The SIS bio-patches were purchased from Beijing Biosis Healing Biological Technology Co., Ltd. (China).

Eight-week-old male wild-type C57BL/6 mice were obtained from the Guangdong Medical Laboratory Animal Center and housed at a controlled temperature (22–23 °C) with a 12-h light/dark cycle. The mice had unrestricted access to a standard diet and water in the pathogen-free facilities of the First Affiliated Hospital Animal Center at Guangdong Pharmaceutical University. The study began after a 6-week adaptation period for feeding. All animal procedures were approved by the Animal Ethics Committee of the First Affiliated Hospital of Guangdong Pharmaceutical University (Approval No. 2024004) and were conducted in accordance with institutional guidelines for animal care and use.

Fourteen mice were randomly assigned to either the control group or the SIS bio-patch group (n = 7 per group) and fasted for 18 h before surgery. General anesthesia was induced by intraperitoneal injection of tiletamine–zolazepam (Telazol, VIRBAC) at a dose of 50 mg/kg.

A midline abdominal incision (∼2 cm) was made to expose the gastrointestinal tract. An enterotomy was created at the gastrointestinal junction, followed by in situ anastomotic repair using interrupted sutures, with minor modifications based on previously described protocols (Eyarefe and Amid, 2010). In the SIS group, an SIS bio-patch was applied to cover the anastomotic site prior to suturing, whereas mice in the control group underwent anastomosis using sutures alone without patch reinforcement. Intra-abdominal bleeding was absorbed using sterile cotton balls. The abdominal wall was closed in two layers, including continuous suturing of the muscle layer and subcuticular closure of the skin. Postoperative analgesia was provided using meloxicam, and cefuroxime was administered to prevent infection. Mice were fasted for 24 h after surgery and subsequently resumed normal feeding. Intraoperative blood loss was estimated by measuring the weight difference of cotton balls before and after blood absorption.

Five days after surgery, mice were euthanized by intraperitoneal administration of Telazol (200 mg/kg) under deep anesthesia in accordance with AVMA guidelines. Anastomotic intestinal tissues were harvested for histological analysis, immunofluorescence staining, flow cytometry, enzyme-linked immunosorbent assay (ELISA), bacterial 16S rRNA sequencing, fungal internal transcribed spacer (ITS) sequencing, and metabolomics analysis. Peripheral blood samples were also collected for flow cytometric analysis. Postoperative day 5 was selected as a representative early healing time point based on previous studies demonstrating its critical role in immune-mediated epithelial repair and tissue remodeling (Ellison, 1989; Jain et al., 2021; Wang et al., 2021).

Anastomotic tissues from mice were fixed in 4% PFA overnight, paraffin-embedded, and sectioned for hematoxylin and eosin staining. Tissue pathology was evaluated using a modified scoring system (Bergstrom et al., 2010). Assessments included submucosal edema (0 = no change; 1 = mild; 2 = moderate; 3 = profound), epithelial hyperplasia (0 = no change; 1 = 1–50% increase; 2 = 51–100% increase; 3 = over 100%), epithelial integrity (0 = no change; 1 = up to 10 cells shed; 2 = 11–20 cells shed; 3 = ulceration; 4 = ulceration with severe crypt damage), and immune cell infiltration (0 = none; 1 = mild; 2 = moderate; 3 = severe).

Using established methods (Zhong et al., 2024), deparaffinized anastomotic tissue sections were subjected to heat-induced antigen retrieval and then treated for 30 min with 5% BSA and 0.2% Triton X-100 in PBS at room temperature. Post-blocking, the sections were incubated overnight at 4 °C with primary antibodies targeting ZO1 tight junction (1:100; Abcam, ab96587) and Occludin (1:100; Abcam, ab216327). After extensive PBS washes, the sections were exposed to CY3-labeled secondary antibodies. Nuclei were stained with DAPI, and images were captured using a Pannoramic MIDI fluorescence microscope. Fluorescence intensity was quantified with ImageJ software.

Mouse TNF-α, interleukin (IL)-6, IL-17A, and IL-22 concentrations were measured using ELISA MAX™ Deluxe Sets (BioLegend, United States of America) in accordance with the manufacturer’s instructions.

Anastomotic intestinal tissues from mice were harvested, rinsed with PBS to eliminate fecal matter, and the mesenteric adipose was removed. The intestines were longitudinally opened and incubated in an extraction solution containing RPMI-1640, 2% FBS, 10 mM DTT, and 1 mM EDTA at 37 °C, shaking at 180 rpm for 15 min. The tissues were then washed with PBS, cut into 1 mm pieces, and digested in a solution of RPMI-1640, 2% FBS, 1 mg/mL collagenase type II, and 0.5 mg/mL Dispase at 37 °C with constant shaking at 180 rpm for 45 min. After digestion, the tissues were filtered through a 70 μm strainer, and the supernatant was centrifuged to isolate lamina propria leukocytes.

Mouse peripheral blood mononuclear cells (PBMCs) were isolated by treating whole blood with ACK Lysis Buffer to remove erythrocytes, followed by washing and resuspension in PBS.

To characterize subsets of innate lymphoid cells (ILCs) in mice, cell suspensions were stained with antibodies against CD3ε, CD5, CD11b, CD11c, CD19, TER-119, CD45, CD127, GATA-3, T-BET, IL-17A, IL-22 (all sourced from BioLegend), and RORγt (eBioscience). Intranuclear staining utilized a Foxp3 staining kit from eBioscience, according to the manufacturer’s guidelines.

For the identification of Th17 and Treg cells, cells were stained with antibodies targeting CD3, CD4, CD45, Foxp3 (all from BioLegend), and RORγt (eBioscience). To delineate macrophage subsets, staining was performed using antibodies against CD45, CD11b, F4/80, CD86, and CD206 (all from BioLegend). Stained cells were then analyzed using a CytoFlex flow cytometer (Beckman Coulter), with data processed via CytExpert software (Beckman Coulter).

DNA extraction, sequencing, and analytical procedures were conducted by Shanghai Majorbio Bio-Pharm Technology Co., Ltd. (Shanghai, China) as previously described (Zhong et al., 2023). DNA was extracted from anastomotic intestinal tissues using the E. Z.N.A.® Soil DNA Kit (Omega Bio-tek, Norcross, GA, United States). For bacterial analysis, the V3-V4 regions of the 16S rRNA gene were amplified via polymerase chain reaction (PCR) with primers 338F (ACT​CCT​ACG​GGA​GGC​AGC​AG) and 806R (GGACTACHVGGGTWTCTAAT). For fungal analysis, the Internal Transcribed Spacer region 1 (ITS1) was amplified using primers ITS1F (CTT​GGT​CAT​TTA​GAG​GAA​GTA​A) and ITS2R (GCT​GCG​TTC​TTC​ATC​GAT​GC). Sequencing was performed on an Illumina MiSeq platform, and results were deposited in the NCBI database under accession number PRJNA1149522.

Sequence merging was performed with FLASh software (version 1.2.11) and quality filtering with fastp (version 0.19.6) (Chen et al., 2018; Magoc and Salzberg, 2011). Amplicon sequence variants (ASVs) were generated using DADA2 (Callahan et al., 2016), and taxonomic assignment was completed using QIIME2 and the SILVA 16S rRNA database. All analyses were carried out on the Majorbio Cloud Platform (www.majorbio.com).

A 50 mg anastomotic intestinal tissue sample was prepared for metabolite extraction using a 400 µL methanol solution (4:1, v/v) with 0.02 mg/mL L-2-chlorophenylalanine as an internal standard. The sample was processed using the Wonbio-96c high-throughput tissue crusher (Shanghai Wanbo Biotechnology Co., Ltd.) at −10 °C and 50 Hz for 6 min, followed by ultrasonication at 40 kHz for 30 min at 5 °C. The sample was then cooled to −20 °C for 30 min to precipitate proteins, followed by centrifugation at 13,000 g at 4 °C for 15 min. The supernatant was transferred to vials for LC-MS/MS analysis. To ensure system consistency and quality control, a pooled QC sample was created by mixing equal volumes from all samples.

LC-MS/MS analysis was conducted on a Thermo UHPLC-Q Exactive HF-X system using an ACQUITY HSS T3 column, with mobile phases of 0.1% formic acid in water (95:5, v/v) and acetonitrile: isopropanol (47.5:47.5, v/v). The flow rate was 0.40 mL/min, and the column temperature was set at 40 °C, with an injection volume of 3 µL. Mass spectrometry was performed in both positive and negative modes under optimized settings including auxiliary gas heating, capillary temperature, gas flow rates, and ion-spray voltage. Data was acquired over a mass range of 70–1,050 m/z using Data Dependent Acquisition mode, with full MS resolution at 60,000 and MS/MS resolution at 7,500.

Data processing was performed with Progenesis QI software, which facilitated baseline filtering, peak identification, retention time correction, and peak alignment. The processed data matrix included sample names, m/z values, retention times, and peak intensities. Metabolite identification was carried out using the HMDB, Metlin, and Majorbio databases, with further analysis conducted on the Majorbio cloud platform.

Data were analyzed using GraphPad Prism version 8.0.2 (GraphPad Software Inc., United States), and variables were reported as mean ± standard deviation. The unpaired Student’s t-test or Mann-Whitney U test was employed to compare two groups as applicable. A p-value below 0.05 denoted statistical significance.

To evaluate the effects of SIS bio-patch reinforcement on gastrointestinal anastomosis outcomes, fourteen mice were randomly assigned to either the control group or the SIS group (n = 7 per group). The experimental workflow and representative images of the anastomosis are shown in Figures 1A,B, respectively. Quantification of intraoperative blood loss revealed no significant difference between the SIS-treated and control groups (0.063 ± 0.022 g vs. 0.061 ± 0.022 g, P = 0.906; Figure 1C). In addition, 1 mouse from each group died within the first postoperative day, resulting in an identical survival rate of 85.7% in both groups (Figure 1D). These results indicate that SIS bio-patch application does not significantly affect intraoperative bleeding or early postoperative survival in this mouse model.

Histological assessment of anastomotic tissues at postoperative day 5 demonstrated markedly reduced submucosal edema and inflammatory cell infiltration in the SIS-treated group compared with controls (Figures 2A,B). Consistently, quantitative histological scoring revealed significantly lower inflammation scores in the SIS group. Analysis of cytokine levels in anastomotic tissues showed that SIS treatment significantly decreased the concentrations of the pro-inflammatory cytokines TNF-α and IL-6, while markedly increasing IL-22 levels (Figure 2C). Immunofluorescence staining further revealed enhanced expression of the tight junction proteins ZO-1 and occludin in the SIS-treated group relative to controls (Figures 2D–G). Together, these findings indicate that SIS bio-patch application alleviates local inflammation and promotes restoration of intestinal epithelial barrier integrity at the anastomotic site.

Flow cytometric analysis was performed to characterize immune cell populations at the anastomotic site. The gating strategy for innate lymphoid cell (ILC) subsets is shown in Figure 3A. The proportion of total ILCs among lymphocytes was significantly higher in the SIS-treated group than in the control group (Figure 3B). While the relative proportions of ILC1s and ILC2s were not significantly altered (Figure 3C), the proportion of ILC3s was significantly increased in the SIS group (Figure 3D).

Notably, the frequency of IL-22–producing ILC3s was markedly elevated following SIS bio-patch application (Figure 3E). Analysis of adaptive immune subsets revealed a reduction in both Th17 and Treg cells in the SIS group; however, the decrease in Th17 cells was more pronounced, resulting in a significantly lower Th17/Treg ratio compared with controls (Figure 3F). In contrast, no significant differences were observed in the proportions of M1 or M2 macrophages between groups (Figure 3G). Consistent with local immune changes, the Th17/Th ratio in peripheral blood mononuclear cells was also significantly reduced in SIS-treated mice (Figure 3H). These data suggest that SIS bio-patch application selectively enhances IL-22⁺ ILC3 responses while rebalancing adaptive immune subsets at the anastomotic site.

To determine whether SIS bio-patch application influences the local microbiota, bacterial 16S rRNA gene sequencing was performed on anastomotic tissues. Alpha diversity analysis revealed a trend toward increased microbial richness and diversity in the SIS group, although these differences did not reach statistical significance (Supplementary Figure S1A). Principal Coordinate Analysis (PCoA) demonstrated significant structural differences in the microbiota at the anastomotic sites between mice treated with the SIS bio-patch and those in the control group, accompanied by a marked reduction in the microbial dysbiosis index (Figures 4A,B). The variations in bacterial species and their relative abundances at the anastomotic sites are depicted in Supplementary Figure S1B,C.

Further differential abundance analysis revealed that the SIS bio-patch increased the abundance of beneficial bacteria such as Bifidobacterium and Alloprevotella, while decreasing that of Enterococcus at the anastomotic sites (Figure 4C; Supplementary Figure S1D). Correlation heatmaps indicated that the abundance of Bifidobacterium, Alloprevotella, and Muribaculum on the anastomotic mucosa positively correlated with intestinal ILC3s and IL-22⁺ILC3s, and negatively with the Th17/Treg ratio (Figure 4D), suggesting that the SIS bio-patch may modulate the proportion of mucosal immune cells through the augmentation of these bacteria at the anastomosis.

Predictive functional profiling of the bacterial communities suggested that the SIS bio-patch enhances bacterial functions related to platelet activation, vascular smooth muscle contraction, and protein digestion and absorption, while reducing those associated with Vibrio cholerae infection (Supplementary Table S1). These findings imply that the SIS bio-patch may mitigate anastomotic inflammation by modifying the structural and functional dynamics of the associated bacterial communities.

Fungal ITS sequencing was performed to assess changes in the local mycobiome following SIS treatment. While comparative analyses revealed no significant differences in fungal richness, diversity, or health indices between the cohorts, substantial variations in fungal community structure were apparent (Figures 5A,B and Supplementary Figure S2A). The Venn diagram and community bar plot analysis revealed significant disparities in the composition and proportions of fungal species and genera across the mucosal communities of the cohorts (Supplementary Figure S2B,C). Further investigations into genus abundance discrepancies indicated a notable increase in the abundance Fungi gen. Incertae sedis within the SIS-treated group. This increase was positively correlated with the levels of intestinal IL-22⁺ILC3s and inversely correlated with Th17 cells and the Th17/Treg ratio (Figures 5C,D and Supplementary Figure S2D). These findings suggest that the SIS bio-patch may modulate inflammation at anastomotic sites by influencing the abundance of Fungi gen. Incertae sedis.

Untargeted metabolomic analysis revealed clear separation between SIS-treated and control groups based on orthogonal partial least squares discriminant analysis (OPLS-DA) (Figure 6A). Volcano plots revealed that, compared to the SIS-treated group, the control group exhibited a significant increase in the abundance of 113 metabolites and a decrease in 222 metabolites (Figure 6B). The top ten significantly different metabolites between the two groups were identified using the variable importance in projection (VIP) value, as shown in Figure 6C. Notably, metabolites such as N,N-Bis(2-hydroxyethyl)dodecanamide and 16-Mercaptohexadecanoic acid were significantly reduced in the SIS group, while others including Val, Phe, Heptadecanoic Acid, and Decanoyl-L-Carnitine showed significant increases. Correlations between metabolite abundances and immune cell levels at the anastomotic sites are depicted in Supplementary Figure S3. KEGG pathway analysis of differential metabolites indicated a significant enhancement in Biotin metabolism pathways in the SIS group, whereas pathways such as glutamatergic synapse and bile secretion were significantly weakened (Figure 6D). These findings suggest that the SIS bio-patch may alleviate inflammation at anastomotic sites by modifying the metabolic characteristics of mouse intestinal tissues.