Pe anti rat il 4

Anticoagulated blood (100 μl) and treated blood (100 μl) from each sample were pipetted to the bottom a flow-cytometry sample tube and appropriate amounts of FITC anti-Rat CD4, PE anti-Rat CD8, APC anti-Rat CD3, PE anti-Rat IL-4, and APC anti-Rat IFN-r antibodies (BioLegend, San Diego, CA) were added and mixed. Then samples were incubated for 20 min at room temperature in the dark. Two microliters of 1 × red cell lysate were add to the samples, mixed well, and centrifuged 5 min at 300 ×g to collect supernatant, which was added to 2 ml cell washing buffer, mixed well, and centrifuged 5 min at 300 ×g to collect supernatant. Subsequently, 500 μl cell washing buffer was add to the supernatant and mixed well before flow cytometry (BD LSRII Flow Cytometry Instrument, BD Corporation).

Free total rhubarb anthraquinones were purchased from the Research Institute (Huaian, Jiangsu, China, batch number: 20130211) and dissolved in normal saline (NS). Sodium taurocholate (STC) was purchased from Sigma–Aldrich Co. (St. Louis, MO, United States). Rat TNF-α and IL-1β ELISA Kits were purchased from United States Systems R&D (Long Beach, CA, United States). NO Kit was purchased from Beyotime Biotechnology Research Institute (Shanghai, China). MPO kit was purchased from Nanjing Jiancheng Biological Engineering Institute (Nanjing, Jiangsu, China). Fixative solution, permeabilization wash buffer, Foxp3 fixative permeabilization wash solution, monensin, APC anti-rats CD4, FITC anti-rats IFN-γ, PE anti-rat IL-4, PE anti-rat CD25, Alexa fluor 488 anti-rat Foxp3 and goat anti-rat SIgA were purchased from Biolegend Company (San Diego, CA, United States). Rabbit anti-rat NLRP3, goat anti-rat ASC, mouse anti-rat caspase-1 were purchased from Santa Company (Santa Cruz, CA, United States). Triton, bovine serum albumin (BSA) DAB color liquid, goat super sensitive two-step reagent Kit, mouse one-step kit, TRITC labeled rabbit ant- goat secondary antibodies, fluorescein (FITC) labeled sheep anti-rabbit secondary antibodies were purchased from ZSGB Biological Company (Beijing, China).

Amylase, lipase, D-lactate, soluble tumor necrosis factor-α receptor (sTNF-αR), and secretory immunoglobulin A (sIgA) enzyme-linked immunosorbent assay (ELISA) kits were purchased from Nanjing Jiancheng Biological Engineering Institute (Nanjing, China). The chromogenic limulus amebocyte lysate kit was purchased from Bioendo Technology Co., Ltd (Xiamen, China). Rat interleukin-1beta (IL-1β) and transforming growth factor-β (TGF-β) ELISA kits were purchased from ExCell Biotech Co., Ltd (Shanghai, China). Rat TNF-α and IL-10 ELISA kits were purchased from Cusabio Biotech Co., Ltd (Wuhan, China). Rat IL-6 and IL-18 ELISA kits were purchased from Elabscience Biotech Co., Ltd (Wuhan, China). The rat IL-8 ELISA kit was purchased from Neobioscience Technology Co., Ltd (Shenzhen, China). The rat IL-4 ELISA kit was purchased from Invitrogen (Carlsbad, CA, USA). The specific antibodies against FITC anti-rat CD4, Alexa fluor^®647, anti-rat IFN-γ, PE anti-rat IL-4, and Alexa fluor^® 647 anti-rat Foxp3 were purchased from BioLegend (San Diego, CA, USA). Rabbit anti-rat CD68 and rabbit anti-rat CD103 were purchased from Abcam (Cambridge, MA, UK). PE anti-rat CD25 was purchased from BD Biosciences (San Jose, CA, USA). The sources of other reagents were indicated in the specified methods.