Il 10

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).

The lung and gut tissues were frozen at −80°C. Proteins were extracted from lung and gut tissues using a protein extraction kit (Coolaber, China) in accordance with the manufacturer's instructions. The lung and gut tissues were homogenized on ice with RIPA buffer, and the protein concentrations were determined using a BCA protein assay kit (Coolaber, China). The protein samples were boiled in sample buffer, loaded into each lane, separated by 10% SDS-PAGE, and transferred to PVDF membranes. The membranes were washed with Tris-buffered saline with Tween 20 (TBST) three times and sealed with 5% nonfat milk for 2 h at 25°C. Subsequently, primary antibodies, including GAPDH (1:5000, ABclonal, China), MPO (1:1000, ABclonal, China), TNF-α (1:1000, ABclonal, China), IL-6 (1:1000, ABclonal, China), IL-10 (1:1000, ABclonal, China), α7nAchR (1:1000, ABclonal, China), and NF-κB p65 (1:1000, ABclonal, China) were incubated at 4°C overnight and then incubated at room temperature for 1 h. Finally, the protein levels were quantified using a chemiluminescence kit. The images were quantitatively analyzed using ImageJ analysis software.

Colonic or spleen tissue lyses solution was fabricated using RIPA buffer (Signaling Technology, Inc.). The protein concentration was examined by a BCA kit (Sigma-Aldrich; Merck KGaA). Total protein (30 µg/sample) was separated via 10% SDS-PAGE and nitrocellulose membranes. We used 5% nonfat dried milk to block the membranes. The corresponding protein antibodies were as follows: ghrelin (Hua-bio, Chengdu, China; ER63531; 1/1000), GHSR (Abcam, MA, USA; ab85104; 1/1000), IL-1β (ABclonal, Wuhan, China; A20529; 1/500), IL-6 (ABclonal, Wuhan, China; A0286; 1/500), IL-10 (ABclonal, Wuhan, China; A2171; 1/500), TNF-α (ABclonal, Wuhan, China; A20851; 1/500), and β-actin (Boster, Wuhan, China; BM0627; 1/1000). Then, the membrane washing was performed with Tris-buffered saline/0. 1% Tween (TBST) and incubated for 1.5 hours with an HRP Goat anti-Rabbit IgG (Abcam, ab6721). The band visualization was carried out using the ECL system (Affinity Biosciences, Cincinnati, Ohio, USA), and as an internal control, β-actin was used.