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The prebiotic arabinogalactan of Anoectochilus formosanus prevents
ovariectomy-induced osteoporosis in mice
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Li-Chan Yanga, Ting-Jang Lua, Wen-Chuan Linb,*
a
Institute of Food Science and Technology, National Taiwan University, Taipei, Taiwan.
School of Medicine, Graduate Institute of Basic Medical Science and Tsuzuki Institute for Traditional
Medicine, China Medical University, Taichung, Taiwan
b
*Corresponding author: Department of Pharmacology, China Medical University, No.
91 Hsueh Shih Road, Taichung, Taiwan, R.O.C. Tel +886 4 22053366; fax +886 4
22053764
e-mail address: wclin@mail.cmu.edu.tw (W.C. Lin)
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Keywords: Prebiotic; Arabinogalactan; Osteoporosis; Short chain fatty acids
1
ABSTRACT:
2
anti-osteoporosis and prebiotic properties in previous studies. In this study, these
3
bioactivities were verified and associated with an isolated type II arabinogalactan
4
(AGAF) in ovariectomized (OVX) mice model. Female ICR mice were OVX and
5
administrated AGAF (5 and 15 mg/kg) or inulin (400 mg/kg) orally for 3 weeks.
6
Streptomycin was used for blocking the bioactivities of AGAF. In results, AGAF
7
increased the level of fecal bifidobacteria, cecal soluble Ca and short chain fatty acids.
8
Comparing to OVX control group, AGAF improved bone mineral content, trabecular
9
bone volume, and the number of trabecular significantly. In RT-PCR analysis, AGAF
10
reduced the expression of tartrate-resistant acid phosphatase, cathepsin K, and
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osteocalcin. Streptomycin inhibited both anti-osteoporosis and prebiotic effects of
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AGAF. In vitro experiments revealed butyrate, not AGAF could activate osteoblasts
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and inhibit osteoclasts differentiation. In conclusion, this study showed AGAF
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prevented bone loss in OVX mice through prebiotic effects in vivo and in vitro.
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Anoectochilus
formosanus
(Orchidaceae)
has
exhibited
1
2
1. Introduction
3
4
Osteoporosis is the most common skeletal problem caused by aging, especially in
5
postmenopausal women (Pietschmann, et al., 2009). The deficiency of ovarian
6
hormones is a major factor in postmenopausal osteoporosis. Bone loss may lead to
7
related fractures and high medical costs. Hormone supplements and bisphosphonate
8
are common therapies for osteoporosis; however, these therapies have several side
9
effects. For example, hormone supplements increase the risk of cardiovascular disease
10
and breast cancer; the risk of bisphosphate includes osteonecrosis of the jaw and
11
atypical femur fractures (de Villiers & Stevenson, 2012; Khosla, et al., 2012). The
12
lack of reliable and effective therapies to cure osteoporosis-related fragility fractures
13
remains an important global issue (Datta, 2011). Previous studies have indicated that
14
certain nutritional factors, such as fruit, prebiotics, and minerals, can increase bone
15
mineral density in people diagnosed with osteoporosis (Devareddy, et al. 2008; Chen,
16
et al., 2006; Stransky & Rysava, 2009). Nutrition could prevent and treat osteoporosis
17
with fewer side effects than medicine therapies (Stransky & Rysava, 2009). Prebiotics
18
are food component not absorbed or digested in the small intestine but are fermented
19
by microbiota in the large intestine (Roberfroid, 2005). Several prebiotics, such as
20
inulin, galactooligosaccharides, and fructooligosaccharides, are thought to improve
21
bone health (Roberfroid, 2005;Weaver, 2005). The microbial fermentation products of
22
prebiotics, such as short chain fatty acids (SCFA), are responsible for the increase of
23
calcium (Ca) absorption in the large intestine. The high concentration of SCFA in the
24
cecum leads to a decrease of cecal pH, which increases the concentrations of soluble
25
Ca (Coxam, 2007). In addition, butyrate, one of the SCFA, belongs to a new class of
26
antiosteoporotic agents that may be useful in the treatment of bone loss (Katono, et al.,
1
2008; Rahman, et al., 2003; Schroeder & Westendorf, 2005). Furthermore, numerous
2
reports have indicated that the ingestion of prebiotics or fermentable dietary fibers
3
might be helpful in preventing osteoporosis (Coxam, 2007; Mitamura, et al., 2004).
4
Anoectochilus formosanus (Orchidaceae) is an important ethnomedicinal plant in
5
Taiwan. It has been popularly used as a nutraceutical herbal tea in Taiwan and other
6
Asian countries (Du, et al., 2008). In Taiwan, the aqueous extracts of A. formosanus
7
have been certified as health food for hepatoprotection bioactivity and showed safety
8
in the 13 week oral toxicity study in rats. In additional, Chang, et al. have indicated
9
that A. formosamus plants cultivated by artificial are safe for use as an herbal
10
medicine (Chang, et al., 2007). Several reports have shown that crude extracts of A.
11
formosanus could ameliorate osteoporosis in the ovariectomized (OVX) rat model
12
(Shih, et al.,2001; Masuda, et al., 2008; Yang, et al., in press). Masuda et al. shows
13
that aqueous extracts of A. formosanus suppress bone loss caused by estrogen
14
deficiency by inhibiting osteoclast formation (Masuda, et al., 2008). Our previous
15
study also shows that water extracts of A. formosanus prevent bone loss in rats caused
16
by OVX (Yang, et al., in press). In our previous studies, an indigestible
17
polysaccharide isolated from A. formosanus was shown to be a potent prebiotic (Yang,
18
et al., 2012). the indigestible polysaccharide of A. formosanus was mainly composed
19
of type II arabinogalactan (AGAF). Water extracts of A. formosanus could enhance
20
the level of cecal SCFAs and increase the number of fecal bifidobacteria in rats (Yang,
21
et al., 2012). Therefore, it was suggested that the anti-osteoporosis activity of A.
22
formosanus may be regulated by its prebiotic effect. This study examines the
23
anti-osteoporosis effects of AGAF in OVX mice, and investigates the relationship of
24
prebiotic properties and anti-osteoporosis activity.
25
1
2. Materials and methods
2
3
2.1. Arabinogalactan preparation
4
Cultured A. formosanus was purchased from Yu-Jung Farm (Pu-Li, Taiwan). Fresh
5
plants were homogenized with distilled water, and then partitioned with ethyl acetate
6
(Tedia Company, OH, USA). The aqueous extracts of A. formosanus were added with
7
a 4-fold volume of 95% ethanol to precipitate crude polysaccharides, and then the
8
crude polysaccharide was treated with α-amylase, protease and protease (Megazyme,
9
Wicklow, Ireland) to remove starches and proteins. After enzymic treatment, AGAF
10
was preserved in ethanol until use.
11
The identity and content of type II arabinogalactan in AGAF (> 80%) were
12
analyzed by precipitation with β-glucosyl yariv reagent according to a previous study
13
(Yang et al., 2012). For an in vivo experiment, AGAF was dissolved in distilled water,
14
and concentrations of 0.5 and 1.5 mg/mL were prepared.
15
The yield rate of AGAF was 0.15% from fresh plants. Chemical analyses showed
16
that AGAF contained 95.5% carbohydrates and 1.0% protein. AGAF is mainly
17
composed of a (1→3)-β-D-galactan backbone with a (1→6)-β-D-galactan side chain,
18
and is a type II arabinogalactan with an average molecular weight of 29 kDa. The
19
monosaccharide composition of AGAF was arabinose, galactose, glucose, and
20
mannose with a ratio of 22.4:56.5:15.4:5.4 (Yang, et al., 2012).
21
22
2.2. Animals
23
Eight-week-old female ICR mice were purchased from BioLASCO Co., Ltd. (Yi-Lan,
24
Taiwan). The experimental animals received humane care, and the study protocols
25
complied with the institutional guidelines of China Medical University for the use of
1
laboratory animals. The animals were housed in an air-conditioned room (21–24 °C)
2
under 12 h of light (8:00 a.m. – 8:00 p.m.), and were allowed free access to food
3
pellets and water throughout the study.
4
5
2.3. Anti-osteoporosis effects of AGAF on OVX mice.
6
The experiments were performed on 40 female ICR mice. Bilateral OVX operation
7
was performed under pentobarbital anesthesia (50 mg/kg) on the mice according to
8
the procedure described before (Idris, 2012). Briefly, the mice were laparotomized to
9
excise both ovaries clearly. The mice in the sham-operated group received anesthesia
10
and a laparotomy as OVX operation, and were then sutured without removing their
11
ovaries (Idris, 2012). After 3 days of adaptation after the surgery, the OVX mice were
12
randomly divided into four groups, and were orally administered H2O, AGAF (5, 15
13
mg/kg), or inulin (Alfa Aesar, Heysham, UK) for the positive control for 3 weeks.
14
The sham-operated group was orally treated with H2O. Each group contained 8 mice.
15
The body weight of each animal was measured once a week until the final day of
16
administration.
17
18
2.4. Assessment of prebiotic effect of AGAF
19
On day 7 after AGAF treatment, fresh feces were collected for analysis for
20
bifidobacteria. Fresh feces were homogenized with 0.1% peptone diluent, followed by
21
serial decimal dilutions. The number of bifidobacteria was counted on Bifidobacteria
22
iodoacetate medium-25 agar after incubation at 37 ℃ for 48 h under anaerobic
23
conditions (95% N2, 3% CO2, and 2% H2) (Munoa & Pares, 1988). After incubation, a
24
single colony was counted, and the results were expressed as the log values of the
25
CFUs per gram of wet weight of feces.
1
2.5. Ca concentrations and SCFA
2
After the mice were sacrificed, the ceca were removed and weighed immediately. The
3
cecal contents were collected. The cecal walls were then flushed with 0.9% saline,
4
blotted dry with filter paper, and weighed, and were then stored at -80 ℃ for reverse
5
transcription-polymerase chain reaction (RT-PCR) analysis. The cecal contents were
6
stored at -80 ℃ until SCFA determination using HPLC analysis (Niven, et al., 2004).
7
For SCFA analysis, the cecal content samples were defrosted on ice, and were diluted
8
with 0.0085N sulfuric acid. The cecum samples were shaken and centrifuged at 12000
9
× g for 20 min. The supernatant was diluted to proper concentrations for HPLC
10
analysis. The SCFAs were analyzed by a Transgenomic ICSep Transgenomic (300 ×
11
7.8 mm, Omaha, NE, USA) at 65 °C, and eluted with 0.0085N sulfuric acid at a flow
12
rate of 0.4 mL min-1. The peaks were detected by a Shodex RI-71 detector (Showa
13
Denko, Tokyo, Japan).
14
15
2.6. Bone Ca content
16
Soft tissues were removed from the lumbar vertebra, and were immersed in a mixed
17
solvent (chloroform: methanol = 2:1, Showa, Tokyo, Japan) to remove bone lipids
18
subcutaneously (Honda, 2001). De-fatty lumbar vertebrae were incinerated at 1000 °C
19
for 12 h for ashing. Bone ash was weighed and solved in 6N HCl (Wako, Osaka,
20
Japan) for determination of Ca with the o-cresolphthalein complexone method with
21
commercial kits (Randox, Crumlin, UK). Values were expressed as milligrams of Ca
22
of bone dry weight.
23
24
2.7. Microcomputed tomography (microCT) analyses
25
The right femurs of the mice were preserved in 75% alcohol until scanning. The bone
1
microarchitecture of the distal right femoral metaphysis was measured using a
2
microtomography scanner (SkyScan 1076, Kontizh, Belgium), with an isotropic
3
resolution of 17 m in all 3 spatial dimensions. To analyze the interest volume of
4
trabecular, 100 slices were selected from the edge of distant direction to the proximal
5
direction. The region of interest volume was analyzed without the cortical bone. The
6
bone and tissue volumes were measured directly from the original 3-dimensional
7
images, and the trabecular volume fraction (bone volume/tissue volume, %) was
8
normalized to compare samples of different sizes. The other examined parameters of
9
the trabecular structure were trabecular thickness, trabecular number, and trabecular
10
separation, which were calculated directly from the 3-dimensional images.
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12
2.8. RT-PCR analysis
13
Total RNA was extracted with Trizol (Invitrogen, CA, USA) from the tibia and cecal
14
walls, followed by acid guanidinium thiocyanate-phenol-chloroform extraction, as
15
described by Chomczynski and Sacchi (1987). A 3 g sample of total RNA was
16
subjected to reverse transcription with moloney murine leukemia virus reverse
17
transcriptase in a 50 L reaction volume. The cDNA was amplified by PCR. The
18
primers used for cecal wall RNA extract were CaBP and glyceraldehyde-3-phosphate
19
dehydrogenase
20
CAGAACCGAAGACTAGCGCA-3’
and
21
-3’(product
5’-TGTGTCCGTCGTGGATCTGA-3’
22
5’-CCTGCTTCACCACCTTCTTGA-3’(product size, 76 bp), respectively. The
23
primers for mice tibia RNA extract were TRAP, 5’-CCAATGCCAAAGAGATCGCC
24
-3’ and 5’- TCTGTGCAGAGACGTTGCCAAG-3’(product size, 216 bp), Cathepsin
25
K
,
size,
(GAPDH)
283
5’-
as
bp),and
internal
standard
and
were
5’-
5’-GCACAAAACAAAGTGGGTGC
CTGCCCATAACCTGGAGG-3’
and
and
5’-
size,
230
bp),
OCN,
5’-
1
GCCCTGGTTCTTGACTGG-3’(product
2
AGACCGCCTACAAACGCATC-‘3’
3
-3’(product size, 113 bp), Runx2, 5’-CCGAGAAGTGGTTCCCGGTCCTG-3’ and 5’-
4
CGACAGATCTGGAGCCTGCGGA-3’(product size, 173 bp), and GAPDH as
5
internal standard. . Electrophoresis was performed on 2% agarose gels in a 0.5X TBE
6
buffer (Amersco, OH, USA) for PCR products, and each lane was loaded with a fixed
7
volume of the sample. PCR products were visualized using ethidium bromide
8
staining.
and
5’-ACAGGGAGGATCAAGTCCCG
9
10
2.9. Plasma bone markers
11
At the end of the experiment, the mice were sacrified, and their blood was drawn with
12
heparin. The plasma was separated from the blood samples by centrifugation at 2000
13
× g for 10 min. The plasma was stored at -30 °C until assay. The plasma osteocalcin
14
(OCN) were measured by commercial enzyme-linked immunosorbent assay (ELISA)
15
kits (Biomedical Technologies Inc., Stoughton, MA, USA), and the plasma
16
carboxy-terminal collagen cross-links (CTx) were analyzed using ELISA kits
17
(Immunegiagnostic Systems, AZ, USA).
18
19
2.10. Effects of AGAF on OVX mice with streptomycin supplement
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40 female ICR mice were treated with sham operations or OVX, and OVX mice were
21
allocated into groups that were H2O, AGAF (15 mg/kg po. daily) with or without
22
streptomycin sulfate (SM; Sigma Aldrich, MO, USA) treatment for 3 weeks. Each
23
group contained eight mice. SM was dissolved in the drinking water at a
24
concentration of 2 mg/mL (Asahara, 2001). The sham-operated group was orally
25
administrated H2O. The body weight of each mouse was recorded every week until
1
the end of the experiment. The mice were assessed for prebiotic activity in vivo. At
2
the end of the experiment, mice femurs were removed for microCT analyses.
3
4
2.11. In vitro assay of alkaline phosphatase activity on murine osteoblast MC3T3-E1
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cells
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The cells of murine osteoblast MC3T3-E1 were grown in Dulbecco’s modified
7
Eagle’s medium (DMEM, Hyclone, UT, USA) supplemented with 10% (v/v) FBS
8
(Hyclone, USA), 100 U/mL penicillin, and 100 mg/mL streptomycin. Incubation was
9
conducted in a CO2 incubator (5% CO2, 95% air) at 37 °C. The cells were
10
subcultured every 2 or 3 days by 0.25% (w/v) trypsin plus a 0.02% (w/v)
11
ethylenediaminetetraacetic acid tetrasodium salt solution (Gibco, NY, USA).
12
For the alkaline phosphatase (ALP) activity assay, the MC3T3-E1 cells were
13
seeded in 48-well plates (104 cells/well) containing DMEM in addition to 10% FBS
14
(Iwami & Moriyama, 1993). After the cells attached to the bottom of the wells, the
15
culture medium was changed to DMEM + 10% FBS medium containing 10 mM
16
disodium β-glycerophosphate (Sigma Aldrich), 0.15 mM ascorbic acid (Sigma
17
Aldrich), and 10-8 M dexamethasone (Sigma Aldrich) (Isama & Tsuchiya, 2003).
18
Simultaneously, different concentrations of sodium butyrate or AGAF were added
19
to the culture medium in the well. On day 6 after cultivation, the cells were washed
20
twice with phosphate buffered saline and harvested in a 200 μL/well of a lysis buffer
21
(pH 8.2, 10 mM Tris-HCl, 2 mM MgCl2, and 0.05% Triton X-100). Cells were lysed
22
through an ultrasonic processor (Vibra-Cell, Sonics & Materials, CT, USA) with 30 J
23
in energy. Aliquots were reserved for protein analysis. A total of 300 μL of 8 mM
24
p-nitrophenyl phosphate (Sigma Aldrich) in a 0.1 M sodium carbonate buffer (pH 10)
25
containing 1 mM MgCl2 were added to the reaction mixture, which was incubated at
1
37 °C for 30 min. The reaction was stopped by adding 50 μL of 1.0 N NaOH/well.26
2
The yellow sample solutions containing p-nitrophenol for the reaction product were
3
measured at 405 nm using a microplate reader. A standard curve was prepared using
4
p-nitrophenol phosphate. The total protein contents of cell lysates was measured by a
5
Bradford reagent (Sigma Aldrich) using albumin for the standard (Bradford, 1976).
6
7
2.12. In vitro Osteoclasts differentiation from murine macrophage RAW264.7 cell line
8
RAW264.7 cells were maintained in an α-modified Eagle’s medium (Hyclone),
9
supplemented with 10% FBS (Gibco, CA, USA), 100 U/mL penicillin, and 100
10
mg/mL streptomycin and L-glutamine (Biological industry, Kibbutz Beit-Haemek,
11
Israel). Cells were cultured at a density of 2 × 10 3 cells/mL in a 24-well plate in the
12
presence of a receptor activator of nuclear factor kappa B ligand (RANKL) for 5 days
13
(Rahman, et al., 2003). RAW264.7 cells were incubated with different concentrations
14
of sodium butyrate or AGAF to examine their effects on osteoclast differentiation. On
15
day 5, cells were treated with 4% formaldehyde solution for 10 min, and then stained
16
to obtain the tartrate-resistant acid phosphatase (TRAP) of the osteoclasts. TRAP
17
staining was applied to measure the presence of osteoclasts, and stained with a
18
standard kit (387A-1 kit, Sigma-Aldrich). The TRAP-positive multinucleated
19
osteoclasts (least 3 nuclei) were counted (Parfitt, et al. 1987).
20
21
2.13. Statistical analysis
22
The results were expressed as mean  SD. All experimental data were analyzed using
23
one-way analysis of variance (ANOVA) with the Duncan multiple-range test. Values
24
of p < 0.05 were considered statistically significant.
25
1
2
3
3. Results
4
The number of fecal bifidobacteria was different between the sham and OVX-H2O
5
group. The OVX treatment caused a 9.1% decrease in the number of bifidobacteria for
6
the OVX mice than in the sham group. Administration of AGAF for 7 days led to a
7
20.7% (5 mg/kg) and 21.2% (15 mg/kg) increase in bifidobacteria in the stool
8
compared to the OVX-H2O group (Table 1). In addition, inulin treatment also led to a
9
15.2% increase in the number of fecal bifidobacteria over the OVX-H2O group.
3.1. Assessment of prebiotic effect of AGAF
10
11
3.2. Cecal Ca concentrations and SCFA
12
The cecal Ca concentrations in the OVX-H2O group and sham group were equal. For
13
the level of cecal Ca concentration, the AGAF administrated groups had 35.1% (5
14
mg/kg) and 42.7% (15 mg/kg) greater levels than the OVX-H2O group; administrated
15
with inulin were 39.8% greater than OVX-H2O group significantly (Table 1).
16
The results of SCFA analyses are shown in Table 1. Concentrations of butyric
17
acid in the sham group and OVX-H2O group have significant differences. The
18
OVX-H2O group had a decrease in the level of butyric acid of 12.1% compared to the
19
sham group. In addition, no differences in lactic acid, acetic acid, and propionic acid
20
were observed between the sham group and the OVX-H2O group. The analysis
21
showed significantly higher concentrations of acetic acid, propionic acid, and butyric
22
acid in the OVX-AGAF group (15 mg/kg) compared to the OVX-H2O group (26.3%,
23
66.0%, and 57.7% increases, respectively). The OVX-inulin group showed increases
24
in propionic acid and butyric acid of 53.6% and 16.4% compared to the OVX-H2O
25
group. In this study, total SCFA is the amount of lactic acid, acetic acid, propionic
26
acid, and butyric acid; no differences were observed between the sham and OVX-H2O
1
groups. Moreover, the OVX mice administered AGAF led to a 21.0% (5 mg/kg) and
2
31.25% (15mg/kg) increase; inulin led to a significant 12.8% increase in the total
3
SCFAs in the ceca of the OVX mice, compared to the OVX-H2O group.
4
5
3.3. Bone Ca content
6
The amount of Ca in the lumbar vertebrae was determined using o-cresophthalein
7
complexone. The total Ca content and Ca ratio of lumbar vertebrae was calculated.
8
The results were showed that OVX treatment reduced the Ca content by 11.4% (14.0
9
± 1.3 mg) and the Ca ratio by 16.9% (12.3 ± 0.6%) in the lumbar vertebrae compared
10
to sham group (15.8 ± 1.0 mg in Ca content; 14.8 ± 0.8% in Ca ratio). The results of
11
AGAF treatment (5 mg/kg) were 13.3 ± 0.9 mg in Ca content and 13.1 ± 0.1% in the
12
Ca ratio, and shown no significant difference compared to OVX-H2O group.
13
Administration of AGAF (15 mg/kg) caused a significant 13.6% increase in the Ca
14
content (15.9 ± 0.4 mg), and a 14.6% increase in the Ca ratio (14.1± 0.1%); however,
15
administration of inulin increased the Ca ratio (13.8 ± 0.5%) only, compared to the
16
OVX-H2O group. Bone Ca content of inulin treatment group was 14.9 ± 1.9 mg and
17
displayed a minor increase to OVX-H2O group.
18
19
3.4. MicroCT analyses
20
The OVX procedure caused significant decreases in the trabecular bone volume ratio
21
(12.6%), trabecular number (11.1%), and trabecular thickness (30.6%) in the OVX
22
group compared to the sham group (Figs. 1B, 1C). Although the trabecular separation
23
was 107.0% in the OVX-H2O group compared to the sham group, the data showed no
24
significant difference (Fig. 1C). These results show that OVX induced a loss of
25
trabecular bone in the femurs of the mice (Fig. 1A). Treatment of AGAF (15 mg/kg)
1
and inulin led to a significant 10.9% and 3.5% increase in the loss in trabecular bone
2
volume, respectively. OVX mice administered AGAF had a 24.5% (5 mg/kg) and
3
30.0% (15 mg/kg) increase in trabecular thickness, respectively. Treatment with inulin
4
caused an increase of 12.2% in trabecular thickness. The trabecular number and
5
separation was unaffected in the AGAF or inulin groups.
6
7
3.5. RT-PCR analysis
8
The expression of Ca-binding protein (CaBP) in cecal mucosal cells is shown in Fig. 2.
9
The gene expression of CaBP was significantly decreased in the OVX-H2O group
10
compared to the sham-H2O group (31.6% decrease). The expression of CaBP were
11
1.6- and 2.0-fold increased by the administration of AGAF (5, 15 mg/kg), and rose
12
1.8-fold in the inulin group compared to the OVX-H2O group.
13
Figure 2B showed that all data were presented as relative expressions compared to
14
GAPDH, which was used as the internal control. For mRNA expression of the tibia,
15
the relative expression of osteoclast-related genes involving TRAP, cathepsin K, and
16
osteoblast-associated genes, including OCN, was significantly upregulated by the
17
OVX operation compared to in the sham operation. The expression of TRAP,
18
cathepsin K, and OCN were 4.4-fold, 3.0-fold, and 1.8-fold higher than in the sham
19
group, respectively (Fig. 2). Administration of AGAF (5, 15 mg/kg) and inulin could
20
significantly inhibit the expression of TRAP and cathepsin K compared to the
21
OVX-H2O group. Both AGAF and inulin treatments could prevent the increased
22
expression of OCN. The expression of OCN in the AGAF administrated groups were
23
83.1% (5 mg/kg) and 67.6% (15 mg/kg), and 70.4% in the inulin administrated group
24
compared to the OVX-H2O group. There was no significant difference in the
25
expression of Runx2 between the OVX-H2O and sham groups (Fig. 2). However, only
1
AGAF treatment (15 mg/kg) could significantly suppress the expression of Runx2
2
(72.1%) compared to the OVX-H2O group.
3
4
3.6. Plasma bone markers
5
The results of the plasma CTx assay are shown in Fig. 3A. The concentration of CTx
6
was significantly increased in the OVX-H2O group over the sham group. Treatments
7
of AGAF (15 mg/kg) and inulin in the OVX mice caused decreases of 31.0% and
8
25.3% of the plasma CTx level, respectively. In the OCN assay, the OCN
9
concentration in the OVX-H2O group was 25% higher than in the sham group (Fig.
10
2B). The AGAF treatment (15 mg/kg) significantly lowered the level of OCN by
11
9.3% compared to the OVX-H2O group.
12
13
3.7. Effects of AGAF on OVX mice with streptomycin supplement
14
The assay for prebiotic effects was determinate by fecal number of bifidobacteria on
15
day 7 and day 21 during the experimental period. After 7 days treatment, the number
16
of bifidobacteria (log10CFU/g) showed no difference in sham and OVX-H2O group
17
and was 6.7 ± 0.7 and 6.2 ± 0.1, respectively. The mice administrated AGAF (15
18
mg/kg) has 24.0% increase in the number of bifidobacteria (7.6 ± 0.3 log10CFU/g)
19
compared to OVX-H2O group significantly. However, continuous treatment of mice
20
with SM in the drinking water (2 mg/mL) for 7 days resulted in the fecal
21
bifidobacteria decreasing to an undetectable level. The fecal bifidobacteria counts in
22
OVX-H2O-SM and OVX-AGAF-SM group was < 2.0 and 4.1 ± 2.5 log10CFU/g,
23
respectively.
24
The results of prebiotic assay on day 21 were similar to the results of day 7. The
25
bifidobacteria count of sham, OVX-H2O, OVX-AGAF groups was 6.7 ± 0.1, 6.3 ± 0.2
1
and 7.8 ± 0.4 log10CFU/g feces, respectively. The administration of AGAF promoted
2
the number of bifidobacteria in 24.1% increase than OVX-H2O significantly. In
3
additional, SM also blocked the prebiotic effects of AGAF on day 21. The results of
4
fecal bifidobacteria in OVX-H2O-SM and OVX-AGAF-SM group were showed as
5
undetectable and 3.7 ± 1.6 log10CFU/g, respectively. Though the bifidobacteria
6
number of OVX-AGAF-SM group on day 21 was minor than on day 7, but the data
7
was not significant difference.
8
The levels of plasma bone markers, including CTx and OCN, were shown in Fig.
9
3B. CTx and OCN in the OVX-H2O group increased 2.5-fold and 1.25-fold compared
10
to the sham group, respectively. No differences were noted between the OVX-H2O
11
group and the OVX-H2O+SM group. Administration of AGAF (15 mg/kg) could
12
prevent the increase of CTx and OCN. However, AGAF administration combined
13
with SM did not affect the levels of CTx or OCN.
14
Mice femurs were assessed for microCT. The OVX operation caused significant
15
reductions in trabecular bone volume, trabecular number, and the trabecular thickness
16
of the femur compared to the sham group. Trabecular separation showed no difference
17
among the groups. Adding SM to the drinking water of the OVX mice did not affect
18
the trabecular parameters, compared to the OVX-H2O group without SM. The results
19
showed that administration of AGAF (15 mg/kg) could improve the trabecular bone
20
volume and trabecular thickness of the femur in OVX mice. Treatment of AGAF with
21
SM caused no difference in trabecular bone volume, trabecular number, trabecular
22
thickness, or trabecular separation, compared to the OVX-H2O group (Table 2).
23
24
3.8. In vitro assay of ALP activity on murine osteoblast MC3T3-E1 cells
25
The ALP activity assay was used to evaluate the differentiation of MC3T3-E1. ALP is
1
a representative enzyme that can indicate osteoblast differentiation (Isama & Tsuchiya,
2
2003). Butyric acid is the most changeable SCFA in the cecum in the in vivo prebiotic
3
experiment. In the presence of different concentrations of butyric acid, MC3T3-E1
4
cells produced increasing ALP activity at concentrations from 0.5 mM to 1.0 mM
5
sodium butyrate after 6 days of treatment (Fig. 4A). No obvious differences were
6
observed in the sodium butyrate concentration of 0.1 mM and for all concentrations of
7
AGAF (Fig. 4A). It appears that sodium butyrate can effectively enhance the ALP
8
activity of MC3T3-E1, but AGAF cannot. The ALP activity results strongly suggest
9
that osteoblastic differentiation could be efficiently stimulated in the presence of
10
sodium butyrate.
11
12
3.9. In vitro osteoclasts differentiation from murine macrophage RAW264.7 cell line
13
The effects of sodium butyrate and AGAF on the osteoclast formation in the murine
14
macrophage cell line RAW264.7 cells were examined. On day 5, the number of
15
TRAP-positive cells with more than 3 nuclei was counted (Fig. 4B & 4C).
16
TRAP-positive cells with 3 or more nuclei are counted as osteoclasts after 5 days
17
cultivation. For the TRAP-positive cells, the presence of sodium butyrate (0.1, 0.5,
18
and 1 mM) in the culture medium caused a significant decrease. The presence of
19
AGAF (100 and 150 μg/mL) in the culture medium could also show decreases in the
20
number of TRAP-positive cells by 13.8% and 37.2%, respectively. The osteoclast
21
inhibition was 37.5%, 67.6%, and 86.9% under the treatment of sodium butyrate at
22
concentrations of 0.1, 0.5, and 1 mM, respectively. However, the suppression of
23
osteoclast differentiation of AGAF was not as remarkable as that of sodium butyrate.
24
25
4. Discussion
1
In this study, we showed that AGAF could prevent osteoporosis through its prebiotic
2
effects in OVX mice. Our results showed that AGAF could enhance the number of
3
probiotics and increase cecal SCFA and bone mass density in OVX mice. We
4
observed that the anti-osteoporosis effects of AGAF could be inhibited by SM, a
5
broad-spectrum antibiotic. The results suggested that the prevention effects of AGAF
6
on osteoporosis might cause by its prebiotic effects. Numerous prebiotic isolated from
7
botanic and found as polysaccharides, like inulin, larch wood arabinogalactan,
8
kiwifruit pectin, konjac glucomannan and resistant dextrin (Roberfroid, 2005; Degnan
9
& Macfarlane, 1995; Parkar, et al., 2010; Connolly, et al., 2010; Barczynska, et al.,
10
2012). Those prebiotic carbohydrates are regarded to be hydrolyzed and utilized by
11
probiotics only (Tomasik & Tomasik, 2003). AGAF is a type II arabinogalactan and
12
has been reported as a potent prebiotic polysaccharide in our previous study (Yang, et
13
al., 2012). The previous study reported that AGAF promote the number of
14
bifidobacteria of feces especially and the prebiotic mechanism of AGAF may due to
15
up-regulate the expression of ATP binding cassette transporter, a part of nutrient
16
uptake system.
17
OVX mice mimicked middle-aged postmenopausal women. In this study, no
18
differences were observed in the fecal number of bifidobacteria between OVX-H2O
19
mice and the sham group (Chen, et al.,2009). The results of fecal bifidobacteria were
20
similar to those of our previous study (Chen, et al.,2009). However, AGAF could
21
increase the number of fecal bifidobacteria in OVX mice. A prebiotic is a selectively
22
fermented ingredient that allows specific changes, both in composition and/or activity
23
in gastrointestinal micoflora. Thus, AGAF possesses prebiotic characteristics, as
24
previous reported (Yang, et al. 2012).
25
SCFA are the products of prebiotic fermentation, and have been shown to enhance
1
bone health (Scholz-Ahrens, et al. 2007). Butyric acid, an SCFA and a histone
2
deacetylase inhibitor, can influence osteoblasts and osteoclasts and provide protection
3
from bone density loss (Rahman, et al., 2003; Iwami & Moriyama, 1993). In addition,
4
high concentrations of SCFA in the cecum lead to increases in the concentration of
5
soluble Ca (Lopez, et al., 2000). In our study, AGAF treatment increased the cecal
6
SCFA, especially butyric acid, and increased the cecal soluble-Ca concentration.
7
Cholecalciferol-induced CaBP is a key factor in the intestinal transcellular Ca
8
transport system (Bouillon, et al., 2003). A direct correlation exists between the
9
mucosal amounts of CaBP and the efficiency of Ca absorption (Bronner, 2003).
10
Hence, an increase in mucosal CaBP indicates an increase in Ca absorption in this
11
intestinal segment. A previous study reported that the high level of soluble Ca in the
12
cecum induces the expression of CaBP (Yang, et al., in press). In our study, the
13
soluble Ca level was higher in the cecum after AGAF treatment. Moreover, the
14
expression cecal CaBP was enhanced by AGAF administration in OVX mice. These
15
results showed that AGAF could promote intestinal Ca absorption in OVX mice
16
through prebiotic fermentation.
17
Other studies have also shown that increasing Ca absorption affects bone
18
characteristics, such as bone Ca content and bone strength, in OVX rats (Mitamura, et
19
al. 2004). In our study, an enhancement of Ca absorption caused by administration of
20
AGAF may also cause an increase in the Ca content in the lumbar vertebrae of OVX
21
mice.
22
In microCT analysis, trabecular bone volume, trabecular number, trabecular
23
thickness, and trabecular separation of the distal femur were analyzed. AGAF could
24
cause increases in femur trabecular bone volume and trabecular thickness compared to
25
OVX-H2O mice. Trabecular bone volume and trabecular thickness are important
1
factors in bone strength (Yao, et al., 2005). These results implied that AGAF could
2
decrease bone loss and bone structural fractures induced by OVX operation.
3
In plasma bone markers, CTx is part of the composition of bone. Plasma CTx
4
levels are markedly increased in postmenopausal women with osteoporosis, and their
5
values decrease markedly and rapidly with anti-resorption therapy (Qvist, et al., 2002).
6
Although the concentration of OCN is a sensitive marker of bone formation, the
7
plasma concentration of OCN is a feedback control mechanism during active bone
8
resorption (Swaminathan, 2001). Because a high turnover may lead to bone loss, a
9
downregulation of bone turnover may be beneficial to bone metabolism. Treatment by
10
AGAF and inulin could reduce the plasma levels of both CTx and OCN, which were
11
induced by OVX operations.
12
In RT-PCR analysis, the expression of osteoclast-associated genes, TRAP, and
13
cathepsin K was induced by OVX. TRAP is a di-iron-containing metalloenzyme that
14
is expressed in osteoclasts and in subsets of tissue macrophages and dendritic cells
15
(Walsh, et al., 2003). TRAP expression is dramatically upregulated during osteoclast
16
differentiation. Therefore, TRAP activity is commonly used as an identifying marker
17
in osteoclasts (Walsh, et al., 2003). Cathepsin K, a lysosomal cysteine proteinase, is
18
expressed predominantly in osteoclasts. Cathepsin K cleaves key bone matrix proteins,
19
and is believed to have an important role in degrading the organic phase of bone
20
during bone resorption (Saftig, et al. 2000). In this experiment, the expression of
21
TRAP and cathepsin K was increased in OVX mice. The administration of AGAF in
22
OVX mice could suppress the induction of expression of TRAP and cathepsin K. The
23
results showed that AGAF could down-regulate osteoclast differentiation and bone
24
resorption in OVX mice.
25
Runx2 is an essential factor in osteoblast differentiation. Runx2 triggers the
1
expression of major bone matrix genes during the early stages of osteoblast
2
differentiation, but Runx2 is not essential for the maintenance of these gene
3
expressions in mature osteoblasts (Komori, 2010). Moreover, Runx2 inhibits
4
osteoblast maturation and mature bone formation at the late stage of osteoblast
5
differentiation (Komori, 2010).
6
In this study, no rebound increase of Runx2 was shown in the tibia of the OVX
7
mice compared to the sham mice. The results suggested that the OVX mice might
8
have been in the mid-to-late stage of the bone resorption process. A previous study
9
indicated that a supplement of dried plum could prevent OVX-induced bone loss
10
(Rendina, et al.,2012). In addition, the study also reported that the expression of
11
Runx2 decreased significantly in the presence of dried plums in OVX mice. The
12
results of our study showed that the expression of Runx2 was similar to those of
13
Rendina et al. (2012). This implies that AGAF might promote the transition of
14
osteoblasts into osteocytes in late stages of OVX-induced osteoporosis.
15
OCN is a major noncollagenous protein component of the bone extracellular
16
matrix, and is synthesized and secreted exclusively by osteoblasts in the late stage of
17
maturation; it is considered an indicator of osteoblast differentiation (Sila-Asna, et al.,
18
2007). OCN is also a sensitive bone turnover marker. The expression of OCN showed
19
a rebounded increase in OVX mice caused by the over expression of osteoclasts in
20
this study. Treatment of AGAF significantly inhibited the increased expression of
21
OCN, which indicated that AGAF could suppress the bone turnover marker through
22
the inhibition of osteoclasts. The results of OCN expression were identical to those of
23
plasma OCN concentrations. These results show that bone protection by AGAF might
24
prevent bone resorption by osteoclasts in OVX mice.
25
We evaluated the effects of AGAF in the treatment of postmenopausal
1
osteoporosis using OVX mice. To test the hypothesis that the anti-osteoporosis effects
2
of AGAF were caused by its prebiotics, we supplemented drinking water with SM to
3
investigate the effects on OVX mice. SM in drinking water could significantly
4
eliminate not only the prebiotic effects of AGAF but also the anti-osteoporosis effects
5
in plasma bone markers in OVX mice. The preventative bone loss of AGAF was
6
inhibited by SM. The results showed that the anti-osteoporosis properties of AGAF
7
were derived mainly from its prebiotic effects. Certain studies have reported that
8
inulin, a well-known prebiotic fructan, could promote intestinal mineral absorption
9
and bone health (Scholz-Ahrens, et al. 2007; Kruger, et al. 2003).
10
An in vivo study showed that AGAF is an anti-osteoporosis component through its
11
prebiotic fermentation products. The results of the in vitro study showed that AGAF
12
could not influence the activity of ALP on osteoblast cell line MC3T3-E1, but butyric
13
acid could increase the activity of ALP significantly. Similar results were observed in
14
murine osteoclast differentiation. These results suggested that the mechanism of
15
anti-osteoporosis effects was caused by its prebiotic fermentation.
16
We investigated why AGAF or its fermentation products, such as SCFA, act as
17
anti-osteoporosis materials. The concentration of butyric acid was considerably
18
affected by AGAF administration. Sodium butyrate was used to examine the
19
anti-osteoporosis effects of SCFA. In an osteoblast differentiation study, AGAF did
20
not affect ALP production. ALP is crucial to the mineralization of osteoblasts (Zhao,
21
et al., 2007). Sodium butyrate, but not AGAF, could improve the mineralization
22
activities of osteoblasts. In osteoclast cell line differentiation, we observed that AGAF
23
and sodium butyrate could both influence the formation of osteoclasts. However,
24
AGAF activity in resisting osteoclast formation was considerably weaker than that of
25
sodium butyrate. These in vitro studies showed that sodium butyrate was a major
1
compound in anti-osteoporosis functions. In addition, certain studies have also shown
2
that histone deacetylase inhibitors, such as butyrate, accelerated in vitro osteoblast
3
maturation (Katono, et al., 2008; Schroeder & Westendrof, 2005). Therefore, it is
4
possible that AGAF partially ameliorates osteoporosis in OVX mice by enhancing
5
osteoblast maturation and inhibiting osteoclast differentiation through butyrate
6
production.
7
8
5. Conclusions
9
The results clearly showed that administration of AGAF inhibited bone turnover,
10
elevated intestinal Ca absorption, and prevented Ca loss and deterioration in bone
11
volume in OVX mice. Our study provides evidence that prebiotic activity was
12
involved in the anti-osteoporotic mechanisms of AGAF. In addition, the prebiotic
13
potency and bone protection activities of AGAF were higher than those of inulin,
14
based on the dosage. Daniel et al. demonstrate that the half-life of butyrate in mice
15
and rabbits is less than 5 min (Daniel, et al., 1989). This indicates that the rapid
16
elimination of butyrate is a limiting factor in practical applications (Daniel, et al.,
17
1989). Our results showed that the prebiotic fermentation effects of AGAF might
18
prolong the absorption period of butyrate in OVX mice. The results showed that
19
AGAF could be a new source of food supplements in the promotion of bone health for
20
postmenopausal osteoporosis.
21
22
Acknowledgment.
23
This study was supported by grants from the National Science Council of the
24
Republic of China (NSC 101-2320-B-039-019)
25
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Figure legends
8
9
Fig. 1. MicroCT analyses of metaphysic of the distal femur in mice of different groups.
10
(A) The photos of microCT scans (i) Sham + H2O group. (ii) OVX + H2O group.
11
These mice showed a decrease in the trabecular bone volume. (iii) OVX + AGAF (5
12
mg/kg) group. (iv) OVX + AGAF (15 mg/kg) group. (v) OVX + inulin (400 mg/kg)
13
group. AGAF and inulin significantly prevented bone loss from OVX-induced
14
osteopenia. (B) Trabecular bone volume (BV/TV, %) and trabecular number (1/mm)
15
(C) Trabecular thickness (μm) and trabecular separation (μm). All values were means
16
± SD (n = 8). Values with different letters were significantly different.
17
18
Fig. 2. Effects of AGAF and inulin on cecal mucosa CaBP mRNA expression and
19
tibia mRNA expression of TRAP, cathepsin K, OCN and Runx2 in OVX mice. A,
20
Fragments were amplified by RT-PCR. The fragments reflect the pooled data for eight
21
samples. B, The expression levels of CaBP, TRAP, cathepsin K, OCN and Runx2
22
mRNA were measured and quantified densitometrically. Values were normalized to
23
GAPDH mRNA expression. All values were means ± SD (n = 8). Values with
24
different letters were significantly different.
25
26
Fig. 3. Effects of AGAF and inulin on plasma bone marker in OVX mice. (A) The
27
plasma OCN and CTx in OVX mice were oral administration of AGAF (5, 15 mg/kg)
1
or inulin (400 mg/kg) for 3 weeks. (B) the plasma OCN and CTx in OVX mice were
2
administration of AGAF (15 mg/kg) and supplemented with or without SM in
3
drinking water for 3 weeks. All values were means ± SD (n = 8). Values with different
4
letters were significantly different.
5
6
Fig. 4. Effects of sodium butyrate and AGAF on ALP activity of murine osteoblast
7
MC3T3 E1 cell line and on the osteoclast differentiation from RAW264.7 cell line. (A)
8
ALP activity of murine osteoblast MC3T3 E1 cell under the treatment with sodium
9
butyrate or AGAF for 6 days. Cells were lysed by ultrasonic and analyzed for ALP by
10
p-nitrophenyl phosphate. (B) The number of counted osteoclasts per well (C)
11
Osteoclasts were stained by TRAP. All values were means ± SD (n = 3). Values with
12
different letters were significantly different.
13
14
Table 1. Effects of AGAF and inulin on fecal bifidobacteria, cecal free Ca, cecal butyric acid, lactic acid, acetic acid, propionic acid and total
SCFA levels in OVX mice
Dosage
Fecal Bifidobacteria Cecal Lactic
(mg/kg ) Log10CFU/g feces
Cecal acetic
Cecal Propionic
Cecal butyric
Cecal total
acid (μmol/g)
acid (μmol/g) acid (μmol/g)
acid (μmol/g) SCFA (μmol/g)
Cecal free Ca
(mg/dL)
Sham + H2O
-
6.6  0.2b
16.8  0.3a
41.7  1.8a
4.3  0.3a
20.9  0.9b
83.7  2.6ab
18.1  4.0a
OVX + H2O
-
6.1  0.5a
16.7  0.3a
40.3  2.2a
4.1  0.4a
18.4  0.5a
79.5 1.5a
17.0 2.0a
OVX + AGAF
5
7.0  0.2c
18.6  2.7a
46.6  3.2ab
5.8  0.6b
25.2  0.4c
96.2 5.4cd
23.0 5.7b
OVX + AGAF
15
7.3  0.2c
17.6  2.4a
50.9  0.9b
6.9  0.1b
29.0 0.3d
104.4 0.8d
24.2 3.0b
7.4  0.3bc
19.2  2.1a
42.4  1.4a
6.4  0.3b
21.4 1.1b
89.7 0.9bc
23.8 2.7b
OVX + Inulin
400
All values were means ± SD (n = 8). Values with different letters were significantly different.
Table 2. MicroCT analyses of femur on OVX mice administrated AGAF and with or
without SM supplement in drinking water
Treatments
Trabecular
bone volume
(%)
Trabecular
number
( 1/mm)
Trabecular
thickness
(μm)
Trabecular
separation
(μm)
Sham + H2O + H2O
31.3  2.3b
3.7  0.2c
87.5  8.9c
186  24.0c
OVX + H2O + H2O
27.5  1.0a
3.3  0.1ab
62.7  6.8a
199  16.9c
OVX + AGAF + H2O
29.8  1.7b
3.5  0.2bc
75.0  7.9bc
194  12.8c
OVX + H2O + SM
26.9  2.4a
3.2  0.3a
62.5  5.9a
202  15.4c
OVX + AGAF + SM
27.6  2.1a
3.3  0.3ab
63.2  6.1a
200  14.3c
SM (2 mg/mL) was supplement in drinking water. The mice were administrated H2O
or AGAF (15 mg /kg) for 3 weeks.
All values were means ± SD (n = 8). Values with different letters were significantly
different.