Tnf α

To determine cytokine levels in the colon, colonic tissue lysates were prepared by cutting the colon of each mouse to 1 cm and then homogenizing with a bead beater (Bead Ruptor Elite, OMNI International, USA) in RIPA buffer (150 mM sodium chloride, 50 mM Tris-HCl, pH 7.4, 1 mM ethylenediaminetetraacetic acid, 1 mM phenylmethylsulfonyl fluoride, 1% Triton X-100, 1% sodium deoxycholic acid, 0.1% sodium dodecyl sulfate, and 5 μg/mL of leupeptin; ATTO, Japan). After centrifugation of colonic tissue lysate at 12,000 × g for 1 min, the supernatant was collected and used to estimate cytokine levels of tumor necrosis factor (TNF)-α (Cat. #K0331186P; Koma Biotech, South Korea), interleukin (IL)-6 (Cat. #K0331230; Koma Biotech), and IL-10 using an ELISA kit (Cat. #K0331213P; Koma Biotech), respectively.

High glucose Dulbecco's modified Eagle's medium (DMEM) was obtained from Welgene Inc. (Gyeongsangbuk, Korea), and Iscove's modified Dulbecco's medium (IMDM) acquired from Merck Millipore (Darmstadt, Germany). Fetal bovine serum (FBS) and antibiotics were purchased from Invitrogen Inc. (Carlsbad, CA, USA). 2,4-Dinitrochlorobenzene (DNCB), corticotropin-releasing hormone (CRH), substance P (SP), 1-thioglycerol, and dexamethasone (DEX) were procured from Sigma-Aldrich (St. Louis, MO, USA). Human VEGF and mouse IFN-γ, IL-1β, IL-6, IL-13, TNF-α, VEGF, eotaxin, and IgE ELISA kits were supplied by Koma Biotech Inc. (Seoul, Korea). Human MDC, TARC ELISA kits were obtained from R&D Systems (Minneapolis, MN, USA). The mouse CORT ELISA kits were purchased from Arigo Biolaboratories (Hsinchu, Taiwan), and Mouse CRH and ACTH ELISA kits were procured from LifeSpan BioSciences (Seattle, WA, USA). Antibodies for phospho-p38, phospho-JNK, phospho-ERK, phospho-STAT1, phospho-I-κB-α, p38, JNK, ERK, STAT1, I-κB-α, and NF-κB were acquired from Cell Signaling Technology (Danvers, MA, USA). Antibodies against loricrin, involucrin, and lamin B2 were supplied by Abcam (Cambridge, MA, USA). HRP-conjugated anti-β-actin was obtained from Sigma-Aldrich (St. Louis, MO, USA).

Seven days after SCI, three rats from each group were sacrificed to collect segments of the spinal cord (10 mm) including the lesion center. The segments were then washed with phosphate-buffered saline and homogenized in lysis buffer. Afterwards, they were centrifuged at 13,000 rpm at 4 °C for 15 min. Protein concentrations in the tissue lysates were measured with the aid of a bicinchoninic acid protein assay kit (Thermo Scientific, Rockford, IL, USA). TNF-α, IL-1β, IL-6 (Koma Biotech, Seoul, Korea), and COX-2 (CUSA bio, Wuhan, China) enzyme-linked immunosorbent assay (ELISA) kits were used according to each manufacturer’s directions.

Sandwich ELISA kits were used for quantitative estimation of serum interleukin-10 (IL-10) and TNF-α (Koma Biotech Inc., Seoul, Korea), as well as hepatic NF-κB-p65 (Elabscience, Biotech Inc., USA) concentrations, according to the manufacturers’ instructions.