Tgf β1

Trizol Reagent was purchased from Invitrogen (Carlsbad, CA, USA), and the PrimeScript^TM RT Kit was from Fermentas (Burlington, ON, Canada). The GoTaq^® RT-Qpcr System was obtained from Promega (Madison, WI, USA). All primers used in the present study were synthesized and purified by Sangon Biotech (Shanghai, China). Rabbit anti-human IP-10 polyclonal antibody was purchased from Abcam (Cambridge, MA, USA). Rabbit anti-human IFN-γ, IL-4, TGF-β1, and α-smooth muscle actin (SMA) polyclonal antibodies were purchased from Boster (Wuhan, Hubei, China). Secondary antibodies and 3, 3′-diaminobenzidine were from ZSGB-Bio (Beijing, China). The Quantikine Human IP-10 (sensitivity, 4.46 pg/mL; limit of quantification (LOQ), 7.8–600 pg/mL) and TGF-β1 (sensitivity, 15.4 pg/mL; LOQ, 31.2–2,000 pg/mL) ELISA kits were from R&D Systems (Minneapolis, MN, USA); the IFN-γ (sensitivity, 4 pg/mL; LOQ, 7.8–500 pg/mL) and IL-4 (sensitivity, 2 pg/mL; LOQ 3.9–250 pg/mL) ELISA kits were from BioLegend, Inc. (San Diego, CA, USA).

An ELISA was used to detect the release of TCIPA. The test samples were collected from the serum and lung tissue homogenate supernatants of three mice in each group. Mouse c–c motif chemokine ligand 2/monocyte chemotactic protein-1 (CCL2/MCP-1, catalog no. EK0568), mouse interleukin-1 alpha (IL-1α, catalog no. EK0391), mouse interleukin-1 beta (IL-1β, catalog no. EK0394), mouse macrophage-stimulating factor (M-CSF, catalog no. EK0445), mouse transforming growth factor-beta 1 (TGFβ1, catalog no. EK0515), mouse interferon-gamma (IFN-γ, catalog no. EK0375), mouse interleukin-4 (IL-4, catalog no. EK0405) and interleukin-10 (IL-10, catalog no. EK0417) ELISA kits were purchased from Boster Biological Technology Co., Ltd. (Wuhan, China). Mouse macrophage migration inhibitory factor (MIF, catalog no. H598-M-96, Nanjing, China) and mouse stromal cell-derived factor 1 (SDF-1, catalog no. H398-1-M-96, Nanjing, China) were purchased from Nanjing Jiancheng Bioengineering Institute.

The paraffin-embedded skin sections were incubated with 3% H₂O₂ for 10 min and then 10% normal goat serum albumin (Solarbio, Beijing, China) for 10 min. The sections were stained with mouse monoclonal antibodies against α-SMA (1:200 dilution, BOSTER, Wuhan, China), Vimentin (1:200 dilution, BOSTER), E-cadherin (1:200 dilution, Cell Signaling Technology, Danvers, MA, USA) and TGF-β1 (1:200 dilution, BOSTER) overnight at 4 °C. The sections were incubated with biotinylated secondary antibodies for 30 min at room temperature. Diaminobenzidine solution was used as a chromogen to visualize the immunostaining results. The immunohistochemistry sections were scanned by pannoramic scan. Then, the images were observed and photographed using CaseViewer software at 400 × magnification, and quantified in Image-Pro Plus software (Media Cybernetics, Rockville, MD, USA). Five microscopic fields were selected for each section, and the mean intensity optical density (IOD) was used to quantify the protein expression level.

To detect the levels of inflammatory cytokines, animals were sacrificed at 12, 24, or 48 h after SCI/R, and the spinal cord homogenates were obtained. The concentrations of tumor necrosis factor-α (TNF-α), IL-1β, IL-10 and transforming growth factor-β1 (TGF-β1) were measured using specific ELISA kits according to the manufacturers’ instructions (Boster Biological Technology, Wuhan, China).