Journal of Functional Foods 75 (2020) 104228

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Journal of Functional Foods

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Effects of loach skin collagen peptides in reducing osteoporosis in mice T

a,b,⁎ b b
Haiying Liu , Lu Huang , Jing Wang
a
State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu, People’s Republic of China
b
School of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu, People’s Republic of China

ARTICLE INFO ABSTRACT

Keywords: The effects of loach skin collagen peptides (LCP) and the combination of LCP and sodium selenite on osteoporosis
Collagen peptide in mice were investigated. The results showed that the combination of LCP and sodium selenite alleviated mice
Osteoporosis osteoporosis induced by retinoic acid. It improved bone microarchitecture, increased the number and mass of
Trabecular bone trabecular bone, maintained blood calcium and phosphate balance in mice. The combination of LCP and sodium
micro-CT
selenite inhibited the relative mRNA expression levels of Wnt10, β-catenin, osteogenic transcription factors
Wnt10
Runx2 and Osterix through the Wnt/β-catenin pathway. The ratio of receptor activator for nuclear factor-kB
ligand/Osteoprotegerin (RANKL/OPG) was down-regulated through the OPG/RANKL/NF-kB pathway. Likewise,
the relative mRNA expression levels of tumor necrosis factor receptor associated factor 6 (TRAF6), nuclear factor
kB (NF-kB) and nuclear factor of activated T-cell (NFATC1) were reduced. This study provides useful information
for the use of LCP and sodium selenite in treating and reduction of osteoporosis.

1. Introduction Watanabe-Kamiyama et al. observed an increase of the content of or-

Osteoporosis seriously affects human health and affects the lives of
one-third of women and one-fifth of men worldwide (Pietschmann,
Mechtcheriakova, Meshcheryakova, Fögersamwald, & Ellinger, 2016).
The drugs used to treat osteoporosis are vitamin D, biphosphonates,
raloxifene, calcitonin, and so on. However, the use of these drugs is
often accompanied by major side effects (Tripathi, 2008). Compared
with drugs, food has multiple assets for good compliance. Some reports
claimed that hydrolyzed collagen or gelatin have therapeutic effects on
osteoporosis.
A growing body of evidence indicates that the potential of gelatin or
hydrolyzed collagen intake are beneficial to bone health. From a cel-
lular perspective, osteoporosis occurs due to an imbalance of bone
metabolism mediated by osteoblast (OB) and osteoclast (OC) cells lo-
cated in the lacuna of the bone matrix (Wang, Jiang, Pan, & Sun, 2016).
This imbalance may be due to hypoactivity of bone forming osteoblasts
or hyperactivity of bone resorbing osteoclasts, resulting in bone re-
sorption exceeding bone formation, ultimately leading to osteopenia
and degeneration of bone microarchitecture (Seeman, 2008). Guiller-
minet et al. demonstrated that dietary hydrolyzed collagen increases
osteoblast (OB) activity, which acts on bone remodeling (Guillerminet
et al., 2010). Nomura et al. demonstrated the shark skin gelatin im-
proved type I collagen and bone mineral density in the femur of ovar-
iectomized rats (Nomura, Oohashi, Watanabe, & Kasugai, 2005).
ganic substance in bone after consuming hydrolyzed collagen for a long
time (Watanabe-Kamiyama et al., 2010). Guillerminet et al. also de-
monstrated that the hydrolyzed collagen was effective for the bone
mineral density and bone strength of ovariectomized mice
(Guillerminet et al., 2010).
Collagen peptides are products of collagen or gelatin hydrolysis, and
their molecular weights are generally less than 10,000 Da. Loach
(Misgurnus anguillicaudatus) is a freshwater fish in the loach family
Cobitidae and widely distributed and bred in China. Loach tastes deli-
cious, contains high protein content, so it is a very popular for East
Asians, such as Chinese, Japanese and South Korean. We have reported
that the body's antioxidant capacities were improved after long-term
consumption of loach (Liu et al., 2018). In some products in China, such
as the loach paste, the black skin is removed and discarded as waste.
Therefore, we made further study on the utilization of loach skin, such
as preparation of collagen and collagen peptides (Liu et al., 2018;
Wang, Pei, Liu, & Zhou, 2018), and planed to study the use of edible
collagen peptide to relieve osteoporosis.
Some researchers speculated that taking antioxidants could alleviate
osteoporosis. Desai et al believed that activated OC generated reactive
oxygen species (ROS) and led to bone loss in osteoporosis (Desai,
Babaria, Nakarani, Shah, & Paranjape, 2007). Satpathy et al reported an
antioxidant enriched fraction (AEF) from whole plant of Hygrophila
spinosa for management of menopausal complications like osteoporosis

⁎
Corresponding author at: State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu, People’s Republic of China.
E-mail address: liuhaiying@jiangnan.edu.cn (H. Liu).

https://doi.org/10.1016/j.jff.2020.104228
Received 29 November 2019; Received in revised form 8 September 2020; Accepted 26 September 2020
1756-4646/ © 2020 Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/BY-NC-ND/4.0/).
H. Liu, et al.
and cancer (Satpathy, Patra, Hussain, & Ahirwar, 2018). There was a
report that the selenium combined with vitamin E and vitamin C re-
stores structural alterations of bare-bones in heparin - induced osteo-
porosis (Belma, Belgin, & Ertan, 2003). In addition, Selenium can reg-
ulate cellular processes by Se-dependent antioxidant enzyme which can
eliminate ROS (Lei, Cheng, & McClung, 2007). Manolagas (2010) be-
lieved that the osteoporosis was caused by deficiency in sodium which
increased in ROS levels. Therefore, selenium was used to assist in im-
proving the effect of collagen peptide on relieving osteoporosis in this
study.
Osteoporosis models have been used in many studies to evaluate the
influence of some substances on bone loss in animals. The rats and mice
with ovariectomized model is a widely used osteoporosis model, but it
is also associated with time-consuming and high mortality. Retinoic
acid-induced osteoporosis is one of the most common causes of sec-
ondary osteoporosis. Large doses of retinoic acid could decrease bone
mineral density in 1–3 weeks, which has been frequently used to pre-
pared acute osteoporosis animal model for its easy operation and high
successful rate (Wang et al., 2019; Wu, Huang, & Wang, 1996). It's
important to note that the mechanism of osteoporotic model prepared
by retinoic acid is distinctly different from that by castration. The
mechanism of ovariectomized model is to decrease the apoptosis of
osteoclasts and inhibit osteoblasts formation, eventually leads to os-
teoporosis. While retinoic acid stimulates both osteoclasts and osteo-
blasts, leading to a high bone conversion rate, which leads to bone
resorption over bone formation and ultimately to osteoporosis.
The purpose of this study is to analyze the effects of loach skin
collagen peptides (LCP) and the combination of it and selenium in re-
ducing osteoporosis, so as to provide more dietotherapy methods for
alleviation of osteoporosis. In this study, osteoporosis was induced in
mice by retinoic acid. In order to develop an effective protocol to treat
retinoic acid-induced osteoporosis, LCP was prepared by loach skin
collagen hydrolysis, then tested with and without sodium selenite.
2. Materials and methods
2.1. Preparation of LCP
Loach skin collagen (laboratory made, average molecular weight of
about 357 kDa) (Wang et al., 2018) was hydrolyzed by alkaline pro-
tease (enzyme activity 146,000 U/g, Novoxin (China) Biotechnology
Co., Ltd.). The specific enzymatic conditions were: loach skin collagen-
to-water ratio 1:10 (w/v), pH 9, temperature 52.5 °C, enzyme loading
6,500 U/g, hydrolysis time 2 h. The LCP was obtained by spray drying
the enzymatic hydrolysate. The drying process was determined by pre-
test with an inlet air temperature of 180 ± 10 °C and an outlet air
temperature of 85 ± 10 °C.
2.2. The basic properties of LCP
2.2.1. Analysis of amino acid composition of LCP
LCP (100 mg) was dissolved in 8 mL of 6 mol/L HCl, and the mix-
ture was evacuated with nitrogen, vacuum-sealed, and hydrolyzed at
110 °C for 22 h. The amino acids in the hydrolysate were analyzed using
an auto-analyzer (Agilent1100, Agilent technologies co., Ltd, USA).
2.2.2. Analysis of LCP molecular weight
The relative molecular weight distribution of LCP was determined
using a Waters 600 high performance liquid chromatograph (Waters
Corporation, Milford, United States) equipped with a column of TSK gel
2,000 SWXL 300 mm × 7.8 nm. Gly-Gly-Gly (Mr 189), Gly-Gly-Arg-Tyr
(Mr 451), bacitracin (Mr 1450), aprotinin (Mr 6500) and cytochrome C
(Mr 12500) were used as molecular weight standards (Sigma-Aldrich
Chemical Co., Ltd. St. Louis, MO. USA).
2
Journal of Functional Foods 75 (2020) 104228
2.3. Design of animal experiments
ICR mice, 9–11-week-old weighing about 38.14 ± 1.20 g were
purchased from the Zhaoyan New Drug Research Center Co., Ltd
(Suzhou, China) (Animal certificate number: SCXK (SU) 2013-0003).
Animal experiments were carried out in the animal experiment center
of Jiangnan University (Wuxi, China). All the animal experiments in-
volved had been reviewed and approved by the ethics committee of
experimental animals of Jiangnan University (Ethical review number:
JN. No 20180930C0500110). All animal experiments were carried out
complying with the ARRIVE guidelines, the U.K. Animals (Scientific
Procedures) Act, 1986 and EU Directive 2010/63/EU for animal ex-
periments.
The mice were acclimated for 2 weeks at 22–25 °C. The mice were
randomly divided into six groups (n = 15): control group (C), model
group (M, osteoporosis mouse model was established by retinoic acid),
low dose LCP group (LLCP, model + 1 g/kg/d LCP), high dose LCP
group (HLCP, model + 2 g/kg/d LCP), LCP and low dose sodium se-
lenite group (HLCP + LSe, model + 2 g/kg/d LCP + 0.1 mg/kg/d
sodium selenite), and LCP and high dose sodium selenite group
(HLCP + HSe, model + 2 g/kg/d LCP + 0.2 mg/kg/d sodium sele-
nite). Pure selenium contents in the HLCP + LSe and HLCP + HSe
groups were 46 μg/kg/d and 92 μg/kg/d respectively. There were fif-
teen mice in a group, each mouse was given free access to water and
food. The experimental groups were continuously intragastrically ad-
ministered with 70 mg/kg/d retinoic acid suspension for 2 weeks
(Wang et al., 2019), while the control group were given the same vo-
lume of normal saline. At the same time, experimental groups ad-
ministered with retinoic acid suspension were intragastrically ad-
ministered with LCP or the combination of LCP and sodium selenite
daily, while the control group were given the same volume of normal
saline.
After intragastric administration of LCP and sodium selenite for
6 weeks, the experimental animals were fasted for 18 h with access to
water. The blood was collected from eyeballs under anesthesia and then
the mice were sacrificed. The blood was centrifuged at 3,000 rpm for
10 min, and the supernatant was collected and stored at −60 °C. The
mice bilateral femur and right tibia were collected on an ice bath, and
excess muscle and mucosal tissues attached on the surface were re-
moved. The right tibia was fixed in formalin solution for micro-CT
analysis. The right femur was divided into two parts: the proximal end
was stored at −60 °C for subsequent RNA extraction, while the distal
end was fixed in formalin for sectioning. The left femur was used for
analysis of mouse bone properties.
2.4. Analysis of serum bone metabolism markers in mice
The activity of bone alkaline phosphatase and tartrate-resistant acid
phosphatase in mouse serum, blood calcium and blood phosphorus le-
vels in the mice were measured using a kit (Nanjing Jianchen
Bioengineering Co., Ltd., China).
2.5. Analysis of mouse bone properties
2.5.1. Analysis of bone strength
The left femurs of mice were taken out and immersed in precooled
sterile saline for 4 h at 4 °C. After being dried with filter paper, the bone
diameter and bone length were measured with a vernier caliper (the
midpoint of the bone was the bone diameter, the distance from the
proximal end of the femur to the distal end of the femur was the length
of the femur). The three-point bending test was performed on the fe-
murs using a TA-XT2i texture analyzer (Stable Microsystems,
Haslernere, UK). The femur was fixed on the fixture and the distance
between two end supports was 2 mm, then the probe descended at a
speed of 2 mm/s until the probe was lowered to break the femur. The
maximum load value of the femur obtained at this time was the bone
H. Liu, et al. Journal of Functional Foods 75 (2020) 104228

strength. Table 1
Sequences of primers in quantitative real-time PCR.
2.5.2. Analysis of bone mineral contents Gene name Bidirectional primer sequences Product length
The fractured femurs were collected and the ash contents in the (5́–3́) (bp)
bone were measured by the muffle furnace method (González-Reimers
Wnt10b Primer F 5′ CTCCACTACAGCCCAGAAC 3′ 122
et al., 2011). Primer R 5′ CTCCCAAGAGCCTGACAAG 3′
β-catenin Primer F 5′ TCACGCAAGAGCAAGTAG 3′ 149
2.5.3. Determination of calcium contents in the femurs Primer R 5′ CTGGACATTAGTGGGATGAG 3′
All the femur ashes were transferred to a small beaker and con- Osterix Primer F 5′ TGCCTACTTACCCATCTGAC 3′ 134
Primer R 5′ TTGCCCACTATTGCCAAC 3′
centrated hydrochloric acid was added until the samples were com- Runx2 Primer F 5′ TTTGCCCTCATCCTTCAC 3′ 150
pletely dissolved, then distilled water was slowly added up to 100 mL. Primer R 5′ GCTTCTGCTACCACTCTAAC 3′
The bone calcium contents were measured by EDTA titration RANKL Primer F 5′ ATGAAACTCACAGCCCTCTC 3′ 151
(Gerstenfeld, Feng, Gotoh, & Glimcher, 1994). Primer R 5′ CATCGGAATACCTCTCCCAATC 3′
OPG Primer F 5′ AGGGCATACTTCCTGTTG 3′ 180
Calcium content in the sample was calculated using the equation
Primer R 5′ TTCCTGGGTTGTCCATTC 3′
below: TRAF6 Primer F 5′ CTTGCTTTGCGTCCGTGCGATG 3′ 192
Primer R 5′ CAGGGTCCGAATGGTCCGTTTG
Calcium content in femur (mg/g dry weight) 3′
C × VEDTA × MCa × 100 NF-κB Primer F 5′ CCAGCAGGGAAGCAAATC 3′ 158
= EDTA Primer R 5′ ACACGGGCATAGAGTCAG 3′
25 × mDW
NFATC1 Primer F 5′ CCGTCCAAGTCAGTTTCTATG 3′ 218
Primer R 5′ TGTGAAGAGGCTACAGGTATG 3′
GAPDH Primer F 5′ ATCACTGCCACCCAGAAG 3′ 191
2.5.4. Morphological observation of mouse bone tissue
Primer R 5′ TCCACGACGGACACATTG 3′
The distal ends of the fixed right femurs of mice were decalcified
with decalcifying solution (EDTA aqueous solution, 186 g/l, pH8.0).
After dehydration, paraffin embedding, sectioning and HE staining, the USA). The differences between groups were analyzed by Duncan’s test.
histopathological changes of mouse bone tissues were observed and Statistical significance was p < 0.05.
recorded under a light microscope (Ji et al., 2019).
3. Results and discussion
2.5.5. Observation of microarchitecture of mouse bone tissue
The proximal end of the mice right tibias were fixed in neutral 3.1. Basic properties of LCP
formaldehyde and stored in 75% ethanol after water rinsing. The
samples were scanned using a micro-CT scanner (LaTheta LCT200, The amino acid composition of LCP was determined. The contents of
Hitachi-Aloka, Tokyo, Japan). The scanning conditions are as follows: glycine, proline and hydroxyproline of LCP were 22.66 g/100 g,
voltage 70 kV, current 114 uA, scanning resolution 10 um.1 mm under 13.49 g/100 g, 8.47 g/100 g, respectively, which were consistent with
the epiphyseal line was selected as the area of interest. Three-dimen- the typical characteristics of type I collagen (Nalinanon, Benjakul,
sional network structure of trabecular bone in the target area were Kishimura, & Osako, 2011). Because LCP is prepared directly by hy-
measured and analyzed using the LaTheta software. drolyzing collagen, its amino acid composition was the same as that of
collagen which has been discussed in our previous study (Wang et al.,
2.6. RT-PCR of mouse bone metabolism related genes 2018). LCP was composed of peptides and free amino acids with dif-
ferent molecular weights, and the relative molecular weight of the main
The mouse femur samples were homogenized in an ice bath. RNA components (85.50%) were less than 1000 (Table 2).
was extracted from the homogenate sequentially with chloroform and
isopropanol, then the mixture were centrifuged at 12,000 r/min for
3.2. Effects of LCP on bone metabolism in mice
15 min at 4 °C. The precipitates were repeatedly washed with pre-
cooled 75% ethanol and then dissolved to obtain total RNA solutions.
3.2.1. Effects of LCP on serum bone metabolism markers in mice
The RNA concentration and quality were evaluated with spectro-
There is a dynamic balance maintained between serum calcium,
photometry and agarose-gel electrophoresis. The total RNA (3.0 μg)
phosphorus levels, and bone calcium and phosphorus levels. Serum
from each sample was reverse transcribed to cDNA with the RevertAid
calcium and phosphorus levels can be indicative markers of bone me-
First Strand cDNA Synthesis Kit (TaKaRa Bio Inc., Tokyo, Japan), ac-
tabolism (Laurent et al., 2011). In the present study, the serum calcium
cording to the manufacturer’s instructions.
and phosphorus levels in the mice of each group are shown in Fig. 1A
Real-time quantitative PCR was performed with SYBR Premix Ex
and 1B. The serum calcium and phosphorus levels in the model group
Taq™ (TaKaRa Bio Inc., Tokyo, Japan) in a QuantStudio™ 6 Flex Real-
were increased significantly relative to controls (denoted control
time PCR System (Applied Biosystems, Foster City, CA, USA). The PCR
group).
cycling program was as follows: pre-denaturation at 95 °C for 10 min,
The serum calcium levels in other four groups were significantly
followed by 40 cycles of denaturation at 95 °C for 15 s, annealing at
lower than those in the model group. Similar trends are observable in
60 °C for 1 m, and extension at 72 °C for 30 s. GAPDH was used as an
internal reference, and the relative expression level of target gene was
Table 2
expressed by the ratio of target gene expression level to GAPDH ex- The relative molecular weight distribution of loach skin collagen peptides.
pression level. Primers for Wnt10b, β-catenin, Osterix, Runx2, RANKL,
Peak order Molecular weight (Da) Peak area (%)
OPG, TRAF6, NF-kB, and NFATC1 are shown in Table 1. Primers were
designed and synthesized by Shanghai Univ-bio Co., Shanghai, Ltd. 1 > 3000 1.27
2 2000–3000 2.12
2.7. Data processing 3 1000–2000 11.12
4 500–1000 30.20
5 180–500 45.27
The data are presented as means ± standard deviations (SD) and 6 < 180 10.03
were analyzed with GraphPad Prism 6.0 (GraphPad Software, Inc., CA,

3
H. Liu, et al. Journal of Functional Foods 75 (2020) 104228

A B
3 b 6 b ab

S e r u m p h o s p h o r u s ( m m o l/L )
S e r u m c a lc iu m ( m m o l/L )
a ab
a a a a a
a
2 4 a

1 2

0 0
C P P e e

e
P

e
P
M

C
S

M
C C S

S
C

C
L L L H

H
L

L
L + +

+
H

+
L
P

H
P

P
C C

C
L L

L
H H

H
C D
20 b
30
b
ab
ab ab
ab
ab
15 ab
ab

B A L P ( n g /m L )
20
T R A P (U /L )

a ab

10 a

10
5

0 0
e

e
P

P
C

e
M

P
C

M
S

S
C

S
C

C
L

L
L

H
L

L
+

+
L

+
L

H
P

P
C

C
L

L
H

H
Fig. 1. Effects of LCP and the combination of LCP and sodium selenite on serum bone metabolism markers in mice. The different letters indicate significant difference
among treatments (P < 0.05). C, control group; M, osteoporosis model group; LLCP, model + Low dose LCP; HLCP, model + high dose LCP; HLCP + LSe,
model + high dose LCP + low dose sodium selenite; HLCP + HSe, model + high dose LCP + high dose sodium selenite.

serum phosphorus levels. In HLCP and HLCP + HSe groups, the serum Table 3
phosphorus levels were lower than those in the model group obviously. Effects of LCP and the combination of LCP and sodium selenite on the femur
These data indicated that intragastric administration of LCP, or the parameters of mice.
combination of LCP and sodium selenite helped maintain the calcium Groups Length of Diameter of Calcium Strength of
content of bones. In addition, HLCP and HLCP + HSe helped maintain femur (cm) femur (cm) content of femur (g)
the balance between serum calcium and phosphorus, thus alleviating femur (mg/
osteoporosis. g)

Tartrate-resistant acid phosphatase (TRAP) is a marker of bone re- C 1.73 ± 0.05 0.16 ± 0.06 225 ± 14b 3054 ± 251b
sorption and osteoclast activity (Tauchert et al., 2009). In this way, M 1.71 ± 0.05 0.15 ± 0.01 191 ± 12a 2160 ± 110a
bone metabolic status can be reflected by analyzing serum TRAP ac- LLCP 1.67 ± 0.05 0.15 ± 0.03 221 ± 6b 2524 ± 248ab
tivity. Bone alkaline phosphatase (BALP) is another marker of osteo- HLCP 1.66 ± 0.05 0.15 ± 0.01 228 ± 6b 2805 ± 192b
HLCP + LSe 1.66 ± 0.03 0.15 ± 0.04 227 ± 15b 2800 ± 325b
blast activity (Bergman et al., 2017). Studies (Sardiwal, Gardham, &
HLCP + HSe 1.71 ± 0.06 0.15 ± 0.01 233 ± 6b 2983 ± 360b
Coleman, 2012; Solberg, Stang, Brorson, Andersson, & Reinholt, 2015)
have found that when osteoporosis occurs, osteoblasts will become Note: The different letter in same column means significant difference
active, resulting in an increase of bone resorption, then TRAP and BALP (P < 0.05).
levels will also be correspondingly increased. As shown in Fig. 1C and
Fig. 1D, the serum TRAP and BALP activities in the model group were differences in length and diameter of mouse femur bones were not
significantly increased relative to the control group. The result is con- significantly different between groups. However, bone calcium and
sistent with the study (Zhang et al., 2010), which has shown that the bone strength of mouse femurs in the model group were significantly
addition of all-trans-retinoic acid (ATRA) to mesenchymal stem cells led reduced relative to the control group. The bone calcium content and
to a significant increase in BALP activity. The serum TRAP and BALP bone strength were significantly improved in the groups that were in-
activities in the groups that were intragastrically administered LCP, or tragastrically administered LCP, or the combination of LCP and sodium
the combination of LCP and sodium selenite decreased compared to the selenite, were not significantly different from those in the model group.
model group, but were not significantly.
3.2.3. Effects of LCP on the histomorphology of mouse bone tissue
3.2.2. Effects of LCP on mouse bone properties Osteoporosis patients undergo not only bone loss, but also dete-
The bone parameters of the mice are shown in Table 3. The rioration of the microarchitecture of trabecular bone (Cınar et al.,

4
H. Liu, et al.
Fig. 2. HE staining of mouse femoral tissue sections. A, group C; B, group M; C, group LLCP; D, HLCP; E, group HLCP + LSe; F, group HLCP + HSe. Trabecula is
pointed out by arrow.
2016). The latter mainly affects the bone fragility of patients with os-
teoporosis and increases the risk of fracture (Buitendijk et al., 2019).
Trabecular bone is one of the important parameters to assess bone
quality (Chuang, Chuang, Koo, & Wang, 2017). The bone quality can be
evaluated by observing the microarchitecture of trabecular bone using
sections of the mouse femoral tissues.
HE staining of mouse femoral tissue sections are shown in Fig. 2.
The femoral distal end structure in the control group was normal, and
the trabecular bone structure was dense, uniform, and had good con-
tinuity. In the model group, the trabecular bones in the distal femur
were cracked and thinned moderately. In addition, the intertrabecular
spaces were widened, and the trabecular bone was not seen in some
5
Journal of Functional Foods 75 (2020) 104228
areas. In the LLCP group, the trabecular bones in the distal femur were
cracked and thinned mildly or moderately, and the intertrabecular
spaces were widened. While in the HLCP group, HLCP + LSe group,
and HLCP + HSe group, the trabecular bones in the distal femur were
cracked and thinned mildly, and the intertrabecular spaces were wi-
dened.
These results indicated that mouse models of osteoporosis were
successfully established by retinoic acid, and the intervention of LCP
improved osteoporosis. In this study, high doses had a slightly better
effect than low doses. The intervention of the combination of LCP and
sodium selenite also improved osteoporosis.
H. Liu, et al. Journal of Functional Foods 75 (2020) 104228

Fig. 3. Compared with the control group, the trabecular bone loss in the
A B
model group was severe, and the trabecular network was severely
loosened. In the LLCP group and the HLCP group, the trabecular bone
loss was moderate, and the trabecular network was moderately loo-
sened. In the HLCP + LSe group, the trabecular bone loss in the distal
femur was mild, and the trabecular network was mildly loosened. In the
HLCP + HSe group, the trabecular bone loss was mild, and the trabe-
cular network was mildly loosened.
The results of quantitative analysis of mouse bones trabecular se-
paration are shown in Fig. 3 G. Compared with mice in the control
group, mice in the model group underwent severe bone micro-
architecture loss. This was primarily presented as a significant increase
D in trabecular separation. However, the trabecular separation was de-
C
creased after intragastric administration of LCP, combination of LCP
and sodium selenite, especially the combination of HLCP and high dose
of sodium selenite. These results indicated that mouse models of os-
teoporosis were successfully established by retinoic acid, the interven-
tion of the combination of HLCP and high dose of sodium selenite,
improved the mouse bone microarchitecture.

3.2.5. Effects of LCP on mRNA expression of key genes in Wnt/β-catenin

signaling pathway
Wnt/β-catenin signaling pathway regulates osteoblast proliferation,
differentiation and apoptosis, and participates in bone cell metabolism
E F through WNT protein (Clevers & Nusse, 2012). Wnt eventually causes
inhibition of the phosphorylation process of β - catenin, so that β -
catenin is enriched in the cell and translocated into the nucleus. In the
nucleus, β - catenin binds to LEF1/TCF to initiate transcription and
expression of target genes, Runx2 and Osterix, thereby regulating the
proliferation and differentiation of osteoblasts (Sato, Meijer,
Skaltsounis, Greengard, & Brivanlou, 2004).
The mechanism of osteoporotic model prepared by retinoic acid is
different from that by castration. The mechanism of castration to pre-
pare osteoporosis model is to decrease the apoptosis of osteoclasts and
inhibit osteoblasts formation, which can be manifested as decreased
mRNA expression levels of such as Wnt, β-catenin, Runx2, and Oxsterix
G in Wnt/β-catenin signaling pathway. The mechanism of using retinoic
T r a b e c u la r S p e p a r a t io n /S p a c in g ,T b .s p

acid to prepare osteoporosis model is that retinoic acid stimulates both

0 .5 osteoclasts and osteoblasts, leading to a high rate of bone transforma-
d
tion, making bone absorption exceed bone formation, and finally
bc cd leading to osteoporosis. Zhang et al. (2016) has found that retinoic acid
0 .4 bc
can activate the Wnt/β-catenin Signaling pathway, promote activity of
ab
a β-catenin in mesenchymal stem cells.
0 .3
As shown in Fig. 4, the mRNA expression levels of Wnt10, β - ca-
tenin, Runx2, and Oxsterix in the model group were significantly in-
0 .2
creased relative to the control group. The mRNA expression levels of
Wnt10 and β - catenin in the groups that were intragastrically ad-
0 .1
ministered LCP, or the combination of LCP and sodium selenite, were
greatly decreased. However, only the combination of LCP and sodium
0 .0 selenite groups had obvious effects on Runx2, and Oxsterix. These re-
sults indicated that retinoic acid promoted the proliferation and dif-
P

e
C

e
M

S
C

ferentiation of mouse osteoblasts at the molecular level, and ac-

L

H
L

+
L

celerated bone formation, leading to osteoporosis due to compensatory

C

C
L

L
H

hyperplasia of mouse bone. This was consistent with biochemical in-

Fig. 3. The three-dimensional reconstructions of mouse tibia. A, group C; B, dicators. Intragastric administration of LCP or the combination of LCP
group M; C, group LLCP; D, HLCP; E, group HLCP + LSe; F, group HLCP + HSe. and sodium selenite effectively attenuated high bone turnover rate by
regulating Wnt10, β - catenin expression. Furthermore, the combina-
3.2.4. Effects of LCP on bone microarchitecture of mice tion of LCP and sodium selenite had a more obvious effect.
Micro-computed tomography (Micro-CT) is commonly used to as-
sess bone quality and to evaluate the outcome of experimental therapies 3.2.6. Effects of LCP on mRNA expression of key genes in OPG/RANKL/
in animal models of bone diseases. Micro-CT can provide high-quality NF-kB signaling pathway
2D and 3D reconstructions and is a very accurate and effective tech- The OPG-RANK-RANKL signaling pathway primarily governs cou-
nique for bone microstructure evaluation (Leszczyński, Skrzat, pling between osteoblast and osteoclast activity (Růžičková, 2012).
Kozerska, Wróbel, & Walocha, 2014). Osteoblasts secrete both the receptor activator of nuclear factor kB li-
The three-dimensional reconstructions of mouse bones are shown in gand (RANKL) and osteoprotegerin (OPG). RANKL binds to the receptor
activator of nuclear factor kB (RANK) located on osteoclasts and

6
H. Liu, et al. Journal of Functional Foods 75 (2020) 104228

A B
0.15 b 0.08
b

B - c a te n in /G A P D H
ab 0.06
W n t1 0 b /G A P D H ab
0.10 a ab
a a a
a
0.04 a
a

0.05
0.02

0.00 0.00

e
P

P
C

M
e
P

e
P
C

S
C

C
S

S
C

H
L

L
H
L

+
+

L
+

H
L

P
P

P
P

C
C

C
C
L

L
L

L
H

H
H

H
C D
b
0.4 0.3
ab
b ab ab
a ab
ab
ab

R U N X 2 /G A P D H
0.3
O s te r ix /G A P D H

0.2
a a a

0.2

0.1
0.1

0.0 0.0

e
P

P
C

M
e
P

e
P
C

S
C

C
S

S
C

H
L

L
L

+
+

+
+

L
L

H
H

P
P

P
P
C

C
C

C
L

L
L

L
H

H
Fig. 4. mRNA expression of the key factors of Wnt/β-catenin pathway in mice. The different letters indicate significant difference among treatments (P < 0.05). C,
control group; M, osteoporosis model group; LLCP, model + Low dose LCP; HLCP, model + high dose LCP; HLCP + LSe, model + high dose LCP + low dose sodium
selenite; HLCP + HSe, model + high dose LCP + high dose sodium selenite.

activates the TNF receptor associated factor (TRAF6). This leads to the
enrichment of nuclear factor (NF-kB) and translocation from cytoplasm
to nucleus (Tecalco-Cruz, 2018). When osteoporosis occurs, mRNA
expression levels of RANKL is upregulated in bone marrow cells, re-
sulting in reduced osteocalcin synthesis in bone cells and accelerated
bone resorption (Parfitt, Drezner, Glorieux, & Kanis, 2010). OPG in-
hibits RANKL-induced osteoclast differentiation and maturation by
competing with RANK to bind to RANKL, so the ratio of RANKL/OPG
plays a decisive role in the proliferation and differentiation of osteo-
clasts. Researchers have confirmed that the OPG-RANK-RANKL sig-
naling pathway and the Wnt/β-catenin signaling pathways are inter-
related (Glass et al., 2005). For example, β-catenin regulates the
expression of OPG in osteoblasts and controls bone resorption in-
directly. Under the regulation of these major signaling pathways, os-
teoblasts and osteoclasts together determine bone volume and bone
remodeling.
As shown in Fig. 5, compared with the control group, the mRNA
expression levels of RNAKL in the model group were significantly in-
creased. However, the mRNA expression levels of OPG were sig-
nificantly decreased. Thus, the ratios of RANKL/OPG were significantly
increased. The ratios of RANKL/OPG in the groups that were in-
tragastrically administered LCP, or the combination of LCP and sodium
selenite, were significantly lower than those in the model group.
NF-kappaB (NF-kB) proteins is composed of 5 members of structu-
rally-related transcription factors that are involved in the control of a
large number of normal body function. Complete expression of NF-kB1
and NF-B2 is essential for osteoclast differentiation (Franzoso, Carlson,
Xing, & Poljak, 1997). Nuclear factor-activated T cell 1 (NFATc1) is
another important transcription factor. It is induced by RANKL and
activated, which eventually leads to osteoclast formation and promotes
bone matrix degradation. When RANKL binds to its receptor RNAK, it
can recruit TRAF6 and other transfer molecules to activate signal
transduction pathways, release NF- kB, and transfer it to the nucleus,
activate key transcription factors such as NFATc1, and finally promote
osteoclast formation (Boyle, Simonet, & Lacey, 2003).
Compared with the control group, the mRNA expression levels of
TRAF6, NF-kB and NFATc1 in the OPG/RANKL/NF-kB signaling
pathway in the model group were significantly increased. Conversely,
the mRNA expression levels of NF- kB in the groups that were in-
tragastrically administered LCP, or the combination of LCP and sodium
selenite, were greatly reduced relative to the model group. Similarly, as
shown in Fig. 5 D and F, LCP and the combination of LCP and sodium
selenite can significantly reduce the mRNA expression level of TRAF6,
but only the HLCP + LSe and HLCP + HSe can reduce the mRNA ex-
pression of NFATc1 obviously.
The experimental results indicated that retinoic acid strengthened
the differentiation and maturation of osteoclasts at the molecular level,
thus accelerating bone resorption, while intragastric administration of
LCP, or the combination of LCP and sodium selenite, inhibited the ex-
pression of key genes in OPG/RANKL/NF- kB pathway at the molecular
level, thereby inhibiting the differentiation and maturation of osteo-
clasts.
4. Conclusion
Loach skin collagen peptide (LCP) was prepared in this experi-
mental study. Animal experiments showed that LCP, or the combination
of LCP and sodium selenite, inhibited the expression of related proteins

7
H. Liu, et al. Journal of Functional Foods 75 (2020) 104228

A B
0.5 0.15
ab ab
b b b
0.4 ab
ab
R A N K L /G A P D H
ab
a

O P G /G A P D H
0.10
0.3 a
a
a

0.2
0.05

0.1

0.0 0.00
C P P e e
M S S

e
P

P
C
C C

S
L

C
L L H

H
+ +

L
L H

+
P

+
P

P
C C

C
L L

L
H H

H
C b D
8 0.15

6 b

T R A F 6 /G A P D H
R A N K L /O P G

a 0.10
a a
a a a
4 a a a
a
0.05
2

0 0.00
e

e
P

P
C

e
P

e
S

P
C

M
C

S
C

C
L

H
L

H
L

L
+

+
L

+
L
P

H
P

P
C

C
L

L
H

H
E F
c
0.10 b 0.25
abc bc
ab
0.08 a 0.20
N F A T C 1 /G A P D H

a
N F K B /G A P D H

a a a
0.06 a 0.15 a

0.04 0.10

0.02 0.05

0.00 0.00
C P P e e
M S S
e

e
P

P
C

C C
M

L
C

L L H
L

+ +
L

L H
+

P
+

P
L

C C
C

L L
L

H H
H

Fig. 5. mRNA expression of the key factors of OPG/RANKL/NF-kB pathway in mice. The different letters indicate significant difference among treatments
(P < 0.05). C, control group; M, osteoporosis model group; LLCP, model + Low dose LCP; HLCP, model + high dose LCP; HLCP + LSe, model + high dose
LCP + low dose sodium selenite; HLCP + HSe, model + high dose LCP + high dose sodium selenite.

and genes through the Wnt/β-catenin pathway, thus reducing com- center of Jiangnan University (Wuxi, China). All the animal experi-
pensatory hyperplasia of osteoblasts. LCP, or the combination of LCP ments involved had been reviewed and approved by the ethics com-
and sodium selenite, also inhibited hyperactivity of bone resorbing mittee of experimental animals of Jiangnan University (Ethical review
osteoclasts through OPG/RANKL/NF-kB pathway, improved bone mi- number: JN. No 20180930C0500110). All animal experiments were
croarchitecture, increased the number and quality of mouse trabecular carried out complying with the ARRIVE guidelines, the U.K. Animals
bone, and increased bone strength, thereby improving retinoic acid- (Scientific Procedures) Act, 1986 and EU Directive 2010/63/EU for
induced osteoporosis in mice. In conclusion, proper consumption of animal experiments.
LCP, or the combination of LCP and sodium selenite can alleviate os-
teoporosis. This study seems to provide some new ideas for the devel-
opment of bone health food. Funding

This study was financially supported by Primary Research &

5. Ethics statement Developement Plan of Jiangsu Province, China (grant number
BE2019331).
Animal experiments were carried out in the animal experiment

8
H. Liu, et al.
CRediT authorship contribution statement
Haiying Liu: Conceptualization, Methodology, Project administra-
tion, Resources, Writing - review & editing, Funding acquisition. Lu
Huang: Software, Writing - original draft. Jing Wang: Data curation,
Software, Formal analysis.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
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