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Introduction

Chitin, a cationic amino polysaccharide composed of N-acetylglucosamine linked by β-1,4-glycosidic bonds, is a natural macromolecule with a yield second only to cellulose in nature. It is commonly found in the shells of crustaceans, insect epidermis and fungal cell walls (1). Complete or partial deacetylation of chitin can yield chitosan, a cationic alkaline polysaccharide composed of D-glucosamine linked by β-1,4-glycosidic bonds. Degradation of chitosan produces chitosan oligosaccharides (COSs), characterized by low molecular weight and high biological activity (2). Moreover, chitin degradation results in the production of N-acetylated COS (NACOS), which exhibits good solubility and multiple biological functions, including antioxidant, anti-inflammatory, antitumor, antimicrobial and plant elicitor activities, as well as immunomodulatory and prebiotic effects (3). While chitin and its derivative chitosan are known for their various functional activities, their limited solubility hinders their use in food and biomedical applications. However, hydrolyzed products of chitosan, including COS (3-5) and NACOS, exhibit increased water solubility due to their shorter chain length, lower viscosity and potentially higher absorption rates (6-8).

Numerous studies have shown that COS and its derivatives possess biological activities, including anti-inflammation, anti-tumor, anti-obesity, anti-Alzheimer's disease, immunostimulation, tissue regeneration promotion, drug and DNA delivery enhancement, anti-microbiome and anti-oxidation effects (5,9-12). Some studies also report that COS possesses robust neuroprotective properties, such as β-amyloid and acetylcholinesterase inhibition, anti-neuroinflammation and anti-apoptosis (13,14). COS has demonstrated its potential to mitigate high-fat diet-induced obesity and insulin resistance by modulating gut microbiota dysfunction, mitigating low-grade inflammation and preserving the integrity of the intestinal epithelial barrier (15). Inflammatory factors serve an important role in cardiovascular diseases such as atherosclerosis, myocardial infarction and cardiac failure (16). Several studies have highlighted the anti-inflammatory effects of COS (17,18). The mechanism underlying the anti-inflammatory effects of orally administered COS may involve suppressing inflammatory processes, including regulating the expression of NF-κB, Nrf2, TGF-β1/Smad, inducible nitric oxide synthase and pro-inflammatory cytokines (15). Notably, COS has been shown to extend the survival of mice challenged with lipopolysaccharide (19).

Oxidative stress and inflammation are linked, with their interdependence consistently documented. Emerging evidence implicates oxidative stress in various types of diseases, including diabetes (20), atherosclerosis (21) and cancer (22), as well as aging (23), prompting attention to the role of antioxidants in biological systems. Extensive efforts have been devoted to identifying suitable antioxidant compounds for preventive and therapeutic purposes. Reactive oxygen species (ROS), such as superoxide anion (O2-), H2O2 and hydroxyl radicals (HO-), are continually generated at a substantial rate as by-products of aerobic metabolism and radiation exposure (24). These oxidants not only damage cellular macromolecules, including DNA, protein and lipids, but also contribute to NLRP3 inflammasome activation and mediate inflammatory responses, establishing a cycle of localized inflammation leading to oxidative damage (25). The persistent threat of oxidant-induced damage to cells, tissue and organisms is underscored by cellular defense mechanisms evolved to combat reactive oxidants. Excessive ROS is readily converted to H2O2, which easily diffuses across cell membranes and generates highly reactive and toxic hydroxyl radicals via the heme-catalyzed Fenton reaction, leading to rapid DNA damage and strand breaks when superoxide anions react with nitric oxide (NO) to form peroxynitrite, a reactive nitrogen species (23). In response to oxidative stress-induced DNA damage, processes such as cell cycle arrest and DNA damage repair are initiated as cellular defense mechanisms. However, if DNA damage exceeds the cell repair capacity, it ultimately leads to apoptotic cell death (26). Macrophages are key immune cells for the host defense system to eliminate microbial pathogens. Studies have verified that macrophages can produce ROS when infected by some bacteria, making them a suitable model for antioxidant research (27,28). The mouse macrophage cell line RAW264.7 is the most commonly used mouse macrophage cell line in medical research (29). Therefore, the present study used H2O2-stimulated RAW264.7 cells to establish a cellular oxidative damage model.

COS has shown potential in protecting against oxidative stress-induced cell damage by modulating enzyme activity and gene expression related to antioxidant defense mechanisms (25,30-33), thus exerting health benefits primarily through antioxidant activity. Scholars have highlighted the impact of physicochemical parameters such as molecular weight and degree of acetylation (DA) on the bioactivities of COS (34,35). However, the lack of well-defined and standardized criterion for distinguishing the bioactivity of COS with varying degrees of polymerization is compounded by the shared nomenclature of COS and NACOS, leading to potential confusion. Therefore, the present study aimed to investigate the impact of acetylation on COS biological activity by comparing the protective effects of deacetylated COS and its non-deacetylated form, NACOS, on H2O2-induced apoptosis and oxidative stress in RAW264.7 cells.

Materials and methods

Chemicals and reagents

Dulbecco's modified Eagle's medium (DMEM) medium was purchased from Corning, Inc. Fetal bovine serum (FBS), penicillin and streptomycin were from Gibco (Thermo Fisher Scientific, Inc.). Annexin V-FITC kit and Cell Cycle Analysis kit were from Beyotime Institute of Biotechnology. Colored standards and all the other reagents for SDS-PAGE and immunoblotting were from Bio-Rad Laboratories, Inc. Antibodies against β-actin, Bax, Bcl-2, PARP and cleaved PARP, as well as horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG and HRP-conjugated goat anti-mouse IgG were obtained from Cell Signaling Technology, Inc. All chemicals used were analytical grade unless otherwise stated.

Purification and quantitative analysis of NACOS and COS

COS with a deacetylation degree of 88% and polymerization degree of 2-6 was prepared through enzymatic hydrolysis of chitosan, as described previously (36). NACOS was also prepared as described previously (37). In brief, COS was dissolved in 50 ml water with 3 ml methanol, 0.1 g 4-dimethylaminopyridine and 4.37 ml acetic anhydride. The mixture was incubated at 60˚C for 4 h. Subsequently, 5X the volume of acetone was added to obtain NACOS. DA and polymerization degree were determined using liquid chromatography-mass spectrometry and nuclear magnetic resonance analyses by Institute of Process Engineering, Chinese Academy of Sciences. The mass spectrometry detection conditions were: ESI source; positive ion scanning mode; 3 kV capillary voltage; 60 V cone hole voltage; 150˚C ion source temperature; 500˚C desolvent gas temperature; 50 l/h flow rate of conical hole gas; 800 l/h desolvent gas flow rate; 150-2,000 m/z quality scanning range.

Cell culture

The mouse monocyte macrophage leukemia RAW264.7 cell line was obtained from the Type Culture Collection of the Chinese Academy of Sciences. RAW264.7 cells were cultured in DMEM supplemented with 5% heat-inactivated FBS, 100 U/ml penicillin and 100 µg/ml streptomycin in a humidified atmosphere with 5% CO2 at 37˚C and passaged twice/week. Cells at a confluence of 70-80% were used for subsequent experiments.

Identification of cell viability using MTT assay

Cell viability was determined by MTT colorimetric assay. In brief, cells were treated with NACOS and COS (25, 50 or 100 µg/ml) for 12 h at 37˚C and cell viability was detected by MTT assay. In addition, cells were pretreated with NACOS and COS (25, 50 or 100 µg/ml) for 12 h at 37˚C, followed by 300 µM H2O2 for another 12 h at 37˚C. Treatment with equal volume PBS was included as the vehicle control. A total of 100 µl 0.5 mg/ml MTT PBS solution was added to each well and the samples were incubated for an additional 4 h at 37˚C. The purple-blue MTT formazan precipitate was dissolved in 100 µl dimethyl sulfoxide and the absorbance at 570 nm was measured using a microplate reader.

Identification of cell cycle stage by propidium iodide (PI) staining

Cell cycle analysis was performed by flow cytometry using a fluorescence-activated cell sorting (FACS) MoFlo XDP (Beckman Coulter) following PI staining. In brief, RAW264.7 cells were exposed to 50 µg/ml NACOS and COS for 12 h and stimulated with 300 µM H2O2 for another 12 h at 37˚C. The cells were harvested, adjusted to a concentration of 1x106 cells/ml by cell count and dilution, and fixed in 70% ethanol at 4˚C overnight. The fixed cells were washed twice with cold PBS and incubated with RNase (8 µg/ml) and PI (10 µg/ml) at 4˚C for 30 min. The fluorescent signal was detected through the FL2 channel and the proportion of DNA in various phases was analyzed using ModfitLT Version 3.0 (Verity Software House, Inc.).

Identification of apoptosis by Annexin V/PI staining

A total of 2x105 RAW264.7 cells in 2 ml DMEM complete medium was seeded into 6-well plates and treated with 50 µg/ml NACOS or COS for 12 h and then stimulated with 300 µM H2O2 for another 12 h at 37˚C. Apoptosis of RAW264.7 cells was determined by flow cytometry analysis using a FACS caliber (Becton-Dickinson) and Annexin V-fluorescein isothiocyanate (FITC)/PI kit. Cell apoptosis was quantified using FlowJo software (v.10.1.1; FlowJo LLC). Staining was performed according to the manufacturer's instructions. Annexin V/PI double-negative population represented viable cells, while Annexin V-positive/PI-negative or Annexin V/PI double-positive population represented cells undergoing early or late apoptosis, respectively.

Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis

The total RNA was isolated from cells using RNAisoPlus reagent (Takara Biotechnology Co., Ltd.). Reverse transcription into cDNA was amplified according to the manufacturer's instructions using the PrimeScript RT reagent kit (Takara Biotechnology Co., Ltd.). An ABI 7500 Fast Real-Time PCR System (Applied Biosystems) and the SYBR Premix Ex Tag (Takara Biotechnology Co., Ltd.) was used for PCR. Following an initial denaturation at 95˚C for 5 min, the PCR conditions were as follows: 35 cycles of denaturation at 95˚C for 30 sec, annealing at 55˚C for 30 sec and extension at 72˚C for 30 sec. The 2-ΔΔCq method was used to calculate the mRNA expression levels in each sample (38). Data were normalized to the GAPDH content of the sample. The primer sequences were as follows: L-1β forward, 5'-ATGATGGCTTATTACAGTGGCAA-3' and reverse 5'-GTCGGAGATTCGTAGCTGGA-3'; IL-6 forward, 5'-ACTCACCTCTTCAGAACGAATTG-3' and reverse, 5'-CCATCTTTGGAAGGTTCAGGTTG-3'; IL-8 forward, 5'-ACTGAGAGTGATTGAGAGTGGAC-3' and reverse, 5'-AACCCTCTGCACCCAGTTTTC-3'; TNF-α forward, 5'-CCTCTCTCTAATCAGCCCTCTG-3' and reverse, 5'-GAGGACCTGGGAGTAGATGAG-3'; MCP-1 forward, 5'-CAGCCAGATGCAATCAATGCC-3' and reverse, 5'-TGGAATCCTGAACCCACTTCT-3'; and GAPDH forward, 5'-CACATGGCCTCCAAGGAGTAA-3' and reverse, 5'-TGAGGGTCTCTCTCTTCCTCTTGT-3'.

Western blot analysis

Total protein extracts were obtained using RIPA lysis buffer and concentrations were determined by the BCA assay (both Pierce; Thermo Fisher Scientific, Inc.). Equal amounts of protein (50 µg) from each sample were separated using 10-15% SDS-PAGE and transferred onto PVDF membranes. The membranes were blocked with 5% non-fat milk for 1 h at room temperature and incubated with specific primary antibodies (all 1:1,000) against Bcl-2 (cat. no. 4223), Bax (cat. no. 5023), PARP, cleaved-PARP (cat. no. 9542) and β-actin (cat. no. 4697) from Cell Signaling Technology, Inc., at 4˚C overnight. After incubating with horseradish peroxidase-conjugated secondary antibodies (1:250,000) at room temperature for 2 h, the signals were visualized using enhanced chemiluminescence. Protein bands were visualized using enhanced chemiluminescence reagent (Thermo Fisher Scientific, Inc.). Protein expression levels were semi-quantified using ImageJ software (v2.1.4.7) with β-actin as the loading control.

Statistical analysis

All experiments were performed three times independently. All data are presented as the mean ± SD. Data were analyzed by one-way ANOVA followed by Tukey-Kramer's post hoc test. P<0.05 was considered to indicate a statistically significant difference. All statistical analysis was performed using the SPSS package for Windows (version 17.0; SPSS, Inc.).

Results

Effects of NACOS and COS on viability of H2O2-stimulated RAW264.7 cells

MTT assay was performed to assess the potential cytotoxic effects of NACOS and COS treatments. The results of liquid chromatography-mass spectrometry and nuclear magnetic resonance analyses indicated that obtained COS had a deacetylation degree of 88% and polymerization degree of 2-6, while NACOS had a deacetylation degree of 97% and a polymerization degree of 3-10 (Fig. S1). COS (25, 50 or 100 µg/ml) for 24 h did not lead to a reduction in RAW264.7 cell viability. By contrast, NACOS at 50 and 100 µg/ml remarkably increased cell viability (Fig. 1A). Subsequently, the effects of NACOS and COS on the viability of RAW264.7 cells was assessed following H2O2-treatment. H2O2 led to a substantial decrease in cell viability, reaching 37.60±0.56% compared with untreated controls. However, NACOS and COS treatment effectively attenuated the inhibitory effects of H2O2 on cell viability. However, 100 µg/ml COS had less effect than 50 µg/ml COS (Fig. 1B). NACOS and COS at 100 µg/ml resulted in a significant increase compared with H2O2 alone in terms of cell viability, with values of 8.61±0.34 and 6.27±0.63%, respectively. This protective effect of NACOS/COS may be associated with scavenging of oxygen free radicals. Both NACOS and COS effectively scavenged oxygen free radicals, albeit with varying scavenging abilities and impacts on different types of oxygen free radicals, including ·OH, ·O2-, NO· and DPPH· (Table SI).

Figure 1 Effects of NACOS and COS on viability of RAW264.7 macrophage cells. (A) Cells were treated with NACOS (25-100 µg/ml) or COS (25-100 µg/ml) for 12 h and cell viabilities were determined by MTT assay. (B) Effects of NACOS and COS on H2O2-treated macrophage cells. Cells were pre-treated with NACOS (25-100 µg/ml) or COS (25-100 µg/ml) for 15 h and then exposed to H2O2 (300 µM) for 12 h. After that, cells were collected for viability detection by MTT assay. n=3. ##P<0.01 vs. control; *P<0.05, **P<0.01 vs. H2O2-alone.

NACOS and COS protect against H2O2-induced cellular apoptosis

Flow cytometry showed that 15.74% of cells underwent apoptosis following treatment with H2O2 alone, which was significantly higher than that of untreated control cells. However, only 6.94 and 8.16% of cells that were pretreated with COS or NACOS, respectively, (100 µg/ml) for 24 h underwent apoptosis following H2O2 treatment, showing a significant difference compared with H2O2 alone (Fig. 2). NACOS and COS pretreatment protected against cellular apoptosis induced by oxidative stress. In addition, NACOS and COS decreased mRNA expression of IL-1β, IL-6, TNF-α and MCP-1 in H2O2-stimulated RAW264.7 cells (Fig. S2).

Figure 2 Effects of NACOS and COS on H2O2-induced apoptosis in RAW264.7 macrophage cells. Cells were pre-treated with NACOS or COS (both 100 µg/ml) for 12 h, then exposed to H2O2 (300 µM) for 12 h. Then, cells were labeled with a combination of PI and Annexin V-FITC for flow cytometric analysis. Representative flow cytometric histograms of (A) control, (B) H2O2-alone (C) NACOS, (D) NACOS + H2O2, (E) COS and (F) COS + H2O2 group. (G) Apoptotic rate. n=3. ##P<0.01 vs. control; **P<0.01 vs. H2O2-alone.

Cell cycle changes following NACOS and COS treatment

Effects of NACOS and COS treatment on H2O2-induced G1/S phase arrest of RAW264.7 cells was assessed using FACS analysis. The results showed that COS or NACOS alone had no significant effect on the proportion of cells at different phases. The percentage of S phase cells was increased by up to 10% following treatment with 100 µg/ml NACOS and COS compared with H2O2 alone (Fig. 3), indicating that NACOS and COS prevented H2O2-induced G1/S cell cycle arrest.

Figure 3 Effect of NACOS and COS on H2O2-induced cell cycle arrest. (A) RAW264.7 cells were pre-treated with NACOS or COS (both 100 µg/ml) for 12 h and exposed to H2O2 (300 µM) for 12 h. Cells were collected and incubated with PI for flow cytometric analysis. Representative flow cytometric histograms of (A) control, (B) H2O2-alone (C) NACOS, (D) NACOS + H2O2, (E) COS and (F) COS + H2O2. (G) Cell cycle distribution.

Effect of NACOS and COS on PARP cleavage and Bcl-2 expression

PARP, a 116 kDa nuclear poly(ADP-ribose) polymerase involved in DNA repair and cell proliferation, is be cleaved by caspase-3, serving as an indicator of apoptotic cells (39). H2O2 is widely recognized for inducing PARP cleavage (40). H2O2 treatment increased the levels of cleaved PARP compared with untreated control, confirming that H2O2 triggered cellular apoptosis (Fig. 4). However, pretreatment with COS and NACOS significantly inhibited PARP cleavage, suggesting both COS and NACOS prevented H2O2-induced apoptosis in RAW264.7 cells. Moreover, the effect of NACOS in protecting against cellular apoptosis was superior to that of COS. The rescue of cellular apoptosis is typically associated with the upregulation of anti-apoptotic molecules such as Bcl-2, which function in antioxidant pathways to prevent apoptosis (41). Bax is also an apoptotic-induced protein (42). COS and NACOS upregulated Bcl-2 and downregulated Bax protein expression following H2O2 treatment (Fig. 4). Moreover, NACOS showed a superior protective effect compared with COS.

Figure 4 Effect of NACOS and COS on apoptotic protein expression. (A) Cells were pre-treated with NACOS or COS (both 100 µg/ml) for 12 h and then exposed to H2O2 (300 µM) for 12 h. The expression of Bax, Bcl-2, PARP and cleaved PARP following NACOS and COS treatment in RAW264.7 cells was determined by western blot analysis. (B) Densitometric analysis was performed to determine protein expression fold change. The protein expression of cleaved PARP was normalized to that of PARP. ##P<0.01 vs. control; *P<0.05, **P<0.01 vs. H2O2-alone.

Discussion

The present study compared the antioxidant activities of NACOS and COS against H2O2-induced oxidative damage. NACOS and COS effectively attenuated oxidative stress induced by H2O2. Although COS did not significantly affect cell viability, NACOS treatment significantly increased cell viability. The improvement of cell viability by NACOS may be associated to its antioxidant, antiapoptotic, cell proliferation induction or cell cycle regulation effects, suggesting the need for further investigation into the underlying mechanism. Furthermore, both NACOS and COS significantly attenuated H2O2-induced inhibition of cell viability in a concentration-dependent manner. Cell cycle assay revealed that NACOS and COS reversed H2O2-induced inhibition of G1/S cell cycle arrest and significantly mitigated H2O2-induced cell apoptosis. The protective effects of NACOS and COS may be attributed to their ability to scavenge oxygen free radicals, thereby preventing oxidative damage.

Previous studies have suggested that COS exerts potential free radical scavenging properties through both direct and indirect mechanisms (43,44). Here, both NACOS and COS effectively scavenged oxygen free radicals, albeit with varying scavenging abilities and impacts on different types of oxygen free radicals. Thus, both COS and NACOS inhibited molecular damage by scavenging free radicals in cells.

Furthermore, both NACOS and COS protected against H2O2-induced apoptosis, as observed by flow cytometry. Previous studies have also indicated that COS enhances cell viability following H2O2 treatment and decreases apoptosis rate (45,46). In addition, COS increases extracellular matrix synthesis and prevents its degradation (47). Here, highly acetylated NACOS notably inhibited H2O2-induced apoptosis. Furthermore, the protective effect of NACOS was greater than that of COS. Previous studies have indicated that degree of polymerization plays a critical role in the neuroprotective effects of COS (47). β-amyloid (Aβ) aggregates containing cross-β-sheet structures induce oxidative stress, neuroinflammation and neuronal loss via multiple pathways. COS can directly bind to Aβ42 in a polymerization-dependent manner, ameliorating Aβ42-induced cytotoxicity (48).

Mitochondria serve as central integrators and transducers of various pro-apoptotic signals, with their disruption and subsequent release of pro-apoptotic proteins being a key aspect of the apoptosis process (49,50). The release of these factors from mitochondria is regulated by Bcl-2 family proteins, which are key regulators for cell apoptosis under both normal and oxidative stress conditions (41,51). The present study showed that both NACOS and COS increased Bcl-2 expression and protected against H2O2-induced apoptosis in RAW264.7 cells. Furthermore, activation of effector caspases, such as caspase-3, leads to PARP cleavage, resulting in apoptosis (52). Here, cleaved PARP expression was ameliorated following NACOS and COS treatments in RAW264.7 cells, with the effects of NACOS superior to those of COS. These findings suggested that acetylation may be essential for the protective effects of COS.

The present findings suggest that the anti-apoptotic effect of COS and NACOS was associated with DA. Both COS and NACOS were effective in inhibiting oxidative damage induced by H2O2, with the antagonistic effect of NACOS greater than that of COS. This implied that the degree of deacetylation was positively associated with the protective effects of COS. Acetylation has been reported to facilitate passive diffusion of disaccharides across cell membranes, enabling them to enter the Golgi (53). Thus, acetylation modification may facilitate the entry of NACOS into cells, allowing them to interfere with ROS-induced apoptosis; however, the specific underlying mechanism requires further investigation. DA may be a key factor influencing the ability of COS to prevent oxidative damage-induced cell apoptosis. However, functional gain or loss analysis experiments are needed. NACOS and COS decreased mRNA expression of IL-1β, IL-6, TNF-α and MCP-1 in H2O2 stimulated RAW264.7 cells and the potential anti-inflammatory mechanisms of NACOS and COS may associated with promoting protein O-GlcNA acylation, which significantly inhibits the phosphorylation and nuclear translocation of inflammatory transcription factors (54). However, the specific underlying mechanism requires further investigation.

Supplementary Material

Mass spectrogram of COS and NACOS. Liquid chromatography-mass spectrometry was used to determine the acetylation degree and polymerization degree of COS and NACOS. A represents N-Acetyl glucosamine and D represents glucosamine, while the subsequent numbers represent the number of monosaccharides in these predicted components. NACOS, N-acetylated chitosan oligosaccharide; AU, arbitrary units.

Effect of NACOS and COS on mRNA levels of inflammatory factors. Cells were pre-treated with NACOS or COS (both 100 μg/ml) for 12 h and then exposed to H2O2 (300 μM) for 12 h. mRNA expression of IL-1β, IL-6, IL-8, TNF-α and MCP-1 was determined by quantitative PCR analysis. N=3. *P<0.05 vs. control; #P<0.05 vs. H2O2-alone. NACOS, N-acetylated chitosan oligosaccharide; MCP-1, macrophage chemoattractant protein-1.

Scavenging effects of NACOS and COS on ·OH, ·O2-, NO· and DPPH·.

Acknowledgements

Not applicable.

Funding

Funding: The present study was supported by the Natural Science Foundation of Fujian Province of China (grant no. 2021J05057).

Availability of data and materials

The data generated in the present study may be requested from the corresponding author.

Authors' contributions

QL designed the study, analyzed data and wrote the manuscript. QL, WS and YH performed the experiments. QL and WS confirm the authenticity of all the raw data. All authors have read and approved the final manuscript.

Ethics approval and consent to participate

Not applicable.

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

References