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Jiangsu Engineering Research Center of Cardiovascular Drugs Targeting Endothelial Cells, College of Health Sciences, School of Life Sciences, Jiangsu Normal University, Xuzhou 221116, Jiangsu, PR ChinaSchool of Chemistry and Chemical Engineering, Harbin Institute of Technology, Harbin 150090, Heilongjiang, PR China
Jiangsu Engineering Research Center of Cardiovascular Drugs Targeting Endothelial Cells, College of Health Sciences, School of Life Sciences, Jiangsu Normal University, Xuzhou 221116, Jiangsu, PR China
Jiangsu Engineering Research Center of Cardiovascular Drugs Targeting Endothelial Cells, College of Health Sciences, School of Life Sciences, Jiangsu Normal University, Xuzhou 221116, Jiangsu, PR China
Milk fat globule-EGF factor VIII (MFG-E8) has been identified as an important source of bioactive peptides, which may exert a pivotal role in regulating biologic redox equilibrium. However, the composition of MFG-E8 polypeptides and their mechanisms on mitigating sarcopenia remain unknown. The aim of this study was to identify the composition of MFG-E8 polypeptides and its effects against oxidative stress in dexamethasone-induced L6 cell injury. Simulated digestion in vitro and liquid chromatography-tandem mass spectrometry were used in this investigation. A total of 95 peptides were identified during complete simulated digestion; among them, the contents of 21 peptides were analyzed, having been determined to exceed 1%. Molecular docking assay found that IDLG, KDPG, YYR, and YYK exhibited high binding affinity with keap1. MTT, dichlorodihydrofluorescein diacetate, mito- and lyso-tracker, and transmission electron microscope assay demonstrated that IDLG and KDPG can alleviate oxidative stress-injured L6 cell vitality, mitochondria activity, vacuolation, and function decrease, and increased autophagy, thereby improving mitochondrial homeostasis. From a molecular perspective, IDLG and KDPG can decrease the expression of keap1 and increase the expression of Nrf2, HO-1, and PGC-1α. Therefore, MFG-E8-derived IDLG and KDPG could be potential polypeptides countering oxidative stress in the treatment of sarcopenia, via the keap1/Nrf2/HO-1 signaling pathway.
). Concomitant to physical alteration, changes of energy metabolism, mitochondria function, and occurrence of inflammatory reaction are accompanied by time-dependent deterioration of cellular function (
). Chronic elevation of mitochondrial reactive oxygen species (ROS) induces oxidant/antioxidant imbalance, decreased mitochondrial volume, morphological changes, and attenuated Nrf2 pathway activity, which appears to be a common feature of dexamethasone (dex)-induced skeletal muscle atrophy among elderly people. Inactivation of the keap1/Nrf2/HO-1 system in aged muscle exerts an adverse effect on clearance of ROS and exercise efficacy (
). Moreover, mitochondrial function exerts a crucial role in maintaining cellular energy expenditure and redox equilibrium. Rapid decrements of respiratory capacity and coupling further cause decreased oxidative phosphorylation for ATP production and muscle contraction (
Milk fat globule-EGF factor VIII (MFG-E8) has been identified as a principal component of the milk fat globule, involving 111 pepsin digestion sites and 36 trysin digestion sites (
). Enzymatically, through gastrointestinal digestion in vivo, MFG-E8 releases bioactive peptides containing 2 to 44 AA, which adsorb through intestinal epithelial cell-related transport carriers and exert a proliferative effect on myoblasts. Existing research recognizes the critical role played by whey and soybean proteins in the process, promoting muscle protein synthesis and attenuating muscle atrophy. In particular, whey hydrolysates and soybean protein-derived peptides can reduce oxidative stress, regulate quality and function of skeletal muscle, and promote myoblast proliferation (
Ingestion of whey hydrolysate, casein, or soy protein isolate: Effects on mixed muscle protein synthesis at rest and following resistance exercise in young men.
Small molecular weight soybean protein-derived peptides nutriment attenuates rat burn injury-induced muscle atrophy by modulation of ubiquitin-proteasome system and autophagy signaling pathway.
J. Agric. Food Chem.2018; 66 (29493231): 2724-2734
). Moreover, evidence suggests that, compared with adequate whey protein supplementation, a small amount of MFG-E8 (78.8 times lower than whey protein) can achieve a similar effect, demonstrating that MFG-E8 polypeptides can be considered a potential valuable resource in regulating aging-related skeletal muscle dysfunction (
Although research has been carried out on the antioxidative effects of the MFG-E8 protein, no single study has demonstrated the regulation of peptides derived from MFG-E8 on the keap1-Nrf2 antioxidative system, which is a principal protective regulator of drug detoxification and oxidative and electrophilic stresses (
). In normal cells, Nrf2 is rapidly degraded by the keap1-mediated ubiquitin proteasome system, specifically through binding to its evolutionarily conserved N-terminal Neh2 regulatory domain (
). Upon exposure to oxidative stress, Nrf2 translocates to the nucleus, where it binds to antioxidant responsive elements via heterodimerization with small Maf proteins and activates the expression of target genes [e.g., drug detoxifying enzymes (glutathione S-transferases), antioxidant proteins (HO-1);
]. In this case, keap1-Nrf2 further emerged as mitochondrial function and quality control indicators, and successive activation of Nrf2 and HO-1 can (1) increase generation of carbon monoxide (CO), which binds to the reduced heme of cytochrome c oxidase (COX), inhibits cellular respiration, and generates hydrogen peroxide, further provoking the ROS-mediated activation of NRF-1 and promoting mitochondrial biogenesis; and (2) induce the activation of NAD(P)H quinone oxidoreductase 1 (NQO1), which rescues the mitochondrial membrane potential (ΔΨm), and ROS generation decreases.
The specific objective of this study was to identify the effect of high-abundance MFG-E8 polypeptides on potentially inhibiting keap1 activity and dex-induced oxidative stress injury. The MFG-E8 polypeptide was prepared by simulating gastrointestinal digestion in vitro, and the particle size and changes to zeta potentials during digestion were measured by ZetaSizer (Malvern). Based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) and molecular docking analysis, the composition of the MFG-E8 polypeptide and high-abundance polypeptides with good abilities to bind to keap1 active sites were determined and screened, respectively. The effects of the bioactive peptide MFG-E8 on L6 cell oxidative stress status and mitochondria was visualized by transmission electron microscopy and confocal laser scanning microscopy. The molecular mechanism of the MFG-E8 bioactive peptide in mediating the keap1/Nrf2/HO-1 oxidative stress pathway was examined via western blotting assay. This study aims to contribute to the growing topic of alleviation of oxidative stress and mitochondrial damage by functional peptides.
MATERIALS AND METHODS
Materials and Reagents
No human or animal subjects were used, so this analysis did not require approval by an Institutional Animal Care and Use Committee or Institutional Review Board. MFG-E8 was extracted from milk fat globule membrane cellulose DEAE-52 (Sigma-Aldrich) by our group. Rat skeletal muscle myoblasts (L6 cells) were supplied by ATCC. Dulbecco's modified Eagle's medium and fetal bovine serum were purchased from Gibco. All other chemicals and reagents were of analytical grade. The process of this analysis is described in Figure 1.
Figure 1Flow chart of this study. MFGM = milk fat globule membrane.
). For the gastric phase, MFG-E8 was dissolved in PBS buffer (50 mg/mL) and diluted with simulated gastric fluid containing porcine pepsin (2,000 U/mL) at a 50:50 (vol/vol) ratio. The pH was adjusted to 3 at 37°C in an orbital shaker at 170 rpm for 2 h. Pepsin was then inactivated by raising the pH to 6.5. For the intestinal phase, gastric chyme was mixed (ratio 50:50, vol/vol) with simulated intestinal fluid containing trypsin (100 U/mL) and porcine bile extract (10 mM). Digestion was performed at pH 7, 37°C for 2 h under continuous shaking at 100 rpm. Once the simulated digestion was finished, enzymes were deactivated at 90°C for 10 min. Finally, the mixture was centrifuged (10,000 × g, 15 min, 4°C) and the supernatant was separated for further analysis. The peptide content was measured using the ortho-phthaldialdehyde method (
Effects of different satiety levels on the fate of soymilk protein in gastrointestinal digestion and antigenicity assessed by an in vitro dynamic gastrointestinal model.
Particle sizes and zeta potentials were measured by dynamic and electrophoretic light scattering techniques with a Malvern ZetaSizer Nano at different times of MFG-E8 gastrointestinal simulation. Approximately 1 mg/mL of MFG-E8 digestive juice diluted in deionized water was introduced into the measurement cell of the apparatus at 25°C. Reported particle size and zeta potential were the average for 3 samples, each sample being measured only 3 times to avoid sample degradation due to voltage.
Identification of MFG-E8 Peptides by Ultra-HPLC Quantitative Time-of-Flight MS/MS
The composition of MFG-E8 peptides was determined by ultra-HPLC and electrospray ionization quadrupole orbitrap high-resolution MS (UHPLC/ESI, Q-Orbitrap). The mobile phases of A and B were 0.1% formic acid in water (vol/vol) and 0.1% formic acid in acetonitrile (vol/vol), respectively. Separation was carried out on a Syncronis C18 column (100 mm × 2.1 mm, 1.7 μm) with a flow rate of 0.3 mL/min at column temperature of 35°C. The elution gradient profile was set as 5% B (0–3 min), 5 to 15% B (3.0–8.0 min), 15 to 30% B (8.0–40.0 min), 30 to 100% B (40.0–55.0 min), 100% B (50.0–65.0 min), 100 to 5% B (65.0–70.0 min), and 5% B (70.0–75.0 min). The major peak sequence of MFG-E8 peptides was identified by MS/MS spectra in PEAKS Studio 8.5 (BSI). The MS conditions were optimized as follows: spray voltage, 3,500.0 V; capillary temperature, 320.00°C; sheath gas, 35.00 arb; auxiliary gas, 10.00 arb; spare gas, 0.00 arb; max spray current, 100.00 μA; probe heater temperature, 350.00°C; S-lens radio frequency level, 50.00%; ion source, heated electrospray ionization. The ions were scanned at high resolution (70,000 in MS1; 17,500 in MS2), and the MS scan range was 150 to 2,000 m/z at both MS1 and MS2 levels. All identified peptides were confirmed by comparing with Q95114 (MFGM_BOVIN) from the Uniprot database (https://www.uniprot.org/).
Preparation of Protein and Ligand for Docking
The 3-dimensional structures of keap1 and NF-κB were retrieved from the Protein Data Bank (keap1 PDB ID, 5FZN; resolution, 1.97 Å; NF-κB PDB ID, 1RAM; resolution, 2.7 Å; https://www.rcsb.org). The binding of proteins chains was extracted and polarized hydrogens were added by AutoDock Tools 1.5.6 (Molecular Graphics Laboratory, Scripps Research Institute).
Ligands in this study were high-abundance MFG-E8 polypeptides, which were drawn by ChemDraw 18.0 and assigned with proper 2-dimensional orientation. All 2-dimensional structures were all converted to 3-dimensional structures with minimized energy by Chem3D 18.0 (Perkinelmer).
Molecular Docking
The molecular docking process was implemented via AutoDock Vina software version 1.1.2 (Molecular Graphics Laboratory, Scripps Research Institute), which combines knowledge-based potentials and empirical scoring (
). The binding site parameters (keap1, x = 13.741 Å, y = 65.988 Å, z = 30.792 Å; NF-κB, x = 14.101 Å, y = 29.331 Å, z = 51.517 Å) and the docking box dimensions (18 × 18 × 18 Å) were determined by redocking the co-crystallization ligand in the catalytic cavity. The results of molecular docking were evaluated through the criteria of binding structure, binding energy, and possible interactions between ligand and key protein residues.
Cell Culture and MTT Assay
We measured L6 cell viability by MTT assay. A density of 5 × 104 cells per milliliter were seeded, and corresponding treatments of 1 μM dex and different concentrations of bioactive peptides (100 μg/mL, 200 μg/mL, and 300 μg/mL) were conducted. Cells were cultured for 24 h and 48 h at 37°C and then treated with MTT, and the blue formazan crystals were dissolved by dimethyl sulfoxide for 10 min. Finally, the optical density value was detected at 490 nm by microplate reader (model 550, Bio-Rad).
Mito- and Lyso-Tracker Staining Assay
Mitochondrial and lysosomal staining was performed using mito-tracker red and lyso-tracker green, respectively. Optimal concentrations of dex and MFG-E8 peptides were used for cell treatment. Cells were incubated with mito- and lyso-trackers for 30 min at 37°C, respectively, and washed 3 times with PBS. Cells were then inspected and photographed via confocal microscope (Zeiss).
). The L6 cells treated with dex and peptides were incubated with dichlorodihydrofluorescein diacetate probe at 37°C for 30 min. Fluorescence was then measured using a fluorescence microscope (Olympus) at 488 nm.
Cellular Ultrastructure Observation
Ultrastructure of L6 cells was observed via transmission electron microscopy. The L6 cells were washed, collected, and then fixed with 4% glutaraldehyde at 4°C for 12 h. The fixed cells were centrifuged, washed, and fixed with 2.5% glutaraldehyde for 2 h and 1% osmiumtetraoxide for 2 h. Cells were then dehydrated with increasing gradients of ethanol and embedded in Epon 812 resin. Ultrathin sections (50–70 nm) were stained with 3% uranyl acetate and lead citrate and observed on a Zeiss 900 electron microscope with magnifications of 5,000 to 30,000.
Western Blot
The western blot assay was used as previously described by
, with slight modification. Total protein of L6 cells treated with dex and MFG-E8 polypeptides was collected and homogenized in cold radioimmunoprecipitation assay and phenylmethylsulfonyl fluoride lysis buffer following centrifugation at 9,168 × g for 15 min at 4°C. Supernatant was sonicated, and a 20-μL protein sample was loaded onto a one-dimensional SDS gel. Proteins were then transferred onto a nitrocellulose filter membrane and subsequently blocked with 5% BSA in Tris-buffered saline with 0.1% Tween (TBST) buffer for 30 min and incubated with primary antibodies for 4 h at 37°C. The concentrations of antibodies were as follows: GAPDH, 1:400; keap1, 1:400; Nrf2, 1:400; HO-1, 1:400; and PGC-1α, 1:200. The membrane was washed for 5 min 3 times with TBST, followed by incubation with secondary antibody (1:1,500, Proteintech) at 37°C for 1 h. The membrane was washed with TBST twice and with Tris-buffered saline once, and finally incubated with alkaline phosphatase until an appropriate signal was obtained. Protein bands were detected via ChemiDoc MP Imaging System (Bio-Rad).
Statistical Analysis
All experiments, except when otherwise described, were tested and analyzed in triplicate. We used an ANOVA to determine significant differences (P < 0.05) between means. Statistical analysis was performed using a general linear model procedure with SAS 9.1.3 (SAS Institute Inc.).
RESULTS
Total Ion Current Chromatogram Analysis of MFG-E8 Polypeptides
Identification of MFG-E8 polypeptide composition was carried out via LC-MS/MS. As shown in Table 1 and Figure 2, the relative contents of 21 peptides were determined to exceed 1%, with relative content calculated as the ratio of ion intensity of the peptide to the total ion intensity of all peptides. Binding free energy was implemented using AutoDock Vina by redocking the co-crystallization ligand in the catalytic cavity. Combined with indices of relative contents, stability, and binding free energy, IDLGS, KDPG, YAR, YYK, LAALF, and others were preliminarily screened as MFG-E8 polypeptides that exhibited high abundances of lactadherin-derived polypeptides with stability and good ability to bind to the active sites of keap1 and NF-κB.
Table 1High-abundance peptides in MFG-E8 identified by liquid chromatography-tandem mass spectrometry
Figure 2Liquid chromatography-tandem mass spectrometry analysis of MFG-E8 polypeptides. (A) Total ion current chromatogram. (B) MS spectra of IDLGS. (C) MS spectra of KDPG.
Mass spectrometry data were further analyzed using the protparam tool (https://web.expasy.org/protparam/) and compared with the Bos taurus protein library, which was employed to elucidate the sequence of samples, and peptides were confirmed. Among them, 2 high-abundance peptides were identified as IDLGS and KDPG (Figures 2B and 2C).
Particle Size and Zeta Potential Changes During MFG-E8 Simulated Gastrointestinal Digestion
Particle size distribution can indirectly reflect protein emulsification and system stability (
Measuring particle size distribution by asymmetric flow field flow fractionation: A powerful method for the pre-clinical characterisation of lipid-based nanoparticles.
). The particle size of MFG-E8 exhibited a trend of increasing to a maximum and then decreasing with prolonged digestion time in the simulated gastrointestinal digestive system. As shown in Figure 3, the particle size reached a maximum of 4.82 μm at 60 min (simulated gastric) and minimums of 2.42 μm at 120 min (simulated gastric) and 1.22 μm at 120 min (simulated intestinal). The gradual increase of particle size at the beginning of gastric digestion is due to the pH of the digestive system being maintained at a value close to the isoelectric point, resulting in protein aggregation and formation of large protein micelles. However, in the case of continuous enzymatic hydrolysis of pepsin, the MFG-E8 protein was gradually digested, and the particle size decreased. In the simulated intestinal digestive system, because the pH of the digestive system is always maintained at a value far from the isoelectric point, the particle size of MFG-E8 was smaller than the final particle size of the gastric digestion stage from the beginning of digestion. Within 30 min of intestinal digestion, the repulsion reduced and proteins aggregated under the action of trypsin, which increased the average particle size of MFG-E8. After digestion time reached 60 min, we found no significant change in the average particle size of MFG-E8 compared with 120 min, indicating that the digestion process was basically completed. The decreased particle size of MFG-E8 shows the excellent digestion characteristics of MFG-E8 and gradual production of MFG-E8 polypeptides, which can more easily penetrate the intestinal barrier and act on cells.
Figure 3Changes of particle size and zeta potential of the MFG-E8 protein during gastric and intestinal digestion. The different letters (a–e) among the 5 groups represent significant differences (P < 0.05 represents significant, P < 0.01 represents extremely significant). Error bars indicate SD.
Zeta potential can characterize the strength of electrostatic interaction between proteins, and decreased zeta potential demonstrates the stability of the digestive system (
). The zeta potential changed from a negative value (−30.59 mV) to a positive value (12.2 mV) over time during the gastric digestive process, which is closely related to the changes of particle size. Lower zeta potential leads to aggregation and an increase in particle size of MFG-E8. The positive charge on the surface of the MFG-E8 protein increased at pH 3, which resulted in increase of electrostatic repulsion between proteins and the originally aggregated proteins, which are partially hydrolyzed; additionally, MFG-E8 proteins are repelled and dispersed again, which is more consistent with the change in particle size. Throughout the simulated intestinal digestion process, the zeta potential of MFG-E8 fluctuated between −18.50 and 37.10 mV, especially from 90 to 120 min (−35.52 to 37.10 mV). With further digestion of the MFG-E8 protein, the whole simulated digestion system gradually became stable, with no obvious change in either zeta potential or particle size.
Binding Affinity and Structure Profile of High-Abundance MFG-E8 Polypeptides with Keap1
Inhibition of the keap1-Nrf2 interaction through binding to the keap1 kelch domain is a well-recognized mechanism for Nrf2 activation and action against oxidative stress (
). High-abundance MFG-E8 polypeptides with high binding affinity to keap1 were screened based on molecular docking. The conformation with the lowest docking energy and the desired docking position was considered as the optimum. The free energy of binding and potential binding sites are shown in Table 2, and intermolecular interactions of keap1 with peptides are shown in Figure 4. Taking IDLGS and KDPG as examples, IDLGS bound to the active site of keap1 through hydrophobic interactions with the residues TYR334, ALA556, ASN414, and SER508, and formed hydrogen bonds with the residues SER602 and ARG415, with docking scores of −7 kcal/mol; moreover, KDPG interacted with keap1 with docking scores of −6.7 kcal/mol through hydrophobic interactions with the residues ARG415, PHE577, ASN414, and ALA556, and hydrogen bonds with the residues LEU557, SER555, and SER508.
found that rutaecarpine can inhibit keap1 and activate Nrf2 by forming a hydrophobic interaction with GLY364, TYR334, and ALA556, and its oxygen atoms on the amide group form a hydrogen bond interaction with ARG415. Through investigating 10 tripeptides with the highest antioxidant activity (e.g., SPW from casein, SVW from beef, CNW from egg, and MKW from bovine serum albumin) binding with keap1,
found that most ligands were stabilized by hydrophobic interactions and hydrogen bonds with TYR334, ARG380, ASN382, ARG415, SER555, and ARG415 of keap1. These active binding sites, consistent with our findings, basically interacted with the catalytic trial (e.g., TYR334 and ARG415), which are important AA for keap1 activity.
Table 2Potential binding sites of high-abundance peptides in MFG-E8 combined with keap1
Figure 4Overall 2- and 3-dimensional structures of intermolecular interactions between keap1 with IDLGS, KDPG, YAR, and YYK. In the 2-dimensional profile, different colors represent different intermolecular forces between peptides and keap1. In the 3-dimensional profile, the rod-shaped and pink peptides were wrapped in the main stem of proteins. The AA residues of receptors and ligands are shown in the close-up picture of peptide-keap1 complex. F = front; R = rear.
Peptides IDLGS and KDPG on L6 Cell Oxidative Status and Inner Structure
Molecular docking assay revealed the potential effects of high-abundance MFG-E8 polypeptides interacting with the key oxidative stress regulator keap1. To further demonstrate the role of MFG-E8 polypeptides against oxidative stress, an in vitro cell experiment was conducted. Oxidative stress is an imbalance between free radical production, ROS, and endogenous antioxidant protective mechanisms (
). Under general physiological conditions, ROS can act as normal products of biological reduction of molecular oxygen, and keap1 degrades Nrf2 as the site of action for cullin-dependent E3 ubiquitin ligase. Although ROS overproduction induced by inhibition of mitochondria number and activity beyond the threshold of organism capacity can trigger keap1, it undergoes a conformational change that causes Nrf2 to dissociate, which inhibits Nrf2 aggregated into the nucleus and transcription of multiple antioxidants (
). The ROS level was measured by dichlorodihydrofluorescein diacetate fluorescence, and the inner structures of L6 cells were measured via transmission electron microscope. As shown in Figure 5A, IDLGS and KDPG can significantly decrease fluorescence by 45.42 and 65.63%, respectively (P < 0.01, versus dex group), demonstrating that dex can increase the risk of cells suffering from oxidative stress, whereas bioactive peptides reversed this trend. The ultrastructures of cytoplasmic mitochondria and vacuoles were further observed (Figure 5B), and cytoplasmic vacuoles were enlarged and disorganized, mitochondria were damaged with fuzzy cristae, and a swelling matrix was found in the dex treatment group. Mitochondria in the IDLGS and KDPG groups were displayed with intact outer membrane and dense cristae. These results showed that IDLGS and KDPG bioactive peptides can alleviate dex-induced oxidative stress injury by decreasing ROS production and maintaining normal ultrastructure and content of mitochondria and lysosomes.
Figure 5Oxidative profiles and inner structures of injured L6 myoblasts treated with the MFG-E8 polypeptides IDLGS and KDPG. (A) Dichlorodihydrofluorescein diacetate (DCFH-DA) staining; green arrows indicate reactive oxygen species (ROS) generation. (B) Ultrastructure of L6 myoblasts observed via transmission electron microscope (TEM); red dotted boxes show organelles containing mitochondria, lysosomes, etc.; black arrows indicate mitochondria, and green arrows show vacuolization. Dex = dexamethasone; TEM = transmission electron microscopy.
Peptides IDLGS and KDPG on L6 Cell Vitality, Mitochondria, and Lysosomes
The effect of the high-abundance MFG-E8 polypeptides IDLGS and KDPG on oxidative stress-injured L6 cell vitality was detected. As shown in Figure 6A, an oxidative stress cell model established by dex inhibited cell proliferation by 13.56 and 26.55% at 24 h and 48 h, respectively (P < 0.01). Compared with the dex group, IDLGS reversed oxidative stress-injured L6 cell apoptosis by 12.07, 20.09, and 18.39% at 24 h and 30.24, 36.65, and 31.88% at 48 h, respectively (P < 0.01); additionally, KDPG promoted L6 cell proliferation by 15.84, 28.11, and 21.65% at 24 h and 31.44, 43.48, and 36.66% at 48 h, respectively (P < 0.01). These results showed that the high-abundance peptides IDLGS and KDPG derived from MFG-E8 can alleviate dex-induced cell apoptosis.
Figure 6Effects of the MFG-E8 polypeptides IDLGS and KDPG on injured L6 myoblast vitality, mitochondria, and lysosomes. (A) Vitality of L6 myoblasts. (B) Mito- and lyso-tracker staining; red arrows indicate mitochondria, green arrows indicate lysosomes, blue arrows indicate nuclei. (C) Relative fluorescence intensity of L6 myoblasts, as measured by ImageJ software (National Institutes of Health, ImageJ 1.8). The bar chart represents mean ± SEM. *P < 0.05, **P < 0.01, IDLGS vs. dexamethasone (dex; n = 3); #P < 0.05, ##P < 0.01, KDPG vs. dexamethasone (n = 3).
). Alternation of mitochondria number, morphology, and function can induce oxidative stress and activate autophagic responses, which are particularly important to monitor abnormal generation of ROS by mitochondria-targeted probes (
). Mitochondrial stress or inhibition of mitochondrial respiratory chain-induced increase of lysosomal biogenesis have been observed in pharmacologic cell models, which further induce depressed autophagy and, subsequently, cellular dysfunction (
). Therefore, the contents of mitochondria and lysosomes were visualized by mito-tracker red and lyso-tracker staining, respectively. As shown in Figures 6B and 6C, compared with the control group, dex treatment can significantly reduce red fluorescence and enhance green fluorescence ratios by 61.25% and 1.48-fold (P < 0.01), illustrating increased autophagy of L6 cells and decreased content of mitochondria. Mitochondria in the IDLGS and KDPG groups exhibited a filamentous, elongated morphology, and lysosome contents significantly decreased, which increased the red/green fluorescence ratio by 1.97- and 2.21-fold (compared with the dex group, P < 0.01). Dexamethasone directly inhibited mitochondrial respiratory complex I, which was further accompanied with mitochondrial loss and dysfunction, as well as L6 cell apoptosis. Additionally, IDLGS and KDPG can reverse dex-induced increase of lysosome content in L6 cells. Thus, MFG-E8 reduced intracellular acidic components and number of lysosomes, and further increased the lysosomal pH to alleviate lysosomal membrane permeabilization and autophagy-lysosomal activity in injured L6 cells.
Molecular Mechanisms of IDLGS and KDPG on Regulation of L6 Cell Oxidative Stress Injury
The keap1/Nrf2/HO-1 pathway is a crucial endogenous mechanism against oxidative stress that targets regulation of antioxidant enzymes and inflammation-related disorders, thereby preventing dex-induced myoblast mitochondrial dysfunction (
). Under quiescent conditions, Nrf2 can be degraded by keap1 to maintain low transcriptional activity via the ubiquitin proteasome pathway; however, once exposed to stress, Nrf2, liberated from keap1-mediated repression, translocates into the nucleus and subsequently activates the downstream substrate HO-1, which enhances myoblast antioxidant capacity. Moreover, PGC-1α/Nrf2 signaling can also mediate cellular oxidative stress status and inflammatory response (
Isoliquiritigenin alleviates LPS/D-GalN-induced acute liver failure by activating the PGC-1α/Nrf2 pathway to reduce oxidative stress and inflammatory response.
). The protein expressions of keap1, Nrf2, and HO-1 have been detected to demonstrate the effect of the bioactive peptide YYR in mitigating oxidative stress injury in L6 cells. As shown in Figure 7, compared with the control group, dex significantly increased the expression of keap1, by 62.17%, and decreased the expression of Nrf2, HO-1, and PGC-1α by 62.61, 57.34, and 46.78% (P < 0.01). In contrast, IDLGS and KDPG decreased keap1 by 22.22% and increased Nrf2, HO-1, and PGC-1α by 1.08-fold, 46.51 and 48.11% (IDLGS group versus dex group, P < 0.01); 1.16-fold, 97.67%, 1.07-fold (KDPG group vs. dex group, P < 0.01), respectively. These results indicate that IDLGS and KDPG peptides derived from MFG-E8 mitigate dex-induced oxidative stress and mitochondrial dysfunction via keap1/Nrf2/HO-1 and PGC-1α/Nrf2 signaling cascades.
Figure 7Effects of the MFG-E8 polypeptides IDLGS and KDPG on the protein expression of keap1/Nrf2/HO-1 and PGC-1α (top). Typical autoradiogram and western blotting analysis of keap1, Nrf2, HO-1 and PGC-1α exhibiting the oxidative status of L6 myoblast (bottom). The bar chart represents mean ± SEM. Different letters (a–d) among groups represent significant differences (P < 0.05). Dex = dexamethasone; con = control.
The present study elucidates the effects against oxidative stress of the high-abundance MFG-E8 polypeptides IDLGS and KDPG, in the regulation of dex-induced L6 cell injury. In this work, MFG-E8 was digested by a simulated gastrointestinal digestive system in vitro. Relative contents of 21 MFG-E8 peptides exceeding 1% were analyzed and identified using LC-MS/MS. Among them, IDLGS, KDPG, YAR, and YYK were selected for potential function in regulating oxidative stress by molecular docking. Cell experiments showed that IDLGS and KDPG can alleviate dex-induced L6 cell vitality, mitochondrial number and functional decreases, and increased autophagy. Further experiments proved that IDLGS and KDPG can exert effects against oxidative stress via the keap1/Nrf2/HO-1 signaling pathway. The results displayed in this study, from LC-MS/MS identification, molecular docking, and the in vitro cell perspective, present the first evidence that the MFG-E8-derived peptides IDLGS and KDPG can mitigate dex-induced L6 cell oxidative stress injury, providing a nutritional basis for treatment of sarcopenia.
ACKNOWLEDGMENTS
The authors thank the Natural Science Foundation of Jiangsu Normal University (Jiangsu, China; 20XSRX001) and the Natural Science Foundation of Xuzhou City (Xuzhou, China; No. KC21027). Author contributions are as follows: Li He designed the experiments, Kaifang Guan carried out experiments and wrote the manuscript, Dandan Liu analyzed experimental results, Min Liu collected and reorganized the data, and Canxia He amended the manuscript. The authors have not stated any conflicts of interest.
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Isoliquiritigenin alleviates LPS/D-GalN-induced acute liver failure by activating the PGC-1α/Nrf2 pathway to reduce oxidative stress and inflammatory response.
Effects of different satiety levels on the fate of soymilk protein in gastrointestinal digestion and antigenicity assessed by an in vitro dynamic gastrointestinal model.
Ingestion of whey hydrolysate, casein, or soy protein isolate: Effects on mixed muscle protein synthesis at rest and following resistance exercise in young men.
Small molecular weight soybean protein-derived peptides nutriment attenuates rat burn injury-induced muscle atrophy by modulation of ubiquitin-proteasome system and autophagy signaling pathway.
J. Agric. Food Chem.2018; 66 (29493231): 2724-2734
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