SP was produced in our laboratory using Solenocera crassicornis by-products obtained from Zhoushan Yueyang Food Co., Ltd (Zhoushan, China). DSS [molecular weight (MW) 36–50 KDa] was purchased from MP Biomedicals (Santa Ana, CA, USA). Standard food for mice was obtained from Suzhou Shuangshi Experimental Animal Feed Technology Co., Ltd (Suzhou, China). ELISA kits for IL-6 and TNF-α were purchased from the Wuhan Boshide Biological Engineering Co. LTD (Wuhan, China); ELISA kits for DAO and LPS were purchased from Wuhan Huamei Biological Company (Wuhan, China); ELISA kits for SOD and GSH-Px were purchased from Nanjing Jiancheng Institute of Biological Engineering (Nanjing, China). Primers were designed using Primer Premier 5.0 and synthesized by Sangon Bioengineering (Shanghai, China) Co. Ltd. All other chemicals, solvents, and reagents were of analytically pure grade.

The heads of red shrimp were mixed with 95% ethanol (1:10, w/v) and stirred and degreased for 8 h at 25°C. The filtered residue was dried at 45°C. Next, distilled water (1:2, w/v) was added to the residue and the pH was adjusted to 7.2. Enzymatic hydrolysis at 55°C was performed using 2% alkaline protease for 5 h. The enzyme was inactivated at 90°C for 20 min, and the supernatant was filtered and collected. The hydrolysates were separated using an inorganic 0.5-μm pore size ceramic membrane. The collected permeates were successively passed through 1,000 and 250 Da ultrafiltration membranes, and the permeate and the intercepted solution were collected to obtain 250–1,000 Da components, which were freeze-dried to obtain SP.

Cytochrome c (MW: 12,355 Da), aprotinin (MW: 6,511 Da), bacitracin (MW: 1,422 Da), and ethylene-ethylene-tyrosine-arginine (MW: 451 Da) were weighed and dissolved in the mobile phase to yield standard molecular weight solutions of 0.2 mg/mL. The sample (100 mg) was weighed into a volumetric flask, diluted to 10 mL with the mobile phase, shaken, and filtered through a microporous membrane (0.45 μm) to remove impurities and facilitate sample injection. The MW distribution of SP was determined using high-performance gel filtration chromatography under the following conditions: gel column: TSK gel G2000 SWXL analytical column (7.8 × 300 mm, 5 μm); mobile phase: acetonitrile/water/trifluoroacetic acid (40:60:0.05, v/v/v); flow rate: 0.5 mL/min; detection wavelength: 220 nm; column temperature: 30°C; injection volume: 20 μL.

About 1 g of the sample was weighed and dissolved in 5% trichloroacetic acid. The sample was kept at a constant volume of 25 mL and mixed using ultrasound for 20 min at room temperature. After standing for at least 2 h, the supernatant was filtered using a double-layer filter paper. About 400 μL of the supernatant was refiltered using a 0.22-μm microporous membrane and the amino acid composition was determined using OPA pre-column derivatization and reversed-phase HPLC (Agilent, UK) with UV detection. The operational conditions were as following: gradient dilution with 80.9 mmol/L sodium acetate-methanol-acetonitrile (1:2:2, v/v; pH 7.2). The flow rate was 1.0 mL/min; column temperature was 40°C; proline was detected at 262 nm and other amino acids was detected at 338 nm detection wavelength with UV detector, and. The amino acid content was quantified by external standard method.

Based on our previously reported method (20), in this study, 32 four-week-old male ICR mice were purchased from Shanghai Slack Laboratory Animal Co., Ltd. All mice were fed a standard diet for 1 week in an animal room and were subjected to a 12 h/12 h dark/light cycle, a temperature of 24 ± 1°C, and humidity of 60 ± 5%. At the end of the adaptation period, the mice were randomly divided into 4 groups (8 mice per group). In order to eliminate the influence of differences between cages on intestinal flora, the same cage is a simple and common method to homogenize the microbial community, but the high number of mice in each cage will lead to stress response and affect the test results. Therefore, in order to ensure the representativeness of the samples, mice in each group were randomly divided into 2 cages (4 mice in each cage) and placed in SPF room. Mice in the control group were provided normal water during the experimental period. From the 15th day until the end of the experiment, mice in both the DSS and SP groups were provided drinking water containing 3.5% DSS (treatment for 8 days). Mice in the SP groups were administered SP extract (300 and 600 mg/kg/day) from day 1 to the end of the experimental period on day 22. All drugs were administered via the intragastric route once daily during the experiment. The body weights and disease activity index (DAI) of mice were recorded daily and routinely (Table 1). The procedure followed in this study was consistent with the ethical standards set by the Animal Experiment Committee of the unit in charge (approval number: 2019003).

After the experiment, Mice were euthanized by cervical dislocation. Blood was collected from the eyeball and centrifuged for 10 min at 4°C and 3,500 rpm. The serum was separated and stored in a refrigerator at −80°C until later use. Mice were dissected and the intestine was isolated to remove the mesentery. The colon length was recorded and then cut off 2 cm above the anus. The most obvious lesion tissue was placed in 4% paraformaldehyde to prepare sections for histopathological analysis. The rest of the intestinal tubes were stored at −80°C.

Hematoxylin and Eosin (H&E) staining was performed according to a previously described method (1). Briefly, the middle portions of colon tissues were excised and immobilized in 10% (100 g/L) formalin for 24 h, followed by dehydration with gradient ethanol. Next, the colonic tissues were made transparent with xylene, embedded in paraffin, sliced into 5-μm-thick sections, stained with H&E, and observed using a DP-72 microscope (Olympus, Tokyo, Japan) to assess the morphometry of intestinal epithelial cells, neutrophil density, loss of crypt glands, and lymphocyte infiltration.

To observe the ultrastructure of tissues, the collected colon tissues were sliced into several pieces, fixed with 2.5% glutaraldehyde at 4°C, and then fixed again with 1% osmium tetroxide. Next, the samples were dehydrated using ethanol solutions of different concentrations. Subsequently, a portion of the treated colon samples was affixed to the stage and gold plated for scanning electron microscopy (SEM). Another portion of the sample was impregnated using epoxy resin, sliced into ultrathin sections, and stained with uranium acetate and aluminum citrate for transmission electron microscopy (TEM) (21). The tight junctions (TJ), microvilli, and other structures of colon epithelial cells were observed using a computer-aided image-analysis system following SEM and TEM.

The separated serum was divided into Eppendorf tubes for cryopreservation, placed at room temperature, and processed according to the instructions on the kit. Superoxide dismutase (SOD), glutathione reductase (GSH-Px), IL-6, TNF-α, diamine oxidase (DAO), and lipopolysaccharide (LPS) levels in mouse serum were determined.

Colon tissues (100 mg) from different treatment groups were treated with 1 mL of sterile physiological saline. The intestines were ground to mucus using an electric homogenizer and centrifuged for 10 min at 4,000 r/min in a precooled apparatus at 4°C. The supernatant was divided and stored in a refrigerator at −80°C. A bicinchoninic acid kit was used to determine the protein concentration and GSH-Px, SOD, TNF-α, IL-6, DAO, and LPS levels in the intestinal tract were determined.

Total bacteriological DNA was obtained from fecal samples of each group of mice following the instructions in the MOBIO PowerSoil DNA Isolation kit (MOBIO, United States) (22). DNA integrity was determined using agarose gel electrophoresis, and a QubitTM3 Fluorometer (Invitrogen, USA) was used to determine the DNA concentration in each sample. Next, the primers 341F (5′-ACTCCTACGGGAGGCAGCAG-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′) were used to amplify the V3–V4 high variant region of the 16S rRNA gene using PCR. The amplification products of PCR were purified using Ampure XP magnetic beads (AGENCOURT, Beckman Coulter, US) to remove the non-specific amplification products. An Agilent 2100 Bioanalyzer (Agilent DNA 1000 Reagents) was used to determine the average molecular length of the amplified library and quantify DNA libraries using real-time quantitative PCR. Lastly, the Illumina miseq platform was used to complete the sequencing of the qualified libraries. To ensure accurate and reliable results for subsequent analysis, DNA sample testing, library construction and quality control were performed on the raw sequencing data with subsequent sequencing consigned to Shanghai Meiji Biomedical Technology Co., Ltd. The number of reads per sample is 40–50,000, and rarefaction was used to reflect the microbial diversity of each sample at different sequencing volumes, indicating whether the amount of sequencing data for the sample is reasonable. In addition, the number of species common and unique species (OTU) in multiple groups or samples is calculated by Venn diagrams, which allow OTU classification of all sequences according to different levels of similarity, and usually perform bioinformatic statistical analysis of OTUs at 97% similarity level. PCoA is used to reflect the differences of multiple data sets on a two-dimensional coordinate plot using variance decomposition, with the axes taking the two eigenvalues that best reflect the differences between samples. The original sequencing data were analyzed on the Meiji Biocloud website.

GraphPad Prism 10.01 was used for statistical analysis of the data, which are expressed as mean ± standard deviation. One-way ANOVA analysis with post-hoc independent samples t-tests was used for multiple comparisons to test the significance of differences. Differences were considered statistically significant at P < 0.05.

MW distribution is an important parameter for the evaluation of proteolytic products as it may affect the antioxidant activity and other functional properties. The MW distribution of SP was determined using HPLC under the same chromatographic conditions as those used for standard samples (Figure 1). The MW distribution of SP was mainly below 1,000 Da, and the distribution range of 180–500 and 500–1,000 Da accounted for 35.20 and 26.75%, respectively. Studies show that short peptides with low MWs tend to be better absorbed; thus, SP being a small MW peptide could be easily digested and absorbed.

The amino acid composition of SP is shown in Table 2. SP has 17 amino acids, among which, the essential amino acid content is 54.50%. Lysine, leucine, glutamic acid, and arginine are abundant and account for 11.03, 14.63, 11.65, and 15.13%, respectively. Besides, methionine (Met), threonine (Thr), tryptophan (Trp), and histidine (His) have been identified as antioxidant amino acids. Moreover, their derivatives and peptides containing these amino acids exert antioxidant effects.

Mice in the normal control group had normal stools, shiny fur, sensitive reaction, and normal activity. After DSS induction, the model mice exhibited different degrees of pathological symptoms, including diarrhea, bloody stools, darkening of fur, loss of appetite, and laziness. Mice in the low- and high-dose SP groups also showed changes in stool characteristics after modeling, such as diarrhea and bloody stools, and the symptoms were significantly less intense than those observed in the DSS-treated group, suggesting better activity and diet status, and a reduction in diarrhea and bloody stools. As shown in Figures 2A,B, during the modeling process, mice in the control group showed no overall decrease in body weight; however, mice in the DSS-treated group showed a significant decrease in body weight (P < 0.05) and had higher DAI scores compared with those in the blank control group. After treatment with low-dose and high-dose SP, the loss of body weight in mice with DSS-induced colitis was significantly inhibited and DAI scores were also found to decrease. It was worth noting that low-dose SP (300 mg/kg) showed a better effect in maintaining body weight and DAI score compared with that observed using high-dose SP (600 mg/kg). Furthermore, as shown in Figures 2C,D, the colon length was significantly shorter in mice in the DSS group compared with those in the blank control group (P < 0.05). Moreover, the colon lengths of mice in the low-dose and high-dose SP groups were significantly longer (P < 0.05) compared with those of mice in the DSS group. The colon lengths of mice in the low-dose and high-dose SP groups were not significantly different (P > 0.05). Collectively, these results indicated that treatment with SP could improve the weight decrease and shortened colon length and reduce the DAI scores in mice with DSS-induced UC.

As seen in Figure 3, HE staining of the colon tissue showed that the tissues from the normal control group (Figure 3A) had clear colon walls, intact epithelia, neatly arranged glands, normal crypts, abundant goblet cells, and no inflammatory cell infiltration. The colonic epithelia in the DSS model group (Figure 3B) samples appeared necrotic and shedding. The intestinal wall was thin, glands were messy, ulcer symptoms were severe, and a great extent of inflammatory cell infiltration was found in the mucosal muscle layer. The colonic mucosa and intestinal villi in the tissue samples from the low-dose (Figure 3C) and high-dose SP groups (Figure 3D) appeared less damaged than those in the DSS model group, and the colonic mucosa structure was intact in the SP-treated groups. Furthermore, the improvement achieved after low-dose SP treatment was better than that achieved using the high dose.

Therefore, the DLP (low-dose SP) group was selected and the ultrastructure of the colonic epithelium was studied to determine the mechanism of SP in alleviating intestinal disorders. TEM and SEM were used to observe the ultrastructural changes in the colonic epithelium at magnifications of 12,000 × and 25,000 × , respectively. TEM revealed that the microvilli in the control (normal) group had the same length and an orderly arrangement (Figures 4A–C). The epithelial cell membrane structure was complete and the tight connection between apical cells was clearly visible. However, the microvilli in the DSS group were obviously damaged, disordered and loose, and were ordered in different directions. Moreover, occasional fractures and indistinct tight connections were observed. After treatment with SP, the microvilli showed an obvious recovery with a more orderly arrangement and slight improvements in the tight connections compared with those in the DSS group. SEM findings further confirmed the repairing effect of SP on the gut (Figures 4D–F). Microvilli in the DSS group were found to be arranged loosely with large gaps, indicating damage to the intestinal mucosal barrier. However, microvilli in the blank and SP groups appeared to have a similar length with a small gap. These results suggested that SP treatment improved the colonic microstructure and morphology in mice with DSS-induced colitis.

Inflammatory response, oxidative stress, and damage index of mice with DSS-induced colitis mice were measured using the serum. Serum IL-6 and TNF-α levels were significantly elevated in mice with DSS-induced UC compared with those in the control group (P < 0.05), indicating an inflammatory response (Figures 5A,B). On the other hand, serum TNF-α levels were significantly reduced in mice in the low- and high-dose SP groups compared with those in the DSS group (P < 0.05). IL-6 levels were less altered after SP intervention, and although a decreasing trend was observed, the changes were not statistically significant when compared with the DSS group. With respect to oxidative stress, SOD and GSH-Px activities were significantly reduced in the serum of mice with DSS-induced UC than that in the normal group (P < 0.05). The serum SOD levels of mice in the SP group were not significantly different from those in the DSS group (P > 0.05) (Figure 5C). However, serum GSH-Px levels were significantly higher (P < 0.05) after treatment with both low-dose and high-dose SP (Figure 5D). In the DSS group (Figures 5E,F), the DAO and LPS levels were significantly higher compared with those in the normal control group. A significant improvement was observed (P < 0.05) after SP intervention, especially with the high dose. Collectively, these results suggested that SP had a restorative function in DSS-induced colitis and that a high dose of SP could elicit a better effect.

We determined the inflammatory response, oxidative stress, and damage index in the colonic tissue of mice with DSS-induced colitis. Levels of the inflammatory factors IL-6 and TNF-α were significantly increased in the colon tissue of mice in the DSS group (P < 0.05) compared with those in control mice (Figures 6A,B), which was suggestive of enteritis. Both IL-6 and TNF-α levels in SP-treated mice (P < 0.05) were significantly reduced compared with those in mice in the DSS group, indicating the effective regulation of inflammatory factors by SP in the colonic tissue of mice with colitis. Furthermore, SOD and GSH-Px activities were significantly reduced in the colonic tissues of mice in the DSS group compared with those in the normal group (P < 0.05) (Figures 6C,D). Treatment with low- and high-dose SP could significantly enhance SOD and GSH-Px activity compared with that in the DSS group (P < 0.05), indicating that SP could improve oxidative stress in the mouse model of UC. Findings for the colon damage index (Figures 6E,F) suggested that DAO and LPS levels in the colonic tissues of mice were significantly increased in the DSS group compared with those in the normal group (P < 0.05). DAO and LPS levels in the colonic tissues of mice in all SP-treated groups were significantly lower (P < 0.05) compared with those of mice in the DSS group, where symptoms of inflammation were observed. This finding suggested that SP had a certain recovery effect in UC mice with intestinal damage.