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Effects of Ipriflavone on Caged Layer Bone Metabolism In Vitro and In Vivo1
Effects of Ipriflavone on Caged Layer Bone Metabolism In Vitro and In Vivo1 J. Yao, J. Zhang, and J.-F. Hou2 College of Veterinary Medicine, Nanjing Agricultural University, 210095, China
Key words: ipriflavone, bone metabolism, ISA caged layer, osteoblast, osteoclast 2007 Poultry Science 86:503–507
Eagle’s medium, α modification essential medium, and collangenase were purchased from Gibco-BRL (Rockville, MD). Fetal cattle serum (FCS) came from Sijiqing Co. Ltd (Hangzhou, China). Test kits for alkaline phosphatase (ALP), bone gla-protein (BGP), and estrogen (E2) were purchased from Jiancheng Biotechnology Ltd. (Nanjing, China). The tartrate-resistant acid phosphatase (TRAP) staining kit was from Sigma-Aldrich (St. Louis, MO). All other chemicals and reagents were of the highest analytical grade.
INTRODUCTION Ipriflavone (7-isopropoxyisoflavone), a derivative of natural isoflavone isolated from alfalfa (Medicago sativa L.), prevents bone loss in several experimental models of osteoporosis (Reginster, 1993). The mechanisms underlying the protective action of ipriflavone include direct inhibition of bone resorption in a variety of in vitro systems for culturing mammalian bone tissue (Tsuda et al., 1986; Yamazaki et al., 1986; Benvenuti et al., 1991; Notoya et al., 1993; Albanese et al., 1994). In chickens, previous findings suggest that ipriflavone may affect bone remodeling by inhibiting bone resorption and stimulating bone formation. Regardless of its effect on bone remodeling, ipriflavone is reported to increase growth and optimize egg performance in hens (Zhang et al., 2003). The objective of the present study was to determine the efficacious concentrations of ipriflavone both in vitro and in vivo, to identify a dosage appropriate for clinical treatment of osteoporosis in laying hens.
Chicken Embryonic Osteoblast Culture Calvariae were excised from 15 chicken embryos (15 d of age), dissected free of loose connective tissue, and washed with PBS at pH 7.4. Calvariae were digested with 0.5 mg/ mL of crude collagenase in a solution of 1 mL of trypsinEDTA and 4 mL of PBS for 10 min at 37°C with gentle rocking. The digestion procedure was repeated to provide 5 populations of cells (fraction 5 was digested for 20 min). After each digestion, released cells were removed, and the reaction was stopped with FCS. Cells from populations 2 to 5 were pooled and cultured for confluence in 100-mm dishes at 37°C in a humidified atmosphere of 5% CO2, in Dulbecco’s modified Eagle’s medium with 10% heatinactivated FCS, 100 U/mL of penicillin, and 50 mg/mL of streptomycin. Cells were resuspended and replated in 6-well dishes at 5 × 105 cells per well for 48 h, digested with 0.25% trypsin before use, and then replated in 96well dishes at 3 × 104 cells per well for use. Ipriflavone
MATERIALS AND METHODS Materials Ipriflavone was synthesized and supplied by Binghu Chemical Co. Ltd. (Wuhan, China). Dulbecco’s modified ©2007 Poultry Science Association Inc. Received June 8, 2006. Accepted November 9, 2006. 1 Supported by grants from the National Natural Science Foundation of China (30671546, 30270998) and the Natural Science Foundation of Jiangsu Province (BK2004099, Q19990023). 2 Corresponding author: [email protected] or [email protected].
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that were fed diets containing 0, 15, 25, 50, and 100 ppm of ipriflavone. The experiment lasted 70 d. Egg production increased in hens fed 25 ppm and decreased in hens fed 50 and 100 ppm when compared with the controls and hens fed 15 ppm (P < 0.05). Egg weight, shell quality, BW, and serum P, Ca, estrogen, and bone mineral content were not affected by inclusion of ipriflavone in the diet. Hens consuming 25 ppm of ipriflavone had greater serum alkaline phosphatase and bone gla-protein levels than controls. Adding 25 ppm of ipriflavone to the feed appears to be close to an ideal level for clinical treatment of osteoporosis because of improved egg production while maintaining bone mineral content.
ABSTRACT The effects of ipriflavone on caged layer bone metabolism were examined in vitro and in vivo. Ipriflavone at 10−8 M stimulated the activity of osteoblasts cultured from embryonic chick calvariae, and 10−9 to 10− 7 M inhibited osteoclasts from chick tibias and humeri. Ipriflavone concentrations of 10−4 and 10−5 M inhibited osteoblast activity. These results suggest that ipriflavone influences bone metabolism by regulating the functional balance between osteoblasts and osteoclasts. Based on these in vitro experiments, in vivo studies were conducted to further clarify the effects of ipriflavone. Five hundred 58-wk-old ISA caged layers were divided into 5 groups
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was dissolved in ethanol at a concentration of 10−2 M before use and added at the desired concentration for the designated periods. In the presence of different doses of ipriflavone, 3-(4,5-dimethylthiazol-2-yl)-2,-5-diphenyl tetrazolium (Mosmann, 1983) and 4-nitrophenyl phosphate disodium salt (Farley et al., 1991) assays were used to determine proliferation of osteoblasts and ALP activity of osteoblastic cells. Staining for promatrix (Bonucci and Gherardi, 1975) and mineralized node (Bellows et al., 1986) showed the characterization of osteoblasts.
Chicken Embryonic Osteoclast Culture Tibias and humeri were isolated from 10 chicken embryos (18 d of age) and cleaned of extraneous soft tissue without removing the bone ends, which are replete with osteoclasts (Collin-Osdoby et al., 2003). The marrow was removed from each bone by gripping the bone with alcohol-soaked tweezers, poking several small holes in each end of the bone using a syringe, and quickly flushing the
Figure 3. Mineralized node stained by alizarin staining the calcified extracellular matrix (40×).
marrow out by repeatedly inserting the tip of the syringe filled with Hanks buffered salt solution (HBSS, pH 7.2) into the end of the bone. After all of the marrow was extruded, the bones were placed in a clean dish on ice to split each bone lengthwise using sterile scissors while keeping the bones submersed in HBSS. The split bones were transferred into polypropylene tubes containing HBSS, shaken vigorously for 30 s, and the cell (from the inside surface of bones) supernatants were collected sequentially. The cell pellet was resuspended in α modification essential medium with 15% FCS, 100 U/mL of penicillin, and 50 mg/mL of streptomycin. Cell suspensions were replated at 5 × 105 cells per well in 24-well dishes containing glass coverslips or bovine bone slices. Nonadherent cells were washed off after 2 h, and the medium was changed every 48 h thereafter. The adherent cells were grown for an additional 6 d, during which time ipriflavone was added at prescribed concentrations. The most commonly used stain to visualize osteoclasts was based on their high level of TRAP (Andersson and Marks, 1989) activity, which was upregulated early in OC development and was essential for their resorption of bone. Osteoclasts were capable of excavating resorption pit lacunae on the bovine bone slices; therefore, this step was essential for evaluating the bone resorptive function of fully developed osteoclasts. The number and area of resorption sites were quantified to determine the efficacy of ipriflavone on chicken osteoclasts.
In Vivo Caged Layer Experiment
Figure 2. Cytochemical staining for promatrix in an osteoblast showing deposits in cytoplasm (40×).
Five hundred 58-wk-old ISA caged layers were divided into 5 groups (100 hens per group), raised in 3-tier caged layer houses (2 hens per cage), and fed a basal layer ration according to the standard (NRC, 1994) for 5 d. The control group continued on the basal layer diet, and the remaining groups were fed diets containing 15, 25, 50, and 100 ppm ipriflavone for up to 70 d. The hens had free access to feed and tap water. During the experimental period, they were
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Figure 1. Cytochemical staining for alkaline phosphatase in an osteoblast showing deposits in cytoplasm (100×).
0.2075b ± 0.0158 0.2073b ± 0.0237 0.2263ab ± 0.0235 0.2485a ± 0.0526 Means with different superscripts differ significantly (P < 0.05). Mean ± SD. 2 OD570 = optical density at 570 nm; OD405 = optical density at 405 nm. 1
a–c
0.0775c ± 0.0071 0.1383c ± 0.0126 MTT (OD570) PNPP (OD405)
0.2075b ± 0.0260 0.2076b ± 0.0211
0.14c ± 0.0193 0.1851c ± 0.0256
0.2338ab ± 0.0550 0.2254ab ± 0.0363
0.22ab ± 0.0370 0.2431a ± 0.0495
0.2425a ± 0.0523 0.2576a ± 0.0519
10−10 10−9 10−8 10 10 0
Concentration (M)
10−7 10
−6 −5 −4
Item
Figure 5. Toluidine staining in bone slices showing resorption lacunae (250×).
2
illuminated for 16 h/d. Eggs were picked up by hand at 1700 h.; egg weight and normal egg, soft-shell egg, and broken egg rates were recorded every day. At 60, 64, and 68 wk of age, 20 hens (not in the same cage) randomly selected from each group were weighed, and blood samples (5 mL per bird from wing vein) were collected using 1% heparin as an anticoagulant at 1000 h. The blood samples were frozen at −20°C for subsequent measurements of serum Ca, P, ALP, BGP, and E2 concentrations. In plasma samples, the values of Ca, P, and ALP were determined by an autoanalyzer (Hitachi 7600-020, Hitachi Ltd., Tokyo, Japan). At the same time, BGP (Epstein et al., 1984) and E2 (Brochu et al., 1984) were determined with 125I labeled by radioimmunological machine (SN-695B, China Institute of Atomic Energy, Beijing). In the end of the experiment, 20 hens were killed at random from each group, and the tibias were isolated and cleaned of extraneous soft tissue for measuring bone mineral content (BMC). The tibia was put into a little plastic box that contained a small amount
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Figure 4. Cytochemical staining for tartrate-resistant acid phosphatase in osteoclasts showing deposit in cytoplasm and in nucleus (100×).
Table 1. Effect of ipriflavone on osteoblast proliferation [3-(4,5-dimethylthiazol-2-yl)-2,-5-diphenyl tetrazolium assay (MTT)] and alkaline phosphatase activity [4-nitrophenyl phosphate disodium salt assay (PNPP)]1
EFFECTS OF IPRIFLAVONE ON CAGED LAYER BONE METABOLISM
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YAO ET AL. Table 2. Effect of ipriflavone on the number and area of osteoclast resorption pit lacunae1 Concentration (M) 0
10−7
10−8
10−9
7.8 ± 1.9a 8,299 ± 556a
1.0 ± 0.0b 171 ± 63c
1.0 ± 0.0b 452 ± 109c
2.3 ± 0.5b 2,030 ± 751b
Item Number of lacunae Areas of lacunae (m2)
Means with different superscripts differ significantly (P < 0.05). Mean ± SD; n = 6.
a–c 1
Statistical Analysis For control and treatment groups, the mean and SD were calculated with statistic software (SPSS 11.0, SPSS Inc., Chicago, IL), and values of calculated means among groups were compared using 1-way ANOVA.
RESULTS Osteoblasts were cultured and identified before ipriflavone treatments. The cells exhibited classic characteristics of osteoblasts; they were fibroblastic and were triangular or stellate in shape. Black deposits of ALP (Figure 1) and red deposits of promatrix (Figure 2) were shown by cytochemical staining. Mineralized nodes formed during prolonged culture (Figure 3). The results of the 3-(4,5-dimethylthiazol-2-yl)-2,-5-diphenyl tetrazolium test showed that 10−4 and 10−5 M ipriflavone inhibited (P < 0.05) the proliferation of osteoblasts, whereas 10−8 M ipriflavone increased (P < 0.01) the proliferation of osteoblasts in vitro when compared with the control group (Table 1). Correspondingly, the results of the 4-nitrophenyl phosphate disodium salt test showed that 10−4 and 10−5 M ipriflavone inhibited the synthesis and secretion of ALP in osteoblasts. Conversely, 10−7 to 10−9 M ipriflavone increased (P < 0.01) ALP activity in the osteoblasts (Table 1). Cultured osteoclasts were also identified before adding ipriflavone. These cells were characterized as being multinucleated and stained positive for TRAP (Figure 4). These cells were able to form bone resorption lacunae on bone slices (Figure 5). Adding ipriflavone at 10−7 to 10−9 M reduced the number and area of bone resorption pit lacunae
DISCUSSION Our results confirmed that small doses of ipriflavone could improve the proliferation of osteoblasts and inhibit the activity of mature osteoclasts in vitro. Larger doses of ipriflavone inhibited osteoblast proliferation, indicating the efficacy of ipriflavone on osteoblasts and osteoclasts was dependent on the dosage. The mechanism of this action is not currently known, and further research will be needed to clarify the mechanism of ipriflavone’s function. We also demonstrated that appropriate doses of ipriflavone can improve egg production in laying hens. Biochemical markers of bone metabolism revealed obvious changes in ALP and BGP activity when ipriflavone was fed in this experiment, especially in hens consuming 25 ppm ipriflavone. In the latter stages of egg formation, the cumulative transfer of Ca to the shell of an egg can gradually deplete Ca reserves in hens’ bodies (Clunies et al., 1992). At this time, appropriate doses of ipriflavone could enhance bone formation and help satisfy the Ca needs of
Table 3. Effect of ipriflavone on egg-laying performance of ISA caged layer1 Concentration (ppm) Item Average egg production rate (%) Soft-shell and broken egg rate (%) Average egg weight (g)
0
15
73.35 ± 2.65 1.195 ± 0.513 63.7 ± 1.3 b
72.6 ± 2.54 1.205 ± 0.969 63.1 ± 1.1
Means with different superscripts differ significantly (P < 0.05). Mean ± SD.
a–c 1
25 b
50
77.00 ± 3.38 0.949 ± 0.550 64.0 ± 0.6 a
100
67.68 ± 3.34 0.961 ± 0.741 63.5 ± 1.2 c
69.60 ± 2.08c 1.180 ± 0.639 63.4 ± 1.1
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(P < 0.05), reflecting an overall inhibition of osteoclastic resorption in vitro (Table 2). Hens fed 25 ppm ipriflavone had the highest cumulative rate of egg production when compared with the other 4 groups. Feeding hens higher levels of ipriflavone (50 and 100 ppm) led to the lowest cumulative egg production rates when compared with the control group and the group fed 15 ppm ipriflavone. Ipriflavone at any dose did not significantly affect egg weight and soft-shell and broken egg rate (Table 3). Serum levels of ALP were higher (P < 0.01) in hens consuming 25 ppm, and BGP levels were higher (P < 0.05) in hens consuming 15 and 25 ppm of ipriflavone than in the other groups (Table 4). There were no differences in serum E2 levels among treatment groups. The level of serum Ca and P and BMC also did not differ among groups.
of water, and the middle of each bone was scanned with bone densitometry that contained a 125I radionuclide source. Before scanning the tibia, the instrument was calibrated using the calibration phantom provided by the manufacturer (BMD-400E, China Institute of Atomic Energy).
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EFFECTS OF IPRIFLAVONE ON CAGED LAYER BONE METABOLISM Table 4. Effect of ipriflavone on serum alkaline phosphatase (ALP), bone gla protein (BGP), estrogen (E2), Ca, and P, and bone mineral content (BMC)1 Concentration (ppm) Item
0
15
25
50
100
1.45 ± 2.92 (n = 28) 12.79 ± 5.34b (n = 27)
b
16.56 ± 3.47 (n = 29) 16.39 ± 6.61a (n = 29)
a
19.30 ± 1.64 (n = 26) 17.72 ± 5.67a (n = 29)
b
15.19 ± 4.19 (n = 29) 14.88 ± 5.12b (n = 25)
11.71 ± 2.79c (n = 29) 14.20 ± 4.89b (n = 29)
E2 (pg/mL)
166.93 ± 66.72 (n = 20)
182.47 ± 75.87 (n = 16)
172.81 ± 90.71 (n = 17)
164.75 ± 94.52 (n = 18)
164.44 ± 72.59 (n = 17)
Ca (mg/dL)
13.48 ± 2.37 (n = 34)
11.81 ± 1.82 (n = 27)
13.13 ± 2.29 (n = 30)
14.09 ± 2.64 (n = 27)
13.15 ± 2.33 (n = 30)
5.28 ± 1.33 (n = 33)
5.41 ± 1.23 (n = 27)
4.69 ± 1.07 (n = 30)
5.70 ± 1.71 (n = 25)
5.80 ± 1.43 (n = 28)
0.775 ± 0.086 (n = 20)
0.795 ± 0.114 (n = 20)
0.819 ± 0.087 (n = 20)
0.856 ± 0.157 (n = 20)
0.783 ± 0.111 (n = 20)
ALP (mU/mL) BGP (ng/mL)
P (mg/dL) BMC (g)
Means with different superscripts differ significantly (P < 0.05). Mean ± SD.
a–c 1
shell formation, because ipriflavone has been reported to simulate the Ca absorption from the intestine (Arjmandi et al., 2000). Previous studies showed that ipriflavone had no effect on metabolism (Hocking and Bernar, 2000). In this study, the level of E2 also did not have significant change in the late laying period Thus, the increasing level of E2 probably reflects normal physiological phenomena late in the laying period. In conclusion, our results confirmed that ipriflavone can play a role in bone remodeling in laying hens both in vitro and in vivo. The level of 25 ppm in the feed may be the perfect dose, because it could not only enhance the egg production, but also maintain BMC to prevent osteoporosis.
REFERENCES Albanese, C. V., A. Cudd, L. Argentine, A. Zambonin-Zallone, and I. MacIntyne. 1994. Ipriflavone directly inhibits osteoclastic activity. Biochem. Biophys. Res. Commun. 199:930–936. Andersson, G. N., and S. C. Marks Jr. 1989. Tartrate-resistant acid ATPase as a cytochemical marker for osteoclasts. J. Histochem. Cytochem. 37:115–117. Arjmandi, B. H., D. A. Khalil, and B. W. Hollis. 2000. Ipriflavone, a synthetic phytoestrogen, enhances intestinal calcium transport in vitro. Calcif. Tissue Int. 67:225–229. Bellows, C. G., J. E. Aubin, J. N. Heersche, and M. E. Antosz. 1986. Mineralized bone nodules formed in vitro from enzymatically released rat calvaria cell populations. Calcif. Tissue Int. 38:143–154. Benvenuti, S., A. Tanini, U. Frediani, L. Masi, R. Casano, L. Bufalino, M. Serio, and M. L. Brandi. 1991. Effects of ipriflavone and its metabolites on a clonal osteoblastic cell line. J. Bone Miner. Res. 6:987–996. Bonucci, E., and G. Gherardi. 1975. Histochemical and electron microscopy investigations on medullary bone. Cell Tissue Res. 163:81–97. Brochu, M., R. Veilleux, A. Lorrain, and A. Belanger. 1984. Monoclonal antibodies for use with 125iodine-labeled radioligands in progesterone radioimmunoassay. J. Steroid. Biochem. 21:405–411.
Clunies, M., D. Parks, and S. Leeson. 1992. Calcium and phosphorus metabolism and eggshell formation of hens fed different amounts of calcium. Poult. Sci. 71:482–489. Collin-Osdoby, P., F. Anderson, and P. Osdoby. 2003. Primary isolation and culture of chicken osteoclasts. Page 71 in Bone Research Protocols. M. H. Helfrich and S. H Ralston, ed. Humana Press Inc., Totowa, NJ. Epstein, S., J. Poser, R. McClintock, C. C. Johnston Jr., G. Bryce, and S. Hui. 1984. Differences in serum bone GLA protein with age and sex. Lancet 1:307–310. Farley, J. R., J. E. Wergedal, S. L. Hall, S. Herring, and N. M. Tarbaux. 1991. Calcitonin has direct effects on 3[H]-thymidine incorporation and alkaline phosphatase activity in human osteoblast-line cells. Calcif. Tissue Int. 48:297–301. Hocking, P. M., and R. Bernar. 2000. Effect of the age of male and female broiler breeders on sexual behavior, fertility and hatchability of eggs. Br. Poult. Sci. 41:370–376. Mosmann, T. 1983. Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays. J. Immunol. Methods 65:55–63. Notoya, K., K. Yoshida, S. Taketomi, I. Yamazaki, and M. Kumegawa. 1993. Inhibitory effect of ipriflavone on osteoclast-mediated bone resorption and new osteoclast formation in longterm cultures of mouse unfractionated bone cells. Calcif. Tissue Int. 53:206–209. NRC. 1994. Nutrient Requirements of Poultry. 9th rev. ed. Natl. Acad. Press, Washington, DC. Reginster, J. Y. 1993. Ipriflavone: Pharmacological properties and usefulness in postmenopausal osteoporosis. Bone Miner. 23:223–232. Tsuda, M., T. Kitazaki, T. Ito, and T. Fujita. 1986. The effect of ipriflavone (TC-80) on bone resorption in tissue culture. J. Bone Miner. Res. 1:207–211. Yamazaki, I., A. Shino, Y. Shimizu, R. Tsukuda, Y. Shirakawa, and M. Kinoshita. 1986. Effect of ipriflavone on osteoporosis induced by ovariectomy in rats. J. Bone Miner. Metab. 3:205–210. Zhang, J., X. Y. Zhu, and J. F. Hou. 2003. Effect of ipriflavone on egg laying performance and bone metablism of ISA cage layer during the late of egg production cycle. Chin. J. Vet. Sci. 23:613–615.
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b
Key words: ipriflavone, bone metabolism, ISA caged layer, osteoblast, osteoclast 2007 Poultry Science 86:503–507
Eagle’s medium, α modification essential medium, and collangenase were purchased from Gibco-BRL (Rockville, MD). Fetal cattle serum (FCS) came from Sijiqing Co. Ltd (Hangzhou, China). Test kits for alkaline phosphatase (ALP), bone gla-protein (BGP), and estrogen (E2) were purchased from Jiancheng Biotechnology Ltd. (Nanjing, China). The tartrate-resistant acid phosphatase (TRAP) staining kit was from Sigma-Aldrich (St. Louis, MO). All other chemicals and reagents were of the highest analytical grade.
INTRODUCTION Ipriflavone (7-isopropoxyisoflavone), a derivative of natural isoflavone isolated from alfalfa (Medicago sativa L.), prevents bone loss in several experimental models of osteoporosis (Reginster, 1993). The mechanisms underlying the protective action of ipriflavone include direct inhibition of bone resorption in a variety of in vitro systems for culturing mammalian bone tissue (Tsuda et al., 1986; Yamazaki et al., 1986; Benvenuti et al., 1991; Notoya et al., 1993; Albanese et al., 1994). In chickens, previous findings suggest that ipriflavone may affect bone remodeling by inhibiting bone resorption and stimulating bone formation. Regardless of its effect on bone remodeling, ipriflavone is reported to increase growth and optimize egg performance in hens (Zhang et al., 2003). The objective of the present study was to determine the efficacious concentrations of ipriflavone both in vitro and in vivo, to identify a dosage appropriate for clinical treatment of osteoporosis in laying hens.
Chicken Embryonic Osteoblast Culture Calvariae were excised from 15 chicken embryos (15 d of age), dissected free of loose connective tissue, and washed with PBS at pH 7.4. Calvariae were digested with 0.5 mg/ mL of crude collagenase in a solution of 1 mL of trypsinEDTA and 4 mL of PBS for 10 min at 37°C with gentle rocking. The digestion procedure was repeated to provide 5 populations of cells (fraction 5 was digested for 20 min). After each digestion, released cells were removed, and the reaction was stopped with FCS. Cells from populations 2 to 5 were pooled and cultured for confluence in 100-mm dishes at 37°C in a humidified atmosphere of 5% CO2, in Dulbecco’s modified Eagle’s medium with 10% heatinactivated FCS, 100 U/mL of penicillin, and 50 mg/mL of streptomycin. Cells were resuspended and replated in 6-well dishes at 5 × 105 cells per well for 48 h, digested with 0.25% trypsin before use, and then replated in 96well dishes at 3 × 104 cells per well for use. Ipriflavone
MATERIALS AND METHODS Materials Ipriflavone was synthesized and supplied by Binghu Chemical Co. Ltd. (Wuhan, China). Dulbecco’s modified ©2007 Poultry Science Association Inc. Received June 8, 2006. Accepted November 9, 2006. 1 Supported by grants from the National Natural Science Foundation of China (30671546, 30270998) and the Natural Science Foundation of Jiangsu Province (BK2004099, Q19990023). 2 Corresponding author: [email protected] or [email protected].
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that were fed diets containing 0, 15, 25, 50, and 100 ppm of ipriflavone. The experiment lasted 70 d. Egg production increased in hens fed 25 ppm and decreased in hens fed 50 and 100 ppm when compared with the controls and hens fed 15 ppm (P < 0.05). Egg weight, shell quality, BW, and serum P, Ca, estrogen, and bone mineral content were not affected by inclusion of ipriflavone in the diet. Hens consuming 25 ppm of ipriflavone had greater serum alkaline phosphatase and bone gla-protein levels than controls. Adding 25 ppm of ipriflavone to the feed appears to be close to an ideal level for clinical treatment of osteoporosis because of improved egg production while maintaining bone mineral content.
ABSTRACT The effects of ipriflavone on caged layer bone metabolism were examined in vitro and in vivo. Ipriflavone at 10−8 M stimulated the activity of osteoblasts cultured from embryonic chick calvariae, and 10−9 to 10− 7 M inhibited osteoclasts from chick tibias and humeri. Ipriflavone concentrations of 10−4 and 10−5 M inhibited osteoblast activity. These results suggest that ipriflavone influences bone metabolism by regulating the functional balance between osteoblasts and osteoclasts. Based on these in vitro experiments, in vivo studies were conducted to further clarify the effects of ipriflavone. Five hundred 58-wk-old ISA caged layers were divided into 5 groups
504
YAO ET AL.
was dissolved in ethanol at a concentration of 10−2 M before use and added at the desired concentration for the designated periods. In the presence of different doses of ipriflavone, 3-(4,5-dimethylthiazol-2-yl)-2,-5-diphenyl tetrazolium (Mosmann, 1983) and 4-nitrophenyl phosphate disodium salt (Farley et al., 1991) assays were used to determine proliferation of osteoblasts and ALP activity of osteoblastic cells. Staining for promatrix (Bonucci and Gherardi, 1975) and mineralized node (Bellows et al., 1986) showed the characterization of osteoblasts.
Chicken Embryonic Osteoclast Culture Tibias and humeri were isolated from 10 chicken embryos (18 d of age) and cleaned of extraneous soft tissue without removing the bone ends, which are replete with osteoclasts (Collin-Osdoby et al., 2003). The marrow was removed from each bone by gripping the bone with alcohol-soaked tweezers, poking several small holes in each end of the bone using a syringe, and quickly flushing the
Figure 3. Mineralized node stained by alizarin staining the calcified extracellular matrix (40×).
marrow out by repeatedly inserting the tip of the syringe filled with Hanks buffered salt solution (HBSS, pH 7.2) into the end of the bone. After all of the marrow was extruded, the bones were placed in a clean dish on ice to split each bone lengthwise using sterile scissors while keeping the bones submersed in HBSS. The split bones were transferred into polypropylene tubes containing HBSS, shaken vigorously for 30 s, and the cell (from the inside surface of bones) supernatants were collected sequentially. The cell pellet was resuspended in α modification essential medium with 15% FCS, 100 U/mL of penicillin, and 50 mg/mL of streptomycin. Cell suspensions were replated at 5 × 105 cells per well in 24-well dishes containing glass coverslips or bovine bone slices. Nonadherent cells were washed off after 2 h, and the medium was changed every 48 h thereafter. The adherent cells were grown for an additional 6 d, during which time ipriflavone was added at prescribed concentrations. The most commonly used stain to visualize osteoclasts was based on their high level of TRAP (Andersson and Marks, 1989) activity, which was upregulated early in OC development and was essential for their resorption of bone. Osteoclasts were capable of excavating resorption pit lacunae on the bovine bone slices; therefore, this step was essential for evaluating the bone resorptive function of fully developed osteoclasts. The number and area of resorption sites were quantified to determine the efficacy of ipriflavone on chicken osteoclasts.
In Vivo Caged Layer Experiment
Figure 2. Cytochemical staining for promatrix in an osteoblast showing deposits in cytoplasm (40×).
Five hundred 58-wk-old ISA caged layers were divided into 5 groups (100 hens per group), raised in 3-tier caged layer houses (2 hens per cage), and fed a basal layer ration according to the standard (NRC, 1994) for 5 d. The control group continued on the basal layer diet, and the remaining groups were fed diets containing 15, 25, 50, and 100 ppm ipriflavone for up to 70 d. The hens had free access to feed and tap water. During the experimental period, they were
Downloaded from http://ps.oxfordjournals.org/ at National Chung Hsing University Library on April 10, 2014
Figure 1. Cytochemical staining for alkaline phosphatase in an osteoblast showing deposits in cytoplasm (100×).
0.2075b ± 0.0158 0.2073b ± 0.0237 0.2263ab ± 0.0235 0.2485a ± 0.0526 Means with different superscripts differ significantly (P < 0.05). Mean ± SD. 2 OD570 = optical density at 570 nm; OD405 = optical density at 405 nm. 1
a–c
0.0775c ± 0.0071 0.1383c ± 0.0126 MTT (OD570) PNPP (OD405)
0.2075b ± 0.0260 0.2076b ± 0.0211
0.14c ± 0.0193 0.1851c ± 0.0256
0.2338ab ± 0.0550 0.2254ab ± 0.0363
0.22ab ± 0.0370 0.2431a ± 0.0495
0.2425a ± 0.0523 0.2576a ± 0.0519
10−10 10−9 10−8 10 10 0
Concentration (M)
10−7 10
−6 −5 −4
Item
Figure 5. Toluidine staining in bone slices showing resorption lacunae (250×).
2
illuminated for 16 h/d. Eggs were picked up by hand at 1700 h.; egg weight and normal egg, soft-shell egg, and broken egg rates were recorded every day. At 60, 64, and 68 wk of age, 20 hens (not in the same cage) randomly selected from each group were weighed, and blood samples (5 mL per bird from wing vein) were collected using 1% heparin as an anticoagulant at 1000 h. The blood samples were frozen at −20°C for subsequent measurements of serum Ca, P, ALP, BGP, and E2 concentrations. In plasma samples, the values of Ca, P, and ALP were determined by an autoanalyzer (Hitachi 7600-020, Hitachi Ltd., Tokyo, Japan). At the same time, BGP (Epstein et al., 1984) and E2 (Brochu et al., 1984) were determined with 125I labeled by radioimmunological machine (SN-695B, China Institute of Atomic Energy, Beijing). In the end of the experiment, 20 hens were killed at random from each group, and the tibias were isolated and cleaned of extraneous soft tissue for measuring bone mineral content (BMC). The tibia was put into a little plastic box that contained a small amount
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Figure 4. Cytochemical staining for tartrate-resistant acid phosphatase in osteoclasts showing deposit in cytoplasm and in nucleus (100×).
Table 1. Effect of ipriflavone on osteoblast proliferation [3-(4,5-dimethylthiazol-2-yl)-2,-5-diphenyl tetrazolium assay (MTT)] and alkaline phosphatase activity [4-nitrophenyl phosphate disodium salt assay (PNPP)]1
EFFECTS OF IPRIFLAVONE ON CAGED LAYER BONE METABOLISM
506
YAO ET AL. Table 2. Effect of ipriflavone on the number and area of osteoclast resorption pit lacunae1 Concentration (M) 0
10−7
10−8
10−9
7.8 ± 1.9a 8,299 ± 556a
1.0 ± 0.0b 171 ± 63c
1.0 ± 0.0b 452 ± 109c
2.3 ± 0.5b 2,030 ± 751b
Item Number of lacunae Areas of lacunae (m2)
Means with different superscripts differ significantly (P < 0.05). Mean ± SD; n = 6.
a–c 1
Statistical Analysis For control and treatment groups, the mean and SD were calculated with statistic software (SPSS 11.0, SPSS Inc., Chicago, IL), and values of calculated means among groups were compared using 1-way ANOVA.
RESULTS Osteoblasts were cultured and identified before ipriflavone treatments. The cells exhibited classic characteristics of osteoblasts; they were fibroblastic and were triangular or stellate in shape. Black deposits of ALP (Figure 1) and red deposits of promatrix (Figure 2) were shown by cytochemical staining. Mineralized nodes formed during prolonged culture (Figure 3). The results of the 3-(4,5-dimethylthiazol-2-yl)-2,-5-diphenyl tetrazolium test showed that 10−4 and 10−5 M ipriflavone inhibited (P < 0.05) the proliferation of osteoblasts, whereas 10−8 M ipriflavone increased (P < 0.01) the proliferation of osteoblasts in vitro when compared with the control group (Table 1). Correspondingly, the results of the 4-nitrophenyl phosphate disodium salt test showed that 10−4 and 10−5 M ipriflavone inhibited the synthesis and secretion of ALP in osteoblasts. Conversely, 10−7 to 10−9 M ipriflavone increased (P < 0.01) ALP activity in the osteoblasts (Table 1). Cultured osteoclasts were also identified before adding ipriflavone. These cells were characterized as being multinucleated and stained positive for TRAP (Figure 4). These cells were able to form bone resorption lacunae on bone slices (Figure 5). Adding ipriflavone at 10−7 to 10−9 M reduced the number and area of bone resorption pit lacunae
DISCUSSION Our results confirmed that small doses of ipriflavone could improve the proliferation of osteoblasts and inhibit the activity of mature osteoclasts in vitro. Larger doses of ipriflavone inhibited osteoblast proliferation, indicating the efficacy of ipriflavone on osteoblasts and osteoclasts was dependent on the dosage. The mechanism of this action is not currently known, and further research will be needed to clarify the mechanism of ipriflavone’s function. We also demonstrated that appropriate doses of ipriflavone can improve egg production in laying hens. Biochemical markers of bone metabolism revealed obvious changes in ALP and BGP activity when ipriflavone was fed in this experiment, especially in hens consuming 25 ppm ipriflavone. In the latter stages of egg formation, the cumulative transfer of Ca to the shell of an egg can gradually deplete Ca reserves in hens’ bodies (Clunies et al., 1992). At this time, appropriate doses of ipriflavone could enhance bone formation and help satisfy the Ca needs of
Table 3. Effect of ipriflavone on egg-laying performance of ISA caged layer1 Concentration (ppm) Item Average egg production rate (%) Soft-shell and broken egg rate (%) Average egg weight (g)
0
15
73.35 ± 2.65 1.195 ± 0.513 63.7 ± 1.3 b
72.6 ± 2.54 1.205 ± 0.969 63.1 ± 1.1
Means with different superscripts differ significantly (P < 0.05). Mean ± SD.
a–c 1
25 b
50
77.00 ± 3.38 0.949 ± 0.550 64.0 ± 0.6 a
100
67.68 ± 3.34 0.961 ± 0.741 63.5 ± 1.2 c
69.60 ± 2.08c 1.180 ± 0.639 63.4 ± 1.1
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(P < 0.05), reflecting an overall inhibition of osteoclastic resorption in vitro (Table 2). Hens fed 25 ppm ipriflavone had the highest cumulative rate of egg production when compared with the other 4 groups. Feeding hens higher levels of ipriflavone (50 and 100 ppm) led to the lowest cumulative egg production rates when compared with the control group and the group fed 15 ppm ipriflavone. Ipriflavone at any dose did not significantly affect egg weight and soft-shell and broken egg rate (Table 3). Serum levels of ALP were higher (P < 0.01) in hens consuming 25 ppm, and BGP levels were higher (P < 0.05) in hens consuming 15 and 25 ppm of ipriflavone than in the other groups (Table 4). There were no differences in serum E2 levels among treatment groups. The level of serum Ca and P and BMC also did not differ among groups.
of water, and the middle of each bone was scanned with bone densitometry that contained a 125I radionuclide source. Before scanning the tibia, the instrument was calibrated using the calibration phantom provided by the manufacturer (BMD-400E, China Institute of Atomic Energy).
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EFFECTS OF IPRIFLAVONE ON CAGED LAYER BONE METABOLISM Table 4. Effect of ipriflavone on serum alkaline phosphatase (ALP), bone gla protein (BGP), estrogen (E2), Ca, and P, and bone mineral content (BMC)1 Concentration (ppm) Item
0
15
25
50
100
1.45 ± 2.92 (n = 28) 12.79 ± 5.34b (n = 27)
b
16.56 ± 3.47 (n = 29) 16.39 ± 6.61a (n = 29)
a
19.30 ± 1.64 (n = 26) 17.72 ± 5.67a (n = 29)
b
15.19 ± 4.19 (n = 29) 14.88 ± 5.12b (n = 25)
11.71 ± 2.79c (n = 29) 14.20 ± 4.89b (n = 29)
E2 (pg/mL)
166.93 ± 66.72 (n = 20)
182.47 ± 75.87 (n = 16)
172.81 ± 90.71 (n = 17)
164.75 ± 94.52 (n = 18)
164.44 ± 72.59 (n = 17)
Ca (mg/dL)
13.48 ± 2.37 (n = 34)
11.81 ± 1.82 (n = 27)
13.13 ± 2.29 (n = 30)
14.09 ± 2.64 (n = 27)
13.15 ± 2.33 (n = 30)
5.28 ± 1.33 (n = 33)
5.41 ± 1.23 (n = 27)
4.69 ± 1.07 (n = 30)
5.70 ± 1.71 (n = 25)
5.80 ± 1.43 (n = 28)
0.775 ± 0.086 (n = 20)
0.795 ± 0.114 (n = 20)
0.819 ± 0.087 (n = 20)
0.856 ± 0.157 (n = 20)
0.783 ± 0.111 (n = 20)
ALP (mU/mL) BGP (ng/mL)
P (mg/dL) BMC (g)
Means with different superscripts differ significantly (P < 0.05). Mean ± SD.
a–c 1
shell formation, because ipriflavone has been reported to simulate the Ca absorption from the intestine (Arjmandi et al., 2000). Previous studies showed that ipriflavone had no effect on metabolism (Hocking and Bernar, 2000). In this study, the level of E2 also did not have significant change in the late laying period Thus, the increasing level of E2 probably reflects normal physiological phenomena late in the laying period. In conclusion, our results confirmed that ipriflavone can play a role in bone remodeling in laying hens both in vitro and in vivo. The level of 25 ppm in the feed may be the perfect dose, because it could not only enhance the egg production, but also maintain BMC to prevent osteoporosis.
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b