American Journal of Therapeutics 0, 1–7 (2015)
Effects of Simvastatin and Combination of Simvastatin and Nylestriol on Bone Metabolism in Ovariectomized Rats Xiao-Feng Li, MD, Chun-Bo Lin, MD, Fu-Rong Xie, MD, Wei-Guo Liang, MD, Jing Ji, MD, and Yuan Yang, MD*
We aim to compare the effects of simvastatin and combination of simvastatin and nylestriol on bone metabolism in ovariectomized (OVX) rats. Fifty healthy Wistar female rats were randomly allocated into 5 groups: sham + saline group (group A), OVX + saline group (group B), OVX + simvastatin (5 mg$kg21$d21) (group C), OVX + nylestriol (0.01 mg$kg21$d21) (group D), and OVX + simvastatin (3 mg$kg21$d21) + nylestriol (0.005 mg$kg21$d21) (group E). All mice were orally administrated with saline or medicine dissolved in saline for 10 weeks. Body weight of rats before and after the experiment was measured. Twenty-four hours after the experiment, calcium (Ca), creatinine (Cr), and hydroxyproline in urine were detected. Serum levels of osteocalcin (bone Gla-protein, BGP) and alkaline phosphatase (ALP) were measured. Bone mineral density was detected and trabecular bone was observed after the isolation of femur and tibia. Remarkably decreased serum BGP and increased serum ALP levels were detected in group B compared with those in group A. However, notably increased serum BGP and decreased serum ALP levels were found in groups C, D, and E compared with those in group B; femoral and tibial bone mineral density decreased in group B compared with that in group A, but increased in groups C, D, and E compared with that in group B. Simvastatin and combination of simvastatin and nylestriol promote formation of new bone, increase bone density, and improve bone microstructure damage in OVX rats. Keywords: osteoporosis, ovariectomized rats, simvastatin, nylestriol, bone metabolism, bone formation, bone density, bone microstructure
INTRODUCTION As a systemic skeletal disease, osteoporosis is characterized by low bone mass and bone microarchitectural deterioration with increased possibility of bone fragility and fractures.1 Osteoporotic fractures occurring at the spine, distal radius, and hip could impact on life quality and increase morbidity and mortality.2 The Department of Spinal Surgery, Guangxi Orthopaedics and Traumatology Hospital, Nanning, China. Supported by Appropriate Medical and Health Care Technology Research and Development Project of Guangxi Province (S201402-02). The authors have no conflicts of interest to declare. *Address for correspondence: Guangxi Orthopaedics and Traumatology Hospital, 32 Xinmin Road, Nanning 530012, China. E-mail: [email protected]
incidence of osteoporotic fractures increases in elderly populations and menopausal women.3 Osteoporosis could affect about one-third of women at age over 50 years, combined with a lifetime risk of fracture and cardiovascular disease around 50%.4 According to World Health Organization (WHO) data, osteoporosis affects about 75 million people throughout Japan, Europe, and United States where osteoporosis occurs in 55% of people aged 50 years and over population.5 Imbalanced bone resorption from bone formation caused by overactivities of osteoclasts than those of the osteoblasts could lead to osteoporosis, which could be accelerated in postmenopausal women due to the loss of estrogen.6 Osteoporosis therapy could be classified as 2 classes, antiresorptive drugs that slow down bone resorption, such as estrogens, calcitonin, raloxifene, and bisphosphonates or anabolic drugs, which stimulate bone formation, such as parathyroid
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hormone PTH 1–34 or PTH 1–84.7 Bisphosphonates, with high affinity for bone and stable safety record, could be administered orally or intravenously and be widely used in a broad spectrum of osteoporosis, including postmenopausal steroid-induced and male osteoporosis.8 PTH could improve bone mineral density (BMD) and reduce osteoporosis fractures rate and also improve fracture healing.9 Nowadays, many researches have focused on stains as a new effective drug for their role in both inhibition of bone resorption and stimulation of bone formation.10,11 Simvastatin, a kind of stains, also an inactive lactone, developed from fermentation product, could lower the level of cholesterol and lipid.12 Besides its lipid-lowering effects, simvastatin can also modulate bone regeneration process at both molecular and cellular levels by participating in activation of osteoblast and inhibition of osteoclast.13 Moreover, simvastatin could not only inhibit 3-hydroxy-3-methylglutarylcoenzyme A reductase and the mevalonic acid pathway but also increase the osteoprotegerin expression to antagonize osteoclasts.14 Nylestriol, a derivative of long-acting estriol and a kind of lente estrogen, was synthesized in China in 1984.15 The estrogenic activity of nylestriol is 3 times that of quinestrol, 23 times that of estriol, and 510 times that of estriol when orally administered.15 Nylestriol with adequate calcium and vitamin D intake could increase BMD at the lumbar spine, neck, and hip and decrease bone turnover markers in postmenopausal women.16 To the best of authors’ knowledge, there have been no previous studies on the protective effect of the combination of simvastatin and nylestriol. The purpose of this study was to compare the effects of simvastatin and combination of simvastatin and nylestriol on bone metabolism in ovariectomized (OVX) rats, thus further clarifying the efficacy of statins and combination therapy for the treatment of osteoporosis.
MATERIALS AND METHODS Ethics statement This study was conducted in strict accordance with the recommendations in the guidelines for the use of experimental animals in China. The protocol was approved by Guangxi Orthopaedics and Traumatology Hospital. Experimental subjects and study design The study was conducted on fifty 3-month-old female Wistar rats (provided by Experimental Animal Center of Guangxi Orthopaedics and Traumatology Hospital) with initial body weight of 200 6 30 g. The rats were housed American Journal of Therapeutics (2015) 0(0)
Li et al
in conventionally controlled clean facilities with a temperature of 24 6 2°C and a 12-hour light–dark cycle with relative humidity of 55%. All the 50 healthy Wistar female rats were either bilateral OVX or sham operated. They were randomly and equally allocated into 5 groups. Group A (sham + saline group): saline (1.5 mL/d for each) was given to the rats by gastric tube for 10 weeks since 1 week after the sham operation. Group B (OVX + saline group): saline (1.5 mL/d for each) was given to the OVX rats by gastric tube for 10 weeks since 1 week after the OVX. Group C (OVX + simvastatin): 1 week after the OVX, simvastatin (5 mg$kg21$d21; Hangzhou MSD Pharmaceutical Co, Ltd., Hangzhou, China) was given to the OVX rats with 1.5 mL of saline by gastric tube for 10 weeks. Group D (OVX + nylestriol): 1 week after the OVX, nylestriol (0.01 mg$kg21$d21; Beijing Four-Rings Pharmaceutical Co, Ltd., Beijing, China) was given to the OVX rats with 1.5 mL of saline by gastric tube for 10 weeks. Group E (OVX + simvastatin + nylestriol): 1 week after the OVX, simvastatin (3 mg$kg21$d21) and nylestriol (0.005 mg$kg21$d21) were given to the OVX rats with 1.5 mL of saline by gastric tube for 10 weeks. Body weight of rats before and after the experiment was measured and the dosages of drug administered in groups C, D, and E were calculated according to the most recent weight measurement once a week. Animal model establishment Rats were anesthetized with 2% sodium thiopental (30 mg/kg) by intraperitoneal injection. After anesthesia, the OVX was performed from a dorsal approach with a small midline dorsal skin incision for 2–3 cm, and iodine and alcohol were used for the skin disinfection. The left and right psoas muscles were separated with a vessel clamp and pink ovaries were exposed in the lateral and inferior side of the bilateral kidneys. Both of the ovaries were ligated and removed. The muscle layer was tied and skin incision was closed with silk suture and treated with penicillin. Sham surgery was performed following the steps above with the visualization of the ovaries, but without clamping or removing of the tissue. Then, the skin was sutured. Sample collection Rat urine was collected at 24 hours after the experiment and preserved at 220°C. Blood samples were taken from decapitated rats, and serum was obtained at low speed centrifuge for 15 minutes and stored at 220°C. After isolation of right and left femur and left tibia, left femur and tibia were used for the measurement of BMD, while right femur was used for histomorphology analysis. www.americantherapeutics.com
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Simvastatin, Nylestriol, and Ovariectomized Rats
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Measurement of bone Gla-protein
Measurement of urine creatinine
Serum level of bone Gla-protein (BGP) was measured by radioimmunoassay method and a kit from People’s Liberation Army General Hospital. The standard curve was obtained from BGP standards with different concentration. Hundred-microliter standard serum samples were harvested and 100 mL of antiserum and 100 mL of 125I-BGP were added orderly. Then, the total system was mixed and placed in 4°C for 24 hours. Then, 500mL separating agents were added and mixed and placed in room temperature for 15–20 minutes. After centrifuging in 4°C at 3500 rpm for 20 minutes, the supernate was discarded. Radioactivity value was measured with g counter and the concentration of the sample was calculated according to the standard curve.
Urine Cr was measured with picric acid methods and a kit provided by Baoding Great Wall Clinical Reagents Co, Ltd. (Baoding, China). Twenty-five microliters of urine sample was added to the 10-mL colorimetric tube and distilled water was added to reach 3 mL, and 3 mL distilled water in blank tube. Two milliliters of alkaline picric acid solution was added into each tube and mixed. Five milliliters of water was added into the tubes after 20–30 minutes reaction in room temperature. Absorbancy was measured in wavelength of 490 nm and the colorimetry was finished within 0.5 hours.
Measurement of alkaline phosphatase Serum level of alkaline phosphatase (ALP) was measured by P-nitrophenyl phosphate (PNPP) method and a kit provided by Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Fifty microliters of phenol standard (0.1 mg/mL) was added in standard tubes, 50 mL of serum sample was added in sample tube, and 50 mL of deionized water was added in blank tube. Fifty microliters of buffer solution and 500 mL of substrate in order were added in each tube and mixed and put in 37°C water for 15 minutes. Then, 1.5-mL developer was added into the tubes and mixed immediately. Ultraviolet spectrophotometer was used for measurement. The wave length was 520 nm and the colorimetric optical path was 1 cm. Absorbancy of each tube was measured after zero setting. The serum ALP level was calculated by the formulate (1 King unit was defined as every 1 mg phenol produced by 100 mL of serum reacting with substrate in 37°C for 15 minutes). ALP level (King unit/100 mL) 5 (absorbancy of measuring tube/absorbancy of standard tube) 3 (phenol content in the standard tube/0.005 mg) 3 (100 mL/0.05 mL).
Measurement of hydroxyproline HOP was measured with digestion method and a kit provided by Nanjing Jiancheng Bioengineering Institute. Reagent was prepared according to the manufacturer’s instruction. Then, 0.25 mL of test solution was added in test tube, 0.25 mL of standard solution in standard tube, and 0.25 mL of distilled water in blank tube. The digestion kit was added into each tube in order and mixed. After 60°C water bath for 15 minutes, the solutions were cooled and centrifuged at 3500 rpm for 10 minutes. The supernatant was taken and absorbancy was measured in wavelength of 550 nm, 1 cm optical path, and zero setting was dealt with steamed water. Measurement of BMD The left femur and tibia were cleaned of the attached muscle and connective tissue and were put in the thin-walled organic glass box full of deionized water (to mimic the soft tissue around bone). The BMD (grams per square centimeter) of left femur and tibia was measured by dual-energy x-ray absorptiometry (DPX-L6843; Lunar Co Ltd., Madison, WI) using the small animal scan model.
Measurement of urine calcium
Femur metaphysis histomorphology
Urine calcium (Ca) was measured by methyl thymol blue colorimetric analysis and a kit provided by Nanjing Jiancheng Bioengineering Institute. Fifty microliters of calcium standard (2.5 mol/L) was added in standard tube, 50 mL of sample in sample tube, and 50 mL of deionized water in blank tube. One milliliter of methyl thymol blue reagent and 2 mL of alkaline solution were added orderly into each tube and mixed and standing for 15 minutes. Ultraviolet spectrophotometer was used for measurement and distilled water was used for zero setting. The wave length was 610 nm and the colorimetric optical path was 1 cm.
The right femur of rat was fixated in 10% formalin solution, decalcified with formic acid, and embedded in paraffin. The paraffin-embedded tissues were cut into 5-mm sections with a microtome and performed with hematoxylin and eosin staining. The change in trabeculae of femur metaphysis was observed under light microscope.
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Statistical analysis All data were performed using SPSS 17.0 statistical software package. Measurement data were expressed as mean 6 SD. Differences between groups were analyzed by analysis of variance and a P , 0.05 was considered statistically significant. American Journal of Therapeutics (2015) 0(0)
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Li et al
Table 1. Body weight of rats before and after the experiment in the sham group, OVX group, and drug-treated groups. Weight (g) Groups
Pre-experiment
A B C D E
203.25 202.52 201.31 197.05 205.53
6 6 6 6 6
Postexperiment
16.85 19.89 21.18 19.31 22.35
239.67 287.12 248.65 240.91 251.64
6 6 6 6 6
16.38 9.83* 10.61† 10.15† 20.16†
*Compared with group A, P , 0.01. †Compared with group B, P , 0.01.
RESULTS Changes of body weight of rats before and after the experiment Before experiments, there were no statistical differences among each 5 groups (all P . 0.05). After experiments, body weight in group B increased significantly than that in group A, whereas body weight in groups C, D, and E decreased significantly compared with that in group B (all P , 0.01). However, there were no significant differences in body weight among groups C, D, and E (all P . 0.05) (Table 1). Changes of indicators of bone formation and absorption Remarkably decreased serum BGP and increased serum ALP levels were detected in group B compared with those in group A. However, notably increased serum BGP and decreased serum ALP levels were found in groups C, D, and E compared with those in group B (all P , 0.05). While there were no significant differences in serum levels of BGP and ALP among groups C, D, and E (all P . 0.05). Compared with group A, the Ca/Cr ratio and the HOP/Cr ratio in group B increased
without significant differences, while compared with group B, the Ca/Cr ratio and the HOP/Cr ratio and in groups C, D, and E decreased slightly (all P . 0.05). Still no significant differences in these 4 indicators among groups C, D, and E were found (all P . 0.05) (Table 2). Changes in bone density Compared with group A, the BMD of rat femur lowered significantly in group B, but the BMD of rat femur increased notably in groups C, D, and E compared with group B (all P , 0.05). The BMD of rat femur showed no significant difference among groups C, D, and E (all P . 0.05). In addition, there existed a downward trend on BMD of rat femur in group B compared with group A, whereas an upward trend on BMD of rat femur in groups C, D, and E compared with group B (all P , 0.05) (Table 3). Histomorphological observation The sections of the femur were examined for any histological changes under LM. The rats of group A revealed closely and orderly arranged trabeculae with the normal morphology and mature structure under epiphyseal plate. The intertrabecular spaces were filled with red marrows (Figure 1A). The animals in group B showed sparse and quantity decreased trabeculae under epiphyseal plate. The connectivity between trabeculae was weak, fracture section can be observed and the thickness of the trabecula wall was uneven. Marrow content between trabeculae was significantly reduced (Figure 1B). Group C exhibited closely arranged and highly organized trabeculae with near-normal morphology and acceptable maturity under epiphyseal plate. The intertrabecular spaces were reticularly connected and the thickness of trabeculae wall was even. The amount of red marrow between trabeculae was much more (Figure 1C). Histomorphology of group D and group E was similar to group C (Figures 1D, E).
Table 2. Serum level of BGP, ALP, and urinary Ca/Cr ratio and HOP/Cr ratio in the sham group, OVX group, and drug-treated groups. Groups A B C D E
BGP (mg/L) 1.441 1.029 1.478 1.436 1.449
6 6 6 6 6
ALP (King unit/100 mL)
0.379 0.308* 0.260† 0.212† 0.189†
15.39 22.23 16.48 14.12 15.16
6 6 6 6 6
3.85 4.52* 5.11† 4.35† 4.58†
Ca/Cr 0.381 0.558 0.442 0.381 0.478
6 6 6 6 6
0.213 0.330 0.251 0.211 0.259
Hop/Cr (mg$L/mgprot$mmol) 24.08 31.29 25.52 25.24 27.12
6 6 6 6 6
7.28 5.67 9.26 7.34 8.78
*Compared with group A, P , 0.05. †Compared with group B, P , 0.05.
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Simvastatin, Nylestriol, and Ovariectomized Rats Table 3. Femoral and tibial BMD in the sham group, OVX group, and drug-treated groups.
Groups
Femoral BMD (g/cm2)
A B C D E
0.251 0.228 0.246 0.245 0.243
6 6 6 6 6
0.010 0.006* 0.007† 0.005† 0.006†
Tibial bone density (g/cm2) 0.217 0.204 0.212 0.215 0.214
6 6 6 6 6
0.007 0.006* 0.010† 0.008† 0.006†
*Compared with group A, P , 0.05. †Compared with group B, P , 0.05.
DISCUSSION Current treatments for osteoporosis mainly include 2 types, antiresorptive drugs aims at prevention of bone loss and anabolic drugs designed to increase bone volume. But either of the drugs could not fully treat the osteoporosis and have some side effects in clinical therapy.17 Simvastatin has reported to have an enhancing effect on the osteogenic genes, such as ALP, BMP2, and osteocalcin in osteoblastic cells.18 In addition, simvastatin is also connected with the decrease in osteoclast number, suggesting the decreased activity of
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osteoclast.19 Thus, we conducted the study to elucidate the efficacy of simvastatin on the treatment of osteoporosis, and we established an OVX rat model to simulate the osteoporosis process in human. The deficiency of estrogen caused by OVX could lead to an increased rate and an imbalance of bone resorption and formation.20 As a kind of estrogen, nylestriol could also promote growth of osteoblasts and osteocytes, thus treat the osteoporosis due to menopause and decreased estrogen level.21 In this study, we also studied the combination of simvastatin and nylestriol in the treatment of osteoporosis. Our study showed that OVX could increase the body weight, whereas both simvastatin and nylestriol could inhibit the gained body weight induced by OVX. Increased body weight has been reported in ovarian hormone deficiency and could be cured by estrogen administration.22 The present data also showed that OVX could decrease serum BGP and increase serum ALP levels. And simvastatin and nylestriol had the opposite effect in the regulation of serum BGP and ALP levels. BGP, a calcium-binding protein of bone, is a sensitive marker for bone formation and bone turnover, suggesting the activity of osteoblasts.23 Thus, the OVX could inhibit the activity of osteoblasts and the bone formation, whereas the 2 kinds of drugs could improve the situation by increase the serum
FIGURE 1. Representative images of hematoxylin and eosin (H&E)-stained specimen of the rat femur (A–E). A, There are some marrow cavities among bone trabeculae in group A. B, Broken bone trabeculae are arranged disorderly in group B. C–E, Bone trabeculae become thick in groups C–E. Magnification: 3200. www.americantherapeutics.com
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BGP. Serum ALP, the most commonly used biomarker for bone formation, is a key enzyme for osteoid formation and bone mineralization.24,25 However, there were no significant differences in the Ca/Cr ratio and HOP/ Cr ratio among different treatment, suggesting the relative safety of the drug use. Our findings also showed that femoral and tibial BMD decreased in the OVX group, but increased after the treatment of simvastatin or nylestriol, suggesting the role of these drugs in preventing bone loss and deficiency of estrogen in osteoporosis treatment. The measurement of BMD by DXA plays an essential role in the osteoporosis and fracture risk assessment.26 BMD value even used to define the osteoporosis is defined by the World Health Organization with BMD 2.5 SDs or more below the young female adult mean.6 Our results also in accordance with the study of Oxlund and Andreassen,27 which demonstrated that simvastatin could partially prevent the loss of cancellous bone after OXV both in proximal tibias and vertebral bodies, increase BMD and lower fractures risk. Histomorphological examination results showed that OVX could decrease the number of trabeculae, weaken the connectivity between trabeculae, cause the formation of fracture section, and reduce the red marrows. While the treatment with simvastatin or combination of simvastatin and nylestriol could reverse the harm caused by OVX by showing closely arranged and highly organized trabeculae. Taken together, our studies showed that simvastatin and combination of simvastatin and nylestriol could promote the formation of new bone, increase bone density, and improve bone microstructure damage in OVX rats. However, we did not observe the bone formation in our histomorphological examination results, thus we could not clarify the changes of BMD is due to bone formation or bone resorption. Thus, further mechanisms related to some signaling ways should be studied before the drugs be tested in humans as an antiosteoporosis therapy.
ACKNOWLEDGMENTS The authors thank their researchers for their hard work and reviewers for their valuable advice.
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Li et al 2. Kawate H, Takayanagi R. Efficacy and safety of bazedoxifene for postmenopausal osteoporosis. Clin Interv Aging. 2011;6:151–160. 3. Cooper C, Cole ZA, Holroyd CR, et al. Secular trends in the incidence of hip and other osteoporotic fractures. Osteoporos Int. 2011;22:1277–1288. 4. Rizzoli R, Reginster JY, Boonen S, et al. Adverse reactions and drug-drug interactions in the management of women with postmenopausal osteoporosis. Calcif Tissue Int. 2011;89:91–104. 5. Rakel A, Boucher A, Ste-Marie LG. Role of zoledronic acid in the prevention and treatment of osteoporosis. Clin Interv Aging. 2011;6:89–99. 6. Das S, Crockett JC. Osteoporosis—a current view of pharmacological prevention and treatment. Drug Des Devel Ther. 2013;7:435–448. 7. Silva I, Branco JC. Denosumab: recent update in postmenopausal osteoporosis. Acta Reumatol Port. 2012;37: 302–313. 8. Migliaccio S, Fornari R, Greco EA, et al. New therapeutical horizons in the management of postmenopausal osteoporosis. Aging Clin Exp Res. 2013;25(suppl 1):S117– S119. 9. Peichl P, Holzer LA, Maier R, et al. Parathyroid hormone 1-84 accelerates fracture-healing in pubic bones of elderly osteoporotic women. J Bone Joint Surg Am. 2011;93: 1583–1587. 10. Xu XC, Chen H, Zhang X, et al. Simvastatin prevents alveolar bone loss in an experimental rat model of periodontitis after ovariectomy. J Transl Med. 2014; 12:284. 11. Ayukawa Y, Ogino Y, Moriyama Y, et al. Simvastatin enhances bone formation around titanium implants in rat tibiae. J Oral Rehabil. 2010;37:123–130. 12. Murtaza G. Solubility enhancement of simvastatin: a review. Acta Pol Pharm. 2012;69:581–590. 13. Montero J, Manzano G, Albaladejo A. The role of topical simvastatin on bone regeneration: a systematic review. J Clin Exp Dent. 2014;6:e286–e290. 14. Tsartsalis AN, Dokos C, Kaiafa GD, et al. Statins, bone formation and osteoporosis: hope or hype? Hormones (Athens). 2012;11:126–139. 15. Liao EY, Luo XH, Deng XG, et al. The effect of low dose nylestriol-levonorgestrel replacement therapy on bone mineral density in women with postmenopausal osteoporosis. Endocr Res. 2003;29:217–226. 16. Zang H, Shi H, Speroff L. Low-dose hormone therapy in postmenopausal women in China. Climacteric. 2010;13: 544–552. 17. Yao W, Farmer R, Cooper R, et al. Simvastatin did not prevent nor restore ovariectomy-induced bone loss in adult rats. J Musculoskelet Neuronal Interact. 2006;6: 277–283. 18. Ho ML, Chen YH, Liao HJ, et al. Simvastatin increases osteoblasts and osteogenic proteins in ovariectomized rats. Eur J Clin Invest. 2009;39:296–303. 19. Park JB. The use of simvastatin in bone regeneration. Med Oral Patol Oral Cir Bucal. 2009;14:e485–e488. www.americantherapeutics.com
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Simvastatin, Nylestriol, and Ovariectomized Rats 20. Marie PJ, Felsenberg D, Brandi ML. How strontium ranelate, via opposite effects on bone resorption and formation, prevents osteoporosis. Osteoporos Int. 2011;22: 1659–1667. 21. Zhao Y, Zou B, Shi Z, et al. The effe ct of 3hydroxybutyrate on the in vitro differentiation of murine osteoblast MC3T3-E1 and in vivo bone formation in ovariectomized rats. Biomaterials. 2007;28:3063–3073. 22. Roesch DM. Effects of selective estrogen receptor agonists on food intake and body weight gain in rats. Physiol Behav. 2006;87:39–44. 23. Song YE, Tan H, Liu KJ, et al. Effect of fluoride exposure on bone metabolism indicators ALP, BALP, and BGP. Environ Health Prev Med. 2011;16:158–163. 24. Bhattarai T, Bhattacharya K, Chaudhuri P, et al. Correlation
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7 of common biochemical markers for bone turnover, serum calcium, and alkaline phosphatase in post-menopausal women. Malays J Med Sci. 2014;21:58–61. 25. Chen H, Li X, Yue R, et al. The effects of diabetes mellitus and diabetic nephropathy on bone and mineral metabolism in T2DM patients. Diabetes Res Clin Pract. 2013;100: 272–276. 26. Gregson CL, Hardcastle SA, Cooper C, et al. Friend or foe: high bone mineral density on routine bone density scanning, a review of causes and management. Rheumatology (Oxford). 2013;52:968–985. 27. Oxlund H, Andreassen TT. Simvastatin treatment partially prevents ovariectomy-induced bone loss while increasing cortical bone formation. Bone. 2004;34:609–618.
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Effects of Simvastatin and Combination of Simvastatin and Nylestriol on Bone Metabolism in Ovariectomized Rats Xiao-Feng Li, MD, Chun-Bo Lin, MD, Fu-Rong Xie, MD, Wei-Guo Liang, MD, Jing Ji, MD, and Yuan Yang, MD*
We aim to compare the effects of simvastatin and combination of simvastatin and nylestriol on bone metabolism in ovariectomized (OVX) rats. Fifty healthy Wistar female rats were randomly allocated into 5 groups: sham + saline group (group A), OVX + saline group (group B), OVX + simvastatin (5 mg$kg21$d21) (group C), OVX + nylestriol (0.01 mg$kg21$d21) (group D), and OVX + simvastatin (3 mg$kg21$d21) + nylestriol (0.005 mg$kg21$d21) (group E). All mice were orally administrated with saline or medicine dissolved in saline for 10 weeks. Body weight of rats before and after the experiment was measured. Twenty-four hours after the experiment, calcium (Ca), creatinine (Cr), and hydroxyproline in urine were detected. Serum levels of osteocalcin (bone Gla-protein, BGP) and alkaline phosphatase (ALP) were measured. Bone mineral density was detected and trabecular bone was observed after the isolation of femur and tibia. Remarkably decreased serum BGP and increased serum ALP levels were detected in group B compared with those in group A. However, notably increased serum BGP and decreased serum ALP levels were found in groups C, D, and E compared with those in group B; femoral and tibial bone mineral density decreased in group B compared with that in group A, but increased in groups C, D, and E compared with that in group B. Simvastatin and combination of simvastatin and nylestriol promote formation of new bone, increase bone density, and improve bone microstructure damage in OVX rats. Keywords: osteoporosis, ovariectomized rats, simvastatin, nylestriol, bone metabolism, bone formation, bone density, bone microstructure
INTRODUCTION As a systemic skeletal disease, osteoporosis is characterized by low bone mass and bone microarchitectural deterioration with increased possibility of bone fragility and fractures.1 Osteoporotic fractures occurring at the spine, distal radius, and hip could impact on life quality and increase morbidity and mortality.2 The Department of Spinal Surgery, Guangxi Orthopaedics and Traumatology Hospital, Nanning, China. Supported by Appropriate Medical and Health Care Technology Research and Development Project of Guangxi Province (S201402-02). The authors have no conflicts of interest to declare. *Address for correspondence: Guangxi Orthopaedics and Traumatology Hospital, 32 Xinmin Road, Nanning 530012, China. E-mail: [email protected]
incidence of osteoporotic fractures increases in elderly populations and menopausal women.3 Osteoporosis could affect about one-third of women at age over 50 years, combined with a lifetime risk of fracture and cardiovascular disease around 50%.4 According to World Health Organization (WHO) data, osteoporosis affects about 75 million people throughout Japan, Europe, and United States where osteoporosis occurs in 55% of people aged 50 years and over population.5 Imbalanced bone resorption from bone formation caused by overactivities of osteoclasts than those of the osteoblasts could lead to osteoporosis, which could be accelerated in postmenopausal women due to the loss of estrogen.6 Osteoporosis therapy could be classified as 2 classes, antiresorptive drugs that slow down bone resorption, such as estrogens, calcitonin, raloxifene, and bisphosphonates or anabolic drugs, which stimulate bone formation, such as parathyroid
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hormone PTH 1–34 or PTH 1–84.7 Bisphosphonates, with high affinity for bone and stable safety record, could be administered orally or intravenously and be widely used in a broad spectrum of osteoporosis, including postmenopausal steroid-induced and male osteoporosis.8 PTH could improve bone mineral density (BMD) and reduce osteoporosis fractures rate and also improve fracture healing.9 Nowadays, many researches have focused on stains as a new effective drug for their role in both inhibition of bone resorption and stimulation of bone formation.10,11 Simvastatin, a kind of stains, also an inactive lactone, developed from fermentation product, could lower the level of cholesterol and lipid.12 Besides its lipid-lowering effects, simvastatin can also modulate bone regeneration process at both molecular and cellular levels by participating in activation of osteoblast and inhibition of osteoclast.13 Moreover, simvastatin could not only inhibit 3-hydroxy-3-methylglutarylcoenzyme A reductase and the mevalonic acid pathway but also increase the osteoprotegerin expression to antagonize osteoclasts.14 Nylestriol, a derivative of long-acting estriol and a kind of lente estrogen, was synthesized in China in 1984.15 The estrogenic activity of nylestriol is 3 times that of quinestrol, 23 times that of estriol, and 510 times that of estriol when orally administered.15 Nylestriol with adequate calcium and vitamin D intake could increase BMD at the lumbar spine, neck, and hip and decrease bone turnover markers in postmenopausal women.16 To the best of authors’ knowledge, there have been no previous studies on the protective effect of the combination of simvastatin and nylestriol. The purpose of this study was to compare the effects of simvastatin and combination of simvastatin and nylestriol on bone metabolism in ovariectomized (OVX) rats, thus further clarifying the efficacy of statins and combination therapy for the treatment of osteoporosis.
MATERIALS AND METHODS Ethics statement This study was conducted in strict accordance with the recommendations in the guidelines for the use of experimental animals in China. The protocol was approved by Guangxi Orthopaedics and Traumatology Hospital. Experimental subjects and study design The study was conducted on fifty 3-month-old female Wistar rats (provided by Experimental Animal Center of Guangxi Orthopaedics and Traumatology Hospital) with initial body weight of 200 6 30 g. The rats were housed American Journal of Therapeutics (2015) 0(0)
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in conventionally controlled clean facilities with a temperature of 24 6 2°C and a 12-hour light–dark cycle with relative humidity of 55%. All the 50 healthy Wistar female rats were either bilateral OVX or sham operated. They were randomly and equally allocated into 5 groups. Group A (sham + saline group): saline (1.5 mL/d for each) was given to the rats by gastric tube for 10 weeks since 1 week after the sham operation. Group B (OVX + saline group): saline (1.5 mL/d for each) was given to the OVX rats by gastric tube for 10 weeks since 1 week after the OVX. Group C (OVX + simvastatin): 1 week after the OVX, simvastatin (5 mg$kg21$d21; Hangzhou MSD Pharmaceutical Co, Ltd., Hangzhou, China) was given to the OVX rats with 1.5 mL of saline by gastric tube for 10 weeks. Group D (OVX + nylestriol): 1 week after the OVX, nylestriol (0.01 mg$kg21$d21; Beijing Four-Rings Pharmaceutical Co, Ltd., Beijing, China) was given to the OVX rats with 1.5 mL of saline by gastric tube for 10 weeks. Group E (OVX + simvastatin + nylestriol): 1 week after the OVX, simvastatin (3 mg$kg21$d21) and nylestriol (0.005 mg$kg21$d21) were given to the OVX rats with 1.5 mL of saline by gastric tube for 10 weeks. Body weight of rats before and after the experiment was measured and the dosages of drug administered in groups C, D, and E were calculated according to the most recent weight measurement once a week. Animal model establishment Rats were anesthetized with 2% sodium thiopental (30 mg/kg) by intraperitoneal injection. After anesthesia, the OVX was performed from a dorsal approach with a small midline dorsal skin incision for 2–3 cm, and iodine and alcohol were used for the skin disinfection. The left and right psoas muscles were separated with a vessel clamp and pink ovaries were exposed in the lateral and inferior side of the bilateral kidneys. Both of the ovaries were ligated and removed. The muscle layer was tied and skin incision was closed with silk suture and treated with penicillin. Sham surgery was performed following the steps above with the visualization of the ovaries, but without clamping or removing of the tissue. Then, the skin was sutured. Sample collection Rat urine was collected at 24 hours after the experiment and preserved at 220°C. Blood samples were taken from decapitated rats, and serum was obtained at low speed centrifuge for 15 minutes and stored at 220°C. After isolation of right and left femur and left tibia, left femur and tibia were used for the measurement of BMD, while right femur was used for histomorphology analysis. www.americantherapeutics.com
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Simvastatin, Nylestriol, and Ovariectomized Rats
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Measurement of bone Gla-protein
Measurement of urine creatinine
Serum level of bone Gla-protein (BGP) was measured by radioimmunoassay method and a kit from People’s Liberation Army General Hospital. The standard curve was obtained from BGP standards with different concentration. Hundred-microliter standard serum samples were harvested and 100 mL of antiserum and 100 mL of 125I-BGP were added orderly. Then, the total system was mixed and placed in 4°C for 24 hours. Then, 500mL separating agents were added and mixed and placed in room temperature for 15–20 minutes. After centrifuging in 4°C at 3500 rpm for 20 minutes, the supernate was discarded. Radioactivity value was measured with g counter and the concentration of the sample was calculated according to the standard curve.
Urine Cr was measured with picric acid methods and a kit provided by Baoding Great Wall Clinical Reagents Co, Ltd. (Baoding, China). Twenty-five microliters of urine sample was added to the 10-mL colorimetric tube and distilled water was added to reach 3 mL, and 3 mL distilled water in blank tube. Two milliliters of alkaline picric acid solution was added into each tube and mixed. Five milliliters of water was added into the tubes after 20–30 minutes reaction in room temperature. Absorbancy was measured in wavelength of 490 nm and the colorimetry was finished within 0.5 hours.
Measurement of alkaline phosphatase Serum level of alkaline phosphatase (ALP) was measured by P-nitrophenyl phosphate (PNPP) method and a kit provided by Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Fifty microliters of phenol standard (0.1 mg/mL) was added in standard tubes, 50 mL of serum sample was added in sample tube, and 50 mL of deionized water was added in blank tube. Fifty microliters of buffer solution and 500 mL of substrate in order were added in each tube and mixed and put in 37°C water for 15 minutes. Then, 1.5-mL developer was added into the tubes and mixed immediately. Ultraviolet spectrophotometer was used for measurement. The wave length was 520 nm and the colorimetric optical path was 1 cm. Absorbancy of each tube was measured after zero setting. The serum ALP level was calculated by the formulate (1 King unit was defined as every 1 mg phenol produced by 100 mL of serum reacting with substrate in 37°C for 15 minutes). ALP level (King unit/100 mL) 5 (absorbancy of measuring tube/absorbancy of standard tube) 3 (phenol content in the standard tube/0.005 mg) 3 (100 mL/0.05 mL).
Measurement of hydroxyproline HOP was measured with digestion method and a kit provided by Nanjing Jiancheng Bioengineering Institute. Reagent was prepared according to the manufacturer’s instruction. Then, 0.25 mL of test solution was added in test tube, 0.25 mL of standard solution in standard tube, and 0.25 mL of distilled water in blank tube. The digestion kit was added into each tube in order and mixed. After 60°C water bath for 15 minutes, the solutions were cooled and centrifuged at 3500 rpm for 10 minutes. The supernatant was taken and absorbancy was measured in wavelength of 550 nm, 1 cm optical path, and zero setting was dealt with steamed water. Measurement of BMD The left femur and tibia were cleaned of the attached muscle and connective tissue and were put in the thin-walled organic glass box full of deionized water (to mimic the soft tissue around bone). The BMD (grams per square centimeter) of left femur and tibia was measured by dual-energy x-ray absorptiometry (DPX-L6843; Lunar Co Ltd., Madison, WI) using the small animal scan model.
Measurement of urine calcium
Femur metaphysis histomorphology
Urine calcium (Ca) was measured by methyl thymol blue colorimetric analysis and a kit provided by Nanjing Jiancheng Bioengineering Institute. Fifty microliters of calcium standard (2.5 mol/L) was added in standard tube, 50 mL of sample in sample tube, and 50 mL of deionized water in blank tube. One milliliter of methyl thymol blue reagent and 2 mL of alkaline solution were added orderly into each tube and mixed and standing for 15 minutes. Ultraviolet spectrophotometer was used for measurement and distilled water was used for zero setting. The wave length was 610 nm and the colorimetric optical path was 1 cm.
The right femur of rat was fixated in 10% formalin solution, decalcified with formic acid, and embedded in paraffin. The paraffin-embedded tissues were cut into 5-mm sections with a microtome and performed with hematoxylin and eosin staining. The change in trabeculae of femur metaphysis was observed under light microscope.
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Statistical analysis All data were performed using SPSS 17.0 statistical software package. Measurement data were expressed as mean 6 SD. Differences between groups were analyzed by analysis of variance and a P , 0.05 was considered statistically significant. American Journal of Therapeutics (2015) 0(0)
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Li et al
Table 1. Body weight of rats before and after the experiment in the sham group, OVX group, and drug-treated groups. Weight (g) Groups
Pre-experiment
A B C D E
203.25 202.52 201.31 197.05 205.53
6 6 6 6 6
Postexperiment
16.85 19.89 21.18 19.31 22.35
239.67 287.12 248.65 240.91 251.64
6 6 6 6 6
16.38 9.83* 10.61† 10.15† 20.16†
*Compared with group A, P , 0.01. †Compared with group B, P , 0.01.
RESULTS Changes of body weight of rats before and after the experiment Before experiments, there were no statistical differences among each 5 groups (all P . 0.05). After experiments, body weight in group B increased significantly than that in group A, whereas body weight in groups C, D, and E decreased significantly compared with that in group B (all P , 0.01). However, there were no significant differences in body weight among groups C, D, and E (all P . 0.05) (Table 1). Changes of indicators of bone formation and absorption Remarkably decreased serum BGP and increased serum ALP levels were detected in group B compared with those in group A. However, notably increased serum BGP and decreased serum ALP levels were found in groups C, D, and E compared with those in group B (all P , 0.05). While there were no significant differences in serum levels of BGP and ALP among groups C, D, and E (all P . 0.05). Compared with group A, the Ca/Cr ratio and the HOP/Cr ratio in group B increased
without significant differences, while compared with group B, the Ca/Cr ratio and the HOP/Cr ratio and in groups C, D, and E decreased slightly (all P . 0.05). Still no significant differences in these 4 indicators among groups C, D, and E were found (all P . 0.05) (Table 2). Changes in bone density Compared with group A, the BMD of rat femur lowered significantly in group B, but the BMD of rat femur increased notably in groups C, D, and E compared with group B (all P , 0.05). The BMD of rat femur showed no significant difference among groups C, D, and E (all P . 0.05). In addition, there existed a downward trend on BMD of rat femur in group B compared with group A, whereas an upward trend on BMD of rat femur in groups C, D, and E compared with group B (all P , 0.05) (Table 3). Histomorphological observation The sections of the femur were examined for any histological changes under LM. The rats of group A revealed closely and orderly arranged trabeculae with the normal morphology and mature structure under epiphyseal plate. The intertrabecular spaces were filled with red marrows (Figure 1A). The animals in group B showed sparse and quantity decreased trabeculae under epiphyseal plate. The connectivity between trabeculae was weak, fracture section can be observed and the thickness of the trabecula wall was uneven. Marrow content between trabeculae was significantly reduced (Figure 1B). Group C exhibited closely arranged and highly organized trabeculae with near-normal morphology and acceptable maturity under epiphyseal plate. The intertrabecular spaces were reticularly connected and the thickness of trabeculae wall was even. The amount of red marrow between trabeculae was much more (Figure 1C). Histomorphology of group D and group E was similar to group C (Figures 1D, E).
Table 2. Serum level of BGP, ALP, and urinary Ca/Cr ratio and HOP/Cr ratio in the sham group, OVX group, and drug-treated groups. Groups A B C D E
BGP (mg/L) 1.441 1.029 1.478 1.436 1.449
6 6 6 6 6
ALP (King unit/100 mL)
0.379 0.308* 0.260† 0.212† 0.189†
15.39 22.23 16.48 14.12 15.16
6 6 6 6 6
3.85 4.52* 5.11† 4.35† 4.58†
Ca/Cr 0.381 0.558 0.442 0.381 0.478
6 6 6 6 6
0.213 0.330 0.251 0.211 0.259
Hop/Cr (mg$L/mgprot$mmol) 24.08 31.29 25.52 25.24 27.12
6 6 6 6 6
7.28 5.67 9.26 7.34 8.78
*Compared with group A, P , 0.05. †Compared with group B, P , 0.05.
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Simvastatin, Nylestriol, and Ovariectomized Rats Table 3. Femoral and tibial BMD in the sham group, OVX group, and drug-treated groups.
Groups
Femoral BMD (g/cm2)
A B C D E
0.251 0.228 0.246 0.245 0.243
6 6 6 6 6
0.010 0.006* 0.007† 0.005† 0.006†
Tibial bone density (g/cm2) 0.217 0.204 0.212 0.215 0.214
6 6 6 6 6
0.007 0.006* 0.010† 0.008† 0.006†
*Compared with group A, P , 0.05. †Compared with group B, P , 0.05.
DISCUSSION Current treatments for osteoporosis mainly include 2 types, antiresorptive drugs aims at prevention of bone loss and anabolic drugs designed to increase bone volume. But either of the drugs could not fully treat the osteoporosis and have some side effects in clinical therapy.17 Simvastatin has reported to have an enhancing effect on the osteogenic genes, such as ALP, BMP2, and osteocalcin in osteoblastic cells.18 In addition, simvastatin is also connected with the decrease in osteoclast number, suggesting the decreased activity of
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osteoclast.19 Thus, we conducted the study to elucidate the efficacy of simvastatin on the treatment of osteoporosis, and we established an OVX rat model to simulate the osteoporosis process in human. The deficiency of estrogen caused by OVX could lead to an increased rate and an imbalance of bone resorption and formation.20 As a kind of estrogen, nylestriol could also promote growth of osteoblasts and osteocytes, thus treat the osteoporosis due to menopause and decreased estrogen level.21 In this study, we also studied the combination of simvastatin and nylestriol in the treatment of osteoporosis. Our study showed that OVX could increase the body weight, whereas both simvastatin and nylestriol could inhibit the gained body weight induced by OVX. Increased body weight has been reported in ovarian hormone deficiency and could be cured by estrogen administration.22 The present data also showed that OVX could decrease serum BGP and increase serum ALP levels. And simvastatin and nylestriol had the opposite effect in the regulation of serum BGP and ALP levels. BGP, a calcium-binding protein of bone, is a sensitive marker for bone formation and bone turnover, suggesting the activity of osteoblasts.23 Thus, the OVX could inhibit the activity of osteoblasts and the bone formation, whereas the 2 kinds of drugs could improve the situation by increase the serum
FIGURE 1. Representative images of hematoxylin and eosin (H&E)-stained specimen of the rat femur (A–E). A, There are some marrow cavities among bone trabeculae in group A. B, Broken bone trabeculae are arranged disorderly in group B. C–E, Bone trabeculae become thick in groups C–E. Magnification: 3200. www.americantherapeutics.com
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BGP. Serum ALP, the most commonly used biomarker for bone formation, is a key enzyme for osteoid formation and bone mineralization.24,25 However, there were no significant differences in the Ca/Cr ratio and HOP/ Cr ratio among different treatment, suggesting the relative safety of the drug use. Our findings also showed that femoral and tibial BMD decreased in the OVX group, but increased after the treatment of simvastatin or nylestriol, suggesting the role of these drugs in preventing bone loss and deficiency of estrogen in osteoporosis treatment. The measurement of BMD by DXA plays an essential role in the osteoporosis and fracture risk assessment.26 BMD value even used to define the osteoporosis is defined by the World Health Organization with BMD 2.5 SDs or more below the young female adult mean.6 Our results also in accordance with the study of Oxlund and Andreassen,27 which demonstrated that simvastatin could partially prevent the loss of cancellous bone after OXV both in proximal tibias and vertebral bodies, increase BMD and lower fractures risk. Histomorphological examination results showed that OVX could decrease the number of trabeculae, weaken the connectivity between trabeculae, cause the formation of fracture section, and reduce the red marrows. While the treatment with simvastatin or combination of simvastatin and nylestriol could reverse the harm caused by OVX by showing closely arranged and highly organized trabeculae. Taken together, our studies showed that simvastatin and combination of simvastatin and nylestriol could promote the formation of new bone, increase bone density, and improve bone microstructure damage in OVX rats. However, we did not observe the bone formation in our histomorphological examination results, thus we could not clarify the changes of BMD is due to bone formation or bone resorption. Thus, further mechanisms related to some signaling ways should be studied before the drugs be tested in humans as an antiosteoporosis therapy.
ACKNOWLEDGMENTS The authors thank their researchers for their hard work and reviewers for their valuable advice.
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