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Undenatured Type II Collagen Relieves Bone Impairment through Improving Inflammation and Oxidative Stress in Ageing db/db Mice
Ageing-related bone impairment due to exposure to hyperglycemic environment is scarcely researched. The aim was to confirm the improvement effects of undenatured type II collagen (UC II) on bone impairment in ageing db/db mice, and the ageing model was established by normal feeding for 48-week-old. Then, the ageing db/db mice were randomly assigned to UC II intervention, the ageing model, and the chondroitin sulfate + glucosamine hydrochloride control groups.
Article
Undenatured Type II Collagen Relieves Bone Impairment
through Improving Inflammation and Oxidative Stress in
Ageing db/db Mice
Rui Fan, Yuntao Hao, Xinran Liu, Jiawei Kang, Jiani Hu , Ruixue Mao, Rui Liu, Na Zhu, Meihong Xu
and Yong Li *
Abstract: Ageing-related bone impairment due to exposure to hyperglycemic environment is scarcely
researched. The aim was to confirm the improvement effects of undenatured type II collagen
(UC II) on bone impairment in ageing db/db mice, and the ageing model was established by
normal feeding for 48-week-old. Then, the ageing db/db mice were randomly assigned to UC II
intervention, the ageing model, and the chondroitin sulfate + glucosamine hydrochloride control
groups. After 12 weeks of treatment, femoral microarchitecture and biomechanical parameters were
observed, biomarkers including bone metabolism, inflammatory cytokines, and oxidative stress were
measured, and the gastrocnemius function and expressions of interleukin (IL) 1β, receptor activator
of nuclear factor (NF)-κB ligand (RANKL), and tartrate-resistant acid phosphatase (TRAP) were
analyzed. The results showed that the mice in the UC II intervention group showed significantly
superior bone and gastrocnemius properties than those in the ageing model group, including bone
mineral density (287.65 ± 72.77 vs. 186.97 ± 32.2 mg/cm3), gastrocnemius index (0.46 ± 0.07 vs.
0.18 ± 0.01%), muscle fiber diameter (0.0415 ± 0.005 vs. 0.0330 ± 0.002 mm), and cross-sectional area
(0.0011 ± 0.00007 vs. 0.00038 ± 0.00004 mm2). The UC II intervention elevated bone mineralization
and formation and decreased bone resorption, inflammatory cytokines, and the oxidative stress. In
addition, lower protein expression of IL-1β, RANKL, and TRAP in the UC II intervention group was
observed. These findings suggested that UC II improved bones impaired by T2DM during ageing,
and the likely mechanism was partly due to inhibition of inflammation and oxidative stress.
Keywords: bone impairment; db/db mice; undenatured type II collagen (UC II); inflammation;
oxidative stress; T2DM
1. Introduction
Diabetes mellitus (DM), caused by dysfunctional glucose metabolism, is a chronic
endocrine disorder. Type 2 DM (T2DM) is a multifactorial disease, the central pathome-
chanisms of which are hyperinsulinemia, insulin resistance, and chronic inflammation,
and progressive secondary β-cell failure occurs at the later stages [1,2]. With the in-
creasing duration of DM, patients suffer from the complications including retinopathy,
nephropathy, neuropathy, and vascular disease. DM accelerates material and microstruc-
tural bone deficits, finally leading to some bone diseases, including osteoporosis and
osteoarthritis [3–5]. Now, bone damage is known as a complication of DM , which is
associated with significant morbidity, mortality, and reduction in quality of life . Hence,
the development of effective management of promoting early intervention and prevention
of bone degeneration to improve the quality of life among elderly T2DM patients is needed.
There is a close relevance between bone and glucose metabolism . Osteocalcin
is a good example; it is produced by osteoblasts and odontoblasts and also plays an im-
portant role as an endogenous insulin sensitizer . Thus, bones are both influenced by
glucose metabolism and are capable of modulating it . T2DM was reported to impair
bone health by unbalancing a series of processes including bone formation and resorption,
collagen formation and crosslinking due to hyperglycemia, high level of reactive oxygen
species (ROS), inflammation and advanced glycation end products (AGEs) [11–14]. In ad-
dition, insulinopenia and lack of IGF-1 also influenced osteoblasts [15,16]. It is well-known
that skeletal muscles are the main site for insulin-mediated glucose disposal and energy
metabolism. It has been reported that persons with DM have accelerated muscle loss . In
fact, skeletal muscles and bones have a coupled and cross-talk relationship, which shows
the secreting myokines including IGF-1 and acting for monokine communication .
The accumulation of these pathomechanisms ultimately leads to decreased bone quality
in T2DM. Therefore, what we need to improve is the understanding of the factors that
determine bone health in people with T2DM, especially in elderly patients. The current
research revealed the mechanism of influence of UC II on bone loss in ageing db/db mice
in the musculoskeletal system for the first time.
Collagen, the main component of bones and cartilages, possesses an important func-
tion. Type II collagen, the main part of cartilages, is an interconnected network of collagen
and proteoglycans that are crucial in maintaining joint flexibility and resistance to stress
and fractures [18,19]. Several studies have suggested that a diet with a high percentage
of collagen peptides improves bone collagen metabolism and the complications of hyper-
glycemia [20,21]. According to many reports, type II collagen can treat arthritis due to its
anti-inflammatory properties [22,23]. Undenatured type II collagen (UC II), showing intact
biological activity, receiving much attention , was reported to be effective in rheumatoid
arthritis and arthritis [25,26] due to reducing joint inflammation and promoting cartilage
repair [27,28]. The potential mechanism is due to the oral tolerance. When consumed, UC
II is believed to be taken up by Peyer’s patches, where it activates immune cells. When they
recognize type II collagen in joint cartilages, Treg cells secrete anti-inflammatory mediators
(cytokines). This action helps reduce joint inflammation . Unexpectedly, a prior report
found UC II could augment bone mineral density, bone volume, and trabecular number,
decrease trabecular separation, and improve intense bone resorption, which aids in the
maintenance of cancellous bone, although this was a non-significant trend .
On the basis of the role of various cytokines in T2DM coupled with the previous
findings, it is speculated that supplementation with UC II could relieve bone impairment
among T2DM patients. In order to simulate a real situation of bone impairment during
the developing process of T2DM, 48-week-old db/db mice normally fed with a basic
feed were used as the ageing model, a model established for the first time in this study.
We investigated the effects of UC II on bone quality, microstructure, biomechanics, and
metabolism properties in ageing db/db mice and explored the underlying mechanisms
with a comprehensive and systematic perspective of the musculoskeletal system for the
first time. This study could identify possible evidence of nutritional solutions for bone
impairment in the future research on elderly T2DM patients.
2. Results
2.1. General Condition and Disease State
The mice in the NC group exhibited smooth, shiny, and lustrous hair, energy, and fecal
columnar formation, whereas the mice in the AM and YM groups featured weak, coarse,
and dull hair, unresponsiveness, and decreased activity. Compared with the AM and YM
groups, the CG and UC groups showed more activity.
Figure 1 shows the body weight; the weight in the AM group was significantly larger
than in the YM and NC groups (p < 0.05), while the body weight difference between the
AM and intervention (CG and UC) groups showed no statistical significance (p> 0.05).
The gastrocnemius index in the AM group was the lowest, which presented significant
differences between the NC, YM, CG, and UC groups (p < 0.05). The gastrocnemius index
in the UC group was significantly higher than in the NC and CG groups (p < 0.05), which
indicated that UC II resulted in an obvious increase in the gastrocnemius index.
differences between the NC, YM, CG, and UC groups (p < 0.05). The gastrocnemius index
in the UC group was significantly higher than in the NC and CG groups (p < 0.05), which
indicated that UC II resulted in an obvious increase in the gastrocnemius index.
FBG levels were less than 9.0 mmol/L in the CG and UC groups, i.e., lower than those
in the AM and YM groups (>15 mmol/L; p < 0.05), but the levels were slightly larger than
in the NC group (<7.5 mmol/L; p> 0.05). There was no significant difference in FINS be-
tween the different groups. Homeostatic model assessment of insulin resistance (HOMA-
IR) is an index that evaluates the level of insulin resistance. The NC group showed the
lowest HOMA-IR, and the HOMA-IR index of the UC group was significantly lower than
in the AM group (p < 0.05).
Figure 1. The effect of UC II on the body weight, gastrocnemius index, plasma glucose, and insulin level in the five groups.
The data are the means ± SD, (n = 5); a p < 0.05 versus the AM group, b p < 0.05 versus the NC group, c p < 0.05 versus the
YM group, d p < 0.05 versus the CG group.
2.2. Micro-CT Femoral Analysis
Micro-CT imaging was performed to quantitatively measure the bone mass on the
basis of BMD and the most common microarchitectural parameters of cancellous bone
including: BV/TV, Tb.N, Tb.Th, Tb.Sp, which reflect bone microvolume, trabecular bone
number, thickness, and spacing, respectively. Micro-CT analysis results in Figure 2 show
that the effects of UC II on femoral BMD and histomorphometry were significant. BMD
values in the AM and NC groups were relatively small and significantly smaller than
those in the YM and UC groups (p < 0.05). BMD in the UC group was the largest among
the five groups, 2.07, 1.84, and 1.39 times higher compared with those values in the AM,
NC, and CG groups, respectively. BV/TV represents the fraction of a given volume of in-
terest, total volume, or tissue volume that is occupied by bone. The effect of UC on the
BV/TV level was similar to that on BMD, which exhibited a significantly larger level in the
YM, CG, and UC groups than in the AM and NC groups (p < 0.05). For BS/BV, the level
order was as follows: AM group> NC group> YM group> CG group> UC group, with
the BS/BV values in the AM group significantly larger than in other groups (p < 0.05); the
values in the CG and UC groups were significantly smaller than those in the AM and NC
groups (p < 0.05). In addition, the smallest values of Tb.Th and Tb.N were observed in the
AM group, and they significantly differed from those in the CG, YM, and UC groups (p <
Figure 1. The effect of UC II on the body weight, gastrocnemius index, plasma glucose, and insulin level in the five groups.
The data are the means ± SD, (n = 5); a p < 0.05 versus the AM group, b p < 0.05 versus the NC group, c p < 0.05 versus the
YM group, d p < 0.05 versus the CG group.
FBG levels were less than 9.0 mmol/L in the CG and UC groups, i.e., lower than those
in the AM and YM groups (>15 mmol/L; p < 0.05), but the levels were slightly larger than in
the NC group (<7.5 mmol/L; p> 0.05). There was no significant difference in FINS between
the different groups. Homeostatic model assessment of insulin resistance (HOMA-IR) is
an index that evaluates the level of insulin resistance. The NC group showed the lowest
HOMA-IR, and the HOMA-IR index of the UC group was significantly lower than in the
AM group (p < 0.05).
2.2. Micro-CT Femoral Analysis
Micro-CT imaging was performed to quantitatively measure the bone mass on the
basis of BMD and the most common microarchitectural parameters of cancellous bone
including: BV/TV, Tb.N, Tb.Th, Tb.Sp, which reflect bone microvolume, trabecular bone
number, thickness, and spacing, respectively. Micro-CT analysis results in Figure 2 show
that the effects of UC II on femoral BMD and histomorphometry were significant. BMD
values in the AM and NC groups were relatively small and significantly smaller than those
in the YM and UC groups (p < 0.05). BMD in the UC group was the largest among the five
groups, 2.07, 1.84, and 1.39 times higher compared with those values in the AM, NC, and
CG groups, respectively. BV/TV represents the fraction of a given volume of interest, total
volume, or tissue volume that is occupied by bone. The effect of UC on the BV/TV level
was similar to that on BMD, which exhibited a significantly larger level in the YM, CG, and
UC groups than in the AM and NC groups (p < 0.05). For BS/BV, the level order was as
follows: AM group> NC group> YM group> CG group> UC group, with the BS/BV
values in the AM group significantly larger than in other groups (p < 0.05); the values in
the CG and UC groups were significantly smaller than those in the AM and NC groups
(p < 0.05). In addition, the smallest values of Tb.Th and Tb.N were observed in the AM
group, and they significantly differed from those in the CG, YM, and UC groups (p < 0.05).
The largest value of Tb.N was observed in the UC group, which was significantly different
from that in the other groups (p < 0.05), while the AM group showed the largest values of
Tb.Sp, which were significantly larger than those in other groups (p < 0.05).
0.05). The largest value of Tb.N was observed in the UC group, which was significantly
different from that in the other groups (p < 0.05), while the AM group showed the largest
values of Tb.Sp, which were significantly larger than those in other groups (p < 0.05).
Figure 2. The histomorphometry of the femur in the five groups. (A) The images of the changes in bone microarchitecture.
(B) The mineral density and histomorphometry of the femur. The data are expressed as the means ± SD (n = 5 in the AM
group, n = 7 in the YM, CG, NC, and UC groups); a p < 0.05 versus the AM group, b p < 0.05 versus the NC group, c p < 0.05
versus the YM group, d p < 0.05 versus the CG group.
2.3. Dynamic Histomorphometric and Biochemical Markers of Bone Turnover Analysis
Dynamic histomorphometric analyses of representative fluorescence images ob-
tained from the femur are shown in Figure 3A. The green fluorescence in the images is
calcein green, which represents the first mineralization label; the red fluorescence in the
images is alizarin complexone, which represents the second mineralization label. The
width of the gap between the red and the green fluorescence in the images could, to some
extent, represent the bone formation rate. Relatively larger gap widths were observed in
Figure 2. The histomorphometry of the femur in the five groups. (A) The images of the changes in bone microarchitecture.
(B) The mineral density and histomorphometry of the femur. The data are expressed as the means ± SD (n = 5 in the AM
group, n = 7 in the YM, CG, NC, and UC groups); a p < 0.05 versus the AM group, b p < 0.05 versus the NC group, c p < 0.05
versus the YM group, d p < 0.05 versus the CG group.
2.3. Dynamic Histomorphometric and Biochemical Markers of Bone Turnover Analysis
Dynamic histomorphometric analyses of representative fluorescence images obtained
from the femur are shown in Figure 3A. The green fluorescence in the images is calcein
green, which represents the first mineralization label; the red fluorescence in the images
is alizarin complexone, which represents the second mineralization label. The width of
the gap between the red and the green fluorescence in the images could, to some extent,
represent the bone formation rate. Relatively larger gap widths were observed in the UC
group, and relatively smaller ones were, remarkably, observed in the AM and NC groups,
which indicated that UC II treatment led to bone formation, which was consistent with the
results of bone microarchitecture (Figure 3).
the UC group, and relatively smaller ones were, remarkably, observed in the AM and NC
groups, which indicated that UC II treatment led to bone formation, which was consistent
with the results of bone microarchitecture
Bone formation and bone resorption determined bone turnover . In addition, the
serological assessment of bone turnover in the five groups is shown in Figure 3B. AGEs
play an important role in the bone diseases development (such as osteoporosis), especially
among T2DM patients. Their accumulation in the bone alters osteoblasts, inducing en-
hanced osteoclastogenesis and impaired matrix mineralization (downregulation of alka-
line phosphatase and osteocalcin mRNA) . OC, mainly controlling mineralization, and
BALP, representing bone formation, were selected. TRAP, a marker of osteoclast numbers,
was selected to represent bone resorption on the basis of a previous report . As ex-
pected, the serum level of BALP in the UC group was obviously larger than in the AM,
NC, YM, and CG groups (p < 0.05), which was consistent with the histomorphometric
bone analysis. Similarly to the BALP data, the OC serum content in the OM group was
significantly lower than in the UC and CG groups (p < 0.05). Moreover, TRAP serum levels
were the lowest in the UC group (p < 0.05).
2.4. Bone Biomechanical Parameter Analysis
The three-point bending technique was used to assess the mechanical properties
(fracture point, stiffness, and elasticity) of the femur. A constant amount of force was ap-
plied to the midpoint of the femur diaphysis to determine the maximum load that could
Figure 3. Dynamic histomorphometric and biochemical markers of bone turnover analyses. (A) Dynamic histomorphometric
analyses of representative fluorescence images (n = 5); (B) serum levels of biochemical markers of bone turnover (n = 5, 7, 9,
9, and 10 in the AM, CG, UC, NC, and YM groups); a p < 0.05 versus the AM group.
Bone formation and bone resorption determined bone turnover . In addition, the
serological assessment of bone turnover in the five groups is shown in Figure 3B. AGEs
play an important role in the bone diseases development (such as osteoporosis), especially
among T2DM patients. Their accumulation in the bone alters osteoblasts, inducing en-
hanced osteoclastogenesis and impaired matrix mineralization (downregulation of alkaline
phosphatase and osteocalcin mRNA) . OC, mainly controlling mineralization, and
BALP, representing bone formation, were selected. TRAP, a marker of osteoclast numbers,
was selected to represent bone resorption on the basis of a previous report . As expected,
the serum level of BALP in the UC group was obviously larger than in the AM, NC, YM,
and CG groups (p < 0.05), which was consistent with the histomorphometric bone analysis.
Similarly to the BALP data, the OC serum content in the OM group was significantly lower
than in the UC and CG groups (p < 0.05). Moreover, TRAP serum levels were the lowest in
the UC group (p < 0.05).
2.4. Bone Biomechanical Parameter Analysis
The three-point bending technique was used to assess the mechanical properties
(fracture point, stiffness, and elasticity) of the femur. A constant amount of force was
applied to the midpoint of the femur diaphysis to determine the maximum load that could
be placed on the femur until fracture/breakage occurred. Table 1 shows biomechanical
femoral parameters in the five groups. The values of the maximum load in the CG and UC
groups were significantly larger than those in the AM and NC groups (p < 0.05), of which
the value in the UC group was significantly larger than that in the YM group (p < 0.05), and
biomechanical femoral properties, including the energy to ultimate load, Young’s modulus,
stiffness, and breaking energy in the UC group showed significant differences between the
AM, NC, YM, and CG groups (p < 0.05).
Table 1. The bone biomechanical parameters of the femur in the five groups.
Groups Maximum
Load (N)
Energy to Ultimate
Load (J) Young’s Modulus (MPa)
Stiffness
(N/mm)
Breaking Energy
(J/m2)
AM 7.49 ± 1.49 0.0013 ± 0.001 775.18 ± 139.11 25.02 ± 9.19 692.99 ± 127.45
NC 9.69 ± 2.11 0.0037 ± 0.001 a 1205.54 ± 371.03 a 54.85 ± 13.53 a 1605.45 ± 257.17 a
YM 9.89 ± 1.88 0.0045 ± 0.001 a 1190.34 ± 11.49 a 43.32 ± 2.77 a 1136.68 ± 22.99 a,b
CG 15.44 ± 3.84 a,b 0.0058 ± 0.002 a 1603.71 ± 102.08 a,b,c 53.11 ± 12.82 a 1572.01 ± 312.61 a,c
UC 18.01 ± 3.00 a,b,c 0.0110 ± 0.003 a,b,c,d 2875.55 ± 312.98 a,b,c,d 79.94 ± 15.04 a,b,c,d 2210.94 ± 15.09 a,b,c,d
The data are expressed as the means ± SD, n = 5; a p < 0.05 versus the AM group, b p < 0.05 versus the NC group, c p < 0.05 versus the YM
group, d p < 0.05 versus the CG group.
2.5. Inflammation and Oxidative Stress in the Serum
It was approved that increased production of adipocytes and hyperglycemia feed
the cycle of chronic inflammation by producing ROS and inflammatory cytokines, which
induce osteoblast apoptosis to bring the negative effect on bone health. In the current study,
to assess the inflammation and oxidative stress, inflammatory cytokines and the oxidative
stress reaction were investigated. The serological assessment of inflammatory cytokines
in the five groups is shown in Figure 4A. Similar change trends were observed for IL-1β,
IL-6, and tumor necrosis factor (TNF) α. The levers of IL-1β, IL-6, and TNF-α in the NC,
YM, CG and UC groups were significantly smaller in the OM group (p < 0.05). In addition,
IL-1β, IL-6, and TNF-α levels in the UC group were significantly smaller than those in the
other groups (p < 0.05).
In order to investigate the effect of UC II on oxidative stress, the activity of superoxide
dismutase (SOD), glutathione peroxidase (GSH-Px), and malondialdehyde (MDA) in the serum
of the mice was examined. As shown in Figure 4B, compared with the AM group, the other
groups showed obviously stronger activities of SOD and GSH-Px and a significantly lower
MDA level (p < 0.05). Regarding the MDA content, values in the UC group were the smallest
among the five groups (significantly; p < 0.05), whereas there were no significant differences
between the SOD and GSH-Px activity found in the two intervention groups (p> 0.05).
be placed on the femur until fracture/breakage occurred. Table 1 shows biomechanical
femoral parameters in the five groups. The values of the maximum load in the CG and
UC groups were significantly larger than those in the AM and NC groups (p < 0.05), of
which the value in the UC group was significantly larger than that in the YM group (p <
0.05), and biomechanical femoral properties, including the energy to ultimate load,
Young’s modulus, stiffness, and breaking energy in the UC group showed significant dif-
ferences between the AM, NC, YM, and CG groups (p < 0.05).
2.5. Inflammation and Oxidative Stress in the Serum
It was approved that increased production of adipocytes and hyperglycemia feed the
cycle of chronic inflammation by producing ROS and inflammatory cytokines, which in-
duce osteoblast apoptosis to bring the negative effect on bone health. In the current study,
to assess the inflammation and oxidative stress, inflammatory cytokines and the oxidative
stress reaction were investigated. The serological assessment of inflammatory cytokines
in the five groups is shown in Figure 4A. Similar change trends were observed for IL-1β,
IL-6, and tumor necrosis factor (TNF) α. The levers of IL-1β, IL-6, and TNF-α in the NC,
YM, CG and UC groups were significantly smaller in the OM group (p < 0.05). In addition,
IL-1β, IL-6, and TNF-α levels in the UC group were significantly smaller than those in the
other groups (p < 0.05).
In order to investigate the effect of UC II on oxidative stress, the activity of superox-
ide dismutase (SOD), glutathione peroxidase (GSH-Px), and malondialdehyde (MDA) in
the serum of the mice was examined. As shown in Figure 4B, compared with the AM
group, the other groups showed obviously stronger activities of SOD and GSH-Px and a
significantly lower MDA level (p < 0.05). Regarding the MDA content, values in the UC
group were the smallest among the five groups (significantly; p < 0.05), whereas there
were no significant differences between the SOD and GSH-Px activity found in the two
intervention groups (p> 0.05).
2.6. Histopathological Muscle Analysis and its Biomarker
We had known that the gastrocnemius decreased in the AM group and the UC II
intervention could increase the gastrocnemius mass. The gastrocnemius morphological
characteristics and pathology are shown in the Figure 5A,B. Muscle cells in the AM group
lost their normal morphology, the nuclear center migration phenomenon was serious, and
the cells showed remarkable differences in size and had a disordered, loose arrangement.
In addition, the intercellular substance level increased, and there was a large amount of
connective-tissue hyperplasia and inflammatory cell infiltration between muscle cells.
Similarly to the AM group, the intercellular substance level in the CG group increased,
and there was a large amount of fibrous connective-tissue hyperplasia and inflammatory
cell infiltration; however, it was inconsistent because the CG group showed normal cell
morphology, uniform sizes, and a slight nuclear center migration phenomenon. Com-
pared with the AM and CG groups, the NC and YM groups showed less fibrous connec-
tive-tissue hyperplasia and inflammatory cell infiltration, and the cells were uniform in
size and tidily arranged. UC resulted in a normal polygonal cell without degenerating
necrotic cells, uniform size, tightly arranged, and with the disappearance of disorderly
connective tissue and inflammatory cells.
Impaired glucose/insulin metabolism may indirectly affect bones and muscles by al-
tering skeletal muscle signaling, such as IGF-1. It was reported that IGF-1 was associated
with increasing the muscle mass and the muscle fiber area. Figure 5B shows the effect of
UC II on muscle fiber parameters in the five groups. The AM group showed the smallest
values in muscle fiber diameter, number, and cross-sectional area, while the intervention
with UC II significantly increased the muscle fiber diameter, number, and cross-sectional
area (p < 0.05). The improvement effect of positive substances (CS + GH) on the muscle
fiber cross-sectional area was smaller compared with that of the UC II intervention.
Figure 4. Serum levels of inflammatory cytokines and oxidative stress in the five groups. (A) Serum levels of inflammatory
cytokines including IL-1β, IL-6, and TNF-α; (B) serum levels of oxidative stress indexes including MDA, SOD, and GSH-Px.
Serum data are expressed as the means ± SD (n = 5, 7, 9, 9, and 10 in the AM, CG, UC, NC, and YM groups); a p < 0.05
versus the AM group, b p < 0.05 versus the NC group, c p < 0.05 versus the YM group, d p < 0.05 versus the CG group.
2.6. Histopathological Muscle Analysis and its Biomarker
We had known that the gastrocnemius decreased in the AM group and the UC II
intervention could increase the gastrocnemius mass. The gastrocnemius morphological
characteristics and pathology are shown in the Figure 5A,B. Muscle cells in the AM group
lost their normal morphology, the nuclear center migration phenomenon was serious, and
the cells showed remarkable differences in size and had a disordered, loose arrangement.
In addition, the intercellular substance level increased, and there was a large amount
of connective-tissue hyperplasia and inflammatory cell infiltration between muscle cells.
Similarly to the AM group, the intercellular substance level in the CG group increased,
and there was a large amount of fibrous connective-tissue hyperplasia and inflammatory
cell infiltration; however, it was inconsistent because the CG group showed normal cell
morphology, uniform sizes, and a slight nuclear center migration phenomenon. Compared
with the AM and CG groups, the NC and YM groups showed less fibrous connective-tissue
hyperplasia and inflammatory cell infiltration, and the cells were uniform in size and tidily
arranged. UC resulted in a normal polygonal cell without degenerating necrotic cells,
uniform size, tightly arranged, and with the disappearance of disorderly connective tissue
and inflammatory cells.
Impaired glucose/insulin metabolism may indirectly affect bones and muscles by
altering skeletal muscle signaling, such as IGF-1. It was reported that IGF-1 was associated
with increasing the muscle mass and the muscle fiber area. Figure 5B shows the effect of
UC II on muscle fiber parameters in the five groups. The AM group showed the smallest
values in muscle fiber diameter, number, and cross-sectional area, while the intervention
with UC II significantly increased the muscle fiber diameter, number, and cross-sectional
area (p < 0.05). The improvement effect of positive substances (CS + GH) on the muscle
fiber cross-sectional area was smaller compared with that of the UC II intervention.
IGF-1 is known to activate IGF-1R which acts through the PI3K/Akt and the MAPK/ERK
pathways . It has been speculated the function of IGF-1 was partly affected by inflamma-
tion, oxidative stress, and diabetes . In order to investigate the effect of UC II on oxidative
stress in the gastrocnemius muscle of the db/db mice, the activity of SOD, GSH-Px, and
MDA in the gastrocnemius muscle was determined. According to Figure 5C, the activities of
SOD and GSH-Px in the AM group were significantly lower than those in four other groups,
and the UC group showed a significantly higher level than that in the other groups (p < 0.05),
while the MDA content in the UC group was remarkably decreased (p < 0.01), indicating
that the UC intervention exerted protection against SOD and GSH-Px depletion and MDA
accumulation in the gastrocnemius muscle tissue of the db/db mice.
IGF-1, secreted from skeletal muscles, was an important growth factor for skeletal
development . For the IGF-1 levels, the lowest content was found in the AM group,
which was obviously lower than that in the other groups (p < 0.05); an intervention with
UC II increased the IGF-1 content, which was higher than in the AM, YM, and CG groups
and lower than in the NC group (p < 0.05).
IGF-1 is known to activate IGF-1R which acts through the PI3K/Akt and the
MAPK/ERK pathways . It has been speculated the function of IGF-1 was partly af-
fected by inflammation, oxidative stress, and diabetes . In order to investigate the ef-
fect of UC II on oxidative stress in the gastrocnemius muscle of the db/db mice, the activity
of SOD, GSH-Px, and MDA in the gastrocnemius muscle was determined. According to
Figure 5C, the activities of SOD and GSH-Px in the AM group were significantly lower
than those in four other groups, and the UC group showed a significantly higher level
than that in the other groups (p < 0.05), while the MDA content in the UC group was re-
markably decreased (p < 0.01), indicating that the UC intervention exerted protection
against SOD and GSH-Px depletion and MDA accumulation in the gastrocnemius muscle
tissue of the db/db mice.
IGF-1, secreted from skeletal muscles, was an important growth factor for skeletal
development . For the IGF-1 levels, the lowest content was found in the AM group,
which was obviously lower than that in the other groups (p < 0.05); an intervention with
UC II increased the IGF-1 content, which was higher than in the AM, YM, and CG groups
and lower than in the NC group (p < 0.05)
2.7. Immunohistochemical Analysis
It was reported that some proinflammatory cytokines, including IL-1β, IL-6, and
TNF-α, also stimulate osteoclast activity by the actions of RANKL . RANKL, an essen-
tial factor for osteoclast differentiation and function, mediates bone loss by promoting the
bone-resorbing activity of osteoclasts and prolongs their survival [37,38]. The receptor ac-
tivator of (nuclear factor) kappa B ligand (RANKL) signaling pathway was analyzed. Fig-
ure 6A shows that the immunostaining of IL-1β in the AM, NC, YM, and CG groups was
stronger than in the UC group. The areal density of IL-1β in the AM group was signifi-
cantly higher than in the four other groups (p < 0.05), which indicated that IL-1β in the
AM group was remarkably more strongly expressed, while the UC II intervention signif-
icantly decreased the expression of IL-1β (p < 0.05). The RANKL expression is shown in
Figure 6B. A higher expression in the AM group was clearly detected, and there was little
expression in the NC and UC groups. Compared with the areal density in the AM group,
it was significantly decreased in the four other groups (p < 0.05). AOD in the UC group
was obviously lower than in the AM, NC, YM, and CG groups (p < 0.05). Figure 6C shows
that the immunoreactive signal for TRAP in the UC group was weaker than that in the
other groups. AOD indicated that the highest expression was in the AM group, while the
lowest expression was in the UC group; TRAP expression in the UC group was relatively
lower than in the AM, NC, and YM groups (p < 0.05).
Figure 5. H&E staining of the gastrocnemius muscle and its characteristic parameter. (A) H&E staining of the gastrocnemius.
The bar indicates 50 µm. Magnification: 40×. (B) Diameter, number, and cross-sectional area of muscle fibers. (C) Biomarkers
including oxidative stress and monokines IGF-1. The data are expressed as the means ± SD, n = 5; a p < 0.05 versus the AM
group, b p < 0.05 versus the NC group, c p < 0.05 versus the YM group, d p < 0.05 versus the CG group.
2.7. Immunohistochemical Analysis
It was reported that some proinflammatory cytokines, including IL-1β, IL-6, and TNF-
α, also stimulate osteoclast activity by the actions of RANKL . RANKL, an essential
factor for osteoclast differentiation and function, mediates bone loss by promoting the
bone-resorbing activity of osteoclasts and prolongs their survival [37,38]. The receptor
activator of (nuclear factor) kappa B ligand (RANKL) signaling pathway was analyzed.
Figure 6A shows that the immunostaining of IL-1β in the AM, NC, YM, and CG groups
was stronger than in the UC group. The areal density of IL-1β in the AM group was
significantly higher than in the four other groups (p < 0.05), which indicated that IL-1β
in the AM group was remarkably more strongly expressed, while the UC II intervention
significantly decreased the expression of IL-1β (p < 0.05). The RANKL expression is shown
in Figure 6B. A higher expression in the AM group was clearly detected, and there was
little expression in the NC and UC groups. Compared with the areal density in the AM
group, it was significantly decreased in the four other groups (p < 0.05). AOD in the UC
group was obviously lower than in the AM, NC, YM, and CG groups (p < 0.05). Figure 6C
shows that the immunoreactive signal for TRAP in the UC group was weaker than that
in the other groups. AOD indicated that the highest expression was in the AM group,
while the lowest expression was in the UC group; TRAP expression in the UC group was
relatively lower than in the AM, NC, and YM groups (p < 0.05).
3. Materials and Methods
3.1. Materials
Undenatured type II collagen (UC II), with the conformational integrity of the triple-
helical structure remaining intact, was purchased from SEMNL Biotechnology Co. Ltd.
(Beijing, China). UC II, prepared from a chicken sternum at a low temperature, showed
the molecular weight of 300 kDa, and its content was as follows: collagen, 263.0 mg/g;
hydroxyproline, 32.9 mg/g. The triple-helical structure and the second structure (17.2% of
the α helix, 21.1% of the β sheet, 44.0% of the β turn, and 17.1% of the nonregular coil) are
shown in the Supplementary Materials. The amino acid composition is shown in Table 2.
Chondroitin sulfate (CS) and glucosamine hydrochloride (GH) were obtained from Wilke
Resources (Lenexa, KS, USA).
Figure 6. Immunohistochemistry analysis of the five groups. Magnification: ×400. IHC quantification is shown in the
bar graphs. (A) Protein expressions of IL-1β in the femur; (B) protein expressions of RANKL in the femur; (C) protein
expressions of TRAP in the femur. The data are the means ± SD (n = 5, 7, 9, 9, and 10 in the AM, CG, UC, NC, and YM
groups); a p < 0.05 versus the AM group, b p < 0.05 versus the NC group, c p < 0.05 versus the YM group, d p < 0.05 versus
the CG group.
3. Materials and Methods
3.1. Materials
Undenatured type II collagen (UC II), with the conformational integrity of the triple-
helical structure remaining intact, was purchased from SEMNL Biotechnology Co. Ltd.
(Beijing, China). UC II, prepared from a chicken sternum at a low temperature, showed
the molecular weight of 300 kDa, and its content was as follows: collagen, 263.0 mg/g;
hydroxyproline, 32.9 mg/g. The triple-helical structure and the second structure (17.2% of
the α helix, 21.1% of the β sheet, 44.0% of the β turn, and 17.1% of the nonregular coil) are
shown in the Supplementary Materials. The amino acid composition is shown in Table 2.
Chondroitin sulfate (CS) and glucosamine hydrochloride (GH) were obtained from Wilke
Resources (Lenexa, KS, USA).
The detection kits of superoxide dismutase (SOD), glutathione peroxidase (GSH-
Px), malondialdehyde (MDA), tumor necrosis factor α (TNF-α), interleukin (IL) 6 and
IL-1β were purchased from Shanghai Lengton Biotechnology Co., Ltd. (Shanghai, China).
Insulin was bought from the Beyotime Institute of Biotechnology (Beijing, China). The
detection kits of alkaline phosphatase (BALP), tartrate-resistant acid phosphatase (TRAP),
and osteocalcin (OC) were bought from USCN Life Science Inc. IL-1β, IL-6, TRAP, and
receptor activator of nuclear factor (NF)-κB ligand (RANKL) antigens (mouse monoclonal
antibody) were purchased from R&D Systems, USA. The basic feed, which met the national
standard (GB 14924.3-2010), was bought from Beijing Keao Xieli Co. Ltd. (Beijing, China).
All the other reagents were analytically pure.
3.2. Animals and Experiment Design
3.2.1. Animal Feeding Conditions and Ageing db/db Mice Model Establishment
Male C57BL/KsJ-leprdb/leprdb diabetic (db/db, 10-week-old) mice and male non-
diabetic littermate (db/m) mice were obtained from the Animal Service of the Health
Science Center (Peking University, Beijing, China), production certificate No. SCXK (Bei-
jing, China) 2016–0010, use license No. SYXK (Beijing, China) 2016-0041. The environment
conditions involved constant temperature (21–25 ◦C), relative air humidity (50–60%), and
12 h light/dark cycles.
All the mice had free access to the standard food (American Institute of Nutrition Ro-
dent Diet 93G) and water. The protocols were reviewed and approved by the Institutional
Animal Care and Use Committee of Peking University (approval No. LA2015094).
Considering bone impairment associated with T2DM and ageing, we used, for the first
time, 48-week-old db/db mice normally fed with a basic feed representing the approach of
ageing as the ageing model to explore the effect of the UC II intervention begun in their
later life on the ageing-related bone impairment, meanwhile, 25-week-old db/db (the age
considered to represent the end of growth and development) mice normally fed with a
basic feed as the young model to explore the alterations of bone impairment during the
process of T2DM and ageing, which could lead to a deeper understanding of the occurrence
and development of bone impairment in T2DM. Consequently, the ageing and the young
models of db/db mice is the highlight of this work (the timepoints were selected on the
basis of previous research coupled with the observation of the general condition and the
disease state including intake, drinking, weights and fasting blood glucose provided in
Figures S3–S5 from the Supplementary Materials) [39,40].
3.2.2. Study Design
Forty-five 48-week-old db/db mice (72 ± 3 g) with plasma glucose above 11.1 mmol/L
were randomly subdivided into the ageing model group (AM; n = 15), the UC II intervention
group (UC; n = 15), and the chondroitin sulfate + glucosamine hydrochloride (CS + GH)
control group (CG; n = 15). Meanwhile, 48-week-old db/m and 25-week-old db/db mice
were used as the normal control (NC; n = 15) and the young model (YM; n = 15) groups,
respectively. The UC group was administered UC II through drinking at the dose of
6 mg/kg body weight, whereas the CG group was treated with 180 mg CS + 225 mg
GH/kg body weight through drinking. The AM, NC, and YM groups were administered
distilled water. The drinking feeding mode was a gentle feeding mode for elderly mice,
whose strict quality control and accuracy were confirmed by our team with solid results in
earlier reports [41,42].
After dividing the group, the general condition, including the coat color, mental state,
and daily activities, was monitored daily (at 10:00) till the end stage of the experiment, and
food intake, water intake, and body weight were regularly recorded each week.
All the mice were exposed to the intervention for 12 weeks. In the later stage of
the intervention period, weight loss showing a sharp decrease (approximately>20% loss
of body weight) was an indication of the dying stage, and if a mouse appeared to be
dying due to weight loss or injury, it was euthanized with CO2 to minimize suffering.
One-quarter of all the mice or one-third of one group approaching death were considered
to signify the end of the experiment (here, the intervention lasted 12 weeks). At the end
of the experiment, the surviving mice (n = 10, 13, 14, 14, and 15 in the AM, CG, UC, NC,
and YM groups, respectively) were euthanized with CO2, and blood was obtained from
the retrobulbar plexus using heparinized anticoagulant tubes for further measurements.
After euthanasia, the main organs were weighed to calculate organ coefficients. Some
separated right femurs from the mice injected with a dye (n = 5 in each group) were fixed
in 10% neutral buffered formalin for dynamic histomorphometry; the left femurs of these
mice were packed in a gauze soaked with phosphate-buffered saline and stored at −20 ◦C
to carry out the three-point test; the right gastrocnemius of these mice was separated
and stored at –80 ◦C to perform biomarker measurements, including oxidative stress and
myokines; and the left gastrocnemius of these mice was fixed in 10% neutral buffered
formalin for histopathological analysis. The mice without an injection in each group
provided serum and femurs to measure biomarkers, and for microcomputed tomography
(CT) and histopathological analysis; histopathological analysis for the right femur also
required decalcification with 10% EDTA (changed each week). The schematic diagram of
the research protocol is shown in Figure 7.
due to weight loss or injury, it was euthanized with CO2 to minimize suffering. One-quar-
ter of all the mice or one-third of one group approaching death were considered to signify
the end of the experiment (here, the intervention lasted 12 weeks). At the end of the ex-
periment, the surviving mice (n = 10, 13, 14, 14, and 15 in the AM, CG, UC, NC, and YM
groups, respectively) were euthanized with CO2, and blood was obtained from the
retrobulbar plexus using heparinized anticoagulant tubes for further measurements. After
euthanasia, the main organs were weighed to calculate organ coefficients. Some separated
right femurs from the mice injected with a dye (n = 5 in each group) were fixed in 10%
neutral buffered formalin for dynamic histomorphometry; the left femurs of these mice
were packed in a gauze soaked with phosphate-buffered saline and stored at –20 °C to
carry out the three-point test; the right gastrocnemius of these mice was separated and
stored at –80 °C to perform biomarker measurements, including oxidative stress and my-
okines; and the left gastrocnemius of these mice was fixed in 10% neutral buffered forma-
lin for histopathological analysis. The mice without an injection in each group provided
serum and femurs to measure biomarkers, and for microcomputed tomography (CT) and
histopathological analysis; histopathological analysis for the right femur also required de-
calcification with 10% EDTA (changed each week). The schematic diagram of the research
protocol is shown in Figure 7.
Figure 7. Schematic plot and the timeline of the research protocol.
Normal deaths were marked with blue. Injection of alizarin-3-methyliminodiacetic
acid and calcein was performed in one-week intervals (n = 5 mice per group per timepoint)
(marked with yellow). Fasting blood glucose and insulin measurements were performed
each week (in all the survived mice per group per timepoint) and at the end stage (n = 5
injected mice per group).
3.3. Fasting Plasma Glucose and HOMA-IR Measurements
Fasting plasma glucose (FBG) and insulin (FINS) in the tail vein were measured using
an Accu-Check glucometer (Roche Diagnostics) and an ELISA kit after fasting for 5 h (n =
5 injected mice of each group). The level of insulin resistance was calculated using the
following formula: (fasting glucose (mmol/L) × FINS (uIU/mL))/22.5 and expressed
as HOMA-IR.
3.4. Micro-CT Analysis
The mass and microarchitecture of undecalcified femurs were measured with micro-
CT (Inveon MM system, Siemens, Munich, Germany), and the parameters were calculated
using an Inveon Research Workplace. The parameters, including 8.82 μm for pixel size,
80 kV for voltage, 500 μA for current, and 1500 ms for exposure time were set. The trabec-
ular region of about 1–2 mm distal to the proximal epiphysis was selected. Under these
conditions, the parameters including bone volume/total volume (BV/TV), bone surface
area/bone volume (BS/BV), trabecular thickness (Tb.Th), trabecular number (Tb.N), tra-
becular separation (Tb.Sp), and bone mineral density (BMD) were obtained.
Figure 7. Schematic plot and the timeline of the research protocol.
Normal deaths were marked with blue. Injection of alizarin-3-methyliminodiacetic
acid and calcein was performed in one-week intervals (n = 5 mice per group per timepoint)
(marked with yellow). Fasting blood glucose and insulin measurements were performed
each week (in all the survived mice per group per timepoint) and at the end stage (n = 5
injected mice per group).
3.3. Fasting Plasma Glucose and HOMA-IR Measurements
Fasting plasma glucose (FBG) and insulin (FINS) in the tail vein were measured using
an Accu-Check glucometer (Roche Diagnostics) and an ELISA kit after fasting for 5 h
(n = 5 injected mice of each group). The level of insulin resistance was calculated using the
following formula: (fasting glucose (mmol/L) × FINS (uIU/mL))/22.5 and expressed
as HOMA-IR.
3.4. Micro-CT Analysis
The mass and microarchitecture of undecalcified femurs were measured with micro-
CT (Inveon MM system, Siemens, Munich, Germany), and the parameters were calculated
using an Inveon Research Workplace. The parameters, including 8.82 µm for pixel size,
80 kV for voltage, 500 µA for current, and 1500 ms for exposure time were set. The
trabecular region of about 1–2 mm distal to the proximal epiphysis was selected. Under
these conditions, the parameters including bone volume/total volume (BV/TV), bone
surface area/bone volume (BS/BV), trabecular thickness (Tb.Th), trabecular number (Tb.N),
trabecular separation (Tb.Sp), and bone mineral density (BMD) were obtained.
3.5. Biomechanical Bone Parameters
In order to evaluate the biomechanical properties of the femurs, three-point bend-
ing tests were performed. The left femurs of the injected mice in each group were cen-
trally loaded with a speed of 1 mm/s using a universal testing machine (Instron 4501,
Instron, Canton, MA, USA). The parameters of the bone samples, namely, stiffness, ultimate
strength, Young’s modulus, and ultimate stress were determined and calculated based on
the deformation curve .
3.6. Slicing of Undecalcified Bones and Dynamic Histomorphometric Analysis
Seven days before euthanasia, five mice per group were intraperitoneally injected with
alizarin red (30 mg/kg body weight); 4 days later, an intraperitoneal injection of 20 mg/kg
body weight calcein was carried out . After euthanasia, the right femurs were fixed
and dehydrated and embedded in destabilized methyl methacrylate resin. The samples
were ground and polished to 40–60 µm and H&E stained. The samples were then observed
under a laser scanning confocal microscope (Leica TCS SP-2, Frankfurt, Germany) set on
534 nm (calcein) and 357 nm (alizarin red) .
3.7. Biochemical Marker Assay
TRAP, a marker of the osteoclast number, represented bone resorption. OC, mainly
controlling for mineralization, and BALP, representing bone formation, were selected. The
levels of BALP, TRAP, OC, common inflammatory cytokines (TNF-α, IL-1b, IL-6), and
common oxidative stress markers (SOD, GSH-Px, MDA) in the serum of the mice without
a dye injection were measured using relevant enzyme-linked immunoassay (ELISA) kits
according to the product instructions.
SOD, MDA, GSH-Px, and IGF-1 activities in the right gastrocnemius muscle (n = 5 in each
group) were measured using the relevant ELISA kits according to the product instructions.
3.8. Histopathological Analysis
The left gastrocnemius of the five injected mice in each group was fixed in 10% forma-
lin. The samples embedded in paraffin were stained with hematoxylin and eosin (H&E).
In order to evaluate the status of the muscles, gastrocnemius slides were microscopically
observed with an inverted microscope (Olympus IX70, Olympus, Tokyo, Japan). Then, the
cross-sectional area of muscle fiber diameters was measured and calculated with Image-Pro
Plus 6.0 (Media Cybernetics, Inc., Rockville, MD, USA).
Immunohistochemistry (IHC) was used to deduce the involvement of the related key
pathways, performed on the right femur of the mice without a dye injection. IL-1β, IL-6,
tartrate-resistant acid phosphatase (TRAP), and receptor activator of (nuclear factor) NF-κB
ligand (RANKL) were applied with the following antibodies: IL-1β and IL-6 antigens
(both 1:100 dilution) for proinflammatory cytokine expression; TRAP (1:100 dilution) and
RAKNL (1:100 dilution) antigens for osteoclast formation. Immunohistochemistry was
carried out on 3–5 µm-thick paraffin sections, followed by overnight incubation with the
primary antibody at 4 ◦C. After staining with DAB, the slides were counterstained with
Mayer’s hematoxylin and dehydrated through a graded ethanol series.
Quantitative image analysis was performed with the Image-Pro Plus 6.0 software
(Media Cybernetics, Inc., Rockville, MD, USA). Cumulative optical density (IOD) and
pixel area (area) values of the tissue of each positive image were obtained. Statistical
analysis of the results was expressed as the average optical density (AOD) (areal density,
AOD = IOD/area).
3.9. Statistical Analysis
The data are expressed as the means ± SD. One-way ANOVA with post-hoc LSD
(equal variances assumed) or Dunnett’s T3 test (equal variances not assumed) were per-
formed with SPSS 18.0, and p-values less than 0.05 were considered to be significant.
4. Discussion
Diabetes-associated bone disease leads to impaired bone quality and increased fracture
risk. This study addressed the alterations of bone quality-impaired T2DM and ageing,
including bone microarchitecture, remodelling, and biomechanical quality. In order to
evaluate the effect of UC II on bone quality impairment in the ageing db/db mice and its
mechanism of action, we comprehensively explored bone metabolism alterations on the
basis of inflammation and oxidative stress in the serum, bone, and skeletal muscle for the
first time. In addition, the T2DM status was examined.
BMD values in the AM group were similar to those in the NC group, which was
consistent with the previous research finding normal or even mildly elevated BMD in
T2DM patients compared to that in those without T2DM . In fact, fracture risk is
increased; although there is normal and even elevated BMD in T2DM, the evidence led to
the hypothesis that there are diabetes-associated alterations in skeletal properties . In
this study, alterations including bone microarchitecture, metabolism, and biomechanical
quality caused by T2DM were investigated. The lowest levels of BV/TV, Tb.N, and Tb.Th
were in the ageing db/db mice (AM group), which was consistent with the previous results
obtained in diabetic rats and humans with T2DM [48,49]; this deteriorated morphological
structure was sharply improved by UC II. Moreover, after the UC II intervention, the thick-
ness of the cortical bone was observed, and cortical porosity and trabecular heterogeneity
were improved. There were morphological and histological changes of bone tissue in
this study, which led to significant worsening of the mechanical properties shown in the
three-point bending test (Table 1). The mice in the AM group showed the smallest values in
the maximum load, the energy to ultimate load, Young’s modulus, stiffness, and breaking
energy, which were similar to those in another report [50,51].
In addition, the alterations of the bone metabolism were analyzed. Serum levels of
BALP and OC in the AM group were the lowest, whereas its TRAP levels were significantly
higher compared to those in the other groups. Similar conclusions were reported in
previous studies, both in rodents and people with T2DM [52–54], while the increased value
in TRAP observed in the AM group was not consistent with other reports . In fact, the
studies examining the effect of diabetes mellitus on bone resorption were not conclusive,
including unaltered, inhibited, and increased in vitro and vivo studies on animals and
human patients [55–58].
The abovementioned alterations in microarchitecture, mechanical properties, and
bone metabolism were easy to understand on the basis of both indirect and direct T2DM
effects on bone impairment. A disorder in glucose and insulin metabolism decreases the
activity of osteoblasts and osteoclasts. Hyperglycemia changes gene expression associated
with osteoblast activity, and insulin resistance may impact bone health . Inflammation
and oxidative stress induced by T2DM have negative effects on bone metabolism. These
accumulation pathomechanisms ultimately result in decreased bone formation or bone
resorption, leading to decreased bone quality [8,35]. In this study, the UC II interven-
tion relieved bone impairment in ageing db/db mice, reflected in improving the bone
microarchitecture, increasing bone formation, and decreasing bone resorption. UC II
also improved inflammation, oxidative stress, and muscle properties, which might have
elevated the disease status of T2DM.
What is the possible reason for UC II improving bone impairment induced by T2DM?
A possible mechanism was the decrease in inflammation and oxidative stress, which could
directly regulate bone metabolism through bones, and indirectly through muscles and
blood glucose.
From a comprehensive and systematic point of view, skeletal muscles and bones have
a coupled and cross-talk relationship, where bones act as a lever and muscles act as a
pulley to move the organism and for monokine communication. The mice in the AM
group exhibited lower values of BV/TV, Tb.N, and Tb.Th and showed smaller values in the
muscle mass, muscle fiber diameter, and cross-sectional area (Figures 3 and 6). In addition,
impaired glucose or insulin metabolism may impact bones by changing skeletal muscle
signaling. IGF-1, secreted from skeletal muscles, is an important growth factor for bone
development. Some investigations provided evidence that muscle IGF-1 can modulate
bone formation and maintain bone structure, which was correspondingly shown in this
study; we found higher content of IGF-1 in the CP group, which showed a higher level
of BV/TV and trabecular number (Tb.N) [28,60–63]. IGF-1 is also an important growth
factor for muscles . There are some reports that elaborated that IGF-1 is associated with
an increase in the muscle mass and muscle fiber area, even inhibiting osteopenia [65,66].
The same finding can be observed in Figures 2 and 6. It was speculated the IGF-1 function
is partly affected by inflammation, oxidative stress, and diabetes [34,35]. In this study,
treating the ageing db/db mice with UC II decreased oxidative stress (Figure 4), leading to
an increased IGF-1 content (Figure 5). This finding was first reported in this study. This
pathway might be the mechanism for an improvement of UC II in bone impairment.
The indirect effect of muscles on bone health was carried out through glucose metab-
olism. Skeletal muscles are recognized as the main site for insulin-mediated glucose
disposal and energy metabolism. Muscle wasting can exacerbate insulin resistance, and
higher muscle mass and strength are associated with a lower level of insulin resistance .
On the molecular level, skeletal homeostasis is linked to insulin sensitivity through nu-
clear receptor peroxisome proliferator-activated receptor (PPAR)γ . IR increases the
FOXO1 expression through PI3-K/MAPK, which could regulate osteoblast proliferation
through ATF4 and p53 signaling . Prolonged inflammation due to IR also stimulates
the expression of proapoptotic genes such as the bcl-2-like protein (Bax). This reduces the
expression of genes that stimulate osteoblast formation, such as the Fos-related antigen
(FRA-1) and the Runt-related transcription factor (RUNX2), resulting in decreased bone
formation. In T2DM, other proteins such as AGEs, proinflammatory cytokines, and ROS
are increased . UC II decreased oxidative stress both in the muscles and the serum
(Figures 5 and 6), indicating partly improved muscle IR (Figure 1), which was speculated
to stimulate membrane translocation of GLUT4 through skeletal muscle phosphorylated
PI3K protein expression, thereby increasing skeletal muscle glucose intake, leading to IR
improvement . UC II increased IGF-1 production by decreasing oxidative stress in the
muscle to improve muscle foundation, glucose metabolism, and insulin signaling, thereby
leading to bone formation.
On the other hand, hyperglycemia leads to the accelerated formation of AGEs and
inflammation cytokines, inhibiting osteoblasts . Therefore, inflammation and oxidative
stress were speculated to be trigger factors of aggravating bone quality impairment. This
study also found that UC II improved oxidative stress in the serum, which likely led to
decreased AGE formation and cross-linking with collagen fibers. Interfering with the
development and function of osteoblasts by upregulating the cell surface receptor for
advanced glycation end products (RAGE) , these receptors increase the production
of proinflammatory cytokines, which may feed a cycle of increased bone resorption and
chronic inflammation .
Figure 4 shows that TNF-α, IL-6, and IL-1β levels in the serum were significantly
decreased in the UC group. In previous cases of osteoarthritis, UC II was documented
to promote a significant reduction in inflammation . Figures 4, 5 and 7 show the re-
lationship between inflammation and bone resorption (TRAP). Therefore, the osteoclasts
overactivated by inflammation play a vital role in imbalanced bone metabolism . Thus,
inhibiting osteoclastogenesis through suppressing inflammation is an important strategy
for improving bone degeneration . Correspondingly, the positive expression of Il-1β in
the CP group was decreased compared with that in the AM group (Figure 6A). IL-1β, IL-6,
and TNF-α, common proinflammatory cytokines, can stimulate osteoclast activity with the
macrophage colony-stimulating factor (M-CSF) and its receptor, c-Fms, which modulate
the pool of available precursor cells for differentiation via the actions of RANKL . These
osteoclast-activating factors interact with a proven final common mediator of osteoclast
differentiation and activation, receptor activator of nuclear factor-kB (RANK) and its func-
tional ligand (RANKL). RANK is a membrane-bound TNF receptor expressed on osteoblast
precursor cells that recognizes RANKL through direct cell–cell interactions, an essential
process for the differentiation of osteoclasts from their precursor cells. RANKL, an essential
factor for osteoclast differentiation and function, is also expressed by lymphocytes and syn-
ovial fibroblasts and may mediate bone loss associated with inflammatory conditions ,
which promotes the bone-resorbing activity of osteoclasts and prolongs their survival .
Similarly, the immunohistochemical analysis showed RANKL had a higher expression in
the AM group, and low expression was observed in the CG and UC groups. The same
tendency was shown in TRAP expression, which means that UC II decreased RANKL
and TRAP expression. In this study, UC II decreased bone resorption by inhibiting serum
cytokines, as well as IL-1 and IL-6 expression, and then inhibited the expression of RANKL
and TRAP. In this study, it was speculated that UC II could improve bone impairment due
to increasing bone mineralization and formation, decreasing bone resorption, which partly
resulted from muscle function and IR improvement through decreasing oxidative stress
and inflammation (Figure 8). This conclusion was partly proved in other studies [28,77].
and its functional ligand (RANKL). RANK is a membrane-bound TNF receptor expressed
on osteoblast precursor cells that recognizes RANKL through direct cell–cell interactions,
an essential process for the differentiation of osteoclasts from their precursor cells.
RANKL, an essential factor for osteoclast differentiation and function, is also expressed
by lymphocytes and synovial fibroblasts and may mediate bone loss associated with in-
flammatory conditions , which promotes the bone-resorbing activity of osteoclasts and
prolongs their survival . Similarly, the immunohistochemical analysis showed
RANKL had a higher expression in the AM group, and low expression was observed in
the CG and UC groups. The same tendency was shown in TRAP expression, which means
that UC II decreased RANKL and TRAP expression. In this study, UC II decreased bone
resorption by inhibiting serum cytokines, as well as IL-1 and IL-6 expression, and then
inhibited the expression of RANKL and TRAP. In this study, it was speculated that UC II
could improve bone impairment due to increasing bone mineralization and formation,
decreasing bone resorption, which partly resulted from muscle function and IR improve-
ment through decreasing oxidative stress and inflammation (Figure 8). This conclusion
was partly proved in other studies [28,77].
This study has some limitations. First, the exact mechanisms of action of UC II on the
bones impaired by T2DM will be explored in future in vitro and in vivo studies. Second,
senescence-accelerated mice (SAM) will be adopted as the experimental model, especially
to demonstrate ageing characteristics from a certain aspect, such as SAMP6 and SAMP10,
and to explore the possible mechanisms of UC II in delaying and improving degenerative
bone diseases.
Figure 8. Graphical summation of the effect of UC II on impaired bone improvement and its likely mechanism.
5. Conclusions
In order to discover effective preventive measures for bone impairment in ageing
coupled with T2DM, UC II was administered in the ageing db/db mice during a period of
Figure 8. Graphical summation of the effect of UC II on impaired bone improvement and its likely mechanism.
This study has some limitations. First, the exact mechanisms of action of UC II on the
bones impaired by T2DM will be explored in future in vitro and in vivo studies. Second,
senescence-accelerated mice (SAM) will be adopted as the experimental model, especially
to demonstrate ageing characteristics from a certain aspect, such as SAMP6 and SAMP10,
and to explore the possible mechanisms of UC II in delaying and improving degenerative
bone diseases.
5. Conclusions
In order to discover effective preventive measures for bone impairment in ageing
coupled with T2DM, UC II was administered in the ageing db/db mice during a period
of 3 months. The microarchitecture, mechanical properties, and bone metabolism were
evaluated. In order to explore the likely mechanism of action of UC II, the T2DM disease
status, biomarkers, and the gastrocnemius function were analyzed. The results showed
that the UC II intervention elevated BMD and the biomechanical parameters. Our results
also demonstrated that UC II increased bone mineralization and formation and decreased
bone resorption. On the one hand, this function was partly due to decreasing inflamma-
tion, leading to inhibited RANKL and TRAP expression. On the other hand, UC II also
improved oxidative stress in both the serum and the gastrocnemius muscle, leading to
IGF-1 secretion, which facilitated bone formation and muscle growth, ultimately leading
to improved IR. Of course, the improvement of hyperglycemia and IR due to UC II could
decrease inflammation and the oxidative stress status, which feed an indirect cycle of
bone impairment.
Supplementary Materials: The following are available online, Figure S1: The image of Undenatured
type II collagen with transmission electron microscope; Figure S2: The infrared spectroscopy of
Undenatured type II collagen; Figure S3: The food intake of db/db mice and db/m mice during the
whole life in the experiments; Figure S4: The weight of db/db mice and db/m mice during the whole
life in the experiments; Figure S5: The FBG of db/db mice and db/m mice during the whole life in
the experiments.
Author Contributions: Conceptualization, Y.L. and R.F.; writing—review and editing, Y.L. and R.F.;
project administration, Y.L.; supervision, Y.L.; investigation, R.F., Y.H., X.L., J.K., J.H., R.M., R.L., N.Z.,
M.X.; writing—original draft preparation, R.F. and Y.H.; methodology, R.F. and Y.H. All authors have
read and agreed to the published version of the manuscript.
Funding: This research received no external funding.
Institutional Review Board Statement: The protocols were reviewed and approved by the Institu-
tional Animal Care and Use Committee of Peking University (approval No. LA2015094).
Informed Consent Statement: This research article described a study exclude humans.
Data Availability Statement: The data presented in this study are available on request from the
corresponding author. The data are not publicly available due to graduation thesis based on this
relevant research results is still in the confidentiality period.
Conflicts of Interest: The authors declare no conflict of interest.
Sample Availability: No Samples of the compounds are available from the authors. All the com-
pounds were bought.
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