Hatched Eggshell Membrane Can Be a Novel Source of Antioxidant Hydrolysates to Protect against H2O2-Induced Oxidative Stress in Human Chondrocytes

Natural antioxidants derived from agricultural by-products have great promise and ecological advantages in the treatment of oxidative stress-related diseases. The eggshell membrane (ESM) from hatched eggs, i.e., the hatched ESM, is a globally abundant agricultural byproduct, and its high-value utilization has been rarely studied compared to the well-studied ESM from fresh eggs. In this research, we systematically characterized the hatched ESM as a novel source of antioxidant hydrolysates and explored their potential role in H2O2-induced human chondrocytes. The results showed that the hatched ESM is a protein-rich fibrous mesh material with a significantly different structure and composition from those of fresh ESM. Enzymatic hydrolysis of hatched ESM can produce antioxidant hydrolysates rich in low molecular weight (MW) peptides, which mainly derived from the Lysyl oxidase homolog by Nano-LC-MS/MS analysis. The peptide fraction with MW < 3 kDa (HEMH-I) exhibited the highest DPPH radical scavenging, Fe2+-chelating, and Fe3+-reducing abilities. In H2O2-induced human SW1353 chondrocytes, HEMH-I treatment significantly increased the cell viability and ameliorated oxidative stress, inflammatory response, and cartilage matrix degradation by reducing the level of ROS, matrix metalloprotease 3 (MMP3), MMP13, and IL-6, and by promoting the expression of SOD and type II collagen, potentially through activating the cellular Keap1/Nrf2/HO-1 pathway. This study provides a theoretical basis for the value-added application of hatched ESM waste to produce antioxidant hydrolysates and indicates their potential as functional food and pharmaceuticals.


Introduction
Oxidative stress occurs when the production of reactive oxygen species (ROS) in the body is excessive and overwhelms the defense capacity of living cells, leading to tissue damage and various chronic diseases, including osteoarthritis (OA), cardiovascular disease, and cancer [1,2]. Among them, OA is a degenerative joint disease induced by oxidative stress in chondrocytes [3,4] and has been considered a major public health concern worldwide [5]. Oxidative stress generated by the accumulation of ROS in chondrocytes can trigger impaired cartilage extracellular matrix (ECM) metabolism and overproduction of proteolytic enzymes and inflammatory cytokines, thereby accelerating cartilage degeneration [4,6]. Therefore, the suppression of oxidative stress injury to chondrocytes is proposed as a potential treatment strategy in OA [3,7]. Due to their high bioactivity and superior safety profile over synthetic antioxidants and drugs, antioxidant hydrolysates or peptides hold great

Degree of Hydrolysis (DH)
The DH of ESM hydrolysates was determined using a pH-stat method as previously reported by Liu et al. [28] using the following equation: where α is the average degree of dissociation of the α-NH 2 groups, and h tot is the total number of peptide bonds per gram of protein with 7.84 mmol/g and 7.87 mmol/g for fresh and hatched ESM, respectively, calculated based on their amino acid composition.

Determination of Molecular Weight (MW) Distribution
Referring to the method reported by Jia et al. [27], the MW distribution was measured using a high-performance liquid chromatography (HPLC) system (Agilent, Santa Clara County, CA, USA) equipped with a TSK gel G2000 column (300 mm × 7.8 mm; Tosoh, Tokyo, Japan). A total of 10 µL of the sample was separated at a flow rate of 0.5 mL min-1 at 30 • C and monitored at 220 nm. Bovine insulin (5733.49 Da), Bacitracin (1422 Da), Gly-Gly-Tyr-Arg (451 Da), and Gly-Gly-Gly (189 Da) were used to plot the standard curve. The data were analyzed using Breeze software (Waters, MA, USA). The peaks of each sample were integrated and divided into 4 ranges (MW > 5000, 3000-5000, 1000-3000, and <1000 Da). The relative content of each molecular weight range was expressed as the corresponding peak area for each region. The logarithm of the relative molecular mass (log MW) was linearly related to the retention time (Rt) with the following equation: Rt = −0.311558(log MW) + 8.51004 (R 2 = 0.9867, p < 0.05).

Nano-LC-ESI-MS/MS Analysis
The major peptides in the HEMH were sequenced by nano-LC-ESI-MS/MS using an Easy-nLC HPLC system with a reversed-phase C18-A2 column (75 µm × 10 cm, Thermo scientific EASY column,). The loaded samples were eluted with a linear gradient at a flow rate of 300 nL/min using 0.1% formic acid aqueous solution (A) and 0.1% formic acid 84% acetonitrile (B) as mobile phases. Linear gradient: from 0% to 35% B for 50 min, from 35% to 100% B for 5 min, and 100% B for 5 min, were adopted. MS scans were obtained from 2.9. Determinati on of Chemical Antioxidative Activity 2.9.1. DPPH Radical Scavenging Activity Assay The scavenging activity of DPPH radicals was determined using the method of Zhang et al. [29] with some modifications. A total of 100 µL of the sample solution and 100 µL of ethanol DPPH (0.2 Mm) were added to a 96-well plate. The mixture was then shaken and reacted at room temperature for 30 min away from light. The absorbance was measured at 517 nm using a microplate reader. Ethanol was used instead of DPPH for the blank, and water was used instead of the sample for the control. The ability to scavenge DPPH radicals was calculated according to the following equation: The scavenging activity of DPPH (%) = [1 − (A sample − A blank )/A control ] × 100% A blank , A control , and A sample represent absorbance for blank, control, and sample, respectively. Trolox standard curve was applied to determine the Trolox equivalent antioxidant capacity. The results were expressed as Trolox equivalents (µmol TE/g sample).

Fe 2+ -Chelating Activity Assay
The Fe 2+ -chelating activity was determined using the method of Zhao et al. [15] with some modifications. One mL of sample solution was blended with 0.1 mL of a FeCl 2 solution (2 mM) and 4.5 mL of absolute ethanol while shaking. Then, 0.2 mL of a ferrozine solution (5 mM) was added to the solution previously mixed and left to react at room temperature for 10 min. An equal volume of distilled water was substituted for the sample to serve as a control group. The absorbance was measured by spectrophotometry at 562 nm and the Fe 2+ -chelating activity was calculated according to the following equation: The Fe 2+ -chelating activity (%) = (1 − A S /A C ) × 100% A S and A C representing the sample and the blank absorbance, respectively.

Reducing Power Assay
The reducing power of the ESM hydrolysates was determined according to Shi et al. [17]. After incubation, the absorbance of the mixed solution was determined at 700 nm. The increased absorbance of the reaction mixture indicated an increase in the energy added to the reduced iron.

Determination of Intracellular ROS
The level of ROS in SW1353 cells was determined using a DCFH-DA probe to evaluate the oxidative stress in cells. According to the method of Wang et al. [31], cells were pretreated with HEMH-I (0.25, 0.5 and 0.75 mg/mL) for 24 h, then exposed to H 2 O 2 (200 µM) for 60 min. After two washes with PBS, SW1353 cells were incubated in the dark for 30 min with 100 µL 2',7'-dichlorofluorescein diacetate (DCFH-DA) (Nanjing Jian cheng Bioengineering Institute, Nanjing, China) at a concentration of 10 µM. A fluorescence microscope (Olympus X71, Tokyo, Japan) was used to observe the fluorescence images, and the fluorescence intensity was quantified using the Image-J software.

Statistical Analysis
All treatments in the experiment were performed at least in triplicate. Significant differences (p < 0.05) in results were determined by analysis of variance (ANOVA) followed by Duncan's multiple range test or by Student's t-test for two-group comparisons applying Statistix software 9.0 (Analytical Software, Tallahassee, FL, USA). All results of the experiment are reported as mean ± standard deviation (SD).

Structure and Chemical Composition of ESM from Two Sources of Chicken Eggs
It is well recognized that the structure and composition of protein substrates affect the efficiency of enzyme hydrolysis and the activity of hydrolysis products [32,33]. Therefore, it was necessary to characterize the hatched ESM as a novel source of protein material prior to the hydrolysis process. The fresh ESM, a well-studied material to generate bioactive peptides, was used as a control.

Electron Microscopic Scanning of ESM
The scanning electron micrographs (SEM) of the hatched and fresh ESM are shown in Figure 1. The SEM pictures revealed that the main structure for both ESM is a meshwork of highly cross-linked fibers, in agreement with previous studies establishing that ESM is formed by interlaced insoluble protein fibers [34,35]; however, the surface morphology of the hatched ESM differs from that of the fresh ESM. Many aggregates, defined as the residual mammillary cones [36], were deposited on the surface of the outer hatched ESM fibers in a quasi-periodic pattern, consistent with previous studies [37]. In contrast, no cone tips remained on the fresh ESM fibers. This difference may be due to the calcium resorption that occurred during embryonic development, resulting in the complete detachment of the hatched ESM with attached mammillary cones from the eggshells, which led to the residual mammillary cones existed on the hatched ESM [37,38]; whereas for fresh eggs, the cones were removed together with the eggshell when the membranes were mechanically pulled from the shell interior [37]. Since the mammillary cones are rich in proteins [36], the hatched ESM should also contain some specific mammillary cone proteins, making its protein composition different from that of the fresh ESM. In addition, the difference between the hatched and fresh ESM may also be related to the incubation process. Rath et al. [22] reported that embryonic development may induce some dynamic changes in the egg proteome and alter the membrane structure to facilitate the hatching process. An increase in oxygen permeability of ESM has been reported during incubation [39], which may lead to a decrease in density of the hatched In addition, the difference between the hatched and fresh ESM may also be related to the incubation process. Rath et al. [22] reported that embryonic development may induce some dynamic changes in the egg proteome and alter the membrane structure to facilitate the hatching process. An increase in oxygen permeability of ESM has been reported during incubation [39], which may lead to a decrease in density of the hatched ESM structures. In addition, the epithelial cells in the chorioallantoic membrane secrete protons to dissolve the calcium reserve [36,38]. The released protons may dissolve the acid-soluble proteins of the ESM, leading to a decrease in the density of the three-dimensional structure of the hatched ESM [40]. In line with these previous findings, the network of the hatched ESM appeared different and much flatter in the present study, probably due to the physical and physiological changes in the ESMs occurring during the embryonic development [39]. Less intensity of insoluble protein fibers may lead to a better efficiency of enzymatic hydrolysis, which will be validated in the next part.

Proximate Composition of ESM
The chemical composition of the hatched and fresh ESM was established, where the results for protein, ash, calcium, and saccharide were expressed as g/100 g dry matter (Table 1). Protein was the main component for both ESMs, ranging from 92.98 to 96.00, in agreement with the 88-96% protein range of fresh ESM in dry matter reported by Huang et al. [16]. Due to their high protein content, ESM can be directly used as a protein material to produce bioactive peptides without any additional protein extraction step [15]. Values are expressed as mean ± SD (n = 3). Means with different letters (a, b) for the same parameter are significantly different (p < 0.05).
Some differences in composition were noted between the two ESMs. The hatched ESM contained significantly lower protein levels than the fresh ESM. This may be due to multiple factors, including the acidic environment that dissolves eggshell calcium during incubation [21], potentially leading to the dissolution of acid-soluble proteins in the ESM and their absorption by the embryo. It was reported that clusterin, a major component of the egg proteome in fresh ESM, was potentially absorbed completely during incubation [22]. The higher ash and calcium contents in the hatched ESM were consistent with the observations of SEM.

Amino Acid Composition of ESM
The amino acid compositions of the fresh and hatched ESMs are shown in Table 2. Glu, Asp, Cys, and Pro were the main constituents of the two ESMs. A previous study of fresh ESM reported a similar amino acid composition [41]. Despite some similarities, there were significant differences in amino acid composition between the two ESMs, especially in Cys, Pro, Ala, Leu, and Lys content. In particular, Cys and Pro have been reported as the key constituent amino acids of cysteine-rich eggshell membrane proteins (CREMPs) and collagen, the structural proteins in ESM [23,41]. The content in Cys and Pro were noticeably higher in fresh ESM than in hatched ESM, implying a much stronger protein network structure in the fresh ESM, which is consistent with our observation of the SEM. The content in Leu, Ala, and Lys, known as hydrophobic amino acids (HAA) [42], were higher in the hatched ESM than the fresh ones. HAA play an important role in scavenging free radicals by increasing the solubility of peptides in the lipid phase and facilitating the interaction between peptides and free radicals [8]. The difference in amino acid profiles between the two ESMs indicated that their hydrolysates will differ both in nutritional values and bioactivities. The hatched and fresh ESMs contain all essential amino acids (EAA), accounting for 30.57% and 29.04%, respectively, values similar to or even higher than those for many other food source proteins, including walnut protein (26.98%), and cashew protein (29.92%) [28,43]. EAA, essential amino acids (Ile, Leu, Lys, Met, Phe, Thr, and Val); TAA, total amino acids; Values are expressed as mean ± SD (n = 3). Different superscripts (a-b) within the same row indicate significant differences (p < 0.05). "*" represents "the key constituent amino acids of CREMPs and collagen, the main structural proteins in eggshell membranes".
The above results further suggested that the hatched ESM can potentially serve as a novel protein source, with a balanced amino acid composition and high nutrition value, to prepare hydrolysates for food products. The different structure and chemical composition between the two sources of ESMs were expected considering the qualitative and quantitative changes in ESM during incubation and passive storage [21,22,44]. These differences might also give rise to significantly different proteolytic properties and hydrolysate bioactivities. Therefore, we further analyzed the hydrolytic properties of the hatched ESM as a novel raw material to obtain bioactive peptides and studied the activity of their hydrolysates, using the fresh ESM as a control.

Enzymatic Hydrolysis of ESM
Enzymatic hydrolysis is considered the most efficient method to produce bioactive peptides from protein precursors, allowing to retain their full nutritional value [45], and avoiding the presence of residual organic solvents or toxic chemicals in the final product, thus making it a widely used practice in the food industry [46]. In this experiment, alcalase, a widely used protease to prepare bioactive peptides from natural protein sources [2,29], was chosen to prepare ESM protein hydrolysates since our previous studies [15,16] demonstrated its value to generate fresh ESM hydrolysates with strong antioxidant properties, which was better than those of hydrolysates obtained with other commonly used proteases [15].

Degree of Hydrolysis (DH)
DH is an essential parameter of hydrolysis, for it can affect the size of peptides, and thus regulating their biological activities [46,47]. In addition, a high DH indicates a high peptide content obtained during hydrolysis, which increased the potential for recovering the protein source for use as a food additive [46]. Figure 2, the hydrolysis kinetics of the hatched and fresh ESM obtained with alcalase reveal an increase in DH with time, corresponding to the gradual release of peptide fragments during hydrolysis. The DH curve for both ESMs is a typical hyperbolic trend with a high rate during the initial stage of hydrolysis followed by a stationary phase until reaching a steady state after 12 h. Similar DH curves were also observed for the hydrolysates of different protein sources such as Bambara bean and rice bran [47,48]. The hatched ESM treated under the same enzymatic hydrolysis conditions possessed a markedly higher degree of hydrolysis (9.28 to 15.46%) during the entire hydrolysis period when compared with the fresh ESM (4.38 to 11.70%), indicating a higher hydrolysis efficiency of the hatched than the fresh ESM. The nature of the protein substrates is known to affect the release of bioactive peptides [47]. Under the same hydrolysis conditions, the difference in DH between the two ESMs may therefore be attributed to their different chemical composition and structure. These DH results were consistent with the structural differences in SEM and amino acid analysis, indicating that the hatched ESM potentially possessed a weaker protein network structure, thus more conducive to enzymatic hydrolysis. The protein hydrolysates can be categorized based on DH values. When the DH value is greater than 10%, it represents high hydrolysis, and highly hydrolyzed protein hydrolysates offer more potential to be used as nutritional supplements or nutraceuticals [4]. Therefore, the hatched ESM hydrolysates obtained in this study were considered highly hydrolyzed and possessed potential applications in functional foods.

Molecular Weight Distributions of ESM Hydrolysates
The molecular weight (MW) distributions of hydrolysates from the two ESMs were determined by HPLC. As shown in Figures 3 and S2, both ESM hydrolysates were mainly composed of low MW peptides (<3000 Da), confirming that small molecular bioactive peptides were released from both ESMs during the enzymatic process. However, the MW distribution of the FEMH and HEMH also exhibited significant differences. The HEMH contained more peptides with small MW (<1000 Da), up to 84.44%, than those of the FEMH, up to 72.58% (p < 0.01). The higher content of low-MW peptides in the HEMH is consistent with the higher DH found in the HEMH. The nature of the protein substrates is known to affect the release of bioactive peptides [47]. Under the same hydrolysis conditions, the difference in DH between the two ESMs may therefore be attributed to their different chemical composition and structure. These DH results were consistent with the structural differences in SEM and amino acid analysis, indicating that the hatched ESM potentially possessed a weaker protein network structure, thus more conducive to enzymatic hydrolysis. The protein hydrolysates can be categorized based on DH values. When the DH value is greater than 10%, it represents high hydrolysis, and highly hydrolyzed protein hydrolysates offer more potential to be used as nutritional supplements or nutraceuticals [4]. Therefore, the hatched ESM hydrolysates obtained in this study were considered highly hydrolyzed and possessed potential applications in functional foods.

Molecular Weight Distributions of ESM Hydrolysates
The molecular weight (MW) distributions of hydrolysates from the two ESMs were determined by HPLC. As shown in Figures 3 and S2, both ESM hydrolysates were mainly composed of low MW peptides (<3000 Da), confirming that small molecular bioactive peptides were released from both ESMs during the enzymatic process. However, the MW distribution of the FEMH and HEMH also exhibited significant differences. The HEMH contained more peptides with small MW (<1000 Da), up to 84.44%, than those of the FEMH, up to 72.58% (p < 0.01). The higher content of low-MW peptides in the HEMH is consistent with the higher DH found in the HEMH. Similarly, Habinshuti et al. [49] and Zhang et al. [50] reported that higher DH values produce low MW peptides. It is well known that hydrolysates containing a higher percentage of lower MW peptides tend to have a higher antioxidant activity [51]. In addition, Mune et al. [47] reported that peptides with small MW (<1000 Da) presented the highest biological activity. Thus, the higher content of low MW peptides in the HEMH may result in higher biological activity than in the FEMH.

Amino Acid Composition of HEMH and FEMH Fractions
The amino acids present in the HEMH and FEMH are presented in Figure S3. Since the biological activity of protein hydrolysates mainly depends on the type and amount of amino acids in their protein sequences [28,52], the amino acid profiles were analyzed. Generally, the HEMH had an amino acid profile similar to the FEMH but differed in the content of various amino acids. Both profiles showed a high level of hydrophobic amino acids (Leu, Val, Ala, Pro, Phe, His, Trp, Gly, Lys, and Ile) and aromatic amino acids (Phe, Trp, and Tyr), previously reported to contribute to the antioxidant and other bioactive properties of peptides [8]. Asp and Gly were the most abundant amino acids of HEMH and FEMH, with contents of 6.93-7.07 g/100 g protein and 5.57-5.75 g/100 g protein, respectively. Jain and Anal [14] and Shi et al. [52] have reported similar results for the hydrolysates derived from fresh ESM. These two amino acids are recognized as strong electron donors or chelators for metal ions, which can improve the antioxidant potential of hydrolysates [53]. In addition, compared to the FEMH, the HEMH presented significantly (p < 0.05) higher contents of Met, Lys, and Try, which have been reported to have antioxidant effects [54]. Finally, a higher content of EAA (17.02 g/100 g) was observed in the HEMH than in the FEMH (15.95 g/100 g).
Overall, the above characterization results of hydrolysates from hatched ESM, using the hydrolysates of the well-studied fresh ESM as a control, indicated that the hydrolysates from hatched ESM have a high potential for antioxidant and nutritional properties. In the following experiments, the peptide composition and antioxidant activity of HEMH were further analyzed.

Nano-LC-ESI-MS/MS Analysis of HEMH
It is well known that the biological activity of a specific protein hydrolysate is closely related to its peptide composition [2]. In order to quantitatively identify the HEMH pep- Similarly, Habinshuti et al. [49] and Zhang et al. [50] reported that higher DH values produce low MW peptides. It is well known that hydrolysates containing a higher percentage of lower MW peptides tend to have a higher antioxidant activity [51]. In addition, Mune et al. [47] reported that peptides with small MW (<1000 Da) presented the highest biological activity. Thus, the higher content of low MW peptides in the HEMH may result in higher biological activity than in the FEMH.

Amino Acid Composition of HEMH and FEMH Fractions
The amino acids present in the HEMH and FEMH are presented in Figure S3. Since the biological activity of protein hydrolysates mainly depends on the type and amount of amino acids in their protein sequences [28,52], the amino acid profiles were analyzed. Generally, the HEMH had an amino acid profile similar to the FEMH but differed in the content of various amino acids. Both profiles showed a high level of hydrophobic amino acids (Leu, Val, Ala, Pro, Phe, His, Trp, Gly, Lys, and Ile) and aromatic amino acids (Phe, Trp, and Tyr), previously reported to contribute to the antioxidant and other bioactive properties of peptides [8]. Asp and Gly were the most abundant amino acids of HEMH and FEMH, with contents of 6.93-7.07 g/100 g protein and 5.57-5.75 g/100 g protein, respectively. Jain and Anal [14] and Shi et al. [52] have reported similar results for the hydrolysates derived from fresh ESM. These two amino acids are recognized as strong electron donors or chelators for metal ions, which can improve the antioxidant potential of hydrolysates [53]. In addition, compared to the FEMH, the HEMH presented significantly (p < 0.05) higher contents of Met, Lys, and Try, which have been reported to have antioxidant effects [54]. Finally, a higher content of EAA (17.02 g/100 g) was observed in the HEMH than in the FEMH (15.95 g/100 g).
Overall, the above characterization results of hydrolysates from hatched ESM, using the hydrolysates of the well-studied fresh ESM as a control, indicated that the hydrolysates from hatched ESM have a high potential for antioxidant and nutritional properties. In the following experiments, the peptide composition and antioxidant activity of HEMH were further analyzed.

Nano-LC-ESI-MS/MS Analysis of HEMH
It is well known that the biological activity of a specific protein hydrolysate is closely related to its peptide composition [2]. In order to quantitatively identify the HEMH peptides, the nano-LC-ESI-MS/MS analysis using Maxquant software was conducted. As shown in the total ion chromatogram ( Figure S4), the LC-MS/MS analysis was completed within 60 min, showing typical peaks corresponding to specific peptides, and 249 peptide sequences derived from HEMH were identified. Figure 4 presents the percentage distribution of peptides from specific parent proteins, including lysyl oxidase homolog (34.60%), VWFD domain-containing protein (21.00%), lysozyme C (16.24%), ovocleidin-116 (8.75%), collagen alpha-1(X) chain (7.35%), and calcium-transporting ATPase (4.14%). This result indicated that most abundant peptides present in HEMH are derived from the parent protein, Lysyl oxidase homolog. It is an enzyme that catalyzes the covalent linkage between collagen and elastin, and plays a critical role in the formation of ESM fiber as a major protein component of ESM [23,41]. Other parent proteins are also reported to have specific biological activity, such as lysozyme C, which is known to possess antibacterial and anti-inflammatory activities [12]. In addition, to understand the potential bioactivity of the identified peptides, 10 major peptide sequences (854-1385 Da) in HEMH were evaluated for bioactivity using PeptideRanker (Table S1), a server for the prediction of bioactive peptides based on a novel N-to-1 neural network and scores greater than 0.5 were considered bioactive [15,24]. The results showed that 60% of the 10 major peptides had scores above 0.5, indicating potentially high biological activity of HEMH. tides, the nano-LC-ESI-MS/MS analysis using Maxquant software was conducted. As shown in the total ion chromatogram ( Figure S4), the LC-MS/MS analysis was completed within 60 min, showing typical peaks corresponding to specific peptides, and 249 peptide sequences derived from HEMH were identified. Figure 4 presents the percentage distribution of peptides from specific parent proteins, including lysyl oxidase homolog (34.60%), VWFD domain-containing protein (21.00%), lysozyme C (16.24%), ovocleidin-116 (8.75%), collagen alpha-1(X) chain (7.35%), and calcium-transporting ATPase (4.14%). This result indicated that most abundant peptides present in HEMH are derived from the parent protein, Lysyl oxidase homolog. It is an enzyme that catalyzes the covalent linkage between collagen and elastin, and plays a critical role in the formation of ESM fiber as a major protein component of ESM [23,41]. Other parent proteins are also reported to have specific biological activity, such as lysozyme C, which is known to possess antibacterial and anti-inflammatory activities [12]. In addition, to understand the potential bioactivity of the identified peptides, 10 major peptide sequences (854-1385 Da) in HEMH were evaluated for bioactivity using PeptideRanker (Table S1), a server for the prediction of bioactive peptides based on a novel N-to-1 neural network and scores greater than 0.5 were considered bioactive [15,24]. The results showed that 60% of the 10 major peptides had scores above 0.5, indicating potentially high biological activity of HEMH.

The Antioxidation Activity of HEMH and Ultrafiltration Fractions
In chemical antioxidant activity assays, the antioxidation of peptides or protein hydrolysates depends on their free radical scavenging ability, metal ion chelating ability, and reducing ability [52,55]. The DPPH free radical scavenging assay is an antioxidant activity evaluation method based on the electron transfer mechanism and is commonly used to assess the antioxidant capacity of natural compounds [42]. Chelation of metal ions is an essential pathway for antioxidant action, as Fe 2+ ions are catalysts for the chain reaction of lipid peroxidation, leading to the oxidation of unsaturated lipids [56]. The reducing power represents the ability to contribute electrons or hydrogen, and the ferric ion reducing power determination involves a redox reaction in which the compound reduces Fe 3+ to Fe 2+ [50].
To further investigate their antioxidant activities, our study determined the DPPH radical scavenging activity, Fe 2+ -chelating activity, and Fe 3+ -reduction capacity of the HEMH and FEMH. As shown in Table 3 the HEMH possessed better antioxidant activities in DPPH radical scavenging ability (153.51 ± 12.63 μmol TE/g), Fe 2+ -chelating activities (80.11 ± 0.30%), and reducing power (0.67 ± 0.005) than the FEMH (51.93 ± 2.47 μmol TE/g, 56.51 ± 1.17%, and 0.10 ± 0.01%, respectively). These results were consistent with the finding that the HEMH exhibited a higher DH value and a higher content in small molecule peptides, when compared to the FEMH.

The Antioxidation Activity of HEMH and Ultrafiltration Fractions
In chemical antioxidant activity assays, the antioxidation of peptides or protein hydrolysates depends on their free radical scavenging ability, metal ion chelating ability, and reducing ability [52,55]. The DPPH free radical scavenging assay is an antioxidant activity evaluation method based on the electron transfer mechanism and is commonly used to assess the antioxidant capacity of natural compounds [42]. Chelation of metal ions is an essential pathway for antioxidant action, as Fe 2+ ions are catalysts for the chain reaction of lipid peroxidation, leading to the oxidation of unsaturated lipids [56]. The reducing power represents the ability to contribute electrons or hydrogen, and the ferric ion reducing power determination involves a redox reaction in which the compound reduces Fe 3+ to Fe 2+ [50].
To further investigate their antioxidant activities, our study determined the DPPH radical scavenging activity, Fe 2+ -chelating activity, and Fe 3+ -reduction capacity of the HEMH and FEMH. As shown in Table 3 the HEMH possessed better antioxidant activities in DPPH radical scavenging ability (153.51 ± 12.63 µmol TE/g), Fe 2+ -chelating activities (80.11 ± 0.30%), and reducing power (0.67 ± 0.005) than the FEMH (51.93 ± 2.47 µmol TE/g, 56.51 ± 1.17%, and 0.10 ± 0.01%, respectively). These results were consistent with the finding that the HEMH exhibited a higher DH value and a higher content in small molecule peptides, when compared to the FEMH. In order to investigate the relationship between the antioxidant activity and the MW of the HEMH, as well as to obtain a purified fraction with a higher bioactivity, the antioxidant activity of ultrafiltration fractions of the HEMH with different MW was further determined. It was found that the peptide fraction with the smallest MW (HEMH-I, MW < 3 kDa) had the highest antioxidant activity, whereas the HEMH-IV (MW > 30 kDa), the largest MW fraction, exhibited the weakest antioxidant effect, in agreement with our previously reported results [24]. Several studies have reported that the antioxidant activity of peptides is related to MW distribution [42,57], and that lower MW peptides are considered to have higher antioxidant activity than higher MW peptides [58]. Altogether, the HEMH has a higher DH value, a higher amount of low molecular weight peptides, and a higher antioxidant activity than the FEMH under the same enzymatic hydrolysis conditions, indicating that the hatched ESM showed more potential than the fresh ESM to release antioxidant peptides. The HEMH-I fraction with the highest antioxidant activity was then evaluated for its protective effects against oxidative stress in SW1353 chondrocytes.

Cytoprotective Effect of HEMH-I on H 2 O 2 -Induced SW1353 Human Chondrocytes
SW1353 human chondrocytes are one of the most common and established cell lines used for substitution of primary chondrocytes [59]. H 2 O 2 is an important factor involved in the pathogenesis of OA. In chondrocytes, H 2 O 2 can induce the cellular production of ROS, inhibit proteoglycan synthesis and promote ECM degradation, causing the expression of inflammatory cytokines and matrix metalloproteinases (MMPs) [60]. Therefore, H 2 O 2 -induced SW1353 human chondrocytes are an representative in vitro OA model [3], replacing articular chondrocytes and animal models in early stages of development of novel OA therapeutics while meeting the need for a simplified approach to understand the pathophysiology of OA [59,61]. In this study, we used H 2 O 2 -induced SW1353 cells to investigate the potential role of HEMH-I in protecting chondrocytes against cellular oxidative stress.

Effects of HEMH-I on Cell Viability of H 2 O 2 -Induced SW1353 Human Chondrocytes
In order to obtain an insight into the response of SW1353 chondrocytes to H 2 O 2 treatment, as well as to establish the optimal conditions for H 2 O 2 induction, cells were cultured in the presence of different concentrations of H 2 O 2 (100, 200, 400, 600, and 800 µM) for 1 h. As seen in Figure 5A Figure 5B demonstrated no toxicity on SW1353 cells for HEMH-I at concentrations up to 0.75 μg/mL. To evaluate the protective effect of HEMH-I against oxidative stress in chondrocytes, the effects of HEMH-I at the level of 0.125, 0.25, 0.5, and 0.75 mg/mL on the cell viability of H2O2-damaged SW1353 cells were measured. As displayed in Figure 5C, all concentrations of HEMH-I could significantly reverse the H2O2-induced injury in SW1353 cells (p < 0.05), and the viability of the cells was increased by 34.59-51.32% after HEMH-I treatment. This result indicated that HEMH-I possessed a protective effect against H2O2-induced cell damage in the in vitro OA model.

Effects of HEMH-I on ROS Levels in H2O2-Induced SW1353 Human Chondrocytes
The oxidative stress levels in cells can be assessed using ROS measurement. Inhibiting cellular damage to chondrocytes by regulating ROS production is an important therapeutic strategy [3]. In this study, the ROS levels of different treatment groups were determined by applying DCFH-DA fluorescence dye. As shown in Figure 6A, the H2O2 model group exhibited the brightest fluorescent signal, while the control group exhibited the lowest fluorescent signal, indicating that H2O2-induced oxidative stress in SW1353 cells by accumulating ROS. Compared with the H2O2 damage group, the fluorescence level of SW1353 cells was significantly reduced after pretreatment with different concentrations of HEMH-I. The fluorescence intensities of all groups were also quantified using Image J software ( Figure 6B). The ROS level of the model group was 2.04-fold higher than that of the control group (p < 0.05). Meanwhile, the pretreatment with HEMH-I (0.25, 0.5 and 0.75 mg/mL) significantly reduced the intracellular fluorescence intensity of SW1353 by 28%, 43%, and 51%, respectively (p < 0.05), suggesting that

Effects of HEMH-I on ROS Levels in H 2 O 2 -Induced SW1353 Human Chondrocytes
The oxidative stress levels in cells can be assessed using ROS measurement. Inhibiting cellular damage to chondrocytes by regulating ROS production is an important therapeutic strategy [3]. In this study, the ROS levels of different treatment groups were determined by applying DCFH-DA fluorescence dye. As shown in Figure 6A, the H 2 O 2 model group exhibited the brightest fluorescent signal, while the control group exhibited the lowest fluorescent signal, indicating that H 2 O 2 -induced oxidative stress in SW1353 cells by accumulating ROS. Compared with the H 2 O 2 damage group, the fluorescence level of SW1353 cells was significantly reduced after pretreatment with different concentrations of HEMH-I. The fluorescence intensities of all groups were also quantified using Image J software ( Figure 6B). The ROS level of the model group was 2.04-fold higher than that of the control group (p < 0.05). Meanwhile, the pretreatment with HEMH-I (0.25, 0.5 and 0.75 mg/mL) significantly reduced the intracellular fluorescence intensity of SW1353 by 28%, 43%, and 51%, respectively (p < 0.05), suggesting that HEMH-I could dose-dependently reduce H 2 O 2 -induced ROS production in SW1353 cells and thus exerting protection against cellular oxidative stress damage.

FOR PEER REVIEW 15 of 22
HEMH-I could dose-dependently reduce H2O2-induced ROS production in SW1353 cells and thus exerting protection against cellular oxidative stress damage.

Effects of HEMH-I on Antioxidant Enzymes and Inflammatory Factor Expression in SW1353 Human Chondrocytes
As the first line of defense against ROS, superoxide dismutase (SOD) plays an important role in maintaining the dynamic balance of oxidation and antioxidation in the body [62]. IL-6 is one of the most important inflammatory markers in OA and represents the level of oxidative stress-induced inflammation in chondrocytes [63]. In order to evaluate the effect of HEMH-I on oxidative stress and inflammatory response in an in vitro OA model, the expression levels of SOD1 and IL-6 in SW1353 cells were determined using western blot analysis.
The results are shown in Figure 7. H2O2 treatment significantly reduced the expres-

Effects of HEMH-I on Antioxidant Enzymes and Inflammatory Factor Expression in SW1353 Human Chondrocytes
As the first line of defense against ROS, superoxide dismutase (SOD) plays an important role in maintaining the dynamic balance of oxidation and antioxidation in the body [62]. IL-6 is one of the most important inflammatory markers in OA and represents the level of oxidative stress-induced inflammation in chondrocytes [63]. In order to evaluate the effect of HEMH-I on oxidative stress and inflammatory response in an in vitro OA model, the expression levels of SOD1 and IL-6 in SW1353 cells were determined using western blot analysis.
The results are shown in Figure 7. H 2 O 2 treatment significantly reduced the expression of SOD1 and promoted the expression of the inflammatory factor IL-6 (p < 0.05), indicating that H 2 O 2 -induced an increase in oxidative stress and inflammation in SW1353 cells. In contrast, 0.25 mg/mL and 0.75 mg/mL of HEMH-I treatment markedly increased the expression of SOD1 by 1.03-and 1.37-fold, respectively, when compared with the model group (p < 0.05). Meanwhile, low-dose and high-dose HEMH-I significantly reduced the H 2 O 2 -induced IL-6 expression by 48.38% and 55.85%, respectively (p < 0.05). The above results indicated that HEMH-I could effectively reduce cellular oxidative stress and inflammatory responses in the H 2 O 2 -induced SW1353 human chondrocytes. Increased ROS in chondrocytes can lead to excessive degradation of the ECM, which plays a central role in the progression and development of OA [31]. Type II collagen is a major constituent of articular cartilage [5]. The overexpression of protein hydrolases, including MMPs, by chondrocytes leads to excessive degradation of type II collagen and other ECM proteins, which is a pivotal event in the pathogenesis of OA [64]. As one of the important MMPs, MMP3 can activate other MMPs (MMP2, MMP9, MMP13, etc.) from the proMMPs and thus plays a key role in the progression of ECM degradation [3,65]. In addition, MMP13 is a major enzyme hydrolyzing type II collagen [60]. Accordingly, the effects of HEMH-I on type II collagen, MMP3 and MMP13 expression in H2O2-induced SW1353 cells were evaluated to investigate the protective role of HEMH-I against ECM degradation.
As shown in Figure 8, the exposure to H2O2 significantly increased the expression of MMP3 and MMP13 while decreasing the level of type II collagen, indicating that oxidative stress induced by H2O2 stimulated ECM degradation in SW1353 human chondrocytes, in agreement with Kim et al. [3] who reported that H2O2-induced ROS could reduce the role of chondrocytes in the formation and maintenance of cartilage ECM. However, HEMH-I treatment at 0.25 and 0.75 mg/mL significantly decreased MMP3 and MMP13 expression compared to the model group. Analogous results for MMP3 and MMP13 expression were observed in previous studies of bioactive substances ameliorating ECM degradation in chondrocytes [66,67]. Additionally, type II collagen protein levels were increased by 1.29-fold and 1.90-fold, respectively. These results suggested that HEMH-I could attenuate ECM degradation by effectively regulating type II collagen, MMP3 and MMP13 expression in SW1353 chondrocytes under oxidative stress. Increased ROS in chondrocytes can lead to excessive degradation of the ECM, which plays a central role in the progression and development of OA [31]. Type II collagen is a major constituent of articular cartilage [5]. The overexpression of protein hydrolases, including MMPs, by chondrocytes leads to excessive degradation of type II collagen and other ECM proteins, which is a pivotal event in the pathogenesis of OA [64]. As one of the important MMPs, MMP3 can activate other MMPs (MMP2, MMP9, MMP13, etc.) from the proMMPs and thus plays a key role in the progression of ECM degradation [3,65]. In addition, MMP13 is a major enzyme hydrolyzing type II collagen [60]. Accordingly, the effects of HEMH-I on type II collagen, MMP3 and MMP13 expression in H 2 O 2 -induced SW1353 cells were evaluated to investigate the protective role of HEMH-I against ECM degradation.
As shown in Figure 8, the exposure to H 2 O 2 significantly increased the expression of MMP3 and MMP13 while decreasing the level of type II collagen, indicating that oxidative stress induced by H 2 O 2 stimulated ECM degradation in SW1353 human chondrocytes, in agreement with Kim et al. [3] who reported that H 2 O 2 -induced ROS could reduce the role of chondrocytes in the formation and maintenance of cartilage ECM. However, HEMH-I treatment at 0.25 and 0.75 mg/mL significantly decreased MMP3 and MMP13 expression compared to the model group. Analogous results for MMP3 and MMP13 expression were observed in previous studies of bioactive substances ameliorating ECM degradation in chondrocytes [66,67]. Additionally, type II collagen protein levels were increased by 1.29-fold and 1.90-fold, respectively. These results suggested that HEMH-I could attenuate ECM degradation by effectively regulating type II collagen, MMP3 and MMP13 expression in SW1353 chondrocytes under oxidative stress. The Keap1/Nrf2/HO-1 signaling pathway is an essential regulatory pathway cellular resistance to oxidative stress induced by various exogenous and endogen factors [25,68]. In this pathway, when cells are under stressed conditions or in the p ence of certain bioactive molecules, nuclear factor erythroid 2-associated factor 2 (N will separate from its inhibitor Kelch-like ECH-associated protein 1 (Keap1) and bin the antioxidant response element, initiating the expression of heme oxygenase-1 (HO and leading to the activation of the defense system against oxidative stress [65]. Rece this pathway has been identified as a novel therapeutic target against OA [1,6]. M studies have supported the important role of Nrf2 and HO-1 expression in this signa pathway to reduce oxidative stress, inflammatory response, and cartilage degradatio OA [69,70]. Our previous study has shown that HEMH-I can regulate the Keap1/N signaling pathway to reduce oxidative stress in RAW264.7 cells [24]. However, whe HEMH-I exerts a regulatory effect on Keap1/Nrf2/HO-1 pathway in OA chondrocyte vitro is not known. Therefore, the expression of Keap1, Nrf2, and HO-1, the key prot of this signaling pathway, were further determined in SW1353 human chondrocytes. As shown in Figure 9, H2O2 treatment resulted in a 34.24% increase in the exp sion of Keap1 and a significant decrease in the expression of Nrf2 and HO-1 in SW1 The Keap1/Nrf2/HO-1 signaling pathway is an essential regulatory pathway for cellular resistance to oxidative stress induced by various exogenous and endogenous factors [25,68]. In this pathway, when cells are under stressed conditions or in the presence of certain bioactive molecules, nuclear factor erythroid 2-associated factor 2 (Nrf2) will separate from its inhibitor Kelch-like ECH-associated protein 1 (Keap1) and bind to the antioxidant response element, initiating the expression of heme oxygenase-1 (HO-1) and leading to the activation of the defense system against oxidative stress [65]. Recently, this pathway has been identified as a novel therapeutic target against OA [1,6]. Many studies have supported the important role of Nrf2 and HO-1 expression in this signaling pathway to reduce oxidative stress, inflammatory response, and cartilage degradation in OA [69,70]. Our previous study has shown that HEMH-I can regulate the Keap1/Nrf2 signaling pathway to reduce oxidative stress in RAW264.7 cells [24]. However, whether HEMH-I exerts a regulatory effect on Keap1/Nrf2/HO-1 pathway in OA chondrocytes in vitro is not known. Therefore, the expression of Keap1, Nrf2, and HO-1, the key proteins of this signaling pathway, were further determined in SW1353 human chondrocytes.
As shown in Figure 9, H 2 O 2 treatment resulted in a 34.24% increase in the expression of Keap1 and a significant decrease in the expression of Nrf2 and HO-1 in SW1353 chondrocytes when compared to the control group (p < 0.05). Both low-dose and high-dose HEMH-I treatments significantly decreased the expression of Keap1 while significantly increasing the expression of Nrf2 and HO-1, the downstream antioxidant enzyme of this pathway, by 1.35-fold and 1.77-fold, respectively (p < 0.05). These results indicated that the mechanism exhibited by HEMH-I to protect chondrocytes against oxidative damage was related to the activation of the antioxidant signaling pathway Keap1/Nrf2/HO-1. Similar studies of many natural bioactive components such as allicin, sulforaphane, and lycopene were reported to reduce oxidative stress, decrease inflammatory marker expression, and increase chondrogenic markers in the H2O2-induced chondrocytes by activating this pathway [3,67].
Taken together, the cell experiment results indicated a novel beneficial role of HEMH-I in ameliorating oxidative stress, attenuating inflammatory factor expression, promoting type II collagen synthesis, and modulating the Keap1/Nrf2/HO-1 signaling pathways in H2O2-induced SW1353 chondrocytes. Notably, even though the SW1353 cells is widely used as an alternative to primary chondrocytes for in vitro OA study [3,59], our results could not be fully translated to primary OA chondrocytes due to the differences between SW1353 cells and human primary chondrocytes [66], which is a limitation of this study and warrants further investigation.

Conclusions
The hatched ESM is a high protein (92.98%) byproduct resource with significantly different structural properties and chemical composition from fresh ESM. It can be used to prepare antioxidant hydrolysates rich in low-MW peptides (94.63%, MW < 3 kDa) by enzymatic hydrolysis with alcalase. Compared with the fresh ESM, the hatched ESM potentially exhibited higher enzymatic hydrolysis efficiency and antioxidant activity of hydrolysates.
The hatched ESM hydrolysates with MW < 3 kDa, HEMH-I, showed the highest antioxidant activity and exerted cytoprotective activity to ameliorate the H2O2-induced oxidative stress, inflammatory response, and cartilage ECM degradation in SW1353 human chondrocytes, which may related to the activation of the cellular Keap1/Nrf2/HO-1 pathway.
This study provides a theoretical foundation for developing hatched ESM waste as a These results indicated that the mechanism exhibited by HEMH-I to protect chondrocytes against oxidative damage was related to the activation of the antioxidant signaling pathway Keap1/Nrf2/HO-1. Similar studies of many natural bioactive components such as allicin, sulforaphane, and lycopene were reported to reduce oxidative stress, decrease inflammatory marker expression, and increase chondrogenic markers in the H 2 O 2 -induced chondrocytes by activating this pathway [3,67].
Taken together, the cell experiment results indicated a novel beneficial role of HEMH-I in ameliorating oxidative stress, attenuating inflammatory factor expression, promoting type II collagen synthesis, and modulating the Keap1/Nrf2/HO-1 signaling pathways in H 2 O 2 -induced SW1353 chondrocytes. Notably, even though the SW1353 cells is widely used as an alternative to primary chondrocytes for in vitro OA study [3,59], our results could not be fully translated to primary OA chondrocytes due to the differences between SW1353 cells and human primary chondrocytes [66], which is a limitation of this study and warrants further investigation.

Conclusions
The hatched ESM is a high protein (92.98%) byproduct resource with significantly different structural properties and chemical composition from fresh ESM. It can be used to prepare antioxidant hydrolysates rich in low-MW peptides (94.63%, MW < 3 kDa) by enzymatic hydrolysis with alcalase. Compared with the fresh ESM, the hatched ESM potentially exhibited higher enzymatic hydrolysis efficiency and antioxidant activity of hydrolysates.
The hatched ESM hydrolysates with MW < 3 kDa, HEMH-I, showed the highest antioxidant activity and exerted cytoprotective activity to ameliorate the H 2 O 2 -induced oxidative stress, inflammatory response, and cartilage ECM degradation in SW1353 human chondrocytes, which may related to the activation of the cellular Keap1/Nrf2/HO-1 pathway.
This study provides a theoretical foundation for developing hatched ESM waste as a novel natural source to prepare antioxidant hydrolysates, as well as new insights into protective effect of HEMH-I against oxidative stress in chondrocytes. Further studies using primary chondrocytes and in vivo models are necessary to confirm the beneficial effects of HEMH-I on OA cartilage protection and the unique mechanisms behind them.

Supplementary Materials:
The following are available online at https://www.mdpi.com/article/ 10.3390/antiox11122428/s1, Figure S1: The flow chart of the research strategy of the present work. Figure S2: Molecular weight distribution of hatched eggshell membrane hydrolysates (A) and fresh eggshell membrane hydrolysates (B). Figure S3: The composition of amino acids (g/100 g) in hydrolysates of hatched ESM (HEMH) and fresh ESM (FEMH). The significance levels of * p < 0.05 and ** p < 0.01 are for FEMH versus HEMH at the same amino acid. Figure S4