Tgf β

Naïve CD4 + T cells were isolated from the spleens of male C57BL/6J mice using Naïve CD4 + T cells isolation kit (Miltenyi, Germany) (Au-Flaherty et al. 2015 ; Duan et al. 2019 (link)) and then cultured in 1640 medium containing 10% fetal bovine serum with plate-bound anti-CD3 (2 μg/mL, #14-0031-86, eBioscience, USA) and anti-CD28 (1 μg/mL, #14-0281-85, eBioscience, USA) antibodies for 4 days. IL-2 (200 U/mL, Cusabio, China) and TGF-β (0.5 ng/mL, Cusabio, China) were added to the culture medium for Treg cell differentiation; while IL-6 (10 ng/mL, Cusabio, China), IL-23 (10 ng/mL, Sinobiological, China) and TGF-β (2.5 ng/mL, Cusabio, China) were added to the culture medium for Th17 cell differentiation. After replacement of culture medium, cells were cultured with 200 µM PA and/or 100 nM exenatide in the presence or absence of 10 µM Foxrkhead box protein O1 (FoxO1) inhibitor (AS1842856, MedChemExpress, USA) for 24 h.

With MDA (#A003-1, Nanjing jiancheng Bioengineering Institute), SOD (#A001-3, Nanjing jiancheng Bioengineering Institute), TNF-α (#CSB-E04741m, CUSABIO), IL-1β (#CSB-E08054m, CUSABIO), HMGB1 (#CSB-E08225m, CUSABIO), IL-6 (#CSB-E04639m, CUSABIO), TGF-β (#CSB-E04726m, CUSABIO), and PGE2 (#CSB-E07965h, CUSABIO) quantitative ELISA kits, related factors were tested according to instructions. The DYNATECHMR7000 microplate reader was used to calculate the concentration of each indicator by forming the standard curve through the constant value provided.

Total proteins from cells were extracted using the PRO-PREP Protein Extraction Kit (iNtRON Biotechnology, Seongnam, South Korea) and measured using the Bio-Rad DC Protein Assay Kit (Bio-Rad Laboratories, Hercules, CA, USA). The total protein content in each 20 μg sample was subjected to SDS-PAGE and immunoblotting with antibodies against IL-10 (ABclonal Tech, MA, USA), TGF-β (Cusabio Biotech, Wuhan, China), PD-L1(Santa Cruz Biotechnology, CA, USA) and β-actin Santa Cruz Biotechnology, CA, USA).