Pharmaceutical Biology
ISSN: 1388-0209 (Print) 1744-5116 (Online) Journal homepage: http://www.tandfonline.com/loi/iphb20
Anti-osteoporotic activity of puerarin 6″-Oxyloside on ovariectomized mice and its potential mechanism Hai Li, Bingpu Chen, Guangfu Pang, Jianhai Chen, Jisheng Xie & Hailing Huang To cite this article: Hai Li, Bingpu Chen, Guangfu Pang, Jianhai Chen, Jisheng Xie & Hailing Huang (2015): Anti-osteoporotic activity of puerarin 6″-O-xyloside on ovariectomized mice and its potential mechanism, Pharmaceutical Biology To link to this article: http://dx.doi.org/10.3109/13880209.2015.1017885
Published online: 10 Apr 2015.
Submit your article to this journal
Article views: 38
View related articles
View Crossmark data
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=iphb20 Download by: [University of Otago]
Date: 27 September 2015, At: 14:42
http://informahealthcare.com/phb ISSN 1388-0209 print/ISSN 1744-5116 online Editor-in-Chief: John M. Pezzuto Pharm Biol, Early Online: 1–7 ! 2015 Informa Healthcare USA, Inc. DOI: 10.3109/13880209.2015.1017885
ORIGINAL ARTICLE
Anti-osteoporotic activity of puerarin 600 -O-xyloside on ovariectomized mice and its potential mechanism Hai Li1, Bingpu Chen2, Guangfu Pang3, Jianhai Chen1, Jisheng Xie1, and Hailing Huang1 Department of Histology and Embryology, 2Department of Anatomy, and 3Department of Laboratory Medicine, Youjiang Medical University for Nationalities, Baise, China
Downloaded by [University of Otago] at 14:42 27 September 2015
1
Abstract
Keywords
Context: Osteoporosis is one of the most common bone diseases, and radix of Pueraria lobata (Willd.) Ohwi possesses an obvious therapeutical effect on postmenopausal osteoporosis. Objective: This study investigates the anti-osteoporotic activity of the puerarin 600 -O-xyloside (PXY) on ovariectomized mice and its related mechanism. Materials and methods: Osteoporotic mice model was established by ovariectomy (OVX). A total of 50 mice were divided into five groups (n ¼ 10): sham, OVX group, PXY treatment groups (20, 40, and 60 mg/kg/d, i.p.). After 12 weeks’ treatment, body weights were recorded. Then, mice were sacrificed, and serum samples were collected to determine the blood calcium, blood phosphorus, alkaline phosphatase (ALP), and osteoprotegerin (OPG) concentrations and uterine index was assayed. The thigh-bones of mice were collected to evaluate histopathological changes. In the in vitro experiment, the effect of PXY on osteoblasts’ proliferation was evaluated and western blotting was performed to determine expressions of OPG and the receptor activators of NF-B ligand (RANKL), as well as the ratio of OPG/RANKL. Results: PXY (40 and 60 mg/kg/d, i.p.) obviously decreased body weights and increased uterine index of OVX (p50.05), and improved osteoporotic syndromes of OVX mice; PXY also significantly increased the concentrations of blood calcium, blood phosphorus, ALP, and OPG of OVX mice (p50.05); moreover, PXY obviously up-regulated the ratio of OPG/RANKL (p50.05). Conclusion: Our results demonstrated that the puerarin 600 -O-xyloside possesses significant anti-osteoporotic activity on ovariectomy mice.
OPG/RANKL, osteoblasts, OVX, radix of Pueraria lobata (willd.) ohwi
Introduction Osteoporosis is one of the common bone diseases characterized by a systemic impairment of bone mass and high risk of fractures (Qin et al., 2008; Zhang et al., 2008). It is estimated that about 30% of postmenopausal women in the USA and Europe have osteoporosis, and osteoporosis is one of the major health problems of elderly women (Bock & Felsenberg, 2008; Tu¨zu¨n et al., 2013). Osteoporosis is associated with ovarian hormone deficiency following menopause, and is the most common cause of age-related bone loss (Nian & Qin, 2006; Riggs & Melton, 1986). The estrogen replacement therapy (ERT) is one of the commonly used treatments for preventing osteoporosis in postmenopausal women, and is considered to be the most effective therapy for osteoporosis (Ma et al., 2013; Qin et al., 2008). However, serious and undesirable adverse effects were found in ERT treatment, including endometrial cancer, breast carcinoma, and Correspondence: Hai Li, Department of Histology and Embryology, Youjiang Medical University for Nationalities, 98 Chengxiang Road, Baise, Guangxi 533000, China. Tel/Fax: +86 776 2849559. E-mail: [email protected]
History Received 25 June 2014 Revised 20 November 2014 Accepted 6 February 2015 Published online 10 April 2015
cardiovascular diseases (Du et al., 2011). Furthermore, there are many other synthetic agents that could be used to prevent or treat osteoporosis, but many side effects also exist as ERT (Canalis et al., 1998; Genant et al., 1998). Thus, it is urgent and important to find new drugs to treat osteoporosis with low side-effects. The natural plant-derived compounds are rich sources for screening novel drugs with low adverse effect, and in recent years, lots of extracts or monomers have been isolated from the plant medicines with significant bio-activities (Bereswill et al., 2010; Li & Vederas, 2009; Peng et al., 2014; Zhu et al., 2010). In addition, there are many investigations reported that radix of Pueraria lobata (Willd.) Ohwi, which is a famous traditional Chinese medicine, has significantly therapeutical effect on postmenopausal osteoporosis, and the active constituents would be the iso-flavones in radix of P. lobata (Li et al., 2011; Wang et al., 2013; Zheng et al., 2002). Puerarin 600 -O-xyloside (PXY, Figure 1) is one of the major iso-flavones of the radix of P. lobata; however, there are few reports regarding the bioactivities of PXY. In our preliminary experiment, we found that PXY showed obvious proliferative effect on osteoblasts in vitro. Therefore, in our present study,
2
H. Li et al.
Pharm Biol, Early Online: 1–7
Experimental protocol and preparation of osteoporotic model by ovariectomy
Figure 1. Chemical structure of puerarin 600 -O-xyloside (PXY).
we established the osteoporosis model of ovariectomized mice to systemically investigate the anti-osteoporotic activity of PXY and its possible mechanism.
Downloaded by [University of Otago] at 14:42 27 September 2015
Materials and methods Chemicals and regents Puerarin 600 -O-xyloside was purchased from the Shanghai Tauto Biotech Ltd. Co. (Shanghai, China); trypsinase, collagenase (type II), a-modified minimum essential medium (a-MEM), and fetal bovine serum (FBS) were purchased from the Gibco Life Tech. (Grand Island, NY); 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and dimethyl sulfoxide were purchased from Sigma (St. Louis, MO).
A total of 50 female mice were used in our experiment, and OVX mice were prepared as described above and randomly divided into five groups (n ¼ 10): Sham group, ovariectomy (OVX) group, PXY treatments groups (20, 40, and 60 mg/kg/d, i.p., for 12 weeks). After 12 weeks treatment of PXY, body weights of the mice were recorded. Then, mice were sacrificed, and the blood samples were collected and centrifuged to obtain serum for further determination of biochemical parameters. Moreover, the uterus were also removed and weighed immediately to calculate the uterine index. Additionally, the thigh-bones of mice were collected and tissue sections were dissected and fixed in 10% formalin; then processed routinely, embedded in paraffin, sectioned to 5 mm thickness, de-paraffinized, rehydrated using standard techniques, stained with hematoxylin and eosin (H&E). The histopathological changes were evaluated with microscope (Olympus, Tokyo, Japan). Determination of ALP, blood calcium, and blood phosphorus in serum An automatic biochemistry analyzer (Siemens ADVIL 1650, Munich, Germany) was used to measure the contents of serum alkaline phosphatase (ALP), blood calcium, and blood phosphorus in serum of OVX mice.
Animals
Determination of the concentration of OPG
ICR mice (female, ages of 3-month-old) and newborn SD rats (3 d old) were purchased from the Shanghai Laboratory Animal Centre (Shanghai, China), and used in our present investigations. Each animal was used only once in the experiment. The experimental protocols were approved by the Animal Care and Use Committee of our university and the procedures employed were strictly according to generally accepted international rules and regulations.
The serum osteoprotegerin (OPG) contents were determined by using an ELISA kit according to the instructions of the manufacturer and quantified using a microplate reader (Bio-Tek ELX800, Bio-Tek, Winooski, VT) at 450 nm.
Toxicity tests Sixty ICR mice were divided into six groups randomly and each group containing 10 mice. Mice of groups 1–5 were intraperitoneal injection (i.p.) of 10, 20, 40, 60, and 80 mg/kg of PXY, respectively, and the sixth group received 10 ml/kg of normal saline. The mortality rates of the mice within a 24 h period were observed and recorded. Establishment of OVX mice The osteoporotic mice model was established by ovariectomy operation described previously (Li et al., 2011; Xue et al., 2012). In brief, mice were general anesthetized with sodium pentobarbital (1.5%, 50 mg/kg). Under sterile condition, bilateral ovaries were resected through the incision made on back of the mice, and then incisions were sutured layer by layer. After surgery, mice were injected with gentamycin for 3 d to prevent wound infection. One week later, mice were administered with drugs. For the sham-operated group, mice were underwent the same surgical procedures, but no ovaries resection was performed.
Effects of PYX on osteoblasts in vitro Cell culture The osteoblasts were isolated from calvarias of newborn SD rats (3 d old) according to the method reported in previous investigation with minor modification (Liu et al., 1995). Briefly, calvarias were isolated from the newborn rats under sterile condition, and cut up by using ophthalmic scissors after washed with PBS for three times. Then, 0.25% trypsinase was added to the bone tissues, and cultivated for 30 min at 37 C. Bone tissues were washed with PBS for three times after discarding the supernatant; then, the digestion solution containing 0.05% trypsinase and 0.02% collagenase (type II) was added to the bone tissues, and cultivated for 1 h at 37 C. The digestion solution was collected and centrifugated for 10 min (1000 rpm) to separated the fresh osteoblasts. Osteoblasts were cultured in a-MEM supplemented with 10% FBS at 37 C in 5% CO2/95% air. Assay for proliferation of osteoblasts Cells (2 104/ml) were seeded in 96-well plates and treated on the following day with indicated concentrations of PYX (5, 10, and 20 mM) for 24, 48, and 72 h, respectively. After that, MTT assay was carried out, and the optical density (OD)
DOI: 10.3109/13880209.2015.1017885
was read at 550 nm using a 96-well plate reader (Bio-Tek ELX800, Bio-Tek, Winooski, VT). Western blotting
Downloaded by [University of Otago] at 14:42 27 September 2015
Cells treated with or without PYX (5, 10, and 20 mM) for 48 h were harvested. Then, total proteins of the cells were extracted, and then equal amounts of protein (40 mg) were separated by sodium dodecyl sulfate/polyacrylamidegel electrophoresis (SDS/PAGE), blotted on polyvinylidene difluoride (PVDF), and probed with anti-RANKL rabbit polyclonal IgG and anti-OPG rabbit polyclonal IgG (Wuhan Boster Bioengineering Ltd. Co., Wuhan, China), and subsequently with goat anti-rabbit/HRP (Wuhan Boster Bio-engineering Ltd. Co., Wuhan, China), and detected by chemiluminescence. To measure protein loading, antibodies directed against b-actin were used.
Anti-osteoporotic activity of PXY
3
phenomenon at all the tested doses compared with the OVX group (p50.05, p50.01, p50.01 for the doses of 20, 40, and 60 mg/kg/d, respectively), in a dose-dependent manner (Figure 2B). Effects of PXY on body blood calcium and blood phosphorus contents As can be seen from Figure 3, after being removed the ovaries, the blood calcium and blood phosphorus contents in the serum decreased dramatically (p50.01). However, the results of our present study indicated that treating with PXY at the doses of 20, 40, and 60 mg/kg/d (i.p.) were able to increase not only the blood calcium contents obviously (p50.05) but also the blood phosphorus contents in the serum significantly (p50.05, p50.01, p50.01) in a dose-dependent manner, compared with the OVX group.
Statistical analysis
Effects of PXY on ALP and OPG contents
Data were expressed as mean ± standard deviation (SD). Statistical analyses were performed using two-tailed Student’s t-test with a significance level of p50.05.
ALP is one of the key marker enzymes for functional osteoblasts, and the differentiation of osteoblasts was evaluated by ALP activity. As can be seen from Figure 4(A), ALP levels of OVX mice were increased 12 weeks after operation. Moreover, the PXY at the doses at 40 and 60 mg/kg/d (i.p.) can increase the ALP content in the serum obviously compared with the OVX group (p50.01, p50.01). Additionally, the OPG levels of the mice suffered from OVX were significantly decreased at the end of the observation (p50.01) (Figure 4B). Interestingly, at the tested doses (20, 40, and 60 mg/kg/d, i.p.), the PXY obviously increased the OPG content in the serum 12 weeks after operation compared with the OVX group (p50.05, p50.01, p50.01), in a dose-dependent manner.
Results Toxicity study results In the results of toxicity investigation, neither death nor any abnormal neuro-behaviors were observed. The 50% lethal dose (LD50) was not obtained due to lack of obvious toxicity at all the tested doses. Effects of PXY on weight changes of body weight and uterus After 12 weeks of OVX operation, the mice in the OVX group were significantly fatter than mice in the sham group (p50.05). However, as can be seen from our present study (Figure 2A), at the doses of 40 and 60 mg/kg/d (i.p.), the weights of mice obviously decreased compared with the OVX mice (p50.05). Additionally, at the end of the observation, the uterus’s weights of mice suffered from OVX were decreased sharply in the OVX group (p50.01, compared with the sham), whereas PXY can inhibit obviously this
Effect of PXY on femur osseous tissues As shown in Figure 5(A), in the sham-operated group, the compact bone was dense, osteocytes were arranged orderly. In the OVX group, the bone marrow cavity was enlarged; trabecular bone was sparse and slender, fractured in large fragments, and poorly arranged (Figure 5B). However, after treatment with PXY at the tested doses (20, 40, and 60 mg/kg/d, i.p.) for 12 weeks, the pathological changes of osteoporosis
Figure 2. Effects of PXY body on weight and uterine weight of OVX mice. Each column represented as mean ± standard deviation (n ¼ 10). The vehicle (10 ml/kg) and PXY (20, 40, and 60 mg/kg) were administered intraperitoneally. #p50.05, ##p50.01, compared with the sham group; *p50.05, **p50.01, compared with the OVX group.
4
H. Li et al.
Pharm Biol, Early Online: 1–7
Downloaded by [University of Otago] at 14:42 27 September 2015
Figure 3. Effects of PXY on body blood calcium and blood phosphorus contents of OVX mice. Each column represented as mean ± standard deviation (n ¼ 10). The vehicle (10 ml/kg) and PXY (20, 40, and 60 mg/kg) were administered intraperitoneally. ##p50.01, compared with the sham group; *p50.05, **p50.01, compared with the OVX group.
Figure 4. Effects of PXY on ALP and OPG contents of OVX mice. Each column represented as mean ± standard deviation (n ¼ 10). The vehicle (10 ml/kg) and PXY (20, 40, and 60 mg/kg) were administered intraperitoneally. #p50.05, ##p50.01, compared with the sham group; *p50.05, **p50.01, compared with the OVX group.
were obviously improved (Figure 5C–E), compact bone was thickened, trabecular bone became thick and compact. In particular, the pathological changes of mice treated with PXY at the dose of 60 mg/kg/d were nearly normal (Figure 5E). Effect of PXY on proliferation of osteoblasts It is no doubt that MTT assay was a classical method that used as an index of proliferation. As can be seen from Table 1, the PXY at the concentrations of 5, 10, and 20 mM slightly enhanced the proliferation of osteoblasts after 24 h culture. However, after culturing more than 48 h, PXY at concentrations of 5, 10, and 20 mM showed significant proliferative effects on osteoblasts, in a dose-dependent manner. Effect of PXY on expressions of OPG and RANKL in osteoblasts The expressions of OPG and receptor activators of NF-B ligand (RANKL) proteins were determined by western blotting (Figure 6). As can be seen from our results, the expressions of RANKL proteins in osteoblasts were decreased significantly by treating with PXY at concentrations of 5, 10, and 20 mM, compared with the control group
(p50.01, p50.01, p50.01). On the contrary, the PXY at concentrations of 10 and 20 mM can significantly up-regulate the expression of OPG proteins, compared with the control group (p50.01, p50.01). In addition, the ratio of OPG/ RANKL was up-regulated by the treatment of PXY at concentrations of 5, 10, and 20 mM, compared with the control group (p50.01, p50.01, p50.01). The relative protein level was normalized to the Western blot intensity of b-actin.
Discussion In our present study, we investigated the effects of PXY on OVX-induced osteoporosis in mice and its potential mechanism. The results of our present study clearly demonstrated the beneficial effects of PXY in the prevention of bone loss induced by ovariectomy for the first time. Estrogen deficiency is an important risk factor in the pathogenesis of osteoporosis (Li et al., 2013). The PXY is one of the main iso-flavones in radix of P. lobata as puerarin, and the structure of PXY is similar to the soy isoflavone which was reported to be beneficial for women’s postmenopausal osteoporosis (Chin & Ima-Nirwana, 2013). Iso-flavones are
Anti-osteoporotic activity of PXY
DOI: 10.3109/13880209.2015.1017885
5
Downloaded by [University of Otago] at 14:42 27 September 2015
Figure 5. Effect of PXY on femur osseous tissue in ovariectomized mice. (A–E) The Sham, ovariectomized model, and PXY treatment (20, 40, and 60 mg/kg) are represented, respectively. The vehicle (10 ml/kg) and PXY were administered intraperitoneally.
Table 1. Effects of PXY on the proliferation of osteoblasts. OD550 24 h
48 h
72 h
0.32 ± 0.04 0.38 ± 0.05*
0.47 ± 0.06 0.56 ± 0.08*
0.67 ± 0.09 0.84 ± 0.12**
0.43 ± 0.05* 0.46 ± 0.06*
0.61 ± 0.09** 0.67 ± 0.10**
0.89 ± 0.15** 0.91 ± 0.14**
Control 5 mM PXY 10 mM 20 mM
Effects of PXY on the proliferation of osteoblasts. Cells were treated with PXY treatment (5, 10, and 20 mM) for 24, 48, and 72 h, respectively. Data were presented as mean ± standard deviation (n ¼ 4).
considered as the natural estrogens, and showed significant anti-osteoporotic activity (Ishida et al., 1998). Therefore, the unique iso-flavones structure might be one important reason for response to the anti-osteoporotic activity of PXY. The osteoporotic mice model induced by ovariectomy is one of the most common and reliable used models for studying osteoporosis, and this model can simulate commendably the clinical syndromes of women’s postmenopausal osteoporosis. Furthermore, the phenomenon of menopause is a signal of ovarian failure, which also means reduction of estrogen, resulting in bone loss increasingly
(Kalu, 1991; Li et al., 2013). In previous investigations, it is reported that ovariectomy can lead to a significantly increase in body weight and decrease in uterus’ weights, and these changes partly due to estrogen deficiency (Li et al., 2013; Nian et al., 2009). The results of our present study demonstrated that OVX decreased the uterine index and increased the body weight compared with the Sham group, which clearly showed the successful establishment of osteoporotic mice model. In addition, PXY treatments (i.p., for 12 weeks) at the tested doses significantly increased the uterine index and decreased the body weight in osteoporotic mice compared with osteoporotic model mice, which indicated that PXY could improve the osteoporotic syndrome of mice suffered from ovariectomy. The contents of blood calcium and blood phosphorus can reflect the degree of bone loss (Li et al., 2011). Our results showed that in the OVX group, levels of blood calcium and blood phosphorus decreased sharply compared with the sham group, but the PXY at the tested doses (i.p., for 12 weeks) can increase the contents of blood calcium and blood phosphorus in serum of mice suffered from ovariectomy. In addition, our results also showed that PXY can obviously improve the osseous tissues’ condition in osteoporotic mice. This result is another evidence for demonstrating that PXY could improve the osteoporotic syndrome of OVX mice.
6
H. Li et al.
Pharm Biol, Early Online: 1–7
Downloaded by [University of Otago] at 14:42 27 September 2015
Figure 6. Effect of PXY on expressions of OPG and RANKL in osteoblasts. Osteoblastic cells from neonatal rat calvarias were treated with PXY treatment (5, 10, and 20 mM) for 48 h. Protein expressions of OPG and RANKL were determined by western blotting, and b-actin was used as an internal reference. Data were presented as mean ± standard deviation (n ¼ 4), **p50.01, compared with control.
ALP is an important enzyme in the bone remodeling process, and it promotes mineralization of matrix by decomposing the phosphoric ester into inorganic phosphorous in order to increase the phosphorous concentration (Jia et al., 2009). In our present study, we found that the PXY at the doses of 40 and 60 mg/kg/d (i.p., for 12 weeks) can obviously increase the contents of ALP. The RANKL and OPG secreted by osteoblast play the important roles in osteoclastic formation, differentiation, and bone resorbing (Steeve et al., 2004; Xue et al., 2012). Furthermore, RANKL can activate the osteoclast progenitors through the receptor activator of NFB (RANK) to induce osteoclasts’ differentiation and function. Importantly, OPG can block the communication between RANKL and RANK receptor, which means that OPG can suppress osteoclastogenesis and bone resorption (Liu et al., 2005; Xue et al., 2012). Thus, the relative ratio of OPG/ RANKL is very important for the bone remodeling. In our present study, PXY at the tested concentrations can significantly decrease the expression of RANKL, and increase the expression of OPG and relative ratio of OPG/RANKL, indicating that PXY can suppress the bone loss and resorption by up-regulating ratio value of OPG/RANKL in osteoblasts. Collectively, our present study demonstrated that the PXY could improve the osteoporotic syndrome of mice suffered from ovariectomy, and the possible mechanisms are regarding increasing the blood calcium, blood phosphorus, and ALP contents and up-regulating the ratio value of OPG/RANKL in osteoblasts.
Declaration of interest The authors report that they have no conflicts of interest. This work was supported by the Guangxi Natural Science Foundation (Nos. 2010GXNSFA013268 and 2013 GXNSFAA019241).
References Bereswill S, Munoz1 M, Fischer A, et al. (2010). Anti-inflammatory effects of resveratrol, curcumin and simvastatin in acute small intestinal inflammation. PLoS One 5:e15099.
Bock O, Felsenberg D. (2008). Bisphosphonates in the management of postmenopausal osteoporosis-optimizing efficacy in clinical practice. Clin Interv Aging 3:279–97. Canalis H, McCarthy T, Centrella M. (1998). Growth factors and the regulation on bone remodeling. J Clin Invest 81:277–81. Chin KY, Ima-Nirwana S. (2013). Can soy prevent male osteoporosis? A review of the current evidence. Curr Drug Targets 14:1632–41. Du J, Wei YJ, Peng C, et al. (2011). Establishment of a luciferase assaybased screening system for detecting estrogen receptor agonists in plant extracts. Bone 49:572–9. Genant HK, Bay DJ, Gallagher JC. (1998). Estrogens in the prevention of osteoporosis in post menopausal women. Am J Obstet Gynecol 161: 1842–6. Ishida H, Uesugi T, Hirai K, et al. (1998). Preventive effects of the plant isoflavones, daidzin and genistin, on bone loss in ovariectomized rats fed a calcium-deficient diet. Biol Pharm Bull 21:62–6. Jia L, Cao DP, Qin LP, et al. (2009). Antiosteoporotic activity of phenolic compounds from Curculigo orchioides. Phytomedicine 16: 874–81. Kalu DN. (1991). The ovariectomized rat model of postmenopausal bone loss. Bone Miner 15:175–91. Li F, Yang XL, Yang YN, et al. (2013). Antiosteoporotic activity of echinacoside in ovariectomized rats. Phytomedicine 20:549–57. Li H, Wang JH, Huang HL, Li B. (2011). Puerarin combined with estradiol for treatment of osteoporosis in ovariectomized rats. J Clin Rehab Tiss Eng Res 15:8698–701. Li JWH, Vederas JC. (2009). Drug discovery and natural products: End of an era or an endless frontier? Science 325:161–5. Liu XH, Kirschenbaum A, Yao S, Levine AC. (2005). Crosstalk between the interleukin-6 and prostaglandin E2 signaling systems results in enhancement of osteoclastogenesis through effects on the osteoprotegerin/receptor activator of nuclear factor-kB (RANK) ligand/RANK system. Endocrinology 146:1991–8. Liu ZD, Zang HS, Ou-Yang YP. (1995). Biological study of growth pattern of newborn rat calvaria osteoblastic cell in vitro. Acta Anatom Sin 26:157–9. Ma XQ, Zheng CJ, Zhang Y, et al. (2013). Antiosteoporotic flavonoids from Podocarpium podocarpum. Phytochem Lett 6:118–22. Nian H, Ma MH, Nian SS, Xu L. (2009). Antiosteoporotic activity of icariin in ovariectomized rats. Phytomedicine 16:320–6. Nian H, Qin LP. (2006). Antiosteoporotic activity of Er-Xian decoction, a traditional Chinese herbal formula, in ovariectomized rats. J Ethnopharmacol 108:96–102. Peng W, Ming QL, Han P, et al. (2014). Anti-allergic rhinitis effect of caffeoylxanthiazonoside isolated from fruits of Xanthium strumarium L. in rodent animals. Phytomedicine 15:824–9. Qin LP, Han T, Zhang QY, et al. (2008). Antiosteoporotic chemical constituents from Er-Xian decoction, a traditional Chinese herbal formula. J Ethnopharmacol 118:271–9.
DOI: 10.3109/13880209.2015.1017885
Downloaded by [University of Otago] at 14:42 27 September 2015
Riggs BL, Melton LJ. (1986). Involutional osteoporosis. New Engl J Med 314:1676–86. Steeve KT, Marc P, Sandrine T, et al. (2004). IL-6, RANKL, TNF-a/ IL-1: Interrelations in bone resorption pathophysiology. Cytokine Growth Factor Rev 15:49–60. Tu¨zu¨n S, Akyu¨z G, Eskiyurt N, et al. (2013). Impact of the training on the compliance and persistence of weekly bisphosphonate treatment in postmenopausal osteoporosis: A randomized controlled study. Int J Mol Sci 10:1880–7. Wang Y, Wang WL, Xie WL, et al. (2013). Puerarin stimulates proliferation and differentiation and protects against cell death in human osteoblastic MG-63 cells via ER-dependent MEK/ERK andPI3K/Akt activation. Phytomedicine 20:787–96.
Anti-osteoporotic activity of PXY
7
Xue LM, Jiao L, Wang Y, et al. (2012). Effects and interaction of icariin, curculigoside, and berberine in Er-Xian decoction, a traditional Chinese Medicinal formula, on osteoclastic bone resorption. Evid Based Complement Alternat Med 2012:490843. Zhang H, Xing WW, Li YS, et al. (2008). Effects of a traditional Chinese herbal preparation on osteoblasts and osteoclasts. Maturitas 61:334–9. Zheng GL, Zhang XY, Meng QH, Gong WG. (2002). Protective effect of total isoflavones from pueraria lobata on secondary osteoporosis induced by dexamethasone in rats. J Chin Med Mater 25:643–6. Zhu Z, Xue LM, Han T, et al. (2010). Antiosteoporotic effects and proteomic characterization of the target and mechanism of an Er-Xian decoction on osteoblastic UMR-106 and osteoclasts induced from RAW264.7. Molecules 15:4695–710.
ISSN: 1388-0209 (Print) 1744-5116 (Online) Journal homepage: http://www.tandfonline.com/loi/iphb20
Anti-osteoporotic activity of puerarin 6″-Oxyloside on ovariectomized mice and its potential mechanism Hai Li, Bingpu Chen, Guangfu Pang, Jianhai Chen, Jisheng Xie & Hailing Huang To cite this article: Hai Li, Bingpu Chen, Guangfu Pang, Jianhai Chen, Jisheng Xie & Hailing Huang (2015): Anti-osteoporotic activity of puerarin 6″-O-xyloside on ovariectomized mice and its potential mechanism, Pharmaceutical Biology To link to this article: http://dx.doi.org/10.3109/13880209.2015.1017885
Published online: 10 Apr 2015.
Submit your article to this journal
Article views: 38
View related articles
View Crossmark data
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=iphb20 Download by: [University of Otago]
Date: 27 September 2015, At: 14:42
http://informahealthcare.com/phb ISSN 1388-0209 print/ISSN 1744-5116 online Editor-in-Chief: John M. Pezzuto Pharm Biol, Early Online: 1–7 ! 2015 Informa Healthcare USA, Inc. DOI: 10.3109/13880209.2015.1017885
ORIGINAL ARTICLE
Anti-osteoporotic activity of puerarin 600 -O-xyloside on ovariectomized mice and its potential mechanism Hai Li1, Bingpu Chen2, Guangfu Pang3, Jianhai Chen1, Jisheng Xie1, and Hailing Huang1 Department of Histology and Embryology, 2Department of Anatomy, and 3Department of Laboratory Medicine, Youjiang Medical University for Nationalities, Baise, China
Downloaded by [University of Otago] at 14:42 27 September 2015
1
Abstract
Keywords
Context: Osteoporosis is one of the most common bone diseases, and radix of Pueraria lobata (Willd.) Ohwi possesses an obvious therapeutical effect on postmenopausal osteoporosis. Objective: This study investigates the anti-osteoporotic activity of the puerarin 600 -O-xyloside (PXY) on ovariectomized mice and its related mechanism. Materials and methods: Osteoporotic mice model was established by ovariectomy (OVX). A total of 50 mice were divided into five groups (n ¼ 10): sham, OVX group, PXY treatment groups (20, 40, and 60 mg/kg/d, i.p.). After 12 weeks’ treatment, body weights were recorded. Then, mice were sacrificed, and serum samples were collected to determine the blood calcium, blood phosphorus, alkaline phosphatase (ALP), and osteoprotegerin (OPG) concentrations and uterine index was assayed. The thigh-bones of mice were collected to evaluate histopathological changes. In the in vitro experiment, the effect of PXY on osteoblasts’ proliferation was evaluated and western blotting was performed to determine expressions of OPG and the receptor activators of NF-B ligand (RANKL), as well as the ratio of OPG/RANKL. Results: PXY (40 and 60 mg/kg/d, i.p.) obviously decreased body weights and increased uterine index of OVX (p50.05), and improved osteoporotic syndromes of OVX mice; PXY also significantly increased the concentrations of blood calcium, blood phosphorus, ALP, and OPG of OVX mice (p50.05); moreover, PXY obviously up-regulated the ratio of OPG/RANKL (p50.05). Conclusion: Our results demonstrated that the puerarin 600 -O-xyloside possesses significant anti-osteoporotic activity on ovariectomy mice.
OPG/RANKL, osteoblasts, OVX, radix of Pueraria lobata (willd.) ohwi
Introduction Osteoporosis is one of the common bone diseases characterized by a systemic impairment of bone mass and high risk of fractures (Qin et al., 2008; Zhang et al., 2008). It is estimated that about 30% of postmenopausal women in the USA and Europe have osteoporosis, and osteoporosis is one of the major health problems of elderly women (Bock & Felsenberg, 2008; Tu¨zu¨n et al., 2013). Osteoporosis is associated with ovarian hormone deficiency following menopause, and is the most common cause of age-related bone loss (Nian & Qin, 2006; Riggs & Melton, 1986). The estrogen replacement therapy (ERT) is one of the commonly used treatments for preventing osteoporosis in postmenopausal women, and is considered to be the most effective therapy for osteoporosis (Ma et al., 2013; Qin et al., 2008). However, serious and undesirable adverse effects were found in ERT treatment, including endometrial cancer, breast carcinoma, and Correspondence: Hai Li, Department of Histology and Embryology, Youjiang Medical University for Nationalities, 98 Chengxiang Road, Baise, Guangxi 533000, China. Tel/Fax: +86 776 2849559. E-mail: [email protected]
History Received 25 June 2014 Revised 20 November 2014 Accepted 6 February 2015 Published online 10 April 2015
cardiovascular diseases (Du et al., 2011). Furthermore, there are many other synthetic agents that could be used to prevent or treat osteoporosis, but many side effects also exist as ERT (Canalis et al., 1998; Genant et al., 1998). Thus, it is urgent and important to find new drugs to treat osteoporosis with low side-effects. The natural plant-derived compounds are rich sources for screening novel drugs with low adverse effect, and in recent years, lots of extracts or monomers have been isolated from the plant medicines with significant bio-activities (Bereswill et al., 2010; Li & Vederas, 2009; Peng et al., 2014; Zhu et al., 2010). In addition, there are many investigations reported that radix of Pueraria lobata (Willd.) Ohwi, which is a famous traditional Chinese medicine, has significantly therapeutical effect on postmenopausal osteoporosis, and the active constituents would be the iso-flavones in radix of P. lobata (Li et al., 2011; Wang et al., 2013; Zheng et al., 2002). Puerarin 600 -O-xyloside (PXY, Figure 1) is one of the major iso-flavones of the radix of P. lobata; however, there are few reports regarding the bioactivities of PXY. In our preliminary experiment, we found that PXY showed obvious proliferative effect on osteoblasts in vitro. Therefore, in our present study,
2
H. Li et al.
Pharm Biol, Early Online: 1–7
Experimental protocol and preparation of osteoporotic model by ovariectomy
Figure 1. Chemical structure of puerarin 600 -O-xyloside (PXY).
we established the osteoporosis model of ovariectomized mice to systemically investigate the anti-osteoporotic activity of PXY and its possible mechanism.
Downloaded by [University of Otago] at 14:42 27 September 2015
Materials and methods Chemicals and regents Puerarin 600 -O-xyloside was purchased from the Shanghai Tauto Biotech Ltd. Co. (Shanghai, China); trypsinase, collagenase (type II), a-modified minimum essential medium (a-MEM), and fetal bovine serum (FBS) were purchased from the Gibco Life Tech. (Grand Island, NY); 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and dimethyl sulfoxide were purchased from Sigma (St. Louis, MO).
A total of 50 female mice were used in our experiment, and OVX mice were prepared as described above and randomly divided into five groups (n ¼ 10): Sham group, ovariectomy (OVX) group, PXY treatments groups (20, 40, and 60 mg/kg/d, i.p., for 12 weeks). After 12 weeks treatment of PXY, body weights of the mice were recorded. Then, mice were sacrificed, and the blood samples were collected and centrifuged to obtain serum for further determination of biochemical parameters. Moreover, the uterus were also removed and weighed immediately to calculate the uterine index. Additionally, the thigh-bones of mice were collected and tissue sections were dissected and fixed in 10% formalin; then processed routinely, embedded in paraffin, sectioned to 5 mm thickness, de-paraffinized, rehydrated using standard techniques, stained with hematoxylin and eosin (H&E). The histopathological changes were evaluated with microscope (Olympus, Tokyo, Japan). Determination of ALP, blood calcium, and blood phosphorus in serum An automatic biochemistry analyzer (Siemens ADVIL 1650, Munich, Germany) was used to measure the contents of serum alkaline phosphatase (ALP), blood calcium, and blood phosphorus in serum of OVX mice.
Animals
Determination of the concentration of OPG
ICR mice (female, ages of 3-month-old) and newborn SD rats (3 d old) were purchased from the Shanghai Laboratory Animal Centre (Shanghai, China), and used in our present investigations. Each animal was used only once in the experiment. The experimental protocols were approved by the Animal Care and Use Committee of our university and the procedures employed were strictly according to generally accepted international rules and regulations.
The serum osteoprotegerin (OPG) contents were determined by using an ELISA kit according to the instructions of the manufacturer and quantified using a microplate reader (Bio-Tek ELX800, Bio-Tek, Winooski, VT) at 450 nm.
Toxicity tests Sixty ICR mice were divided into six groups randomly and each group containing 10 mice. Mice of groups 1–5 were intraperitoneal injection (i.p.) of 10, 20, 40, 60, and 80 mg/kg of PXY, respectively, and the sixth group received 10 ml/kg of normal saline. The mortality rates of the mice within a 24 h period were observed and recorded. Establishment of OVX mice The osteoporotic mice model was established by ovariectomy operation described previously (Li et al., 2011; Xue et al., 2012). In brief, mice were general anesthetized with sodium pentobarbital (1.5%, 50 mg/kg). Under sterile condition, bilateral ovaries were resected through the incision made on back of the mice, and then incisions were sutured layer by layer. After surgery, mice were injected with gentamycin for 3 d to prevent wound infection. One week later, mice were administered with drugs. For the sham-operated group, mice were underwent the same surgical procedures, but no ovaries resection was performed.
Effects of PYX on osteoblasts in vitro Cell culture The osteoblasts were isolated from calvarias of newborn SD rats (3 d old) according to the method reported in previous investigation with minor modification (Liu et al., 1995). Briefly, calvarias were isolated from the newborn rats under sterile condition, and cut up by using ophthalmic scissors after washed with PBS for three times. Then, 0.25% trypsinase was added to the bone tissues, and cultivated for 30 min at 37 C. Bone tissues were washed with PBS for three times after discarding the supernatant; then, the digestion solution containing 0.05% trypsinase and 0.02% collagenase (type II) was added to the bone tissues, and cultivated for 1 h at 37 C. The digestion solution was collected and centrifugated for 10 min (1000 rpm) to separated the fresh osteoblasts. Osteoblasts were cultured in a-MEM supplemented with 10% FBS at 37 C in 5% CO2/95% air. Assay for proliferation of osteoblasts Cells (2 104/ml) were seeded in 96-well plates and treated on the following day with indicated concentrations of PYX (5, 10, and 20 mM) for 24, 48, and 72 h, respectively. After that, MTT assay was carried out, and the optical density (OD)
DOI: 10.3109/13880209.2015.1017885
was read at 550 nm using a 96-well plate reader (Bio-Tek ELX800, Bio-Tek, Winooski, VT). Western blotting
Downloaded by [University of Otago] at 14:42 27 September 2015
Cells treated with or without PYX (5, 10, and 20 mM) for 48 h were harvested. Then, total proteins of the cells were extracted, and then equal amounts of protein (40 mg) were separated by sodium dodecyl sulfate/polyacrylamidegel electrophoresis (SDS/PAGE), blotted on polyvinylidene difluoride (PVDF), and probed with anti-RANKL rabbit polyclonal IgG and anti-OPG rabbit polyclonal IgG (Wuhan Boster Bioengineering Ltd. Co., Wuhan, China), and subsequently with goat anti-rabbit/HRP (Wuhan Boster Bio-engineering Ltd. Co., Wuhan, China), and detected by chemiluminescence. To measure protein loading, antibodies directed against b-actin were used.
Anti-osteoporotic activity of PXY
3
phenomenon at all the tested doses compared with the OVX group (p50.05, p50.01, p50.01 for the doses of 20, 40, and 60 mg/kg/d, respectively), in a dose-dependent manner (Figure 2B). Effects of PXY on body blood calcium and blood phosphorus contents As can be seen from Figure 3, after being removed the ovaries, the blood calcium and blood phosphorus contents in the serum decreased dramatically (p50.01). However, the results of our present study indicated that treating with PXY at the doses of 20, 40, and 60 mg/kg/d (i.p.) were able to increase not only the blood calcium contents obviously (p50.05) but also the blood phosphorus contents in the serum significantly (p50.05, p50.01, p50.01) in a dose-dependent manner, compared with the OVX group.
Statistical analysis
Effects of PXY on ALP and OPG contents
Data were expressed as mean ± standard deviation (SD). Statistical analyses were performed using two-tailed Student’s t-test with a significance level of p50.05.
ALP is one of the key marker enzymes for functional osteoblasts, and the differentiation of osteoblasts was evaluated by ALP activity. As can be seen from Figure 4(A), ALP levels of OVX mice were increased 12 weeks after operation. Moreover, the PXY at the doses at 40 and 60 mg/kg/d (i.p.) can increase the ALP content in the serum obviously compared with the OVX group (p50.01, p50.01). Additionally, the OPG levels of the mice suffered from OVX were significantly decreased at the end of the observation (p50.01) (Figure 4B). Interestingly, at the tested doses (20, 40, and 60 mg/kg/d, i.p.), the PXY obviously increased the OPG content in the serum 12 weeks after operation compared with the OVX group (p50.05, p50.01, p50.01), in a dose-dependent manner.
Results Toxicity study results In the results of toxicity investigation, neither death nor any abnormal neuro-behaviors were observed. The 50% lethal dose (LD50) was not obtained due to lack of obvious toxicity at all the tested doses. Effects of PXY on weight changes of body weight and uterus After 12 weeks of OVX operation, the mice in the OVX group were significantly fatter than mice in the sham group (p50.05). However, as can be seen from our present study (Figure 2A), at the doses of 40 and 60 mg/kg/d (i.p.), the weights of mice obviously decreased compared with the OVX mice (p50.05). Additionally, at the end of the observation, the uterus’s weights of mice suffered from OVX were decreased sharply in the OVX group (p50.01, compared with the sham), whereas PXY can inhibit obviously this
Effect of PXY on femur osseous tissues As shown in Figure 5(A), in the sham-operated group, the compact bone was dense, osteocytes were arranged orderly. In the OVX group, the bone marrow cavity was enlarged; trabecular bone was sparse and slender, fractured in large fragments, and poorly arranged (Figure 5B). However, after treatment with PXY at the tested doses (20, 40, and 60 mg/kg/d, i.p.) for 12 weeks, the pathological changes of osteoporosis
Figure 2. Effects of PXY body on weight and uterine weight of OVX mice. Each column represented as mean ± standard deviation (n ¼ 10). The vehicle (10 ml/kg) and PXY (20, 40, and 60 mg/kg) were administered intraperitoneally. #p50.05, ##p50.01, compared with the sham group; *p50.05, **p50.01, compared with the OVX group.
4
H. Li et al.
Pharm Biol, Early Online: 1–7
Downloaded by [University of Otago] at 14:42 27 September 2015
Figure 3. Effects of PXY on body blood calcium and blood phosphorus contents of OVX mice. Each column represented as mean ± standard deviation (n ¼ 10). The vehicle (10 ml/kg) and PXY (20, 40, and 60 mg/kg) were administered intraperitoneally. ##p50.01, compared with the sham group; *p50.05, **p50.01, compared with the OVX group.
Figure 4. Effects of PXY on ALP and OPG contents of OVX mice. Each column represented as mean ± standard deviation (n ¼ 10). The vehicle (10 ml/kg) and PXY (20, 40, and 60 mg/kg) were administered intraperitoneally. #p50.05, ##p50.01, compared with the sham group; *p50.05, **p50.01, compared with the OVX group.
were obviously improved (Figure 5C–E), compact bone was thickened, trabecular bone became thick and compact. In particular, the pathological changes of mice treated with PXY at the dose of 60 mg/kg/d were nearly normal (Figure 5E). Effect of PXY on proliferation of osteoblasts It is no doubt that MTT assay was a classical method that used as an index of proliferation. As can be seen from Table 1, the PXY at the concentrations of 5, 10, and 20 mM slightly enhanced the proliferation of osteoblasts after 24 h culture. However, after culturing more than 48 h, PXY at concentrations of 5, 10, and 20 mM showed significant proliferative effects on osteoblasts, in a dose-dependent manner. Effect of PXY on expressions of OPG and RANKL in osteoblasts The expressions of OPG and receptor activators of NF-B ligand (RANKL) proteins were determined by western blotting (Figure 6). As can be seen from our results, the expressions of RANKL proteins in osteoblasts were decreased significantly by treating with PXY at concentrations of 5, 10, and 20 mM, compared with the control group
(p50.01, p50.01, p50.01). On the contrary, the PXY at concentrations of 10 and 20 mM can significantly up-regulate the expression of OPG proteins, compared with the control group (p50.01, p50.01). In addition, the ratio of OPG/ RANKL was up-regulated by the treatment of PXY at concentrations of 5, 10, and 20 mM, compared with the control group (p50.01, p50.01, p50.01). The relative protein level was normalized to the Western blot intensity of b-actin.
Discussion In our present study, we investigated the effects of PXY on OVX-induced osteoporosis in mice and its potential mechanism. The results of our present study clearly demonstrated the beneficial effects of PXY in the prevention of bone loss induced by ovariectomy for the first time. Estrogen deficiency is an important risk factor in the pathogenesis of osteoporosis (Li et al., 2013). The PXY is one of the main iso-flavones in radix of P. lobata as puerarin, and the structure of PXY is similar to the soy isoflavone which was reported to be beneficial for women’s postmenopausal osteoporosis (Chin & Ima-Nirwana, 2013). Iso-flavones are
Anti-osteoporotic activity of PXY
DOI: 10.3109/13880209.2015.1017885
5
Downloaded by [University of Otago] at 14:42 27 September 2015
Figure 5. Effect of PXY on femur osseous tissue in ovariectomized mice. (A–E) The Sham, ovariectomized model, and PXY treatment (20, 40, and 60 mg/kg) are represented, respectively. The vehicle (10 ml/kg) and PXY were administered intraperitoneally.
Table 1. Effects of PXY on the proliferation of osteoblasts. OD550 24 h
48 h
72 h
0.32 ± 0.04 0.38 ± 0.05*
0.47 ± 0.06 0.56 ± 0.08*
0.67 ± 0.09 0.84 ± 0.12**
0.43 ± 0.05* 0.46 ± 0.06*
0.61 ± 0.09** 0.67 ± 0.10**
0.89 ± 0.15** 0.91 ± 0.14**
Control 5 mM PXY 10 mM 20 mM
Effects of PXY on the proliferation of osteoblasts. Cells were treated with PXY treatment (5, 10, and 20 mM) for 24, 48, and 72 h, respectively. Data were presented as mean ± standard deviation (n ¼ 4).
considered as the natural estrogens, and showed significant anti-osteoporotic activity (Ishida et al., 1998). Therefore, the unique iso-flavones structure might be one important reason for response to the anti-osteoporotic activity of PXY. The osteoporotic mice model induced by ovariectomy is one of the most common and reliable used models for studying osteoporosis, and this model can simulate commendably the clinical syndromes of women’s postmenopausal osteoporosis. Furthermore, the phenomenon of menopause is a signal of ovarian failure, which also means reduction of estrogen, resulting in bone loss increasingly
(Kalu, 1991; Li et al., 2013). In previous investigations, it is reported that ovariectomy can lead to a significantly increase in body weight and decrease in uterus’ weights, and these changes partly due to estrogen deficiency (Li et al., 2013; Nian et al., 2009). The results of our present study demonstrated that OVX decreased the uterine index and increased the body weight compared with the Sham group, which clearly showed the successful establishment of osteoporotic mice model. In addition, PXY treatments (i.p., for 12 weeks) at the tested doses significantly increased the uterine index and decreased the body weight in osteoporotic mice compared with osteoporotic model mice, which indicated that PXY could improve the osteoporotic syndrome of mice suffered from ovariectomy. The contents of blood calcium and blood phosphorus can reflect the degree of bone loss (Li et al., 2011). Our results showed that in the OVX group, levels of blood calcium and blood phosphorus decreased sharply compared with the sham group, but the PXY at the tested doses (i.p., for 12 weeks) can increase the contents of blood calcium and blood phosphorus in serum of mice suffered from ovariectomy. In addition, our results also showed that PXY can obviously improve the osseous tissues’ condition in osteoporotic mice. This result is another evidence for demonstrating that PXY could improve the osteoporotic syndrome of OVX mice.
6
H. Li et al.
Pharm Biol, Early Online: 1–7
Downloaded by [University of Otago] at 14:42 27 September 2015
Figure 6. Effect of PXY on expressions of OPG and RANKL in osteoblasts. Osteoblastic cells from neonatal rat calvarias were treated with PXY treatment (5, 10, and 20 mM) for 48 h. Protein expressions of OPG and RANKL were determined by western blotting, and b-actin was used as an internal reference. Data were presented as mean ± standard deviation (n ¼ 4), **p50.01, compared with control.
ALP is an important enzyme in the bone remodeling process, and it promotes mineralization of matrix by decomposing the phosphoric ester into inorganic phosphorous in order to increase the phosphorous concentration (Jia et al., 2009). In our present study, we found that the PXY at the doses of 40 and 60 mg/kg/d (i.p., for 12 weeks) can obviously increase the contents of ALP. The RANKL and OPG secreted by osteoblast play the important roles in osteoclastic formation, differentiation, and bone resorbing (Steeve et al., 2004; Xue et al., 2012). Furthermore, RANKL can activate the osteoclast progenitors through the receptor activator of NFB (RANK) to induce osteoclasts’ differentiation and function. Importantly, OPG can block the communication between RANKL and RANK receptor, which means that OPG can suppress osteoclastogenesis and bone resorption (Liu et al., 2005; Xue et al., 2012). Thus, the relative ratio of OPG/ RANKL is very important for the bone remodeling. In our present study, PXY at the tested concentrations can significantly decrease the expression of RANKL, and increase the expression of OPG and relative ratio of OPG/RANKL, indicating that PXY can suppress the bone loss and resorption by up-regulating ratio value of OPG/RANKL in osteoblasts. Collectively, our present study demonstrated that the PXY could improve the osteoporotic syndrome of mice suffered from ovariectomy, and the possible mechanisms are regarding increasing the blood calcium, blood phosphorus, and ALP contents and up-regulating the ratio value of OPG/RANKL in osteoblasts.
Declaration of interest The authors report that they have no conflicts of interest. This work was supported by the Guangxi Natural Science Foundation (Nos. 2010GXNSFA013268 and 2013 GXNSFAA019241).
References Bereswill S, Munoz1 M, Fischer A, et al. (2010). Anti-inflammatory effects of resveratrol, curcumin and simvastatin in acute small intestinal inflammation. PLoS One 5:e15099.
Bock O, Felsenberg D. (2008). Bisphosphonates in the management of postmenopausal osteoporosis-optimizing efficacy in clinical practice. Clin Interv Aging 3:279–97. Canalis H, McCarthy T, Centrella M. (1998). Growth factors and the regulation on bone remodeling. J Clin Invest 81:277–81. Chin KY, Ima-Nirwana S. (2013). Can soy prevent male osteoporosis? A review of the current evidence. Curr Drug Targets 14:1632–41. Du J, Wei YJ, Peng C, et al. (2011). Establishment of a luciferase assaybased screening system for detecting estrogen receptor agonists in plant extracts. Bone 49:572–9. Genant HK, Bay DJ, Gallagher JC. (1998). Estrogens in the prevention of osteoporosis in post menopausal women. Am J Obstet Gynecol 161: 1842–6. Ishida H, Uesugi T, Hirai K, et al. (1998). Preventive effects of the plant isoflavones, daidzin and genistin, on bone loss in ovariectomized rats fed a calcium-deficient diet. Biol Pharm Bull 21:62–6. Jia L, Cao DP, Qin LP, et al. (2009). Antiosteoporotic activity of phenolic compounds from Curculigo orchioides. Phytomedicine 16: 874–81. Kalu DN. (1991). The ovariectomized rat model of postmenopausal bone loss. Bone Miner 15:175–91. Li F, Yang XL, Yang YN, et al. (2013). Antiosteoporotic activity of echinacoside in ovariectomized rats. Phytomedicine 20:549–57. Li H, Wang JH, Huang HL, Li B. (2011). Puerarin combined with estradiol for treatment of osteoporosis in ovariectomized rats. J Clin Rehab Tiss Eng Res 15:8698–701. Li JWH, Vederas JC. (2009). Drug discovery and natural products: End of an era or an endless frontier? Science 325:161–5. Liu XH, Kirschenbaum A, Yao S, Levine AC. (2005). Crosstalk between the interleukin-6 and prostaglandin E2 signaling systems results in enhancement of osteoclastogenesis through effects on the osteoprotegerin/receptor activator of nuclear factor-kB (RANK) ligand/RANK system. Endocrinology 146:1991–8. Liu ZD, Zang HS, Ou-Yang YP. (1995). Biological study of growth pattern of newborn rat calvaria osteoblastic cell in vitro. Acta Anatom Sin 26:157–9. Ma XQ, Zheng CJ, Zhang Y, et al. (2013). Antiosteoporotic flavonoids from Podocarpium podocarpum. Phytochem Lett 6:118–22. Nian H, Ma MH, Nian SS, Xu L. (2009). Antiosteoporotic activity of icariin in ovariectomized rats. Phytomedicine 16:320–6. Nian H, Qin LP. (2006). Antiosteoporotic activity of Er-Xian decoction, a traditional Chinese herbal formula, in ovariectomized rats. J Ethnopharmacol 108:96–102. Peng W, Ming QL, Han P, et al. (2014). Anti-allergic rhinitis effect of caffeoylxanthiazonoside isolated from fruits of Xanthium strumarium L. in rodent animals. Phytomedicine 15:824–9. Qin LP, Han T, Zhang QY, et al. (2008). Antiosteoporotic chemical constituents from Er-Xian decoction, a traditional Chinese herbal formula. J Ethnopharmacol 118:271–9.
DOI: 10.3109/13880209.2015.1017885
Downloaded by [University of Otago] at 14:42 27 September 2015
Riggs BL, Melton LJ. (1986). Involutional osteoporosis. New Engl J Med 314:1676–86. Steeve KT, Marc P, Sandrine T, et al. (2004). IL-6, RANKL, TNF-a/ IL-1: Interrelations in bone resorption pathophysiology. Cytokine Growth Factor Rev 15:49–60. Tu¨zu¨n S, Akyu¨z G, Eskiyurt N, et al. (2013). Impact of the training on the compliance and persistence of weekly bisphosphonate treatment in postmenopausal osteoporosis: A randomized controlled study. Int J Mol Sci 10:1880–7. Wang Y, Wang WL, Xie WL, et al. (2013). Puerarin stimulates proliferation and differentiation and protects against cell death in human osteoblastic MG-63 cells via ER-dependent MEK/ERK andPI3K/Akt activation. Phytomedicine 20:787–96.
Anti-osteoporotic activity of PXY
7
Xue LM, Jiao L, Wang Y, et al. (2012). Effects and interaction of icariin, curculigoside, and berberine in Er-Xian decoction, a traditional Chinese Medicinal formula, on osteoclastic bone resorption. Evid Based Complement Alternat Med 2012:490843. Zhang H, Xing WW, Li YS, et al. (2008). Effects of a traditional Chinese herbal preparation on osteoblasts and osteoclasts. Maturitas 61:334–9. Zheng GL, Zhang XY, Meng QH, Gong WG. (2002). Protective effect of total isoflavones from pueraria lobata on secondary osteoporosis induced by dexamethasone in rats. J Chin Med Mater 25:643–6. Zhu Z, Xue LM, Han T, et al. (2010). Antiosteoporotic effects and proteomic characterization of the target and mechanism of an Er-Xian decoction on osteoblastic UMR-106 and osteoclasts induced from RAW264.7. Molecules 15:4695–710.
