Human umbilical vein endothelial cells H926 (ATCC, USA) were maintained in HG-DMEM medium (Life Technologies, USA) supplemented with 10% fetal bovine serum in a humidified atmosphere containing 5% CO2 at 37 °C.
AS-1 was dissolved in DMSO, and stored at −20 °C. For the cell viability test, H926 cells were stimulated with 0, 25, 50, and 100 μmol/L of AS-1 for 72 hours. To evaluate the protective effect of AS-1, H926 cells were pretreated with 50 μmol/L of AS-1 or DMSO for 30 minutes. H/R group cells were placed in a humidified atmosphere containing 1% O2 and 5% CO2 for 4 hours, and then 20% O2 and 5% CO2 for required time.
The normal, necrotic, and border areas of the skin flap were fixed with 4% formalin. HE (Beyotime, China) and TUNEL (Roche, Germany) staining were performed according to the manufacturer's instructions. The skin flap tissue was obtained from Taixing People's Hospital, and the study was approved by Ethical Committee of Taixing People's Hospital.
Cytoplasmic proteins were prepared from harvested cells. Western blotting was performed as described previously. Briefly, the cytoplasmic proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred onto PVDF membranes (Cell Signaling Technology, USA). The membranes were incubated with the appropriate primary antibodies followed by incubation with peroxidase-conjugated secondary antibodies. The signals were detected using the ECL system. The same membranes were probed with antibodies for GAPDH (1:5 000, Sigma, USA). The signals were quantified by scanning densitometry. Computer-assisted image analysis was performed using Image J. The primary antibodies included anti-cleaved caspase-3, anti-Bax, anti-Bcl-2, anti-phospho-p38, anti-phospho-IκB, anti-Tublin, and anti-LaminB (all diluted in 1:1 000, Cell Signaling Technology).
The assay was performed according to the manufacturer's instruction. Briefly, both treated and untreated cells were harvested, washed with PBS, suspended in Annexin V binding buffer (10 mmol/L HEPES, pH 7.4; 2.5 mmol/L CaCl2, 140 mmol/L NaCl), stained with Annexin V-FITC (BD Biosciences, USA), and evaluated by the FACScalibur flow cytometer.
H926 cells were seeded in a 96-well plate (5 000 cells/well), pre-incubated for 24 hours in a humidified incubator (37 °C, 5% CO2), and treated as above. Then after 10 μL of CCK-8 solution was added into each well, the plate was incubated for 4 hours. The absorbance was measured using a microplate reader at 450 nm.
H926 cells were seeded in six-well plates and allowed to proliferate to a density of 80% (the cells nearly attaching to each other). Then the cell monolayer was scratched using a 1 mL pipette tip. Each well was washed once with growth media to remove cell debris. To record scratch wound closure, the images were captured at 0, 12, 24, and 48 hours until the wound was closed.
RAW264.7 cell solution was plated on top of the filter membrane in a transwell insert and incubated at 37 °C and 5% CO2. H926 cells were plated on the bottom of the lower chamber in a 24-well plate and treated as described above. The transwell insert was added without generating bubbles, incubated for 24 hours, and then removed from the plate. A cotton-tipped applicator was used to carefully remove the media. The cells that had not migrated from the membrane top were retained. Then, 70% ethanol was added into a 24-well plate to fix the cells. After removing the transwell insert, the remaining ethanol was removed from the top of the membrane using a cotton-tipped applicator. The membrane was positioned into a 24-well plate with 0.2% crystal violet and incubated at room temperature for 5–10 minutes for staining. The crystal violet was gently removed from the membrane top with a pipette tip. The cells on the membrane were photographed with SLR camera.
Total RNA was extracted from cells using RNAiso reagent (Takara Biotechnology, Japan) and RT-PCR assays were performed with the Prime-ScriptTM RT detection kit (Takara Biotechnology) as previously described. Amplification and detection of specific products were performed using the ABI Prism 7500 sequence detection system with the cycles indicated by the Prime-ScriptTMRT detection kit. As an internal control, HPRT primers were used for RNA template normalization. qRT-PCR primers were presented in Table 1. Relative levels of the target gene mRNA expression were calculated and expressed as 2−△△Ct.
Name Sequences (5′-3′) IL-1α-F CCAGAAGAAAATGAGGTCGG IL-1α-R AGCGCTCAAGGAGAAGACC MCP-1-F CCCACTCACCTGCTGCTACT MCP-1-R TTCCTTCTTGGG GTC AGC AC TNFα-F AGGGTCTGGGCCATAGAACT TNFα-R CCACCACGCTCTTCTGTCTAC HRPT-F GTTGGATACAGGCCAGACTTTGTT HPRT-R GATTCAACTTGCGCTCATCTTAGGC
Table 1. PCR primer sequences
Cytoplasmic proteins were incubated with anti-IL-1R antibody (Santa Cruz Biotechnology, USA) for 2 hours at 4 °C on a rotator. Then, 20 μL protein A/G beads (Santa Cruz Biotechnology) was added to each sample followed by incubation overnight at 4 °C on a rotator. The samples were centrifuged briefly in a microcentrifuge and washed three times in the 1 mL lysis wash buffer (250 mmol/L NaCl, 1% NP-40, 1 mmol/L PMSF, 5 mg/mL aprotinin, and 5 mg/mL leupeptin) (pH 8.0). Loading buffer (pH 6.8) was added to each sample and boiled for 5 minutes.
All results were expressed as mean±standard deviation (SD). One-way ANOVA was used to test the differences between the groups. Comparisons between two groups were performed using Student's t-test. A P-value <0.05 was considered statistically significant.
Cell culture and reagents
Tissue paraffin section and staining
Western blotting assay
Flow cytometric analysis
Cell viability assay by CCK8
Scratch wound closure assay
Total RNA extraction and qRT-PCR