Animals and diets

All animal experimental procedures were approved and performed in accordance with the ARRIVE guidelines and ethical standards of the Animal Protection and Use Committee of Anhui Medical University (No. LLSC20231041). Sprague–Dawley rats (Wild Type) in this experiment were obtained from (Laboratory Animal Center of Anhui Medical University, Hefei, China; aged 3–4 months; weight 2.0–2.5 kg) and housed in a specific pathogen-free room under a 12/12 h light–dark cycle at room temperature and 50% humidity, with free access to water and standard laboratory diet during the experiment.

Establishment of the AP model and experimental design

Experimental rats were randomly divided into five subgroups (n = 10 per group): control, AP, AP + UTI, AP + ML-385, and AP + UTI + ML-385. The AP model was induced in rats by intraperitoneal injection of 20% L-arginine in phosphate-buffered saline (PBS) at a dose of 2.5 g/kg body-weight (BW) twice with a 1-h interval. Rats in the control group were intraperitoneally injected with PBS in the same manner. Rats in the AP + UTI group were intraperitoneally injected with ulinastatin at a dose of 10000 U/kg BW immediately after the final L-arginine injection, and the ulinastatin injection was repeated at an interval of 24 h. Rats in the AP + ML-385 and AP + UTI + ML-385 groups were administered ML-385 before the first L-arginine injection, whereas those in the AP + UTI + ML-385 group were intraperitoneally injected with ulinastatin in the same manner as the AP + UTI group, and rats in the AP + ML-385 group were intraperitoneally injected with the same volume of PBS. The rats in the AP group were administered the same volume of PBS at the same timepoint.

Biochemical assays

Experimental rats were anesthetized by intraperitoneal injection (2 ml/kg BW) of 3% pentobarbital sodium (P3761, Sigma-Aldrich, Shanghai, China). Blood samples were collected using heparinized syringes from the caudal veins at 0, 24 and 48 h after the initial l-arginine injection, and the upper serum was separated by centrifugation at 4000r/min for 10 min at 4 °C. The Serum was placed in an EP tube and cryopreserved in 80 °C liquid nitrogen for unified detection. Serum levels of amylase (LE-B1215, Lai Er Bio-Tech, Hefei, China), lipase (LE-1–139, Lai Er Bio-Tech, Hefei, China), intestinal fatty acid-binding protein (I-FABP) (LE-B0433, Lai Er Bio-Tech, Hefei, China) and diamine oxidase (DAO) (LE-H1628, Lai Er Bio-Tech, Hefei, China) were determined using ELISA kits. The entire process was performed in accordance with the manufacturer’s instructions and the standard laboratory procedures.

Pathological examination

Experimental rats were intraperitoneally injected with 3% pentobarbital sodium and then sacrificed. Then, Segmental fresh pancreatic and terminal ileal tissues were harvested under sterile conditions, fixed in 4% paraformaldehyde solution (P6148, Sigma-Aldrich, Shanghai, China) at 4 °C, followed by embedding in paraffin, and the sections were dewaxed. Pancreatic and terminal ileal sections were stained with hematoxylin and eosin solution (H&E). Finally, the sections were dehydrated and sealed with optical resin adhesive. H&E-stained sections were randomly assigned to two pathologists blinded to the experimental treatments, and they observed and evaluated the damage in pancreatic and terminal ileal tissues under 200 × optical electron microscopes. The degrees of pancreatic injury were evaluated by scoring the grading system according to Schmidt’s scoring system [17] (Table 1), and the degrees of terminal ileal injury were evaluated by scoring the grading system according to Chiu’s scoring system [18] (Table 2).

Table 1 Histopathologic grades of pancreatic tissue by SchoenbergTable 2 Histopathologic grades of intestinal tissue by ChiuLaser Doppler blood flow meter

The rats were anesthetized with an intraperitoneal injection of 3% pentobarbital sodium. A laparotomy was performed, and the terminal ileum tissue was exposed. The probe was used to touch the mucosa in the terminal ileal tissue without pressure and the blood flow was measured using a laser Doppler blood flow meter (MoorFLPI-2, Moor Instruments Ltd, Devon, UK) connected to a computer with the matching software moorVMS-PC v3.1 to process data. The perfusion unit (PU) refers to blood perfusion within the measured area. The concentration of moving blood cells (CMBC) refers to the number of associated moving blood cells that cause Doppler shift. Velocity is the average movement rate of associated blood cells within the measured area. The relationship between the above three signals is

Transmission electron microscope

Segmental fresh terminal ileal tissue was harvested on ice, and 2 mm sections were fixed for 2 h with buffered glutaraldehyde, post-fixed with 1% OsO4, dehydrated through graded alcohols, infiltrated through Epon12, and then embedded in resin. Sections were cut at 1–2 mm thickness and then stained with uranyl acetate and lead citrate, and photographed using a Hitachi HT-7800 (Hitachi, Tokyo, Japan) electron microscope operated at 75 kV. Morphological and structural changes in the IECs, microvilli, and TJs between IECs were observed using TEM.

Western blot analysis

The Terminal ileal tissue was ground and homogenized with pyrolysis liquid containing PMSF for 30 min on ice, and the homogenized tissue was centrifuged at 4 °C for 15 min. The supernatants were collected and equal amounts of protein were electrophoretically separated using 10% SDS–PAGE and transferred to nitrocellulose membranes. Membranes were blocked with 5% skim milk and incubated with antibodies against rat claudin-1 (SAB4200462, Sigma-Aldrich, Shanghai, China), occludin (SAB5700784, Sigma-Aldrich, Shanghai, China), tumor necrosis factor (TNF-α) (SAB5700698, Sigma-Aldrich, Shanghai, China), interleukin-6 (IL-6) (SAB5700181, Sigma-Aldrich, Shanghai, China), interleukin-1β (IL-1β) (ab315084, Abcam, Shanghai, China), interleukin-10 (IL-10) (ab9969, Abcam, Shanghai, China), nuclear factor erythroid 2-related factor 2 (Nrf2) (SAB4501984, Sigma-Aldrich, Shanghai, China), heme oxygenase-1 (HO-1) (SAB5700731, Sigma-Aldrich, Shanghai, China), iNOS (SAB4502011, Sigma-Aldrich, Shanghai, China), CD86 (SAB5700710, Sigma-Aldrich, Shanghai, China), Arg-1 (SAB5700762, Sigma-Aldrich, Shanghai, China), CD206 (ab64693, Abcam, Shanghai, China), and GAPDH (AB2302, Sigma-Aldrich, Shanghai, China), followed by incubation with secondary goat anti-rabbit antibody or secondary goat anti-mouse antibody conjugated to horseradish peroxidase for 1 h at room temperature. Protein bands were quantified using the mean ratio of the integral optical density normalized to GAPDH expression.

Quantitative reverse transcription PCR (qRT–PCR) analysis

The terminal ileal tissue was ground and dissolved in TRIzol reagent (Life Technologies, Carlsbad, CA, USA). The mixture was transferred to 1.5 mL RNase-free tubes and centrifuged at 4 °C for 15 min in chloroform. The supernatants were collected and centrifuged at 4 °C for 10 min with an equal volume of isopropyl alcohol. Then, the supernatants were removed and 75% ethanol was added to the precipitate and centrifuged at 4 °C for 5 min. Finally, RNA extraction lysis buffer was obtained by dissolving in diethylprocarbonate. Total RNA was extracted using an RNA Prep Pure Micro Kit (DP420, Tiangen Biotech, Beijing, China). RNA concentration was measured using a spectrophotometer (NanoDrop 2000c, Thermo Fisher Scientific, Waltham, MA, USA). Equal amounts of samples were reverse-transcribed using the FastQuant RT Kit (KR-106–02, Tiangen Biotech, Beijing, China). SYBR-based qPCR was performed using the Bestar qPCR Mastermix (DBI-2223, DBI Bioscience, Ludwigshafen, Germany) on an ABI StepOnePlus platform (Thermo Fisher Scientific). Various mRNAs were quantified, and GAPDH was used as an internal control. The mRNA expression levels of Nrf2 and HO-1 in the terminal ileal tissue were measured using the comparative 2−(ΔΔCq) method. The primer sequences of Nrf2 (ZN-YW0758, Zhien Biology, Hefei, China), HO-1 (ZN-YW0531, Zhien Biology, Hefei, China) and GAPDH (ZN-NYW006, Zhien Biology, Hefei, China) used to amplify the mRNAs are listed in Table 3.

Table 3 Primer sequences for RT-PCRImmunofluorescence staining

Histological sections of terminal ileal tissues were deparaffinized in xylene and graded in water using different concentrations of ethanol ranging from 100 to 50%. The sections were blocked with 5% bovine serum albumin for 1 h at room temperature and incubated with primary antibodies (1:100 dilution) against iNOS (ab178945, Abcam, Shanghai, China), CD86 (SAB5700710, Sigma-Aldrich, Shanghai, China), Arg-1 (SAB5700762, Sigma-Aldrich, Shanghai, China), CD206 (ab64693, Abcam, Shanghai, China), and Nrf2 (SAB4501984, Sigma-Aldrich, Shanghai, China) overnight at 4 °C. For co-immunostaining, two primary antibodies from distinct species were incubated with the sections. The sections were washed with PBS and incubated with tetramethylrhodamine-5-(and 6)-isothiocyanate (46950, Sigma-Aldrich, Shanghai, China) conjugated secondary antibodies (1:200 dilution) for 1 h at room temperature. Nuclei were labeled by incubation with 4′,6-diamidino-2-phenylindole (32670, Sigma-Aldrich, Shanghai, China), and fluorescence images of randomly selected fields were observed under a fluorescence microscope, and the photographs were obtained using an Olympus CKX41 camera (Olympus Company, Japan).

Measurement of antioxidants and peroxides activities

Terminal ileal tissue was weighed, homogenized, and centrifuged. The GPx and GR activities were measured by calculating the consumption of NADPH as a cofactor for the reduction of GSSG to GSH. The GST activity was measured using 1-chloro-2,4-dinitrobenzene (1.02427, Sigma-Aldrich, Shanghai, China) as the substrate. The T-SH activity was determined by Ellman’s reaction using 5,5’-dithio-bis-2-nitrobenzoic acid (D8130, Sigma-Aldrich, Shanghai, China). MDA content was measured using thiobarbituric acid reactive substances, and ROS levels were measured using an Oxiselect In Vitro ROS Assay Kit (STA-347-T, Cell Biolabs, San Diego, USA).

Statistical analysis

All data were recorded in Excel 2019, and IBM SPSS (version 21.0) was used for statistical analysis. Graphs were generated using the GraphPad Prism 8 software. Normality and homogeneity of variances were assessed for all key data sets using Shapiro–Wilk and Levene’s tests, respectively. The measured values that followed a normal distribution are expressed as mean ± standard deviation (SD). Statistical significance was determined by one-way ANOVA with Student–Newman–Keuls and Mann–Whitney rank-sum tests. P < 0.01 was regarded as a statistically significant difference.