Novel Peptides from Sturgeon Ovarian Protein Hydrolysates Prevent Oxidative Stress-Induced Dysfunction in Osteoblast Cells: Purification, Identification, and Characterization

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Novel Peptides from Sturgeon Ovarian Protein Hydrolysates Prevent
Oxidative Stress-Induced Dysfunction in Osteoblast Cells:
Puriﬁcation, Identiﬁcation, and Characterization
Ruichang Gao,*Lingling Zhu, Wei Zhang, Wengang Jin, Fan Bai, Peng Xu, Jinlin Wang, Quancai Sun,
Zitao Guo, and Li Yuan*
Cite This: J. Agric. Food Chem. 2024, 72, 10076−10088
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sı Supporting Information
ABSTRACT: This study aimed to explore antioxidant peptides derived from sturgeon (Acipenser schrenckii) ovaries that exhibit
antiosteoporotic eects in oxidative-induced MC3T3-E1 cells. The F3−15 component obtained from sturgeon ovarian protein
hydrolysates (SOPHs) via gel ﬁltration and RP-HPLC signiﬁcantly increased the cell survival rate (from 49.38 ±2.88 to 76.26 ±
2.09%). Two putative antioxidant-acting peptides, FDWDRL (FL6) and FEGPPFKF (FF8), were screened from the F3−15 faction
via liquid chromatography−tandem mass spectrometry (LC−MS/MS) and through prediction by computer simulations. Molecular
docking results indicated that the possible antioxidant mechanisms of FL6 and FF8 involved blocking the active site of human
myeloperoxidase (hMPO). The in vitro tests showed that FL6 and FF8 were equally adept at reducing intracellular ROS levels,
increasing the activity of antioxidant enzymes, and protecting cells from oxidative injuries by inhibiting the mitogen-activated protein
kinase (MAPK) pathway and activating the phosphoinositide-3 kinase (PI3K)/protein kinase B (AKT)/glycogen synthase kinase-3β
(GSK-3β) signaling pathway. Moreover, both peptides could increase dierentiation and mineralization abilities in oxidatively
damaged MC3T3-E1 cells. Furthermore, FF8 exhibited high resistance to pepsin and trypsin, showcasing potential for practical
applications.
KEYWORDS: sturgeon ovary, antioxidant peptides, separation and puriﬁcation, MC3T3-E1 cells, osteoblast activity
■INTRODUCTION
Reactive oxygen species (ROS) are produced during cellular
respiration and play essential roles as mediators in diverse
biological phenomena, encompassing cellular proliferation,
apoptosis, and transmission of molecular signals in organisms.
These ROS entail hydroxyl radicals (•OH) and superoxide
anion radicals (O2•−), representing vital constituents of this
intricate network.
1
However, ROS can lead to oxidative stress,
which results in tissue damage and chronic diseases, including
skin aging, and osteoporosis.
2,3
Mesenchymal stem cells
(MSCs) must undergo osteogenic dierentiation for the growth
and maintenance of bone. Nonetheless, MSCs undergo
metabolic alterations, characterized by a dwindling of glycolytic
activity and a heightened reliance on mitochondrial respiration,
to maintain an adequate energy supply for dierentiation, which
coincides with an upsurge in the production of ROS.
4
On the
other hand, human myeloperoxidase (hMPO), an endogenous
enzyme released from neutrophils, has the potential to intensify
oxidative stress by directly stimulating the generation of ROS
and reactive nitrogen species.
5
Normally, excessive ROS can be
balanced by endogenous antioxidant defense systems, consisting
of enzymatic antioxidant systems in the body.
6
Therefore, the
intrinsic activity of antioxidant enzymes is generally an indicator
used to evaluate the intracellular ROS level and cell vitality of
osteoblasts.
7
Therefore, decreasing ROS production, enhancing
the activity of antioxidant enzymes, and suppressing hMPO
activity via antioxidants could potentially serve as promising
strategies for mitigating bone loss.
In recent years, with the presence of an aging society, research
on methods to prevent and mitigate ROS-induced osteoporosis
or other related bone metabolic disorders has been increasingly
investigated.
3
One eective method is the supplementation of
antioxidants.
8
Bioactive peptides are commonly employed in
research investigations involving anti-inﬂammatory, antioxidant,
and antiallergic eects, because of their diversity of sources, ease
of absorption, safety, and nontoxicity.
9
The antioxidant peptides
produced from pilose antler, casein, and walleye pollock skin
have been shown to successfully reduce oxidative stress in
osteoblasts.
10−12
Various methods are used to screen for
bioactive peptides. However, the puriﬁcation procedures
involved in these studies required substantial eort and achieved
a relatively low eciency. In contrast, technology combining
peptidomics and computer simulation prediction (PeptideR-
anker, molecular docking, etc.) could greatly reduce the isolation
steps and expand the range of peptide screening. These
Received: September 27, 2023
Revised: March 9, 2024
Accepted: April 6, 2024
Published: April 17, 2024
Articlepubs.acs.org/JAFC
© 2024 American Chemical Society 10076
https://doi.org/10.1021/acs.jafc.3c07021
J. Agric. Food Chem. 2024, 72, 10076−10088
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technologies have been successfully used to screen for hMPO

inhibitors.

Sturgeon (Acipenser schrenckii) is famous for its caviar and

skin. However, the rest of the ﬁsh body is usually discarded

instead of exploited, which contradicts the long life cycle and

costly farming of sturgeon. Additionally, sturgeon byproducts

show great potential for utilization and are rich in protein, lipids,

chondroitin sulfate, and other nutrients. Gao et al. reported that

peptides screened from sturgeon meat via column chromatog-

raphy exhibited antioxidative and anti-inﬂammatory activities.

Gao et al. demonstrated the signiﬁcant ecacy of sturgeon skull-

derived chondroitin sulfate in mitigating weight gain among

mice subjected to a high-fat dietary regimen.

Therefore, the

ability to eectively utilize sturgeon byproducts is very

important. Previous studies have shown that the ovarian protein

of sturgeon is abundant in hydrophobic amino acids, and the

amount of hydrophobic amino acids is often used as a basis for

screening antioxidant peptides.

Therefore, ovarian proteins

might be a good source of antioxidant peptides. This study aims

to identify antioxidant peptides from sturgeon ovarian protein

hydrolysates (SOPHs) via continuous chromatography, com-

puter simulation prediction technology (PeptideRanker and

molecular docking), and an MC3T3-E1 oxidative damage cell

model induced by hydrogen peroxide (H2O2), which is the main

substance released by hMPO. Moreover, the molecular

mechanisms of the screened antioxidant peptides in reducing

cell oxidative damage were investigated via the mitogen-

activated protein kinase (MAPK) and phosphoinositide-3

kinase (PI3K)/protein kinase B (AKT)/glycogen synthase

kinase-3β(GSK-3β) signaling pathways. The ﬁndings of this

investigation oer a theoretical foundation for the high-value use

of sturgeon byproducts.

■MATERIALS AND METHODS

Chemicals. Ovaries of the sturgeon were obtained from Quzhou

Xunlong Aquatic Products Sci-tech Development Co., Ltd. (Quzhou,

China) and stored at −20 °C until use. The peptides FDWDRL and

FEGPPFKF (purity >95%) were provided by APeptide Co., Ltd.

(Shanghai, China). Cell Counting Kit-8 (CCK-8) and alcalase were

obtained from Shanghai Yuanye Biotechnology Co., Ltd. (Shanghai,

China). 2,2′-Azino-bis(3-ethylbenzthiazoline)-6-sulfonic acid (ABTS),

pepsin, and trypsin were purchased from Aladdin Industrial

Corporation (CA). Fetal bovine serum (FBS) was obtained from

Invitrogen Biological Industries (CA). Minimum Essential Medium α

Modiﬁcation (α-MEM) was purchased from Biosharp Technology Co.,

Ltd. (Shanghai, China). The bicinchoninic acid (BCA) protein assay kit

was obtained from the Nanjing Jiancheng Institute of Biological

Engineering (Nanjing, China). The alkaline phosphatase (ALP)

activity assay kit, alizarin red S staining kit for osteogenesis, SOD and

CAT detection kits, penicillin, streptomycin, resveratrol (RES),

dimethyl sulfoxide, and phosphate-buered saline (PBS) were obtained

from Beyotime Biotechnology (Shanghai, China). The YF 488-Annexin

V/PI apoptosis kit was purchased from Nanjing Puen Biotechnology

Co., Ltd. (Nanjing, China). Antibodies against c-jun N-terminal kinase

(JNK), phosphorylated JNK (p-JNK), p38 mitogen-activated protein

kinase (p38 MAPK), p-p38 MAPK, extracellular-signal-regulated

protein kinase (ERK), p-ERK, phosphoinositide-3 kinase (PI3K), p-

PI3K, protein kinase B (AKT), p-AKT, glycogen synthase kinase-3β

(GSK-3β), p-GSK-3β(Ser9), and glyceraldehyde-3-phosphate dehy-

drogenase (GAPDH) were supplied by Cell Signaling Technology

(Beverly, MA). Hydrogen peroxide (H2O2), L-ascorbic acid (Vc), and

β-glycerophosphate were obtained from Sinopharm Chemical Reagent

Co., Ltd. (Shanghai, China). All other chemicals used in the

experiments were of analytical grade.

Preparation of SOPHs. The sturgeon ovaries were homogenized in

a 1:3 (w/v) ratio with distilled water, followed by sonication for 30 min.

Subsequently, alcalase (4000 U/g of prot) was incorporated into the

solution, and the pH was regulated to 9.0. The hydrolysis process was

carried out at 55 °C for 5 h. The solution was heated in a water bath at

95 °C for 15 min to terminate the reaction. The supernatant was

obtained through centrifugation (Model TGl-16gR, Shanghai Anting

Scientiﬁc Instrument Factory, China) at 10,000 rpm for 15 min and

subsequently freeze-dried.

ABTS+•Scavenging Activity. The ABTS+•assay was carried out

following a previously described with a slight modiﬁcation.

Samples of

various concentrations (20 μL) were combined with 2 mL of ABTS+•

solution and incubated in the dark at 25 °C for 10 min. Subsequently,

the absorbance was measured at 734 nm. Distilled water was used as a

substitute for the samples in the blank group. The samples were

subjected to triplicate testing with Vc serving as the positive control.

The ABTS+•radical scavenging activity was determined using the

following formula:

A A

ABTS radical scavenging activity (%)

( ) 100%

blank sample

blank

= ×

+•

where Asample and Ablank, respectively, denote the absorbance values of

the sample tube and the blank tube. The IC50 value is deﬁned as the

antioxidant compound concentration necessary to neutralize 50% of

the ABTS+•radical.

Hydroxyl Radical (OH•) Scavenging Activity. The assay was

determined via a method described by Agrawal et al.

First, 0.5 mL of

FeSO4solution (9 mM), 0.5 mL of salicylic acid-ethanol solution (9

mM), and 0.5 mL of sample solution were mixed with 0.5 mL of H2O2

solution (9 mM) and then diluted to 5 mL with distilled water.

Furthermore, the solution was tested for absorbance at 510 nm after

being submerged in a water bath at 37 °C for 30 min. Vc served as a

positive control. OH•scavenging capacity was evaluated using the

following formula:

A A A

hydroxyl radical scavenging capacity (%)

( ) 10

control blank

sample

= ×

where Acontrol,Asample, and Ablank, respectively, represent the absorbance

value of the control tube, sample tube, and blank tube.

Separation and Puriﬁcation of SOPHs. In this experiment,

ultraﬁltration and puriﬁcation were carried out in three steps. The

partial enzymatic hydrolysate underwent fractionation via ultraﬁltration

using membranes with dierent molecular weight cutos (100, 10, and

3 kDa, Millipore Co., MA). Subsequently, all fractions (molecular

weight (MW) > 100, 10−100, 3−10, and <3 kDa) were freeze-dried for

subsequent analysis. The fraction with the most potent antioxidative

eects was then chosen for subsequent puriﬁcation. The fraction with

antioxidative properties was processed through a Sephadex G-25 gel

column (1.6 cm ×60 cm, GE Healthcare, DE), in which elution was

performed using distilled water at a ﬂow rate of 1.0 mL/min. The

elution was monitored at 280 nm, and subsequent fractions were

collected and subjected to freeze-drying for further analysis. After

separation by Sephadex G-25 gel ﬁltration, the fraction with the most

potent antioxidative activity was solubilized in deionized water. The

sample solution was injected into the preparation RP-HPLC (Yuexu

Company, Shanghai, China) through a C18 preparative column (2.12

cm ×25 cm, 10 μm, Yuexu Company, Shanghai, China). The column

was subjected to gradient elution using acetonitrile (0−50%) at a ﬂow

rate of 5 mL/min. The elution peak was detected at 280 nm, and the

component exhibiting the most potent antioxidant activity was selected

for subsequent analysis.

Identiﬁcation of Peptide Sequences by Liquid Chromatog-

raphy−Tandem Mass Spectrometry (LC−MS/MS). The peptides

were reconstituted in solvent A (A, 0.1% formic acid in water) and

subsequently evaluated through Orbitrap Fusion linked with an EASY-

nanoLC 1200 system (Thermo Fisher Scientiﬁc, MA). A 3 μL peptide

sample was loaded onto a 25 cm analytical column (75 μm inner

Journal of Agricultural and Food Chemistry pubs.acs.org/JAFC Article

https://doi.org/10.1021/acs.jafc.3c07021

J. Agric. Food Chem. 2024, 72, 10076−10088

10077

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