Journal: International Journal of Molecular Medicine
Article Title: TIM-4 blockade of KCs combined with exogenous TGF-β injection helps to reverse acute rejection and prolong the survival rate of mice receiving liver allografts
Figure Lengend Snippet: Blockade of TIM-4 improves hepatic acute reaction response injury and reduces the secretion of inflammatory cytokines. (A) KCs were isolated from mice in the sham, control mAb and TIM-4 mAb groups on day 7 post-surgery. Cells were stained with specific secondary antibodies labeled with tetramethylrhodamine (red) and DAPI (blue) and then examined using laser confocal microscopy (magnification ×800). (B) KCs were isolated from each group and stained with FITC-TIM-4 mAb for flow cytometry. (C) Blood samples were obtained (0.5 ml/mouse) from murine abdominal aortas in each group on day 7 post-surgery. Levels of AST, ALT and TBIL were determined in a clinical biochemical laboratory. (D-F) Levels of inflammatory cytokines, including TNF-α, IFN-γ, CCL2, CXCL2 and TGF-β were detected using ELISA and western blotting. (G) Representative micrographs of the pathological damage observed in each group post-transplantation following staining with hematoxylin and eosin (magnification ×400). (H) Donor mice received CL treatment to destroy KCs prior to surgery. orthotopic liver transplantation or sham surgery was subsequently performed and mice were treated either with or without TIM-4 mAb. All mice underwent analysis to determine the RAI score on day 7 following surgery. (I) The expression of key phosphoproteins involved in the NF-κB and p38 MAPK signaling pathways, including p65 and p38, respectively, were analyzed using western blotting. Data are presented as the mean ± standard deviation. a P
Article Snippet: The primary antibodies used included: TIM-4 (RMT4-53, cat. no. GTX14149; 1:100 dilution; GeneTex Inc.), tumor necrosis factor-α (TNF-α; 52B38, cat. no. ab1793; 1:100 dilution; Abcam), interferon-γ (IFN-γ; #37895R, cat. no. MAB485R; 1:100 dilution; R & D Systems Inc., Minneapolis, MN, USA), chemokine ligand 2 (CCL2; 2D8, cat. no. GTX60582; 1:100 dilution; GeneTex Inc.), C-X-C motif chemokine ligand 2 (CXCL2; cat. no. GTX74085; 1:200 dilution; GeneTex Inc.), TGF-β (cat. no. ab92486; 1:100 dilution), phosphorylated (p)-signal transducer and activator of transcription 6 (STAT6; phospho Y641, cat. no. ab28829; 1:100 dilution), p-p65 (phospho S536, cat. no. ab86299; 1:100 dilution), p-p38 (phospho Y182, cat. no. ab47363; 1:100 dilution), forkhead box P3 (Foxp3; 236A/E7, cat. no. ab20034; 1:100 dilution), transcription factor gata3 (Gata3; cat. no. ab106625, 1:100 dilution) and β-actin (cat. no. ab8226; 1:100 dilution) (all from Abcam).
Techniques: Isolation, Mouse Assay, Staining, Labeling, Confocal Microscopy, Flow Cytometry, Cytometry, AST Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Transplantation Assay, Expressing, Standard Deviation