Abstract
Background
Osteoporosis (OP) is a chronic, systemic skeletal disorder
characterized by progressive bone loss and microarchitectural
deterioration, which increases fracture susceptibility and presents a
challenging set of global healthcare problems. Current pharmacological
interventions are limited by adverse effects, high costs, and
insufficient long-term efficacy. Here, we identify snow crab
shell-derived polypeptides (SCSP) as a potent osteoprotective agent.
Methods
SCSP were extracted and characterized. Using an ovariectomized (OVX)
mouse osteoporosis model, mice received daily oral SCSP (50, 100 mg/kg)
or saline for 8 weeks. Bone microstructure (micro-CT), histomorphometry
(H&E, Masson, TRAP), immunohistochemistry, and serum bone turnover
markers were analyzed. In vitro, SCSP (100, 200 μg/ml) effects on
osteogenic/adipogenic differentiation in MSCs/preosteoblasts were
assessed via staining (ARS, ALP, Oil Red O) and molecular analyses
(Western blot, qPCR, RNA-Seq).
Results
SCSP, enriched in glutamic acid, aspartic acid, and lysine,
significantly enhances bone mineral density, restores trabecular
architecture, and preserves bone tissue integrity in an
ovariectomy-induced OP mouse model without detectable systemic
toxicity. At the molecular level, SCSP treatment induces the expression
cell cycle regulators and motor protein pathways in osteoblasts while
suppressing pro-inflammatory signaling networks, thereby
re-establishing osteoblast-osteoclast balance and restoring calcium and
phosphorus homeostasis. This combined mechanism promotes osteogenesis
while simultaneously suppressing adipogenesis.
Conclusion
Our findings position SCSP as a promising natural therapeutic for OP
and provide key mechanistic insights that may guide future
bone-targeted interventions.
Keywords: crab shell polypeptides, osteoporosis, calcium dynamics,
osteogenic activity, OP treatment
Introduction
Osteoporosis (OP) is a systemic skeletal disorder characterized by
decreased bone mass, deterioration of bone microarchitecture, and an
increased risk of fragility fractures ([55]Zhang et al., 2022; [56]Tang
et al., 2024). The global number of OP cases between 2030 and 2034 is
estimated to increase to 263.2 million, exacerbating the global
healthcare burden ([57]Zhu et al., 2023). The prevalence of OP is
significantly higher in females than in males, particularly among
postmenopausal women, due to estrogen deficiency-induced bone
resorption ([58]Alswat, 2017). The pathophysiology of OP is primarily
driven by an imbalance between bone resorption and bone formation,
resulting from reduced osteoblast (OB) proliferation and
differentiation ([59]Pan et al., 2018), excessive osteoclasts (OC)
activation ([60]Chen et al., 2024), and dysregulated calcium metabolism
([61]Wang et al., 2024). Current pharmacological interventions for OP
include anti-resorptive agents, such as bisphosphonates ([62]Kuźnik et
al., 2020), and denosumab, anabolic agents, such as parathyroid hormone
analogs ([63]Leder, 2017), and supportive treatments like active
vitamin D and calcium supplements ([64]Capozzi et al., 2020).
Notwithstanding their clinical benefits, these therapies fail to
address the underlying disease mechanisms and are often associated with
significant adverse side effects, high costs, and limited long-term
efficacy.
Crustacean shells are rich in polysaccharides, proteins, lipids, and
minerals such as calcium, phosphorus, and magnesium, as well as
compounds including astaxanthin and β-carotene. These components
exhibit unique bioactive properties, biocompatibility, and low toxicity
([65]Vilasoa-Martínez et al., 2008; [66]Crespo et al., 2006;
[67]Beaulieu et al., 2009; [68]Taksima et al., 2019). For example,
chitin, a key polysaccharide, exhibits potent anti-inflammatory,
antioxidant, antimicrobial, wound-healing ([69]Chotphruethipong et al.,
2023; [70]literature review of pathology et al., 2014), and anti-tumor
capabilities ([71]Younes et al., 2014). Similarly, lipids derived from
crustacean shells have demonstrated anti-inflammatory and
neuroprotetive properties ([72]Tsoupras et al., 2024; [73]Cholidis et
al., 2024; [74]Abraúl et al., 2023). Astaxanthin is recognized for its
antioxidant, anti-inflammatory, and skin-protective and anti-skin
carcinogenesis properties ([75]Chintong et al., 2019; [76]Rao et al.,
2013), while β-Carotene contributes to antioxidant defense, vision
support, and immune modulation ([77]Jiang et al., 2024; [78]Rammuni et
al., 2019; [79]Nair et al., 2023). Nevertheless, the bioactivity and
therapeutic potential of crustacean shell-derived proteins remain
largely unexplored. The process of calcium deposition in crustaceans is
a highly regulated biomineralization process, and matrix proteins
within the shell play critical roles in nucleation, stabilization, and
orchestrated calcium deposition, thereby contributing to the mechanical
strength of the exoskeleton ([80]Nagasawa, 2012; [81]Nagasawa, 2011;
[82]Abehsera et al., 2018; [83]Shaked et al., 2024). To this end, we
hypothesize that proteins derived from crustacean shells may play a
pivotal role in regulating calcium homeostasis in bone tissue.
Here, we extract and characterize the enzymatically hydrolyzed peptides
from snow crab shells, and demonstrate that these snow crab shell
derived polypeptides (SCSP) exhibit potent anti-osteoporotic activity
in a bilateral ovariectomy-induced osteoporosis mouse model.
Mechanistically, SCSP enhances calcium deposition, promotes OB
activity, and inhibits OC function. Based on chemical, biochemical,
bioinformatics, and functional data detailed below, we propose SCSP as
a promising natural candidate for improving bone health and provide new
insights and therapeutic strategies for OP treatment.
Materials and methods
Reagents and antibodies
Antibody against RUNX-2 and COL-1 were purchased from Cell Signaling
Technology. Antibodies against OSX, NFATc1, RANKL, and CTSK were
purchased from Santa Cruz Biotechnology. Antibodies against BMP-2 was
purchased from Servicebio ([84]Table 1). Hematoxylin and Eosin (H&E)
Staining Kit (C0105S) and BCIP/NBT Alkaline Phosphatase (ALP) Color
Development Kit (C3206) were purchased from Beyotime. Masson’s
trichrome staining solution (G1006) and tartrate-resistant acid
phosphatase (TRAP) staining reagents (G1050) were purchased from
Servicebio. Alizarin Red S (ARS) solution (G1452) was purchased from
Solarbio, and Oil Red O staining solution (320-06-5) was purchased from
Sigma-Aldrich.
TABLE 1.
Antibodies used for Western blot (WB) and Immunohistochemistry (IHC).
Antibody Catalog number Dilution (WB) Dilution (IHC) Vendor
RUNX-2 #[85]12556 1:1000 1:100 Cell Signaling Technology
OSX sc-393325 1:1000 — Santa Cruz Biotechnology
NFATc1 sc-7294 1:1000 — Santa Cruz Biotechnology
RANKL sc-377079 1:1000 — Santa Cruz Biotechnology
CTSK sc-48353 1:1000 — Santa Cruz Biotechnology
GAPDH E-AB-48016 1:1000 — Santa Cruz Biotechnology
BMP-2 GB11252 — 1:100 Servicebio
COL-1 #[86]72026 — 1:100 Cell Signaling Technology
[87]Open in a new tab
Extraction and characterization of SCSP
Alaskan snow crab (Genus: Chionoecetes, Species: Opilio) caught wild in
the United States. Fresh snow crab shells were cut into ∼1 cm pieces,
crushed into fragments, washed, pH-adjusted to 10 with 0.2 mol/L KOH,
and hydrolyzed at 70°C under constant stirring for 3 h. The hydrolysate
was filtered, neutralized with acetic acid, hydrolyzed using papain for
4 h at 37°C, vacuum-concentrated, precipitated using ethanol, and
vacuum-dried at 40°C. The molecular weights of SCSP were determined
based on viscosity and retention time using a PL aquagel-OH Mixed-H
Column (8 μm, 7.5 × 300 mm, Agilent) coupled with a Refractive Index
Detector (Agilent) and Multi-Angle Laser Light Scattering Detector
(Agilent) at 45°C. Amino acid composition was analyzed by hydrolyzing
samples in 6M hydrochloric acid at 110°C for 22 h, followed by
chromatography with Sulfonic Acid Cation Exchange Resin Columns
(Agilent). Detections were performed at wavelengths of 570 nm and
440 nm.
Experimental animals
Five-week-old female C57BL/6 mice (20 ± 5 g) were purchased from
Home-SPF Biotechnology Co., Ltd. (Beijing, China). Mice were housed in
an SPF-grade facility at Qingdao University under controlled conditions
(25°C ± 3°C, 60%–70% humidity, 12-h light/dark cycle). All animal
procedures followed ethical guidelines approved by the Shandong
Provincial Laboratory Animal Management Committee and the Experimental
Animal Center of Qingdao University (QDU-AEC-2024418).
Ovariectomy (OVX) mice model
Mice were anesthetized, and the surgical area was shaved and sterilized
with iodine. The skin, mucosa, and muscle layers were incised
sequentially, and a dorsal incision was made approximately 2 cm lateral
to the spine at the level of the last rib. Both ovaries were ligated at
the oviduct and excised. A total of 24 female C57BL/6 mice were
randomly assigned into 4 groups: Sham-operated (Sham), osteoporotic
model (OVX), OVX+50 mg/kg SCSP treatment (Low-SCSP), and OVX+100 mg/kg
SCSP treatment (High-SCSP). The SCSP treatment groups received SCSP via
oral gavage daily, while the Sham and OVX groups received equivalent
volumes of saline. All treatments were continued for 8 weeks before
femur collection.
Micro-CT scanning
Femurs were fixed in 4% paraformaldehyde (PFA) and scanned using a
Micro-CT System (Quantum GX2, PerkinElmer, Japan) at 90 kV and 200 μA.
100 layers at the proximal end of the tibial platform were selected for
statistical analysis of cortical layer thickness, trabecular structure,
and bone marrow cavity volume using Analyzer 12.0 Software
(PerkinElmer). The volume of interest (VOI) was positioned at the
proximal tibial metaphysis, starting precisely 0.5 mm distal to the
growth plate to exclude the primary spongiosa and epiphyseal tissue.
100 layers = 1 mm: 100 layers × 10 μm = 1000 μm (1 mm).
Histological staining
Femurs were decalcified, embedded in paraffin, and sectioned for
histological analysis. For H&E staining, sections were deparaffinized,
stained with hematoxylin for 2 min and eosin for 10 s, washed with
water, and imaged. For masson trichrome staining, sections were stained
with Weigert’s iron hematoxylin for 10 min, sequential stained with
acid ethanol, masson blue, aniline blue, and imaged. For TRAP staining,
sections were incubated with TRAP solution at 37°C for 30 min in the
dark, counterstained with hematoxylin, and imaged. All the histological
staining were imaged with a light microscope (Ni-U, Nikon, USA).
Immunohistochemistry (IHC)
Decalcified bone sections were dewaxed, rehydrated, quenched,
antigen-retrievaled, blocked with 5% BSA, incubated overnight at 4°C
with primary antibodies, washed, incubated with secondary antibodies,
counterstained with hematoxylin, and imaged with a light microscope
(Nikon, USA).
Serum biochemical analysis
Urine was collected and centrifuged at 13,000 rpm for 5 min to obtain
the supernatant. Blood was collected via orbital puncture and
centrifuged at 3,000 rpm for 10 min. The concentrations of calcium
(Ca^2+) and inorganic phosphate (Pi) in serum and urine were measured
using Calcium (Ca^2+) colorimetric Assay Kit (E-BC-K103-M, Elabscience)
and [88]Phosphorus (Pi) Colorimetric Assay Kit (E-BC-K245-M,
Elabscience) according to the manufacturer’s instructions. Absorbance
were recorded at 610 nm (Ca^2+) and 660 nm (Pi) using a microplate
reader (SpectraMax iD3, Molecular Devices, USA).
Cell culture and differentiation
Bone marrow mesenchymal stem cells (MSCs) (CP-M131) was purchased from
Pricella. MSCs were cultured in BC-T4 medium (04304P05, Baso)
supplemented with 10% FBS (A5670701, Gibco). Murine MC3T3-E1
preosteoblasts were cultured in α-MEM media supplemented with 10% FBS.
Cultures were passaged every 3-4 days by adding 0.25% trypsin
(25300054, Gibco) for 5-10 min and re-plating at a 1:4 ratio.
Osteogenic differentiation was induced with DMEM medium containing
10 mM β-glycerophosphate (HY-126304, MCE), 100 nM dexamethasone
([89]HY-14648, MCE), and 50 μM L-ascorbic acid (HY-B0166 MCE) for 21
days ([90]Choi et al., 2009). Adipogenic differentiation was induced
with DMEM containing 100 μg/mL 3-isobutyl-1-methylxanthine, 1 μM
dexamethasone ([91]HY-14648, MCE), and 50 μg/mL ascorbic acid for 12
days ([92]Liu et al., 2023). All cultures were maintained at 37 °C and
5% CO[2]. Cell experiments were divided into 4 groups: untreated group
(Normal), differentiated group (Control), 100 μg/mL SCSP group
(Low-SCSP) and 200 μg/mL SCSP group (High-SCSP).
Cell viability assay
5 × 10^3 cells per well were seeded in a 96-well plate and cultured in
complete medium for 24 h. After SCSP treatment for 48 h, 10 μL of CCK-8
(Yeasen) solution was added to each well, and incubated for 2 h at 37°C
in the dark. Absorbance was measured at 450 nm using a full-wavelength
microplate reader (SpectraMax iD3, Molecular Devices, USA).
Cell staining analysis
Cells were fixed with 4% PFA for 20 min and washed, stained with 1% ARS
solution, BCIP/NBT staining solution, or Oil Red O solution, incubated
at room temperature in the dark for 30 min, washed, and imaged with a
light microscope (Ni-U, Nikon, USA).
Western blot analysis
Cells or tissues were lysed with RIPA buffer containing protease and
phosphatase inhibitors (E-BC-R327, Elabscience), homogenized, and
incubated on ice for 10 min. The supernatant protein concentration was
determined using a BCA assay. Protein lysate was resolved on SDS-PAGE
gel and transferred onto a PVDF membrane (1620177, BIO-RED). Blots were
blocked in 5% non-fat dry milk, incubated with primary antibody
overnight at 4°C, washed, incubated with secondary antibody for 1 h at
room temperature, washed, and developed with Super Excellent
Chemiluminescent Substrate Detection Kit (E-IR-R308, Elabscience).
RNA isolation and qPCR
Total RNA was isolated using FreeZol Reagent (R711-01, Vazyme),
precipitated, washed with 70% ethanol and dissolved in H[2]O. 1 μg of
total RNA was reverse transcribed using random hexamers and Hiscript
III Reverse Transcriptase (R302-01, Vazyme). 20 ng cDNA was used in
each RT-qPCR reaction on a CFX96 instrument using Taq Pro Universal
SYBR qPCR Master Mix (Q712-02, Vazyme). The primers used for qPCR were
listed in [93]Table 2.
TABLE 2.
Primer sequences used for qPCR analysis.
Gene name Forward primer (5'→3′) Reverse primer (5'→3′)
Runx-2 ATGCTTCATTCGCCTCACAAA GCACTCACTGACTCGGTTGG
Bmp2 GGGACCCGCTGTCTTCTAGT TCAACTCAAATTCGCTGAGGAC
Opg ACCCAGAAACTGGTCATCAGC CTGCAATACACACACTCATCACT
Rankl CAGCATCGCTCTGTTCCTGTA CTGCGTTTTCATGGAGTCTCA
Gapdh TCCCACTCTTCCACCTTCGATGC GGGTCTGGGATGGAAATTGTGAGG
[94]Open in a new tab
RNA sequencing (RNA-Seq) analysis
RNA libraries were prepared using the VAHTS^® Universal V8 RNA-Seq
Library Prep Kit (NRM605, Vazyme). Sequencing was performed on the
MGI-SEQ 2000 platform. Reads were aligned to the mouse genome (GRCm38)
using HISAT2, and differential expression analysis was conducted using
DESeq2. GO and KEGG pathway enrichment analyses were performed using
WebGestalt.
Statistical analysis
All the data conform to a normal distribution. Data were represented as
means ± SEM. Statistical comparisons were performed using GraphPad
Prism 9.5, employing one-way ANOVA, two-way ANOVA, or Student’s t-test.
Significance was defined as P < 0.05.
Results
Characterization of SCSP
To characterize SCSP, we first quantified its yield following enzymatic
hydrolysis. After 4 h of hydrolysis, SCSP yield reached 12.2% of the
input snow crab shells, significantly higher than the chitin content
(6.42%). Molecular weight distribution analysis revealed that the
5,000-10,000 Da fraction constituted the highest proportion (47.58%),
followed by the 10,000-20,000 Da fraction (27.56%) ([95]Table 3). These
findings indicate that SCSP predominantly consists of low-to
medium-molecular-weight peptides, a property potentially linked to its
functional stability and biological activity. Amino acid composition
analysis ([96]Table 4) showed that glutamic acid (Glu) was the most
abundant (5.91 g/100 g), followed by aspartic acid (Asp, 4.59 g/100 g)
and lysine (Lys, 3.36 g/100 g). Essential amino acids (EAA) constituted
39.21% of the total, while non-essential amino acids (NEAA) accounted
for 60.78%. The presence of both EAA and NEAA, particularly the
enrichment in Glu, Asp, and Lys, suggests that SCSP may possess
substantial bioactive benefits, such as promoting bone health.
TABLE 3.
The molecular weight distribution of SCSP.
Low Limit MW High Limit MW Percent MW
5,000,000 2,73,380,792 0%
200,000 300,000 0%
100,000 200,000 0.94%
50,000 100,000 3.58%
30,000 50,000 6.7%
20,000 30,000 9.42%
10,000 20,000 27.56%
5,000 10,000 47.58%
4,795 5,000 4.21%
[97]Open in a new tab
TABLE 4.
Amino acid composition of SCSP.
Amino Acid Content (g/100 g)
Glu 5.91
Asp 4.59
Lys 3.36
Leu 3.19
Val 2.93
Arg 2.93
Gly 2.41
Ala 2.39
IIe 2.15
Phe 2.09
Ser 1.97
Thr 1.91
Pro 1.70
Met 1.51
Tyr 1.43
His 0.98
[98]Open in a new tab
SCSP improves bone morphology and bone density in OVX mice
To investigate the therapeutic effects of SCSP on osteoporosis (OP), we
utilized an ovariectomy (OVX)-induced osteoporosis mouse model
([99]Figure 1A) and assessed bone morphology, bone mineral density
(BMD), and trabecular parameters. Micro-CT scanning revealed
significant trabecular degradation and reduced BMD in OVX mice compared
to Sham controls, confirming osteoporotic bone loss. Treatment with
SCSP at 50 mg/kg and 150 mg/kg markedly improved BMD and trabecular
architecture, with the 150 mg/kg group showing bone parameters similar
to those observed in the Sham group ([100]Figure 1B). Quantitative
analyses confirmed these findings, demonstrating that BMD, trabecular
number (Tb.N), trabecular thickness (Tb.Th), and trabecular separation
(Tb.Sp) were significantly reduced in the OVX group compared to the
Sham group, while SCSP group showed notably higher as for these
parameters, highlighting the protective effects of SCSP on bone quality
and morphology ([101]Figure 1C).
FIGURE 1.
[102]Illustration of an experimental timeline, micro-CT scans, bar
graphs, and tissue staining results. (A) Timeline shows SCSP
administration and ovariectomy-induced osteoporosis. (B) Micro-CT
images compare bone structure in different treatment groups. (C) Bar
graphs present differences in bone mineral density (BMD), trabecular
number (Tb.N), thickness (Tb.Th), and separation (Tb.Sp) across groups.
Asterisks indicate statistical significance. (D) H&E and Masson
staining reveal bone and tissue morphology. (E) Histological sections
of heart, liver, spleen, lung, and kidney compare different treatments.
[103]Open in a new tab
Snow crab shell-derived polypeptides (SCSP) improves bone morphology
and bone density in ovariectomy (OVX) Mice. (A) Flow chart of animal
experiment: Mice were administrated with SCSP or saline orally for 7
days, followed by bilateral ovariectomy to establish an osteoporosis
model. Bone assessments were conducted on day 56 post-surgery. (B)
Representative Micro-CT images of microstructure of proximal tibia from
Sham-operated (Sham), osteoporotic model (OVX), OVX+50 mg/kg SCSP
treatment (Low-SCSP), and OVX+100 mg/kg SCSP treatment (High-SCSP)
groups. Scale bar, 1 mm. N = 6 mice/group. (C) Quantitative analysis of
bone mineral density (BMD), trabecular number (Tb.N), trabecular
thickness (Tb.Th), and trabecular separation (Tb.Sp) in microstructure
of proximal tibia from Sham, OVX, Low-SCSP, and High-SCSP mice. Each
parameter was measured 3 times, *p < 0.05, **p < 0.01, N = 6
mice/group. (D) Representative H&E (scale bar, 0.2 mm) and masson
images (scale bar, 0.5 mm) of microstructure of proximal tibia from
Sham, OVX, Low-SCSP, and High-SCSP mice. N = 6 mice/group. (E)
Representative H&E images of heart, liver, spleen, lung and kidney
tissues from Sham, OVX, Low-SCSP, and High-SCSP mice. Scale bar,
0.05 mm. N = 6 mice/group.
Histological analysis using H&E and Masson’s staining further validated
these structural improvements. Bone tissue integrity and collagen fiber
distribution were severely disrupted in OVX mice, whereas SCSP
administration preserved these features, particularly in the 150 mg/kg
group ([104]Figure 1D). Importantly, SCSP treatment did not induce
histological abnormalities in major organs, including the heart, liver,
spleen, lungs, and kidneys, across all experimental groups, as
evidenced by H&E staining ([105]Figure 1E).
These findings demonstrate that SCSP alleviates OVX-induced bone loss
by improving BMD, restoring trabecular architecture, and preserving
bone tissue integrity, without inducing systemic toxicity.
SCSP re-establishes the osteoblast/osteoclast balance in OVX mice
OP is characterized by an imbalance between osteoblast-mediated bone
formation and osteoclast-driven bone resorption. To assess whether SCSP
modulates this balance, we analyzed markers of osteoblast and
osteoclast activity. IHC staining showed significantly reduced
expression of osteoblast markers (BMP-2, RUNX-2, and COL-1) in OVX
mice, reflecting impaired osteoblast function ([106]Figure 2A). SCSP
administration restored these markers in a dose-dependent manner, with
the 150 mg/kg group achieving levels comparable to Sham controls.
Western blot analysis further confirmed increased expression of RUNX-2
and OSX in SCSP-treated groups ([107]Figure 2B). In contrast, TRAP
staining demonstrated a significant increase in osteoclast numbers in
OVX mice, indicative of enhanced bone resorption ([108]Figure 2C). SCSP
treatment significantly reduced osteoclast numbers, particularly at the
150 mg/kg dose, where levels were comparable to Sham controls. Western
blot analysis revealed elevated expression of osteoclast-related
proteins (NFATc1, RANKL, and CTSK) in OVX mice, which was markedly
reduced by SCSP treatment ([109]Figure 2D).
FIGURE 2.
[110]Panel A shows immunohistochemistry images for BMP-2, RUNX-2, and
COL-1 across four groups: Sham, OVX, Low-SCSP, and High-SCSP. Panel B
displays Western blot results for RUNX-2, OSX, and GAPDH, with bar
graphs showing relative protein expression. Panel C includes TRAP
staining images for the same groups. Panel D presents Western blot
results for NFATc1, RANKL, CTSK, and GAPDH, with corresponding bar
graphs of protein expression. Statistical significance is indicated
with asterisks.
[111]Open in a new tab
SCSP maintains the osteoblast/osteoclast balance in OVX Mice. (A)
Representative immunohistochemistry (IHC) images of microstructure of
proximal tibia stained for BMP-2, RUNX-2, and COL-1 from Sham, OVX,
Low-SCSP, and High-SCSP mice. Scale bar, 0.02 mm. N = 6 mice/group. (B)
Western blot analysis of RUNX-2 and OSX expression of microstructure of
proximal tibia from Sham, OVX, Low-SCSP, and High-SCSP mice. All assays
were repeated 3 times, *P < 0.05, **P < 0.01. (C) Representative TRAP
images of microstructure of proximal tibia from Sham, OVX, Low-SCSP,
and High-SCSP mice. Scale bar, 0.05 mm. N = 6 mice/group. (D) Western
blot analysis of NFATc1, RANKL, and CTSK expression of microstructure
of proximal tibia from Sham, OVX, Low-SCSP, and High-SCSP mice. All
assays were repeated 3 times, *P < 0.05, **P < 0.01, ***P < 0.001.
With these data in hand, we examined the effects of SCSP on calcium
homeostasis, which is often disrupted in OP. As expected, OVX mice
exhibited reduced urinary calcium excretion (0.18 ± 0.52 mmol/L) and
elevated serum phosphorus levels (2.46 ± 0.26 mmol/L). SCSP treatment
increased urinary calcium and phosphorus levels above both Sham and OVX
groups ([112]Table 5). Suggesting that SCSP modulates calcium and
phosphorus metabolism, potentially counteracting the metabolic
disruptions induced by ovariectomy.
TABLE 5.
Levels of serum calcium, urinary calcium, serum phosphorus and urinary
phosphorus in mice (
[MATH: X¯
:MATH]
± S).
Group Blood Ca2+ (mmol/L) Urine Ca2+ (mmol/L) Blood Pi (mmol/L) Urine
Pi (mmol/L)
Sham 2.20
[MATH: ± :MATH]
0.16 1.22
[MATH: ± :MATH]
0.18 1.60
[MATH: ± 0 :MATH]
.08 46.74
[MATH: ± :MATH]
1.41
OVX 2.68
[MATH: ± :MATH]
0.05 0.18
[MATH: ± :MATH]
0.52 2.46
[MATH: ± :MATH]
0.26 11.65
[MATH: ± :MATH]
0.93
Low-SCSP 2.36
[MATH: ± :MATH]
0.03 1.05
[MATH: ± :MATH]
0.22 3.92
[MATH: ± :MATH]
0.075 23.53
[MATH: ± :MATH]
2.27
High-SCSP 2.60
[MATH: ± :MATH]
0.13 2.75
[MATH: ± :MATH]
0.31 4.76
[MATH: ± :MATH]
0.57 49.53
[MATH: ± :MATH]
1.03
[113]Open in a new tab
These results demonstrate that SCSP re-establishes the
osteoblast/osteoclast balance by enhancing osteoblast activity,
inhibiting osteoclast-driven bone resorption, and normalizing calcium
and phosphorus metabolism.
SCSP promotes osteogenesis and inhibit adipogenesis
To further elucidate the effects of SCSP on bone regeneration, we
examined its influence on osteoblast proliferation, differentiation,
and mineralization using MC3T3-E1 preosteoblasts cells. CCK-8 assays
revealed a dose-dependent increase in osteoblast viability with SCSP
treatment ([114]Figure 3A). ARS staining indicated enhanced mineralized
nodule formation and calcium deposition in SCSP-treated groups,
particularly at the high dose. ALP staining confirmed enhanced early
osteogenic differentiation ([115]Figure 3B). qPCR analysis demonstrated
upregulation of osteogenic genes, including Runx-2, Bmp-2, and Opg, and
downregulation of Rankl expression ([116]Figure 3C). Western blot
analysis corroborated these findings, showing increased protein levels
of RUNX-2 and OSX and decreased expression of NFATc1, RANKL, and CTSK
([117]Figure 3D), suggest the role of SCSP in maintaining
osteoblast/osteoclast balance.
FIGURE 3.
[118]Panel A displays a bar graph of cell viability percentages at
varying concentrations of SCSP over 24 hours. Panel B includes images
showing the staining of samples using Alizarin Red S and ALP under
different conditions: Normal, Control, Low-SCSP, and High-SCSP. Panel C
features bar graphs depicting mRNA expression of genes Runx-2, Bmp-2,
Opg, and Rankl relative to Normal across the same conditions, marked
with significance levels. Panel D presents protein expression analysis
via Western blots for RUNX-2, OSX, NFATc1, RANKL, CTSK, and GAPDH,
accompanied by corresponding bar graphs for expression levels,
displaying statistical significance.
[119]Open in a new tab
SCSP promotes osteogenic activity of MC3T3-E1 cells. (A) CCK8 analysis
of MC3T3-E1 in the presence of SCSP ranging from 50 to 1,600 μg/mL. All
assays were repeated 3 times, *P < 0.05, **P < 0.01. (B) Representative
Alizarin Red S and ALP images of MC3T3-E1 cells treated with 100 and
200 μg/mL SCSP. All assays were repeated 3 times. Scale bar, 100 μm.
(C) qPCR analysis of Runx-2, Bmp-2, Opg and Rankl expression from
MC3T3-E1 cells treated with 100 and 200 μg/mL SCSP. All assays were
repeated 3 times. *P < 0.05, **P < 0.01. (D) Western blot analysis of
RUNX-2, OSX, NFATc1, RANKL and CTSK expression from MC3T3-E1 cells
treated with 100 and 200 μg/mL SCSP. All assays were repeated 3 times.
*P < 0.05, **P < 0.01.
Similarly, MSCs, which can undergo both osteogenesis and adipogenesis
exhibit enhanced osteogenic differentiation capacity in the presence of
SCSP. ARS staining showed increased mineralized nodule formation in the
SCSP-treated groups, indicating enhanced osteogenesis ([120]Figure 4A).
Western blot analysis confirmed these findings, with increased RUNX-2
and OSX expression and decreased NFATc1, RANKL, and CTSK levels in
SCSP-treated groups ([121]Figure 4B). In the contrast, SCSP treatment
reduced lipid accumulation, suggesting an inhibitory effect on
adipogenic differentiation ([122]Figure 4C).
FIGURE 4.
[123]Panel A shows four images of cell cultures stained with Alizarin
Red S, labeled Normal, Control, Low-SCSP, and High-SCSP, depicting
varying mineralization levels. Panel B presents Western blot results
and graphs indicating protein expression levels of RUNX-2, OSX, NFATc1,
RANKL, and CTSK across the same conditions, with significant
differences marked by asterisks. Panel C contains four images of cell
cultures stained with Oil Red O, demonstrating lipid accumulation under
the same four conditions, with varying intensities.
[124]Open in a new tab
SCSP promotes osteogenic differentiation and inhibits adipogenic
differentiation of mesenchymal stem cells (MSCs). (A) Representative
Alizarin Red S images of MSCs differentiated osteoblasts treated with
100 and 200 μg/mL SCSP. Scale bar, 100 μm. All assays were repeated 3
times. (B) Western blot analysis of RUNX-2, OSX, NFATc1, RANKL, and
CTSK expression from MSCs differentiated osteoblasts treated with 100
and 200 μg/mL SCSP. All assays were repeated 3 times. *P < 0.05, **P <
0.01, ***P < 0.001. (C) Representative Oil Red O images of MSCs
differentiated adipocytes, Scale bar, 50 μm. All assays were repeated 3
times.
These findings highlight SCSP’s capacity to bias MSCs differentiation
toward osteogenesis, promoting bone formation while inhibiting
adipogenesis, and osteoclastgenesis.
SCSP modulate cell cycle prograssion, inflammatory response, and motor
protein activity in osteoblasts
To obtain molecular insights into the observed above mentioned bias
toward to osteogenesis, we performed RNA-Seq analysis on MC3T3-E1
differentiated osteoblast cells treated with and without 200 μg/mL of
SCSP. A total of 2,410 genes were upregulated and 1,837 genes were
downregulated in SCSP-treated cells compared to controls ([125]Figure
5A). KEGG pathway enrichment analysis identified significant
involvement of the Cell Cycle, inflammation, and Motor Proteins
pathways ([126]Figure 5B). Key genes involved in the Cell Cycle pathway
included Ccnb1, Ttk, Ndc80, Ccnb2, Cdc20, Espl1, Plk1, and Cdc25. Among
these, Plk1, Ccnb2, and Ccnb1 are closely associated with the FoxO
signaling pathway, a key regulator of osteoblast survival, oxidative
stress, and bone remodeling. SCSP treatment also modulated inflammatory
responses, altering expression of IL-17 signaling, Toll-like receptor
signaling, and rheumatoid arthritis-related genes, including Ccl2,
Il17re, Fosl1, Mmp13, Ccl5, Tlr1, Il12b, and Atp6v1b1. Notably, SCSP
significantly upregulated genes associated with Motor Protein activity,
including Myo5c, Kif20a, Kif18b, Kif4, Kif23, Kif2c, Cenpe, Kif20b,
Kif14, and Myh7b.
FIGURE 5.
[127]Panel A displays a heatmap showing gene expression levels between
control and SCSP groups, with red indicating upregulation and blue
indicating downregulation. Panel B presents a dot plot illustrating
enriched pathways ranked by rich factor, with color denoting
significance level and size indicating gene count. Pathways include the
cell cycle and motor proteins.
[128]Open in a new tab
SCSP responsive genes/signaling pathways involve in osteoblasts
activity. (A) Heatmap represents differential gene expression in
RNA-Seq analysis of MC3T3-E1 cells treated with 200 μg/mL SCSP. The
data from 3 biological repeat are shown as fold change greater than 2
and p values less than 0.05 were considered differentially expressed.
(B) Analysis of KEGG pathway of differentially expressed genes from
MC3T3-E1 cells treated with or without 200 μg/mL SCSP.
These findings suggest that SCSP enhances osteoblast activity via cell
cycle regulation and immunomodulation, and simultaneously modulating
cytoskeletal function through Motor Proteins.
Discussion
OP has emerged as a significant global public health issue, affecting
millions worldwide ([129]Ma C. et al., 2023). Current therapeutic
strategies offer only short-term symptom relief without addressing the
underlying disease mechanisms ([130]Ma M. et al., 2023). In this study,
we identify SCSP as a promising candidate for OP treatment,
demonstrating its potential to effectively modulate the bone remodeling
process by targeting key molecular pathways involved in
osteoclastogenesis, as well as osteoblast differentiation and
functions.
Our data reveal that SCSP has a molecular weight primarily within the
range of 5,000-10,000 Da. Notably, smaller peptides, such as
dipeptides, tripeptides, and oligopeptides, are more readily absorbed
across the intestinal epithelium compared to larger proteins
([131]Santos et al., 2012), suggesting that SCSP may undergo enzymatic
degradation within the gastrointestinal tract, facilitating its
absorption. The ideal molecular weight range for optimal oral
bioavailability in the context of OP treatment requires further
investigation to enhance SCSP absorption. Strategies such as optimizing
enzymatic hydrolysis conditions or utilizing alternative enzymes to
reduce peptide size could improve bioavailability, thereby enhancing
its therapeutic potential ([132]Saiwong et al., 2023; [133]Nikoo et
al., 2023; [134]Nikoo et al., 2022). The amino acid composition of SCSP
is also noteworthy. Amino acids are pivotal in mitigating age-related
bone loss, enhancing bone mass, and promoting osteoblast proliferation
and differentiation while concurrently suppressing osteoclast activity.
Glu has been shown to be essential for osteoclast differentiation and
function, as it supports the high energy demands of osteoclasts through
its metabolic conversion to α-ketoglutarate, which feeds into the
tricarboxylic acid cycle ([135]Indo et al., 2013). This metabolic
pathway is vital for osteoclast activity. Moreover, studies have
demonstrated that depriving culture media of Glu inhibits osteoclast
differentiation, indicating its critical role in osteoclastogenesis and
bone resorption ([136]Huang et al., 2021). Asp, as part of amino acid
metabolism, may influence overall metabolic balance, indirectly
impacting OP progression. Intriguingly, Lys, as a NEAA, promotes
osteoblastogenesis by facilitating collagen crosslinking, an essential
component of bone matrix formation ([137]Goldberga et al., 2018;
[138]Jenni et al., 2016). Our chemical analysis data revealed that SCSP
is abundant in Glu, Asp, and Lys, which may collectively contribute to
significant bone preservation in OVX-OP models. Of interest, protein
sequence and activity may vary among crustacean species. Therefore,
further characterization of these crustacean shell peptides, including
factors such as structure, charge, hydrophobicity, stability, binding
affinity, and delivery mechanisms, is essential to achieve optimal
therapeutic efficacy ([139]Kannan et al., 2011; [140]Zeng et al., 2021;
[141]Sharayei et al., 2021).
We show that SCSP mitigates OP progression by restoring the
osteoblast/osteoclast balance, which is disrupted due to estrogen
deficiency in OVX mouse model, a central trigger for RANKL/OPG
dysregulation. This imbalance results in: (i) increased osteoclast
activity, as estrogen normally inhibits osteoclast formation and
promotes osteoclast apoptosis; (ii) reduced osteoblast activity, as
estrogen stimulates osteoblast differentiation and function; and (iii)
a net bone loss due to a greater rate of bone resorption than bone
formation. While current treatments predominantly focus on inhibiting
bone resorption to reduce bone loss, anti-resorptive agents alone
cannot restore lost bone structure. In contrast, SCSP treatment
addresses both osteoclast inhibition and osteoblast stimulation, making
it a promising strategy for promoting bone regeneration. SCSP treatment
significantly reduces the RANKL/OPG ratio, suppresses
osteoclastogenesis, and enhances osteoblastic differentiation and
function, as evidenced by the upregulation of osteogenic markers
(RUNX2, OSX) and downregulation of osteoclast markers (NFATc1, RANKL,
CTSK) in MSC and osteoblast models.
Our transcriptome analyses show that SCSP modulates pathways associated
with the cell cycle, inflammatory responses, and motor protein
dynamics. Cell cycle dysregulation is a hallmark of impaired bone
metabolism, with senescent MSCs exhibiting reduced osteogenic potential
and increased adipogenesis ([142]Khosla et al., 2018). Senescent
osteocytes and osteoclasts also secrete senescence-associated secretory
phenotype factors, including pro-inflammatory cytokines, chemokines,
oxidative stress mediators, and proteases, which collectively disrupt
bone homeostasis ([143]Fa et al., 2017; [144]Collison, 2017;
[145]Paccou et al., 2019). We find that SCSP treatment inhibits Plk1
expression, supporting the differentiation and function of bone-forming
cells while preventing premature senescence ([146]Sütterlin et al.,
2001; [147]Peng et al., 2023). In addition, SCSP modulates inflammatory
cascades by attenuating IL-17 signaling ([148]Byravan et al., 2024;
[149]Peng et al., 2024), Toll-like receptor pathways ([150]Carroll et
al., 2025; [151]He et al., 2016), and key genes implicated in
inflammatory bone diseases ([152]Lo et al., 2024), such as Ccl2,
Il17re, Fosl1, Mmp13, Ccl5, Tlr1, Il12b, and Atp6v1b1. These
anti-inflammatory effect likely contributes to the preservation of bone
integrity in inflammatory OP contexts. Intriguingly, motor proteins,
including myosin, dynein, and kinesin, are integral to intracellular
transport ([153]Vale, 2003), mitosis ([154]Celestino et al., 2022), and
cytoskeletal dynamics in osteoblasts and osteoclasts ([155]Mikhajlov et
al., 2025; [156]Qiu et al., 2012; [157]Santos-Ledo et al., 2017).
SCSP’s influence on motor protein expression may enhance cellular
trafficking and division, thereby supporting bone formation and
remodeling processes.
Apart from the potent anti-osteoporotic effects, SCSP presents a
favorable safety profile and cost-effective nature, which further
strengthens its potential as a novel peptide-based therapeutic for OP.
Thus, SCSP holds promise not only for the treatment of OP but also for
broader applications in other skeletal diseases, providing a versatile
therapeutic option for bone health management.
Funding Statement
The author(s) declare that financial support was received for the
research and/or publication of this article. This work was funded by
grants from National Natural Science Foundation of China (81871231 to
BL; 32070859 to ZW; 32200653 to XX), Natural Science Foundation of
Shandong Province (ZR2020MC083 to ZW; ZR202209280042 to BL; ZR2021MH350
to JL), Shandong Taishan Scholars Program of Shandong Province
(TS20190931 to ZW; TSQN202103056 to BL), Qingdao Natural Science
Foundation Key Project (24-8-4-zrjj-8-jch to BL), the Science, and
Education and Industry Integration Innovation Pilot Project from Qilu
University of Technology (Shandong Academy of Sciences) (2024ZDZX14 to
DW), Science and Technology Program Development project of Qingdao city
south district (2023-2-020-YY to JL) and The Youth Fund of Qingdao
University Affiliated Hospital (QDFY + X2023126 to JL).
Data availability statement
The data generated in the present study can be found in the NCBI
Sequence Read Archive database under accession number PRJNA1291493, or
at the following URL:
[158]https://www.ncbi.nlm.nih.gov/sra/PRJNA1291493.
Ethics statement
Ethical approval was not required for the studies on humans in
accordance with the local legislation and institutional requirements
because only commercially available established cell lines were used.
The animal study was approved by Ethics Committee of Qingdao University
Medical Science Center. The study was conducted in accordance with the
local legislation and institutional requirements.
Author contributions
XD: Writing – original draft, Conceptualization, Funding acquisition,
Data curation, Formal analysis. GZ: Data curation, Writing – original
draft. CS: Writing – original draft, Formal analysis. HZ: Writing –
original draft, Investigation. JZ: Writing – original draft,
Investigation. XL: Investigation, Writing – original draft. XX: Writing
– original draft, Methodology. JL: Writing – original draft, Data
curation. XZ: Writing – original draft, Methodology. YZ: Writing –
original draft, Resources. LL: Writing – original draft, Software. ST:
Software, Writing – original draft. DW: Writing – original draft,
Supervision. ZW: Supervision, Validation, Writing – review and editing,
Funding acquisition, Visualization. BL: Writing – review and editing,
Supervision, Visualization, Project administration, Validation.
Conflict of interest
The authors declare that the research was conducted in the absence of
any commercial or financial relationships that could be construed as a
potential conflict of interest.
Generative AI statement
The author(s) declare that no Generative AI was used in the creation of
this manuscript.
Publisher’s note
All claims expressed in this article are solely those of the authors
and do not necessarily represent those of their affiliated
organizations, or those of the publisher, the editors and the
reviewers. Any product that may be evaluated in this article, or claim
that may be made by its manufacturer, is not guaranteed or endorsed by
the publisher.
References