Ionomycin

To measure IFN-γ levels by stimulating splenocytes in vitro, splenocytes were cultured in 12-well plates containing proteins of rPoAMA-1. After 18 h, the cells were simultaneously mixed with various concentrations of phorbol 12-myristate 13-acetate (50 ng/mL PMA; Sigma), ionomycin (1 µg/mL; Solarbio), and brefeldin A (5 µg/mL; Solarbio) for 6 h at 37 °C with 5% CO₂. The cells were washed twice with PBS. Subsequently, 50 µL PBS containing CD4–488 (1:200; BioLegend, California, USA) and CD8-APC (1:300; BioLegend, California, USA) were added to samples at 4 °C for 1 h, and then centrifuged (1500 × g, 10 min) and washed twice with PBS. Then, 100 µL of fixative (BioLegend, California, USA) was added, and the samples were incubated in a dark environment at 4 °C for 20 min. The samples were then washed with membrane washing buffer (BioLegend, California, USA), and 50 μL of IFN-r-PE (1:200; BioLegend, California, USA) diluted in transmembrane washing solution was added in a dark environment at 4 °C overnight. Finally, the samples were analyzed in BD Accuri C6 PLUS flow cytometry (BD, New Jersey, USA).

The standard of berberine (C₂₀H₁₈NO₄, molecular weight: 235.324 kD) was obtained from Solarbio Biotechnology Co., Ltd. (Beijing, China). Dextran sulfate sodium (DSS molecular weight: 36000-50000kD), ciprofloxacin, metronidazole, Phorbol 12-myristate (PMA), ionomycin, and Bravertin A were also obtained from Solarbio Biotechnology Co., Ltd. (Beijing, China). Total DNA extraction kit, total RNA extraction kit, first-stand cDNA reverse transcription kit, polymerase chain reaction kit, and primers were obtained from TianGen Biotechnology Co., Ltd. (Beijing, China). Mouse IL-10 and IL-17 ELISA kits were obtained from Multi Science Biotechnology Co., Ltd. (Hangzhou, China). APC anti-CD4, FITC anti-IL17A, PE anti-CD25, and Alexa Fluor 488 anti-Foxp3 antibodies for flow cytometry were purchased from BD bioscience Co., Ltd. (Franklin Lakes, NJ, United States). BCA, alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN), and creatinine (Cr) test kit were purchased from Nanjing Jiancheng Biological Engineering Institute (Nanjing, China).

To analyze the Th17 cell percentage (CD4⁺IL17⁺ T cells/CD4⁺ T cells%), PBMCs were first stimulated with 50 ng/mL phorbol 12‐myristate 13‐acetate (PMA, cat. no. P6741; Beijing Solarbio Science & Technology Co., Ltd.), 1 μg/mL ionomycin (cat. no. 18800; Beijing Solarbio Science & Technology Co., Ltd.), and 10 μg/mL brefeldin A (cat. no; 7150022; Beijing Solarbio Science & Technology Co., Ltd.) at 37°C under a 5% CO₂ environment for 4 h. Then, cells were stained with APC‐labeled anti‐CD4 antibody (0.8 μg/mL, cat. no. 357408; BioLegend Inc.) in the dark at room temperature for 30 min. After surface staining, the cells were fixed and permeabilized in a dark environment at room temperature and stained with PE‐labeled anti‐IL‐17A antibody for 30 min (2.5 μg/mL, cat. no. 512306; BioLegend Inc.). Flow cytometric analysis was performed on a LSRII flow cytometer (BD Biosciences) and analyzed by FlowJo software (FlowJo‐v 10.8.1.).