Effect of Topical Insulin on Wound Neutrophil Function
Effect of Topical Insulin on Wound Neutrophil Function
The following materials and equipment were used: neutral insulin injection; Tegaderm film (3M, St. Paul, MN); anti-Ly-6G (Gr-1, Gr1); HRP-conjugated secondary antibody; RIPA lysis buffer, BCA protein assay kit, SDS sample loading buffer and ECL reagents; MDA assay kit and MPO assay kit; Trizol® Invitrogen, Grand Island, NY); PrimeScript RT reagent kit and SYBR Premix Ex Taq (Takara, Shiga, Japan); SDS-PAGE running tank and Western blot membrane transferring equipment (BIO-RAD); 7500 real-time PCR system (Applied Biosystems, Carlsbad, CA); Spectra Max®190 (Molecular Devices, Sunnyvale, CA); and cornea trephine.
A total of 42, 6- to 8-week-old mice (SPF C57BL/6J; 21 female and 21 male) were purchased from the Department of Laboratory Animal Science, Fudan University (Shanghai, China). Mice gender was randomly assigned to control or insulin treatment group. Mice were anesthetized with 0.5% pentobarbital sodium (50 mg/kg). Depilatorium (barium sulfide: pulvistalci: washing powder = 2:1:1, volume ratio) was used on the dorsal skin to remove hair after shaving. Six full-thickness wounds on the top, middle, and bottom areas of the dorsal skin, 3 at each side symmetrically, were created using a 5-mm diameter cornea punch. Wounds were treated with 0.03-U insulin in 20-ÎĽL saline or 20-ÎĽL saline immediately after injury; absorption was allowed for 5 minutes and then the wounds were covered with film dressing. Insulin was applied once every day. Tissue from the wounded areas and 5-mm adjacent normal skin tissue (n = 12) were collected at 1, 2, and 3 days after injury, and then were frozen in liquid nitrogen. These samples were later examined by Western blotting, real-time polymerase chain reaction (PCR), myeloperoxidase (MPO), and malondialdehyde (MDA) biochemical quantification assays.
The area without epithelium was identified as the wounded surface, and the period of time between the injury and total epithelium recovery was identified as the wound healing time (n = 6). The wounded surfaces were drawn on transparent tracing paper and scanned for analysis. The pictures were analyzed with ImageJ software to calculate the area of the wounded surface.
Tissue samples from the wounded area were lysed with RIPA lysis buffer. Total protein was detected by bicinchoninic acid assay (BCA). A total of 50 μg of total protein from each sample was mixed with sample buffer and loaded into each well of a sodium dodecyl sulfate polyacrylamide electrophoresis (SDS-PAGE) gel. Samples were then run by SDS-PAGE on 12% gradient gels, and transferred to polyvinylidene fluoride (PVDF) membrane. Transferred PVDF membranes were blocked by 5% BSA in tris-buffered saline and Tween 20 (TBST) at room temperature for 1 hour, followed by overnight incubation with a primary anti-Gr-1 antibody at 4°C. On the second day, membranes were washed 3 times and then incubated in secondary antibody for 2 hours at room temperature. Visualization was processed by ECL reagents and compatible scanners. Blots then were re-probed for glyceraldehyde 3-phosphate dehydrogenase (GAPDH) to show equal loading. Band intensities were analyzed using ImageJ software.
The experimental protocol followed the manufacturer's instructions. The unit of MPO quantification was units/g (wet sheet). The unit of MDA quantification was nmol/mg (protein amount).
Total mRNA was extracted using Trizol (Life Technologies, Grand Island, NY). The ratio of OD 260 nm to OD 280 nm was detected. If the values were higher than 1.8, RNA samples were reverse transcribed. The system volume of reverse transcription was 20 μL containing 1 μg RNA. The reaction conditions of the reverse transcription were 37°C for 15 minutes and 80°C for 5 seconds. The system volume of the real-time PCR was 20 μL containing 10 μL of SYBR Premix Ex Taq II (2x), 8 μL of PCR forward primer (10 μm), 8 μL of PCR reverse primer (10 μm), 0.4 μL of ROX Reference Dye or Dye II (50x), 2 μL of cDNA solution, and 6.8 μl of dH2O. The primers for MIP-2 and GAPDH were designed by BioTNT (Wanchai, Hong Kong). The reaction conditions of real-time PCR were 95°C for 30 seconds, followed by 40 cycles of 95°C for 5 seconds, and 60°C for 30 seconds.
Materials and Methods
Equipment/Materials
The following materials and equipment were used: neutral insulin injection; Tegaderm film (3M, St. Paul, MN); anti-Ly-6G (Gr-1, Gr1); HRP-conjugated secondary antibody; RIPA lysis buffer, BCA protein assay kit, SDS sample loading buffer and ECL reagents; MDA assay kit and MPO assay kit; Trizol® Invitrogen, Grand Island, NY); PrimeScript RT reagent kit and SYBR Premix Ex Taq (Takara, Shiga, Japan); SDS-PAGE running tank and Western blot membrane transferring equipment (BIO-RAD); 7500 real-time PCR system (Applied Biosystems, Carlsbad, CA); Spectra Max®190 (Molecular Devices, Sunnyvale, CA); and cornea trephine.
Experimental Animals and Tissue Samples
A total of 42, 6- to 8-week-old mice (SPF C57BL/6J; 21 female and 21 male) were purchased from the Department of Laboratory Animal Science, Fudan University (Shanghai, China). Mice gender was randomly assigned to control or insulin treatment group. Mice were anesthetized with 0.5% pentobarbital sodium (50 mg/kg). Depilatorium (barium sulfide: pulvistalci: washing powder = 2:1:1, volume ratio) was used on the dorsal skin to remove hair after shaving. Six full-thickness wounds on the top, middle, and bottom areas of the dorsal skin, 3 at each side symmetrically, were created using a 5-mm diameter cornea punch. Wounds were treated with 0.03-U insulin in 20-ÎĽL saline or 20-ÎĽL saline immediately after injury; absorption was allowed for 5 minutes and then the wounds were covered with film dressing. Insulin was applied once every day. Tissue from the wounded areas and 5-mm adjacent normal skin tissue (n = 12) were collected at 1, 2, and 3 days after injury, and then were frozen in liquid nitrogen. These samples were later examined by Western blotting, real-time polymerase chain reaction (PCR), myeloperoxidase (MPO), and malondialdehyde (MDA) biochemical quantification assays.
Healing Times and Healing Rates
The area without epithelium was identified as the wounded surface, and the period of time between the injury and total epithelium recovery was identified as the wound healing time (n = 6). The wounded surfaces were drawn on transparent tracing paper and scanned for analysis. The pictures were analyzed with ImageJ software to calculate the area of the wounded surface.
Western Blot Assay for Gr-1 Expression
Tissue samples from the wounded area were lysed with RIPA lysis buffer. Total protein was detected by bicinchoninic acid assay (BCA). A total of 50 μg of total protein from each sample was mixed with sample buffer and loaded into each well of a sodium dodecyl sulfate polyacrylamide electrophoresis (SDS-PAGE) gel. Samples were then run by SDS-PAGE on 12% gradient gels, and transferred to polyvinylidene fluoride (PVDF) membrane. Transferred PVDF membranes were blocked by 5% BSA in tris-buffered saline and Tween 20 (TBST) at room temperature for 1 hour, followed by overnight incubation with a primary anti-Gr-1 antibody at 4°C. On the second day, membranes were washed 3 times and then incubated in secondary antibody for 2 hours at room temperature. Visualization was processed by ECL reagents and compatible scanners. Blots then were re-probed for glyceraldehyde 3-phosphate dehydrogenase (GAPDH) to show equal loading. Band intensities were analyzed using ImageJ software.
Biochemical Analysis of MPO and MDA Expression
The experimental protocol followed the manufacturer's instructions. The unit of MPO quantification was units/g (wet sheet). The unit of MDA quantification was nmol/mg (protein amount).
Real-Time Quantitative PCR Assay for MIP-2 mRNA Expression
Total mRNA was extracted using Trizol (Life Technologies, Grand Island, NY). The ratio of OD 260 nm to OD 280 nm was detected. If the values were higher than 1.8, RNA samples were reverse transcribed. The system volume of reverse transcription was 20 μL containing 1 μg RNA. The reaction conditions of the reverse transcription were 37°C for 15 minutes and 80°C for 5 seconds. The system volume of the real-time PCR was 20 μL containing 10 μL of SYBR Premix Ex Taq II (2x), 8 μL of PCR forward primer (10 μm), 8 μL of PCR reverse primer (10 μm), 0.4 μL of ROX Reference Dye or Dye II (50x), 2 μL of cDNA solution, and 6.8 μl of dH2O. The primers for MIP-2 and GAPDH were designed by BioTNT (Wanchai, Hong Kong). The reaction conditions of real-time PCR were 95°C for 30 seconds, followed by 40 cycles of 95°C for 5 seconds, and 60°C for 30 seconds.