Con a

Con A was bought from Solarbio Science & Technology Co., Ltd. (Beijing, China, catalog number: C8110). Malondialdehyde (MDA) assay kit (TBA method), Alanine aminotransferase (ALT) Assay Kit, and Aspartate aminotransferase (AST) Assay Kit were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China, catalog number: A003-1-2, C009-2-1 and C010-2-1). Total Nitric Oxide (NO) Assay Kit was gotten from Beyotime Biotechnology (Shanghai, China, catalog number: S0021S). Chloral hydrate, UNlQ-10 Column Total RNA Isolation Kit and 2X SYBR Abstart Master Mix was purchased from Sangon Biotech Co., Ltd. (Shanghai, China, catalog number: A600288, B511321 and B110032). The Total Protein Extraction Kit (BOSTER Biological & Technology Co. Ltd., China, catalog number: AR0146), SDS–polyacrylamide gel electrophoresis (SDS-PAGE, Beyotime Biological Technology Co. Ltd., China, catalog number: P0012A), PVDF membranes (Millipore, USA, catalog number: R1CB73920), High-sig ECL Western Blotting Substrate (Tanon Science & Technology Co., Ltd., China, catalog number:180-501), Anti-p38 Rabbit pab, Mapk3k13 Rabbit pab, HRP-conjugated Goat Anti-Rabbit IgG (Additional file 1: Table S1) were used to detect the expression of target protein.

2 × 10⁶ cells/mL single T and B cell suspensions were seeded into 96-well plates (100 μL/well). After exposure to 5 μg/mL Con A (Solarbio, China) and 10 μg/mL LPS (Solarbio, China), T and B cells were treated with 5, 10, 20, 40, and 80 μg/mL fraction C4 or C5 for 72 h, respectively. 10 μL of CCK-8 (Dojindo, Japan) solution was added to incubate the cells for 1 h. Absorbance at 450 nm wavelength (A₄₅₀) was detected by an enzyme-labeling instrument (Sunrise, Biocell). The inhibition rate (IR) = (1 − average A₄₅₀ of the experimental group/the average A₄₅₀ of the control group) × 100%.

Anti-HA and anti-FLAG primary antibodies were purchased from Sigma-Aldrich (St. Louis, MO, USA). Anti-PARP1 primary antibody was purchased from Abcam (Cambridge, UK). Immunoaffinity-purified rabbit polyclonal antibodies against PD-L1, LMNB1, and GAPDH were obtained from Saier Biotech (Tianjin, China). Horseradish peroxidase-conjugated anti-mouse and anti-rabbit secondary antibodies were purchased from Zhongshan Golden Bridge Biotechnology (Beijing, China). The FITC/TRITC-conjugated secondary antibodies were purchased from Jackson ImmunoResearch Laboratories (West Grove, PA, USA). FITC-conjugated CD69 (human/mouse), APC-conjugated TNF-α (human/mouse), Brilliant Ultraviolet 615-conjugated IFN-γ (human/mouse), PE-Cy7-conjugated CD3 (mouse), PE-conjugated CD8 (mouse), APC-conjugated CD4 (mouse), and FITC-conjugated CD4 (mouse) were purchased from eBioscience (San Diego, CA, USA). Anti-PD-L1 (PE-conjugated CD274, human) was purchased from BD Biosciences (NJ, CA, USA).
Con A was obtained from Solarbio (Beijing, China), and actinomycin D (Act D), cycloheximide (CHX), and puromycin were purchased from Sigma-Aldrich. CellTrace CFSE was purchased from Invitrogen (Carlsbad, CA, USA). The compounds were dissolved in DMSO and used at the indicated concentrations.

Con A was bought from Solarbio Science & Technology Co., Ltd. (Beijing, China), batch number: C8110; chloral hydrate, TRIzol Reagent and 2× SYBR Abstart Master Mix was purchased from Sangon Biotech Co., Ltd. (Shanghai, China), batch number: A600288, B610409 and B110032.

Splenocytes were obtained as described above and adjusted to 5 × 10⁶ cells/mL in RPMI-1640 medium supplemented with 10% FBS (RPMI-1640 complete medium). Cell suspension (100 μL/well) was inoculated into 96-well plates (Corning) and treated with p30 + modified p54 (5 µg/mL or 10 µg/mL) for 72 h. Wells treated with concanavalin A (ConA, 5 μg/mL, Solarbio, Beijing, China), unstimulated wells and equal amount of RPMI-1640 complete medium were established as positive, negative and blank controls, respectively. After addition of 10 µL of Cell Counting Kit-8 (CCK8) assay solution (APE × BIO, Houston, TX, USA) and incubation for 3.5 h, the OD₄₅₀ of each well was then measured. The results are presented as stimulation index (SI, ratio of the OD₄₅₀ of stimulated well to the OD₄₅₀ of unstimulated well).

Con A (batch number: C8110) was gained from Solarbio Science & Technology Co., Ltd. (Beijing, China). Chloral hydrate (batch number: A600288) and UNlQ‐10 Column Total RNA Isolation Kit (batch number: B511321) were obtained from Sangon Biotech Co., Ltd. (Shanghai, China). Maxima Reverse Transcriptase (batch number: EP0743) was bought from Thermo Fisher Scientific (China) Co., Ltd. (Shanghai, China).

Male C57BL/6J mice (18 ± 22) g were purchased from the Animal Center of Anhui Medical University. The mice were kept in a temperature- and humidity-controlled room under standard 12-h light/dark cycles, maintained with administered food and water ad libitum. The Ethics Review Committee approved the animal experimental protocol at the Animal Experimentation of Anhui Medical University.
In this experiment, seven groups were divided at random from the mice: normal group, ConA-induced hepatitis model group, three dosages of CP-25-treated groups (25, 50, and 100 mg/kg), paeoniflorin-treated group (100 mg/kg), bicyclol-treated group (100 mg/kg). The effect of CP-25 and paeoniflorin at the same dose in the treatment of liver injury was compared. Meanwhile, bicyclol (Beijing Union Pharmaceutical Factory, Beijing, China) was served as a positive control. To establish the hepatitis mice model, mice received an intravenous injection of ConA (Solarbio, Beijing, China, C8110) at dose of 25 mg/kg. All drugs were diluted with 0.5% carboxymethyl cellulose sodium (CMC-Na) solution and intragastrically treated for 10 days before ConA injection. Other mice were intragastrically administered with an equivalent volume of CMC-Na solution. Then sacrificed the mice at indicated time point for liver, thymus, spleen and blood samples gathering.