Vascular endothelial growth factor (vegf)

Aminophylline was obtained from Shandong Xinhua Pharmaceutical Co., Ltd. (Zibo, People’s Republic of China). Klebsiella pneumoniae (strain ID: 46114) was purchased from the National Center for Medical Culture Collection (Beijing, People’s Republic of China). Antibodies against interleukin (IL)-6, IL-10, tumor necrosis factor (TNF)-α, soluble TNF-α receptor 2, collagen I, collagen III, collagen IV, endothelin (ET)-1, transforming growth factor (TGF)-β, vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), matrix metalloproteinase (MMP)-2, MMP-9, and tissue inhibitor of MMP (TIMP)-1 were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). The RNeasy kit was obtained from Qiagen (Valencia, CA, USA). Mayer’s hematoxylin and 1% eosin alcohol solution were purchased from MUTO Pure Chemicals (Tokyo, Japan). In all, 42 Sprague Dawley rats (21 male and 21 female; 200±20 g) were purchased from the Experimental Animal Center of Henan Province (Zhengzhou, People’s Republic of China). The animals were housed in cages with free access to food and tap water under standard conditions of humidity (50%±10%), temperature (25°C±2°C), and light (12 hours light/12 hours dark cycle). All animals were handled with humane care throughout the experiment.

The collected PN and bladder were fixed in 4% paraformaldehyde for 24 h at 4°C
before creating a paraffin block, as described previously⁹ . The primary antibodies were used as follows: vascular endothelial growth
factor (VEGF; diluted 1:200; Santa Cruz Biotechnologies, Santa Cruz, CA, USA),
βIII-tubulin (diluted 1:200; Abcam, Cambridge, UK), bFGF (diluted 1:500; Cell
Signaling Technology, Danvers, MA, USA), SDF-1 (diluted 1:200; Abcam, Cambridge,
UK), poly-ADP-ribose polymerase (PARP, diluted 1:500; Abcam, Cambridge, UK), and
6-diamidino-2-phenylindole (DAPI; Vector Laboratories, Inc., Burlingame, CA,
USA) were used to stain the nuclei. Digital images were obtained using a Zeiss
LSM 510 Meta confocal microscope (Zeiss, Oberkochen, Germany), and bladder
images were analyzed using ZEN 2012 (Zeiss, Oberkochen, Germany). The other
resulting images were analyzed using Image J to determine the positive rate for
each figure. In brief, we used Image J to split each color from merged figure,
calculate the intensity of each color, and then added them. The positive was the
ratio of a special color fluorescence intensity to all colors intensity.

At 0, 2, 8, 12 and 24 h post-treatment, the burn tissue samples were collected and homogenized, and centrifuged at 8,000 × g for 15 min at 4°C. The supernatant was used to measure total protein concentrations using a BCA kit. The proteins (50 µg) were separated using 10% SDS polyacrylamide gel electrophoresis and transferred onto a nitrocellulose membrane. The membrane was blocked with 5% milk in TBST for 1 h at 4°C and incubated with the following antibodies: NF-κB/p65 (cat. no. sc-109, 1:2,000; Santa Cruz Biotechnology, Inc., Franklin Lakes, NJ, USA), vascular endothelial growth factor (VEGF; cat. no. sc-13083, 1:4,000; Santa Cruz Biotechnology, Inc.), protease-activated receptor 1 (PAR1; cat. no. SAB5300042, 1:3,000; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) and β-actin (cat. no. sc-7210, 1:5,000; Santa Cruz Biotechnology, Inc.) at 4°C overnight. Subsequently, the membrane was incubated with sheep anti-rabbit or mouse IgG (cat nos. 14708 or 14709, 1:1,000; Cell Signaling Technology, Inc.) at room temperature for 2 h, and bands were revealed using an ECL kit (Cell Signaling Technology, Inc., Danvers, MA, USA) and quantified using Quantity One software version 3.0 (Bio-Rad, Hercules, CA, USA).