Abstract

   Objectives: This study aimed to characterize the basic structure of
   sialylated glycopeptide (SCP) from edible bird’s nest, and to explore
   the intervention effect and mechanism of SCP based on a mouse lung
   inflammation model induced by lipopolysaccharide (LPS). Methods:
   C57BL/6 mice were randomly divided into the control group (CON), model
   group (LPS), EBN group, SCP group, and SA group. Results: The results
   showed that SCP had the typical structures of polypeptides and
   carbohydrates. SCP effectively intervened in the lung inflammation
   response. The number of neutrophils (Neu) in BALF decreased by 41.3%,
   the level of tumor necrosis factor-α (TNF-α) decreased by 36.4%, and
   the W/D ratio of lung tissues decreased by 27.2%, effectively
   preventing pathological changes in lung tissues. A total of 40
   differential metabolites such as choline, linolenic acid, and xanthine
   were screened between the SCP group and the LPS group. These
   differential metabolites were mainly enriched in the metabolic pathways
   of glycerophospholipids, alpha-linolenic acid, and purines.
   Conclusions: The research results support that SCP, as the active
   substance of edible bird’s nest, can effectively improve lung
   inflammation, providing theoretical guidance for the development of
   functional edible bird’s nest products.

   Keywords: edible bird’s nest, sialylated glycopeptide, lung
   inflammation, metabolomics

1. Introduction

   Edible bird’s nest (EBN) is coagulated from the saliva secreted by
   several species of swiftlets in the genus Aerodramus of the Apodidae
   family mixed with other substances. As a traditional precious product
   with both medicinal and edible properties, it has a long history of
   consumption in Asian regions, especially in China [[38]1]. The main
   component of EBN is primarily composed of macromolecular glycoproteins
   rich in sialic acid (SA), which typically exist as terminal residues at
   the non-reducing ends of glycan chains in these glycoproteins [[39]2].
   The SA content in EBN, a pivotal component of its bioactive
   glycopeptides, exhibits notable variability influenced by species
   origin, geographical provenance, and nesting environment [[40]3]. For
   example, Aerodramus fuciphagus nests contain 1.9-fold higher conjugated
   SA (dry weight) than A. maximus nests, reflecting genetic differences
   in salivary glycoprotein biosynthesis. House-farmed EBN from Malaysia
   exhibits 63% higher SA levels than cave-nested counterparts, a
   disparity linked to environmental factors like dietary composition and
   microhabitat [[41]4]. Regionally, Vietnamese EBN harbors elevated
   sialoglycoprotein content (the primary molecular carriers of SA)
   compared to major producing regions such as Malaysia [[42]5]. These
   variations in SA abundance and glycan architecture are critical
   determinants of glycopeptide structures, potentially influencing
   biological activities like inflammation modulation [[43]6]. Compared
   with the untreated EBN, the enzymatically hydrolyzed EBN extract has
   higher biological activities such as anti-inflammatory, antioxidant,
   and immunological activities [[44]7]. Glycopeptides prepared from the
   extraction of many natural foods, including Lycium barbarum and egg
   glycopeptides, have been demonstrated to play anti-inflammatory and
   immune-promoting roles [[45]8,[46]9]. Notably, EBN has the efficacy of
   moistening the lungs and nourishing yin in Chinese medicine. In
   existing studies, EBN can treat respiratory diseases by inhibiting
   inflammation, regulating the immune system, and maintaining oxidative
   balance [[47]10,[48]11]. Currently, research on the anti-inflammatory
   activities of EBN mainly focuses on peptides, but the role of
   glycopeptides has not been emphasized. Previous studies have proven
   that the sialylated glycopeptide (SCP) of the EBN is the main active
   component in edible bird’s nest, and it can exert a neuroprotective
   effect through the gut–brain axis [[49]12]. However, currently, the
   efficacy of SCP in alleviating lung inflammation remains unclear and
   requires further research.

   Lung inflammation is an important pathological basis for many severe
   respiratory diseases, which can significantly affect the metabolic
   homeostasis of the body and weaken the body’s functions. Its
   pathogenesis is extremely complex, involving multiple aspects including
   immune response, oxidative stress, cell apoptosis, and microbial
   infection [[50]13]. When the lungs are invaded by pathogens or other
   harmful stimuli, the immune system is activated, triggering an
   inflammatory response, which subsequently leads to phenomena such as
   immune disorders, tissue damage, and metabolic abnormalities [[51]14].
   Currently, although traditional clinical intervention methods such as
   glucocorticoids and antibiotics can alleviate symptoms, long-term use
   is likely to trigger drug resistance and side effects. In recent years,
   extensive attention has been paid to searching for food-derived
   components with anti-inflammatory properties from the diet. A variety
   of food-derived glycoprotein complexes have been proven to possess
   anti-inflammatory effects [[52]15]. Many natural foods, such as
   Ganoderma lucidum, contain abundant bioactive components and have been
   confirmed to be capable of intervening in lung inflammation [[53]16].
   Lipopolysaccharide (LPS), which serves as a key constituent of the cell
   wall in Gram-negative bacteria, can activate the immune system and
   trigger inflammatory reactions such as the massive release of
   pro-inflammatory cytokines and all kinds of tissue damage [[54]17].
   Thus, LPS is commonly used to induce lung injury [[55]18,[56]19].

   Metabolomics has been extensively utilized to comprehensively
   investigate the patterns of metabolic dynamic responses in organisms
   when they are under exogenous stimuli or endogenous pathological
   changes [[57]20]. It enables the precise identification and
   quantitative investigation of the metabolic components of small
   molecules in biological samples [[58]21]. Subsequently, the matching of
   metabolic pathways and the elucidation of molecular mechanisms can be
   achieved. Existing studies have proven that when pulmonary inflammation
   exists in the organism, it will be accompanied by metabolic disorders.
   Therefore, investigating the metabolic changes in lung tissues helps
   understand the mechanism of lung inflammation induced by LPS and
   evaluate the regulatory effect of SCP.

   In this study, the structure of SCP was characterized by combining the
   chemical composition and primary structure. A mouse model of lung
   inflammation was established using lipopolysaccharide. The intervention
   effect of SCP on lipopolysaccharide-induced lung inflammation in mice
   was evaluated from three aspects: the level of lung inflammation, the
   pathological structure of lung tissues, and the metabolic level of lung
   tissues.

2. Materials and Methods

2.1. Materials and Reagents

   Edible bird’s nests horn (from Malaysia) was purchased from a local
   supermarket in Wuxi. Sialic acid was purchased from Kesitan
   Biotechnology Co., Ltd. (Wuhan, China), and Alcalase 2.4L (2.4 AU/g),
   Papain (800 U/mg) were purchased from Novozymes (Bagsværd, Denmark).
   Beyoblue™ coomassie blue super fast staining solution, prestained color
   protein molecular weight marker (10–170 kDa), SDS-PAGE protein loading
   buffer (2×), periodic acid-Schiff staining kit, and BCA protein assay
   kit, protein precast gel (4–20%, 15 Wells) were purchased from Beyotime
   Biotechnology Co., Ltd. (Shanghai, China). Ethanol, NaHSO[4],
   o-phenylenediamine hydrochloride (OPA), glucose, phenol, sulfuric acid,
   etc., were all purchased from Sinopharm Chemical Reagent Co., Ltd.
   (Shanghai, China). Lipopolysaccharide (LPS) was purchased from
   Sigma-Aldrich (St. Louis, MO, USA). TNF-α, IL-1β, and IL-6 commercial
   kits were purchased from Xiamen Hui Jia Biotechnology Co., Ltd.
   (Xiamen, China). Acetone was procured from Zhejiang Hannuo Chemical
   Technology Co., Ltd. (Jinhua, China).

2.2. Experimental Animals

   Forty 7-week-old male C57BL/6J mice (specific pathogen-free [SPF]
   grade) were obtained from Jiangsu GemPharmatech Co., Ltd. (Nanjing,
   China). The experimental procedures were approved by the Laboratory
   Animal Welfare and Ethics Committee of Jiangnan University (approval
   number: JN.No20230415c0800705[145]).

2.3. Preparation of Sialylated Glycopeptides (SCP) from EBN

   The sequential enzymatic hydrolysis of EBN horn was performed using
   Alcalase 2.4 L followed by papain under optimized conditions. The
   first-step hydrolysis with Alcalase 2.4 L was conducted at 60 °C and pH
   8.5 for 12 h with an enzyme–substrate ratio of 6000 U/g. Subsequently,
   papain hydrolysis was performed at 50 °C and pH 7.0 for 2 h using an
   increased enzyme-substrate ratio of 10,000 U/g. Enzyme inactivation was
   achieved through thermal denaturation (95 °C, 10 min). Finally,
   purification was achieved through our laboratory’s established method
   [[59]12]. After being frozen at −80 °C for 12 h, the extracted SCP
   powder was obtained by a lyophilizer (SCIENTZ-10N, SCIENTZ Biotech Co.,
   Ltd. (Ningbo, China)).

2.4. Structural Characterization of SCP

2.4.1. Neutral Sugar and SA Content Quantification

   The content of neutral sugars was measured via the phenol-sulfuric acid
   approach. To SCP samples, 1 mL of 5% phenol solution was added,
   followed by the rapid addition of 5 mL of concentrated sulfuric acid.
   After thorough mixing, the mixture was incubated in a water bath at 37
   °C for 30 min. Subsequently, the absorbance was measured at 490 nm
   using a Microplate Reader 96 (EPOCH1, Bio Tek Instruments, Inc.,
   Winooski, VT, USA). Glucose (anhydrous, dried to constant weight in an
   oven) was used as the standard reference [[60]22].

   SA content was determined by liquid chromatography (LC-20AT, Shimadzu,
   Kyoto, Japan). SCP sample (50 mg) was hydrolyzed with 5 mL 0.5 M
   NaHSO[4] at 80 °C for 30 min, after which, derivatization was performed
   with 5 mL 10 g/L OPA (in 0.25 M NaHSO[4]) at 80 °C for 40 min. After
   filtration through the 0.22 μm filters, the solution was diluted to 25
   mL and stored in the dark. In the HPLC analysis, the EF-C18Bio column
   (Galaksil, Rugao City, China) was applied at 30 °C. The mobile phase,
   acetonitrile–water (5:95, v/v), flowed at a 1.0 mL/min rate. Detection
   was performed at 230 nm with a 20 μL injection volume [[61]12].

2.4.2. Amino Acid Composition Analysis

   Approximately 100 mg of solid powder was hydrolyzed with 8 mL 6 M HCl
   under N[2] at 120 °C for 22–24 h. After neutralization with 10 M NaOH
   and dilution to 25 mL, the sample was filtered and centrifuged (10,000
   rpm, 10 min). For tryptophan, 8 mL 5 M NaOH was used for initial
   hydrolysis, followed by neutralization with 6.7 mL 6 M NaOH. In the
   HPLC analysis (Agilent, Santa Clara, CA, USA), the ODS column (Agilent
   Hypersil, Santa Clara, CA, USA) was employed with gradient elution
   using mobile phases A (27.6 mmol/L sodium acetate-triethylamine-THF,
   500:0.11:2.5, v/v/v, pH 7.2) and B (80.9 mmol/L sodium
   acetate-methanol-acetonitrile, 1:2:2, v/v/v, pH 7.2), 0–17 min (8–50%
   B), 17–20.1 min (50–100% B), 20.1–24 min (100–0% B). The flow rate was
   1.0 mL/min at 40 °C, with UV detection at 338 nm. Quantification was
   via external calibration.

2.4.3. Molecular Weight Distribution Determination

   SCP sample was vortexed with loading buffer (1:1, v/v), heat-denatured
   (100 °C, 3 min) and then centrifuged (8000 rpm, 3 min) to pellet
   aggregates. The molecular weight of SCP was determined through a
   discontinuous SDS-PAGE system (5% stacking/10% resolving gel) under
   reducing conditions, with a sample loading volume of 10 μL. Coomassie
   Brilliant Blue was used to stain protein, and Periodic acid-Schiff
   (PAS) reagent was used for glycan [[62]23].

2.4.4. FTIR Spectral Analysis

   ATR-FTIR spectroscopy (Thermo Fisher Scientific, Waltham, MA, USA) was
   employed to analyze secondary structures. Lyophilized SCP samples were
   directly placed on the ATR crystal and scanned over 4000–650 cm^−1 with
   32 scans at 4 cm^−1 resolution [[63]24].

2.5. Animal Grouping and Biological Sample Collection

   Mice were kept at Jiangnan University’s Laboratory Animal Centre
   (SYXK-(Su)-2021-0056) in a barrier environment. They experienced a 12 h
   light/dark cycle. The temperature ranged from 20 to 26 °C, and humidity
   was 40–70% under the artificial 12 h light/dark regime. Forty mice were
   first individually tagged with unique ear tags (numbered 1–40), and a
   random sequence was derived from a random number table. Mice were then
   assigned to five experimental groups (n = 8) based on this sequence.
   Mice were acclimated for 1 week in a standard environment with free
   access to food and water before the experiment. During the experiment,
   stress responses were monitored by observing behavioral status, such as
   activity level, food intake, and body weight changes. Procedures were
   performed gently to avoid unnecessary stress. The control group (CON)
   and LPS model group (LPS) were given 0.2 mL of saline by oral gavage
   daily, while the EBN group (EBN) was administered 200 mg/kg/d EBN
   homogenate (dry weight basis), the SCP group (SCP) received
   glycopeptides equivalent to EBN’s sialic acid content, and the SA group
   was given an equimolar free sialic acid solution for 7 consecutive
   weeks. During the last week, all groups except CON were
   intraperitoneally injected with LPS (500 μg/kg/d) for 7 days. After the
   last LPS injection, the mice were fasted overnight. Following weighing,
   they were anesthetized by isoflurane inhalation. Subsequently, the mice
   were euthanized via cervical dislocation and immediately dissected.
   After sacrificing the mice, the bronchoalveolar lavage fluid (BALF) and
   lung tissue were collected (see [64]Figure 1).

Figure 1.

   [65]Figure 1
   [66]Open in a new tab

   Animal grouping and intervention scheme.

   BALF collection: Mice were euthanized, and limbs were secured with
   pins. The chest cavity and tracheal skin were incised, and the right
   lung was gently ligated with silk thread. After exposing the trachea,
   blunt forceps were used to puncture it, followed by the insertion of a
   tracheal cannula. Once stabilized, 0.5 mL of PBS buffer was injected
   via a syringe, held for 15–30 s, and then aspirated into a 1.5 mL
   microcentrifuge tube. This procedure was repeated three times.

   Lung tissue homogenization: Frozen lung tissue was thawed and
   homogenized in PBS buffer (1:100, m/v). After mincing, 2–3 zirconia
   beads were added, and the tissue was disrupted using a tissue
   homogenizer (Jingxin Industrial, Dongguan City, China) at 70 Hz for 90
   s (2–3 cycles). Then, it was centrifuged (4 °C, 4000 rpm, 20 min), and
   the supernatant was collected for biochemical assays.

2.6. Lung Inflammatory Markers

   BALF was used to conduct cell count using an automatic hematology
   analyzer (BC-5000 Vet, Mindray Bio-Medical Electronics Co., Ltd.,
   Dongguan City, China), including white blood cells (WBC), monocytes
   (Mon), neutrophils (Neu), eosinophils (Eos), basophils (Bas), and
   lymphocytes (Lym). Lung tissues were used to determine TNF-α, IL-1β,
   and IL-6 levels by ELISA kits (Xiamen Huijia Biotech, Xiamen, China,
   OD450 nm).

2.7. Lung Histopathology

   Lung tissue was rinsed with PBS buffer, followed by blotting to remove
   surface moisture. The wet weight of the tissue was then measured.
   Subsequently, the tissue was dried at 80 °C for 48 h until a constant
   weight was achieved (dry weight). The wet/dry weight (W/D) ratio of the
   lung tissue was calculated accordingly [[67]25].

   Fresh lung tissues were fixed by immersion in 4% paraformaldehyde for
   24 h. After fixation, the tissues underwent gradient ethanol
   dehydration, xylene clearing, paraffin embedding, and tissue
   sectioning. Subsequently, the sections were deparaffinized, rehydrated,
   and stained with hematoxylin solution for 10 min. Differentiation was
   performed using 1% hydrochloric acid-alcohol for 30 s, followed by
   rinsing with tap water and bluing with ammonia water. After rinsing
   again, the sections were stained with eosin for 3 min. Following
   dehydration, clearing, and mounting, the pathological changes in lung
   tissues from each group were observed under an optical microscope.

2.8. Lung Metabolomics

   Lung tissue metabolomic profiling was performed according to the
   established LC-MS/MS protocol [[68]26]. Chromatographic raw data were
   processed through Compound Discoverer 3.3 (Thermo Fisher Scientific)
   for baseline correction, peak alignment, and feature extraction.
   Subsequent analytical workflows included quantitative analysis of peak
   areas coupled with molecular formula prediction, with spectral matching
   conducted against the mzCloud, mzVault, and MassList databases,
   supplemented by a proprietary compound library (Shanghai Senschip
   Biotech, Shanghai, China).

2.9. Statistical Analysis

   Data were presented as the mean ± standard deviation (SD). For data
   that conformed to a normal distribution, statistical analyses were
   performed as follows: For datasets with three or more groups, one-way
   analysis of variance (ANOVA) was conducted, followed by post-hoc
   pairwise comparisons using the Least Significant Difference (LSD) test
   and Duncan’s multiple range test. For two-group comparisons, a t-test
   was applied. When the data did not meet the assumptions of parametric
   tests, the non-parametric Kruskal–Wallis (KW) rank sum test was
   utilized. A significance level of p < 0.05 was set to determine
   statistical differences among groups. Multivariate statistical analyses
   were carried out using MetaboAnalyst 6.0 and SIMCA 13.0 software.

3. Results

3.1. Chemical Composition Analysis of SCP

   The sialic acid (SA) content in EBN was determined to be 10.25 ± 0.14%,
   with neutral sugars accounting for 11.84 ± 0.28%. In contrast, an
   increase in SA content (11.06 ± 0.22%) and a concomitant rise in
   neutral sugar levels (13.52 ± 0.35%) were observed in SCP ([69]Figure
   2). These results indicated an enriched glycan proportion in SCP
   compared to native EBN.

Figure 2.

   [70]Figure 2
   [71]Open in a new tab

   Neutral sugar contents and sialic acid contents of EBN and SCP.

   An analysis of 18 amino acids in SCP was performed ([72]Table 1). The
   top five amino acids by abundance in SCP were proline (Pro), aspartic
   acid (Asp) or asparagine (Asn), glutamic acid (Glu) or glutamine (Gln),
   valine (Val), and leucine (Leu). Additionally, SCP was rich in nine
   essential amino acids (with histidine (His) being essential for
   infants), which accounted for 42.22 ± 0.02% of the total amino acid
   content. The combined content of Asp or Asn, threonine (Thr), and
   serine (Ser) represented 23.34 ± 0.08% of the total amino acid content,
   suggesting the potential presence of glycosylation modifications in
   SCP.

Table 1.

   Amino acids distribution of SCP.
   Amino Acids Percentage Composition (%) Amino Acids Percentage
   Composition (%)
   Asp 9.97 ± 0.04 Glu 8.25 ± 0.04
   Cys 0.90 ± 0.02 Val * 8.03 ± 0.03
   Ser 7.05 ± 0.02 Met * 1.07 ± 0.01
   His * 3.56 ± 0.06 Phe * 6.89 ± 0.02
   Gly 4.40 ± 0.00 Ile * 3.40 ± 0.01
   Thr * 6.32 ± 0.02 Leu * 7.62 ± 0.01
   Arg 7.14 ± 0.01 Lys * 4.13 ± 0.02
   Ala 3.44 ± 0.01 Pro 10.39 ± 0.06
   Tyr 6.23 ± 0.03 Trp * 1.22 ± 0.02
   [73]Open in a new tab

   Aspartic acid (Asp), glutamic acid (Glu), cysteine (Cys), valine (Val),
   werine (Ser), methionine (Met), histidine (His), phenylalanine (Phe),
   glycine (Gly), isoleucine (Ile), threonine (Thr), leucine (Leu),
   arginine (Arg), lysine (Lys), alanine (Ala), proline (Pro), tyrosine
   (Tyr), tryptophan (Trp), * essential amino acids.

3.2. Primary Structure Analysis of SCP

   FT-IR spectra of EBN and SCP revealed significant differences
   ([74]Figure 3A). In EBN, a broad peak at 3600–3000 cm^−1 was attributed
   to hydroxyl (−OH) stretching vibrations of polysaccharides [[75]27].
   Absorption bands at 1695–1630 cm^−1 (amide I, C=O stretching) and
   1560–1500 cm^−1 (amide II, N-H bending) confirmed the presence of
   proteins/polypeptides [[76]28,[77]29]. For SCP, enhanced absorption
   bands at 1401 cm^−1 (−COO− symmetric stretching), and 1066 cm^−1 (C-O),
   coupled with a carbohydrate-specific peak at 1085–1050 cm^−1,
   demonstrated its glycopeptide nature [[78]29]. Vibrations in the
   1440–1395 cm^−1 region likely corresponded to carboxyl groups from
   sialic acid (SA) and alduronic acids, consistent with characteristic
   glycopeptide spectral profiles. These results further supported the
   glycopeptide structure of SCP.

Figure 3.

   [79]Figure 3
   [80]Open in a new tab

   FT-IR spectral and molecular weight distribution of EBN and SCP. (A)
   The Fourier infrared spectra analysis of EBN and SCP. (B)
   Electrophoresis of EBN and SCP stained with carmine blue. (C) Relative
   molecular weight distribution of EBN and SCP under carmine blue
   staining. (D) Electrophoresis of EBN and SCP under PAS staining. (E)
   Relative molecular weight distribution of EBN and SCP under PAS
   staining. M: Marker, a: EBN, b: SCP.

   The molecular weight profiles were analyzed by SDS-PAGE ([81]Figure
   3B,C). EBN proteins’ molecular weight was mainly between 70–170 kDa
   (69%). Following enzymatic hydrolysis, the proportion of this range was
   reduced to 13%, with a concurrent increase in the 25–70 kDa fraction to
   48% and the emergence of low molecular weight fragments (10–25 kDa,
   38%), confirming effective protein size reduction. PAS staining
   ([82]Figure 3D,E) revealed that glycans uniformly occupied the 70–170
   kDa range. Post-enzymatic treatment, this proportion decreased to 47%,
   while the 25–70 kDa fraction increased to 53%. Notably, glycans were
   not degraded below 25 kDa, suggesting covalent stabilization between
   glycan and protein backbones.

3.3. SCP Alleviated the Changes of LPS-Induced Lung Inflammation Levels

   The counts of inflammatory cells in bronchoalveolar lavage fluid (BALF)
   among different experimental groups were determined ([83]Figure 4A–D).
   LPS induction resulted in a statistically significant increase (p <
   0.01) in the counts of neutrophils (Neu), white blood cells (WBC),
   eosinophils (Eos), and monocytes (Mon), suggesting that LPS
   successfully induced inflammatory cell disorder in BALF. Compared with
   the LPS group, intervention groups involving EBN, SCP, and SA were
   observed to significantly decrease the number of Mon in the BALF (p <
   0.01). Furthermore, a significant reduction in the number of Neu within
   the BALF was also noted following interventions with EBN (p < 0.05),
   SCP (p < 0.01), and SA (p < 0.01). Specifically, under SCP
   intervention, the numbers of Neu in the BALF were reduced by 41.3%. It
   was indicated that EBN, SCP, and SA effectively modulated the
   aggregation of inflammatory cells within the BALF of mice induced by
   LPS. Among these interventions, SCP exhibited the most significant
   inhibitory effect on Neu recruitment.

Figure 4.

   [84]Figure 4
   [85]Open in a new tab

   Changes in lung inflammation levels of mice in each group. (A–D) Counts
   of inflammatory cells (Neu, WBC, EOS, and Mon) in the BALF of mice in
   each group. (E–G) Levels of pro-inflammatory cytokines (TNF-α, IL-1β,
   and IL-6) in the lung tissue of mice in each group. n = 8; results are
   presented as mean ± SD. # p < 0.05, ## p < 0.01 vs. CON group, * p <
   0.05, ** p < 0.01 vs. LPS group.

   Important pro-inflammatory cytokines in lung tissues of all groups,
   such as TNF-α, IL-1β, and IL-6, were determined ([86]Figure 4E–G).
   Compared with the CON group, the levels of TNF-α (p < 0.01), IL-1β (p <
   0.01), and IL-6 (p < 0.05) in the lung tissues of mice significantly
   increased after LPS induction. It was indicated that the
   intraperitoneal injection of LPS successfully induced inflammation in
   the lung tissues of mice. After the intervention of EBN, SCP, and SA,
   the levels of IL-1β and IL-6 in the lung tissues of mice were
   significantly decreased compared with those in the LPS group. Notably,
   compared with the LPS group, the level of TNF-α in the lung tissues
   decreased by 36.4% after the intervention of SCP, which was 3.6 times
   and 3.2 times the reduction ratios of EBN and SA intervention groups,
   respectively. It was suggested that the intervention groups of EBN,
   SCP, and SA all had a certain regulatory effect on the increase in the
   levels of pro-inflammatory cytokines. Among them, the intervention of
   SCP had the most significant effect on improving the level of TNF-α in
   the lung tissues.

3.4. SCP Alleviated the Changes of LPS-Induced Lung Tissue Pathological
Structures

   To evaluate the protective effect of SCP against lung tissue injury
   induced by LPS, the wet-to-dry (W/D) weight ratio of lung tissues was
   measured ([87]Figure 5A). Compared with the CON group, a significant
   increase in the W/D ratio was detected in the LPS group (p < 0.01),
   suggesting that an edema phenomenon might occur in the lung tissue. All
   intervention groups (EBN, SCP, and SA) mitigated this increase (p <
   0.05). Notably, the SCP group showed the most remarkable improvement,
   with a 27.2% decrease in the W/D weight ratio.

Figure 5.

   [88]Figure 5
   [89]Open in a new tab

   Changes in lung tissue pathological structures of mice in each group.
   (A) The ratio of W/D weight in the lung tissue of mice in each group.
   (B) H&E staining in the lung tissue of mice in each group (200×). n =
   8; results are presented as mean ± SD. ## p < 0.01 vs. CON group, * p <
   0.05, ** p < 0.01 vs. LPS group.

   Hematoxylin and Eosin (H&E) staining of lung tissues in each group
   further revealed structural alterations of the lung tissues ([90]Figure
   5B). The CON group exhibited intact alveolar architecture, orderly
   arranged epithelial cells, and no edema [[91]30]. In contrast, the LPS
   group displayed collapsed alveoli, thickened alveolar walls, and
   increased interstitial exudation. Intervention groups (EBN, SCP, and
   SA) showed varying degrees of mitigation in tissue damage. Notably, the
   SCP group demonstrated near-normal alveolar structure with reduced
   inflammatory cell infiltration. All changes in the pathological
   structure of the lung tissues revealed that the intraperitoneal
   injection of LPS successfully led to pathological changes in the
   structure of the lung tissues. Compared with the LPS group, the lung
   tissue structures of the intervention groups of EBN, SCP, and SA all
   showed certain improvements. In addition, the lung tissue structure of
   the SCP intervention group was the closest to that of the CON group.

3.5. SCP Alleviated the Change of LPS-Induced Lung Tissue Metabolic Levels

3.5.1. Analysis of the Metabolic Profiles

   After preprocessing the data and imputing the missing values, 417
   chromatographic peaks were detected in the positive ion mode, and 358
   were found in the negative ion mode. To assess the variations in the
   metabolic profiles, principal component analysis (PCA) and orthogonal
   partial least squares-discriminant analysis (OPLS-DA) models were
   built. In the PCA score plots ([92]Figure 6A,C), the metabolic profile
   of the LPS group showed a significant divergence along the first
   principal component (PC1, x-axis), which verified the successful
   establishment of the inflammation model 30. Along the second principal
   component (PC2, y-axis), the groups treated with EBN, SCP, and SA
   presented distinct metabolic changes compared to the LPS group.
   Significantly, the SCP group was clustered nearest to the CON group,
   suggesting its excellent ability to restore metabolic homeostasis. The
   OPLS-DA model further effectively amplified the differences among the
   groups ([93]Figure 6B,D).

Figure 6.

   [94]Figure 6
   [95]Open in a new tab

   Pattern recognition analysis in positive and negative ion modes. (A,C)
   PCA diagram of positive ion mode and negative ion mode. (B,D) OPLS-DA
   diagram of positive ion mode and negative ion mode.

3.5.2. Screening of Differential Metabolites

   In the metabolomics analysis, the screening of differential metabolites
   is usually based on the variable importance in the projection (VIP)
   values obtained from the OPLS-DA model. VIP describes the overall
   contribution of each variable to the model, as well as the p values
   obtained from the t-test. A threshold of p < 0.05 and VIP > 1 was set
   to screen out the differential metabolites with statistical
   significance. The differential metabolites among groups under the
   positive and negative ion modes were screened respectively ([96]Figure
   7). A total of 35 differential metabolites were identified under the
   positive ion mode, and 45 differential metabolites were identified
   under the negative ion mode. Compared with the CON group, a total of 39
   metabolites in the LPS group showed significant changes in their
   contents (p < 0.05). Among them, the contents of 25 metabolites such as
   L-glutamic acid, xanthine, uric acid, and PC (14:0/18:0) in the LPS
   group increased significantly, while the contents of 14 metabolites
   such as glycerophosphocholine, pyrophosphate, o-toluidine, and GPEtn
   (16:0/18:1) decreased significantly. This indicated that LPS may cause
   metabolic disorders in the lung tissues of mice by interfering with
   these 39 metabolites. Compared with the LPS group, a total of 40
   metabolites in the SCP intervention group showed significant changes in
   their contents (p < 0.05). Among them, the contents of 28 metabolites
   such as linolenic acid, choline, LPC (18:1), and indoline in the SCP
   intervention group increased significantly, while the contents of 12
   metabolites such as glycerophosphocholine, xanthine, inosine, and
   LysoPC (18:1(11Z)/0:0) decreased significantly. This suggested that SCP
   may regulate the inflammation of the lung tissues in mice induced by
   LPS by interfering with these 40 metabolites.

Figure 7.

   [97]Figure 7
   [98]Open in a new tab

   Clustering heat map of the expression of differential metabolites under
   positive and negative ion modes in the lung tissue of mice in each
   group. (A) Cluster heat map of differential metabolites in positive ion
   mode. (B) Cluster heat map of differential metabolites under negative
   ion mode.

3.5.3. Metabolic Pathway Analysis of Differential Metabolites

   Differential metabolites were mapped to the KEGG database to identify
   metabolic pathways with p < 0.05 and Impact > 0 ([99]Figure 8).
   Compared to the CON group, the LPS group showed significant enrichment
   in glycerophospholipid metabolism, purine metabolism, glyoxylate, and
   dicarboxylate metabolism. Compared to the LPS group, the pathways
   enriched in EBN were glycerophospholipid metabolism and alpha-linolenic
   acid metabolism, the pathways enriched in SCP were glycerophospholipid
   metabolism, purine metabolism, and alpha-linolenic acid metabolism, and
   the pathways enriched in SA were glycerophospholipid metabolism,
   alpha-linolenic acid, and linoleic acid metabolism. Notably, the SCP
   group shared more overlapping pathways with the LPS group, suggesting
   its superior regulatory capacity at the metabolic pathway level.

Figure 8.

   [100]Figure 8
   [101]Open in a new tab

   Heat map of differential metabolite expression in positive and negative
   ion modes in the lung tissue of mice in each group. (A–D) Differential
   metabolic pathways between the LPS and CON groups, the EBN and LPS
   groups, the SCP and LPS groups, and the SA and LPS groups. 1:
   glycerophospholipid metabolism, 2: purine metabolism, 3: glyoxylate and
   dicarboxylate metabolism, 4: alpha-linolenic acid metabolism, 5:
   linoleic acid metabolism.

4. Discussion

   EBN proteins and carbohydrates are predominantly present as
   high-molecular-weight mucoproteins in the form of sialylated
   glycoproteins, which are characterized by low solubility and resistance
   to digestion [[102]31]. In this study, EBN was digested using a
   two-step enzymatic hydrolysis protocol with Alcalase 2.4 L and papain.
   The contents of sialic acid (SA) and neutral sugars in SCP were both
   slightly higher than those in EBN, indicating that the optimization of
   the enzymatic hydrolysis process is conducive to the dissolution of
   neutral sugars and sialic acid [[103]32]. Amino acids are the basic
   building blocks of proteins and polypeptides, and their composition and
   proportion are important factors influencing the physicochemical
   properties, biological activities, and other aspects of glycopeptides
   [[104]33]. In this study, the contents of nine essential amino acids in
   SCP were abundant, according to the nutritional standards of essential
   amino acids recommended by the FAO/WHO. SCP has the potential to be an
   excellent choice for supplementing essential amino acids. Studies have
   shown that Asn is the main site where the sugar chain is linked to the
   protein during the N-glycosylation process, and Thr and Ser are the
   main sites where the sugar chain is linked to the protein during the
   O-glycosylation process [[105]34]. The abundant contents of these three
   amino acids in SCP indicated that there may be certain glycosylation
   modifications in SCP.

   It was revealed by electrophoretic analysis that glycoproteins retained
   high molecular weights due to the tight conjugation of glycan and
   protein. After hydrolysis, the molecular weights of glycoproteins were
   reduced, and non-glycan-conjugated proteins were further degraded into
   smaller peptides [[106]22]. This observation suggested that peptide
   bonds in glycoproteins were selectively targeted by enzymatic cleavage;
   low-molecular-weight peptides were released while glycan chains
   covalently bound to residual peptide fragments were preserved. The
   resistance of glycan-peptide conjugated to enzymatic degradation
   implies that the cleavage sites of Alcalase 2.4 L and papain are likely
   to be distant from glycosylation sites in EBN glycoproteins. Thus, the
   integrity of the glycan was maintained during hydrolysis. Furthermore,
   SCP displayed typical peptide and carbohydrate features, with spectral
   profiles resembling those of quinoa and Stichopus japonicus
   glycopeptides [[107]35,[108]36]. Compared to EBN, enhanced 1401 cm^−1
   (−COO− symmetric stretching) and 1066 cm^−1 (carbohydrate C-O)
   absorptions in SCP likely reflected increased SA and total carbohydrate
   content post-purification, confirming successful enrichment of
   glycan-rich moieties consistent with its sialylated glycopeptide
   identity.

   The findings based on the C57BL/6 mouse model shared fundamental
   biological similarities with those based on human lung inflammation in
   LPS-induced mechanisms, and the experimental methods were universally
   used in the field, supporting the results’ reference value for similar
   inflammatory mechanism studies. LPS induced significantly elevated lung
   pro-inflammatory cytokine levels (TNF-α, IL-1β, and IL-6) and
   inflammatory cell counts (Neu, WBC, Eos, and Mon) in BALF, confirming
   the induction of lung inflammation. Increased inflammatory cell
   infiltration can reflect the immune system’s response to inflammatory
   stimuli, with cell counts as direct indicators of inflammatory severity
   [[109]37,[110]38]. In our study, all intervention groups significantly
   attenuated these inflammatory markers, with the SCP group demonstrating
   superior efficacy compared to EBN and SA groups, suggesting SCP as the
   primary bioactive component in EBN. In addition, the W/D ratio of lung
   issues, a key indicator of lung edema, was markedly increased in the
   LPS group, indicative of fluid accumulation in alveolar and
   interstitial spaces [[111]39]. This observation was corroborated by
   histopathological analysis, which revealed alveolar structural damage,
   interstitial exudation, and neutrophil infiltration in the LPS group.
   Intervention groups alleviated lung tissue injury, with SCP exhibiting
   the most pronounced restorative effects. These findings align with
   prior studies showing that glycoprotein-bound SA exhibits enhanced
   anti-inflammatory activity compared to free SA [[112]40].

   Metabolomic profiling was conducted to elucidate SCP-mediated
   regulatory mechanisms on LPS-induced metabolic perturbations in the
   lung [[113]41]. PCA and OPLS-DA demonstrated distinct metabolic profile
   alterations in the LPS group, with a clear separation between LPS and
   intervention groups, indicating metabolic homeostasis restoration. KEGG
   pathway enrichment analysis revealed that SCP primarily modulated
   glycerophospholipid, purine, and alpha-linolenic acid metabolism.

   Glycerophospholipids, essential components of cellular membranes, are
   critically involved in membrane stability and functionality [[114]42].
   This study’s glycerophospholipid-related metabolites accounted for the
   highest proportion of differential metabolites. Moreover, choline and
   lysophosphatidylcholines (LysoPCs) have been identified as biomarkers
   associated with lipid metabolism-related chronic lung diseases
   [[115]43]. Choline, a key precursor for phosphatidylcholine synthesis,
   is integral to membrane integrity. Under LPS-induced lung inflammation,
   elevated choline levels in the LPS group may reflect compensatory
   mechanisms to enhance choline synthesis or uptake for membrane repair
   [[116]44]. Notably, SCP intervention increased choline content,
   suggesting that SCP may indirectly modulate choline metabolism by
   promoting synthesis or suppressing catabolism to support membrane
   homeostasis.

   Purine metabolism dysregulation has been implicated in pulmonary injury
   and inflammation [[117]45]. Xanthine, a key intermediate in purine
   metabolism, exacerbates oxidative stress and inflammatory responses
   when excessively accumulated. This study observed elevated xanthine
   levels in the LPS group, which may be attributable to the LPS-induced
   disruption of purine metabolic pathways, including enzymatic activation
   promoting xanthine synthesis [[118]46]. Concurrently, LPS-mediated
   renal impairment increases uric acid production [[119]47]. Following
   SCP intervention, xanthine levels were significantly reduced,
   suggesting the restoration of purine metabolism toward uric acid
   conversion. However, elevated uric acid levels persisted, potentially
   due to impaired renal clearance capacity. These findings indicate that
   SCP can alleviate LPS-induced lung inflammation partially through
   xanthine level modulation.

   Alpha-linolenic acid, an essential fatty acid critical for maintaining
   physiological homeostasis, exhibits anti-inflammatory and antioxidant
   properties [[120]48]. This study significantly reduced alpha-linolenic
   acid levels in the LPS group, likely due to its extensive consumption
   to counteract LPS-induced inflammatory responses [[121]49]. Notably,
   alpha-linolenic acid levels were restored in the SCP intervention
   group. This restoration may be attributed to the SCP-mediated
   modulation of metabolic enzyme activity, which reduced inflammatory
   cell-driven alpha-linolenic acid depletion. These findings suggested
   that the anti-inflammatory effects of SCP against LPS-induced lung
   inflammation were mediated through the upregulation of endogenous
   alpha-linolenic acid levels.

5. Conclusions

   In this study, SCP extracted from EBN exhibited typical structures of
   polypeptides and carbohydrates. EBN, SCP, and SA exerted effects on the
   level of pulmonary inflammation, the pathological structure of lung
   tissues, and the metabolic level of lung tissues, effectively improving
   the LPS-induced pulmonary inflammatory state in mice. Specifically,
   after SCP intervention, the number of Neu in BALF, the level of TNF-α
   in lung tissues, and the W/D ratio of lung tissues were significantly
   reduced. Moreover, the morphology of mouse lung tissues after SCP
   intervention was most similar to that of the CON group. SCP rectified
   the metabolic profiles of mice with pulmonary inflammation, regulated
   the expression of differential metabolites in lung tissues, such as
   choline, linolenic acid, and xanthine, and promoted the restoration of
   glycerophospholipid metabolism, alpha-linolenic acid metabolism, and
   purine metabolism pathways. However, the mechanism of action of SCP in
   vivo is complex and requires further investigation.

Author Contributions

   Conceptualization, X.C. and D.W.; methodology, S.Q. and Q.L.;
   investigation, Q.L. and C.Z.; data curation, Q.L. and C.Z.;
   writing—original draft preparation, Q.L. and G.F.; writing—review and
   editing, N.Q., S.Q., M.Y., X.C. and D.W.; visualization, S.Q., M.Y. and
   D.W.; supervision, X.C. and D.W. All authors have read and agreed to
   the published version of the manuscript.

Institutional Review Board Statement

   The animal study protocol was approved by the Experimental Animal
   Welfare and Ethics Review Committee of Jiangnan University
   (JN.No20230415c0800705[145], approval date: 15 April 2023).

Informed Consent Statement

   Not applicable.

Data Availability Statement

   The original contributions presented in the study are included in the
   article, further inquiries can be directed to the corresponding author.

Conflicts of Interest

   Hebei Edible Bird’s Nest Fresh Stew Technology Innovation Center
   focuses on technological research and development in the field of
   instant bird’s nest fresh stewing and is responsible for the
   transformation and application of related technologies in the Beijing
   Xiaoxiandun Biotechnology Co., Ltd. (Beijing, China). Jiangnan
   University has carried out collaborative research with the Hebei Edible
   Bird’s Nest Instant Fresh Stew Technology Innovation Center, and the
   relevant work has been advanced based on the independent research plans
   of the cooperative laboratory and the technical service needs of the
   industry. The authors solemnly declare that there are no conflicts of
   interest involved. The research work described in this manuscript was
   carried out in strict accordance with scientific principles and ethical
   standards. All the authors have fulfilled their responsibilities in an
   unbiased manner, ensuring that the presented research findings are
   reliable and free from any undue influence.

Funding Statement

   This research received no external funding.

Footnotes

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References