(ng/L) OC (ng/L) DPD(g/L) CTx(nmol/L) NTx(nmol/L)

Control 0.76 0.29 5.52 0.37 2.48 0.17 1.70 0.11 269.81 34.39 1.88 0.23 0.237 0.015 235.54 18.67 4.90 0.59 12.04 1.92

HLS 2.53 0.43 ## 7.13 0.45 # 2.31 0.25 1.72 0.09 218.24 27.56 ## 2.33 0.71 ## 0.177 0.019 # 391.78 17.91 ## 6.06 0.73 ## 17.11 1.97 ##

Note: Values are means S.D., n ¼ 8, *p < 0:05, **p < 0:01 vs. HLS;

Aln

DZCE

1.58 0.47* 6.51 0.61* 2.40 0.27 1.67 0.15 223.72 29.79 1.92 0.51** 0.180 0.020 367.59 19.54* 5.99 0.45* 14.78 1.74**

1.90 0.32* 6.47 0.53* 2.45 0.32 1.78 0.10 245.68 22.45* 2.12 0.56* 0.212 0.022* 329.62 21.88** 6.02 0.61* 13.25 1.63*

#p

< 0:05,

## p

< 0:01 vs. Control.


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Table 2. Effects of DZCE on Bone Biomechanical Parameters in the Femoral Diaphysis of Rats Parameters

Control

HLS

Aln #

Energy (J) 85.62 7.32 77.47 4.69 ## Young’s Mudulus (MPa) 3443.52 242.89 2342.69 178.64 ## Stiffness (N/mm) 165.19 19.63 97.89 16.74 ## Maximum Stress (MPa) 89.19 14.29 64.29 7.72 ## Maximum Load (N) 111.98 10.75 74.75 9.76

74.97 4.45 76.42 2.95 2870.88 211.19* 3165.38 258.44** 125.38 17.59* 96.48 18.69 63.18 7.84 72.61 12.26* 80.79 9.78* 93.75 9.18**

Note: Values are means S.D., n ¼ 8, *p < 0:05, **p < 0:01 vs. HLS;


DZCE

#p

< 0:05,

## p

< 0:01 vs. Control.

were significantly decreased as compared with the HLS group ( p < 0:05). Similarly, Trap, DPD, CTx and NTx levels were significant increased in the HLS group ( p < 0:01), while they were significantly reversed by both the Aln and DZCE treatments ( p < 0:01 or p < 0:05). The serum level of BALP and OC was significantly decreased in the HLS group ( p < 0:01 or p < 0:05). The DZCE, but not the Aln treatments, significantly increased BALP and OC levels ( p < 0:05), as compared with the HLS control group ( p > 0:05). Biomechanical Test The results of the mechanical testing of excised femora from rats are presented in Table 2. All the parameters significantly decreased in the HLS group as compared with the control group ( p < 0:05 or 0.01). Aln treatment could markedly prevent the HLS-induced decrease in the Young’s mudulus, stiffness and maximum load ( p < 0:05). Meanwhile, DZCE administrations significantly prevented the Young’s mudulus, maximum load and maximum stress decrease induced by HLS ( p < 0:05). MicroCT Analysis Three-dimensional (3D), renderings of the trabecular bone compartment as imaged by microCT are shown in Fig. 3. Further analysis of the properties of trabecular bone at the tibial proximal metaphysis is presented in Table 3. HLS led to the deterioration of trabecular bone microarchitecture as demonstrated by the reduction of Conn.D, Tb.N and Tb.Th

(A)

(B)

(C)

(D)

Figure 3. Representative 3D images of architecture of trabecula bone within the distal metaphyseal femur region under various treatments. (a) Control, (b) HLS, (c) Aln and (d) DZCE.

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Control 2.26 0.32 39.84 2.27 0.17 0.04 0.06 0.02 2.63 0.28

HLS #

2.82 0.37 ## 0.29 0.09 ## 0.04 0.02 0.04 0.02 ## 0.75 0.14

Aln

DZCE

1.35 0.28** 20.66 9.68** 0.17 0.06** 0.07 0.02 2.24 0.97**

2.25 0.45* 34.02 9.08** 0.25 0.05** 0.059 0.02 2.72 0.39**


Note: Values are means S.D., n ¼ 8, *p < 0:05, **p < 0:01 vs. HLS; vs. Control.

#p

< 0:05,

## p

< 0:01

( p < 0:01). In contrast, SMI and Tb.Sp in the HLS vertebra were significantly increased as compared with the control ( p < 0:01). Aln and DZCE administration significantly reversed these defects. Discussion Osteoporosis is a degenerative bone disease that is developed with reduced estrogen levels during and after menopause or induced by long periods of disuse (Brouwers et al., 2009). Although both ovariectomy and disuse deteriorate bone, the actions of ovariectomy and loading on bone structure are independent and in a different manner (Pajamäki et al., 2008; Maïmoun et al., 2012). The bone losses in HLS rats are much more rapid and dramatic than those in ovariectomized rats (Bagi and Miller, 1994; Brouwers et al., 2009). The preventive ability of Du-Zhong cortex or leaf on ovariectomy-induced osteoporosis has been confirmed (Zhang et al., 2009, 2012). To systematically evaluate the efficacy of DZCE on the treatment of osteoporosis, the HLS rat model was used in the present study. Bisphosphonate (alendronate, Aln) was chosen as a reference drug on the effect of bone modeling and remodeling. HLS treatment caused a significant decrease in body weight, which was similar to the previously published study (Qi et al., 2012). The lower body weight in the HLS treatment group might be due to both disuse and lower food intake (Akhter et al., 2011). There was no significant change of the organ index between the DZCE group and control group, which preliminarily indicated that DZCE administration at the current dosage was safe and not toxic to rats. As expected, four weeks of HLS treatment resulted in a significant decrease in the femoral BMD. Both Aln and DZCE treatment could significantly reverse the BMD decrease. Since risedronate treatment could partially prevent bone loss during long-term disuse (Li et al., 2005), our data further indicated that the DZCE administration had more efficacy in suppressing the decrease of BMD than the Aln. This loss of bone mass was accompanied by a significant increase in bone remodeling, which was demonstrated by the changes in the bone turnover markers (Smith et al., 2005). The levels of four bone turnover markers (Trap, DPD, CTx and NTx) tended to increase in the HLS group as compared with the control group, while the BALP and OC was decreased. Treatment with DZCE reversed these markers changed during HLS, which were



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shown by the decreases in the levels of Trap, DPD, CTx and NTx, and increases in BALP and OC levels. Although the levels of Trap, DPD, CTx and NTx were reversed in the Alntreated group, BALP and OC levels failed to increase as compared with the HLS control group. In other words, the change of bone turnover markers in DZCE-treated group was different from the Aln-treated, since the bisphosphonates are incapable of directly stimulating bone formation (Li et al., 2005). These results preliminarily indicated that DZCE prevented the BMD decrease in HLS rats by attenuating bone remodeling that could be of benefit to bone formation. BMD is only a surrogate measurement of bone strength, and increased BMD alone has recently been shown to under-estimate the reduction in fracture risk with bone antiresorptive therapy (Bouxsein, 2003; Delmas and Seeman, 2004). The assay of the mechanical strength of bones might be more relevant. Three-point bending tests on femoral diaphysis are necessary to evaluate the true impact of a treatment on the biomechanical quality of bone. The actual effect of treatment on biomechanical quality can only be fully evaluated if the structural biomechanical parameters (i.e., maximum load and stiffness) are corrected by the changes in geometric properties of the femur midshaft, yielding material biomechanical parameters, such as maximum stress and Young’s mudulus (Ederveen et al., 2001). We found that DZCE administration significantly prevented the decrease of maximum load, maximum stress and Young’s mudulus induced by HLS treatment. The preservation of trabecular microarchitecture might contribute more to the maintenance of bone strength and may reduce fracture risk beyond BMD (Ulrich et al., 1999). Trabecular loss in structure is a normal bone response to disuse (Milstead et al., 2004; Stevens et al., 2006). Imbalance between bone reabsorption and formation contributes to the loss of cancellous bone during HLS, and it is considered to be a result of a reduction in bone formation rather than an increase in reabsorption (Jee et al., 1983; Vico et al., 1988), while the others suggest that the direct effect of accelerate bone loss caused disuse-induced bone loss (Saxena et al., 2011; Nabavi et al., 2011). The effects of DZCE on femoral microarchitecture were investigated by scanning with microCT. The results showed increased trabecular BV/TV, Tb.N, Tb.Th and Conn.D, and decreased Tb. Sp in rats treated with DZCE administration compared with HLS group rats. According to these data analysis and the histomorphometric change, DZCE-treated rats exhibited reductions in bone reabsorption. SMI distinguishes between rods and plates of bony trabeculae. SMI, 0 and 3, represents bone that consists purely of plate or rod-like structures (Wronski et al., 1989). DZCE treatment significantly prevented the trabecular change to the rod-like structures in the HLS model. In the disuse-induced osteoporosis, the lack of stress or load stimulus is the primary cause for bone loss. Mechanical loading induces bone formation through activation of estrogen receptor alpha and this receptor is up-regulated by estrogen therapy (Lee and Lanyon, 2004). Estrogen receptor alpha is a transcription factor for osteoblasts (Riggs et al., 2002); it regulates gene expression when activated by estrogen or phytoestrogens. When the estrogen receptor alpha gene is deficient, stress or load stimulus loses its effectiveness of inducing osteoblasts proliferation (Lee et al., 2004). Strain and estrogen are additive for inducing the proliferation of osteoblasts from male rats (Damien et al., 2000).


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One possible mechanism for DZCE to prevent the disuse-induced osteoporosis might be that the phytoestrogens compounds such as lignans and flavonoids in Du-Zhong are beneficial to bone formation through estrogen receptor alpha. Our previous study showed that total lignans from Du-Zhong cortex could increase the ratio of OPG/RANKL mRNA in a dose-dependent manner (Zhang, 2008). It may affect the osteoblast formation, so that it reduces the bone reabsorption. On one hand, the current study demonstrated the in vivo anti-osteoporotic effect of DZCE, however, the potential molecular mechanisms behind this effect need to be further clarified. On the other hand, DZCE was used in the current study as a mixture of bioactive constituents. Since several chemical constituents have been separated from Du-Zhong including lignans, phenolic acid, and flavonoids (Deyama et al., 2001), it is of great importance to identify the major bioactive components that are responsible for its anti-osteoporotic effect for better understanding the mechanisms. In conclusion, DZCE administration was able to prevent HLS-induced decrease of bone mass and the deterioration of trabecular microarchitecture, while helping to maintain the structural integrity and biomechanical quality of bone. DZCE exhibited a beneficial effect on bone metabolism, which suggested that it will be served as potential alternative medicine for the prevention and treatment of disuse-induced osteoporosis. Acknowledgments We thank Dr. Jun Wang for his assistance of the microCT analysis (Department of Orthopedics, Xijing Hospital, Fourth Military Medical University); and Dr. Zhiguang Duan for his assistance of material testing machine analysis (Northwestern University, Xi’an). This work was financially supported by the National Natural Science Foundation of China (grant no. 81073037; 81202457) as well as the China Postdoctoral Science Foundation funded project (grant no. 2012T50822). References Akhter, M.P., G.K. Alvarez, D.M. Cullen and R.R. Recker. Disuse-related decline in trabecular bone structure. Biomech. Model Mechanobiol. 10: 423–429, 2011. Bagi, C.M. and S.C. Miller. Comparison of osteopenic changes in cancellous bone induced by ovariectomy and/or immobilization in adult rats. Anat. Rec. 239: 243–254, 1994. Bouxsein, M.L. Mechanisms of osteoporosis therapy: a bone strength perspective. Clin. Cornerstone 5(2 Suppl): S13–S21, 2003. Brouwers, J.E., F.M. Lambers, B.V. Rietbergen, K. Ito and R. Huiskes. Comparison of bone loss induced by ovariectomy and neurectomy in rats analyzed by in vivo micro-CT. J. Orthop. Res. 27: 1521–1527, 2009. Chinese Pharmacopoeia Commission. Chinese Pharmacopeia. Chemical Industry Publishing House, Beijing, 2010, pp. 154. Collet, P., D. Uebelhart, L. Vico, L. Moro, D. Hartmann, M. Roth and C. Alexandre. Effects of 1- and 6-month spaceflight on bone mass and biochemistry in two humans. Bone 20: 547–551, 1997. Damien, E., J.S. Price and L.E. Lanyon. Mechanical strain stimulates osteoblast proliferation through the estrogen receptor in males as well as females. J. Bone Miner. Res. 15: 2169–2177, 2000.



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Soluble epoxide hydrolase inhibitory and anti-inflammatory components from the leaves of Eucommia ulmoides Oliver (duzhong).

Effects of Eucommia ulmoides extract on longitudinal bone growth rate in adolescent female rats.

Suppression of splenic lymphocyte proliferation by Eucommia ulmoides and genipin.

Health-Promoting Properties of Eucommia ulmoides: A Review.

NF-κB Pathways in Rats.

Effect of recovery mode following hind-limb suspension on soleus muscle composition in the rat.

Astrocytes and microglia of the mouse spinal cord during hind limb suspension.

Propranolol Prevents Osteoporosis and up-regulates Leptin in Ovariectomized Rats.

Relationship between progeny growth performance and molecular marker-based genetic distances in Eucommia ulmoides parental genotypes.

Inhibitory Effects of Eucommia ulmoides Oliv. Bark on Scopolamine-Induced Learning and Memory Deficits in Mice.

Effervescent Granules Prepared Using Eucommia ulmoides Oliv. and Moso Bamboo Leaves: Hypoglycemic Activity in HepG2 Cells.

Studies on resin purification process optimization of Eucommia ulmoides Oliver and its antihypertensive effect mechanism.

Controllable biosynthesis of gold nanoparticles from a Eucommia ulmoides bark aqueous extract.

Anti-inflammatory activity of iridoid and catechol derivatives from Eucommia ulmoides Oliver.

Effect of quercetin-3-O-sambubioside isolated from Eucommia ulmoides male flowers on spontaneous activity and convulsion rate in mice.

Eucommia ulmoides Oliv.: ethnopharmacology, phytochemistry and pharmacology of an important traditional Chinese medicine.

Eucommia ulmoides cortex, geniposide and aucubin regulate lipotoxicity through the inhibition of lysosomal BAX.

Effect of picroside II on hind limb ischemia reperfusion injury in rats.

A molecular genetic linkage map of Eucommia ulmoides and quantitative trait loci (QTL) analysis for growth traits.

Hind limb ischemia-reperfusion injury in diet-induced obese mice.

Improved on-line high performance liquid chromatography method for detection of antioxidants in Eucommia ulmoides Oliver flower.

Chlorogenic acid-enriched extract from Eucommia ulmoides leaves inhibits hepatic lipid accumulation through regulation of cholesterol metabolism in HepG2 cells.

Lignans from the bark of Eucommia ulmoides inhibited Ang II-stimulated extracellular matrix biosynthesis in mesangial cells.

Treatment of hind limb ischemia using angiogenic peptide nanofibers.