This study aimed to investigate the expression features of Beclin-1 and c-Myc in the skin burn of rats. A total of 48 Sprague-Dawley (SD) rats were randomly divided into the normal group (n=12), the 3-day burn group (n=12), the 5-day burn group (n=12) and the 7-day burn group (n=12). Except for the normal group, the rat models of burn were established in the other three groups, burn wounds were given routine dressing change, and rats were sacrificed at 3, 5 and 7 days after modeling to collect materials. Then, immunohistochemistry was applied to detect the expression of c-Myc and Beclin-1. The expression levels of c-Myc protein and Beclin-1 protein were measured via western blotting. The expression levels of c-Myc messenger ribonucleic acid (mRNA) and Beclin-1 mRNA were detected by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). In comparison with the normal group, three burn groups had significantly increased the expression of c-Myc and Beclin-1, and the differences were statistically significant (P<0.05). Beclin-1 expression in the 5-day burn group was obviously higher than those in the 3 and 7-day burn groups, and the differences were of statistical significance (P<0.05). The expression of c-Myc in the 7-day burn group was overtly higher than those in the 3 and 5-day burn groups, and the differences showed statistical significance (P<0.05). The expression of Beclin-1 and c-Myc in post-burn skin tissues were gradually increased, with the Beclin-1 expression level reaching the peak on the 5th day after burn, and the expression level of c-Myc was the highest on the 7th day after burn.
Burn is a serious accidental injury. In particular, burn caused by fire is a challenge in surgical treatment. According to epidemiological statistics, approximately 450,000 burn patients needed treatment in the United States in 2011, on increase of approximately 340% compared with that in 1995 (
A total of 48 Sprague-Dawley (SD) rats weighing 220±20 g (half male and half female) were purchased from Shanghai Slack Laboratory Animal Co., Ltd. (Shanghai, China) license no. SCXK 2014-0003. The above-mentioned 48 rats were randomly divided into the normal group, the 3-day burn group, the 5-day burn group and the 7-day burn group, with 12 rats in each group Rats were housed in a temperature controlled room (21±2°C) on a 12-h light/dark cycle (lights on at 06:00). All rats had free access to water and food.
This study was approved by the Animal Ethics Committee of Weifang People's Hospital Animal Center (Weifang, China).
Main reagents used in this study included primary antibodies anti-Beclin-1 and anti-c-Myc, immunohistochemical kits (KIT-9710; Maxim, San Jose, CA, USA), AceQ reverse transcription-quantitative polymerase chain reaction (RT-qPCR) SYBR-Green Master mix kits (Q111-02/03) and HiScript II Q RT Sperfect for qPCR [(+genomic deoxyribonucleic acid (gDNA) wiper] kits (R223-01) (both from Vazyme Biotech Co., Ltd., Nanjing, China).
Main experimental equipment used in this study were an optical microscope (Leica DMI 4000B/DFC425C), a fluorescence RT-qPCR instrument (ABI 7500), Image-lab image analysis system, Image-Pro image analysis system (both from Bio-Rad Laboratories, Inc., Hercules, CA, USA) and a digital thermostatic water bath kettle (Changzhou Guohua Electric Appliance Co., Ltd., Changzhou, China).
Rats were intraperitoneally injected with 7% chloral hydrate, and the injection volume was 5 ml/kg. After successful anesthesia, hair on the back of the rats was removed to expose the skin. After disinfection, the back of the rats was immersed in the digital thermostat water bath kettle for 10 sec, resulting in burn wounds.
Burn models were prepared in the 3, 5 and 7-day burn groups according to the above establishment method for burn models. After successful modeling, wounds were given routine dressing change and packing. Then, rats were sacrificed at 3, 5 and 7 days after modeling, respectively, and skin tissues of burn wounds on the back were collected for experiments. However, rats in the normal group received no treatment and were sacrificed directly to collect normal skin tissues on the back for experiments.
After successful anesthesia, 6 rats in each group were fixed with paraformaldehyde, and skin tissues on the back (with an area of ~1 cm2) were collected and placed in 4% paraformaldehyde, followed by fixation at 4°C for 48 h. Then, paraffin tissue sections were made for immunohistochemical detection. Skin tissues on the back (with an area of ~1 cm2) were directly taken from the remaining 6 rats and placed in epoxy resin (EP) tubes for western blotting detection.
Paraffin tissue sections (5 µm) were conventionally dewaxed, hydrated, added with citric acid buffer, and heated in a microwave oven for antigen retrieval. Then, sections were rinsed with phosphate-buffer saline (PBS) solution and added with endogenous peroxidase blocking agent for 10 min of incubation. After that, sections were rinsed with PBS solution and added with goat serum for 20 min of blocking. Then, serum-blocking solution was removed, and anti-Beclin-1 primary antibody (1:200; cat. no. ab62557) and anti-c-Myc primary antibody (1:200; cat. no. ab39688), both from Abcam, Cambridge, MA, USA, were added for incubation overnight at 4°C. After that, sections were rinsed with PBS solution and incubated with secondary goat anti-rabbit (HRP) IgG antibody (dilution, 1:500; cat. no. ab6721; Abcam) for 10 min. Then, PBS solution was used for rinsing, and streptavidin peroxidase was added for 10 min of incubation. Last, sections were subjected to color development via diaminobenzidine (DAB), counterstained with hematoxylin, mounted with neutral balsam, observed under the microscope (Leica) and photographed.
Skin tissues stored at −20°C for standby application were added with lysis solution, followed by ice bath for 60 min. Then, tissues were centrifuged at 14,000 × g for 10 min, and protein was quantified by bicinchoninic acid (BCA) assay. The standard curve and optical density were measured via a microplate reader (Bio-Rad) and the concentration of protein was calculated. After protein was denatured, samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with corresponding concentration. When marker protein reached to the bottom of the glass plate, and sample protein was almost in a straight line at the bottom, gel running was stopped. Then, the sample was transferred onto a polyvinylidene fluoride (PVDF) membrane for blocking, followed by washing with tetrapropyl benzene sulfonate (TPBS) 3 times. After that, membrane was blocked with blocking solution for 1.5 h, added with anti-Beclin-1 primary antibody (1:1,000) and anti-c-Myc primary antibody (1:1,000), rinsed with Tris-buffered saline with Tween-20 (TBST), and added with secondary goat anti-rabbit (HRP) IgG antibody (dilution, 1:1,000; cat. no. ab6721; Abcam), USA), followed by rinsing with TBST. After the secondary antibody was removed by TBST washing, development was started. Finally, a membrane was placed in a chemiluminescence reagent for 1 min of reaction, developed in the dark and analyzed using the gel scanning imaging system.
Total ribonucleic acid (RNA) was extracted from spare bone stored at −20°C using RNA extraction kits. The extracted total RNA was reverse-transcribed into complementary deoxyribonucleic acid (cDNA) through reverse transcription kits, and the volume of the reaction system was 20 µl. Reaction conditions: reaction at 51°C for 2 min, predenaturation at 96°C for 10 min, denaturation at 96°C for 10 sec, annealing at 60°C for 30 sec, for 40 cycles. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control, and relative messenger RNA (mRNA) expression levels of Beclin-1 and c-Myc were calculated. Primer sequences are shown in
The expression levels of Beclin-1 and c-Myc in skin tissues were detected by immunohistochemistry. The protein expression levels of Beclin-1 and c-Myc in skin tissues were detected through western blotting. The mRNA expression levels of Beclin-1 and c-Myc in skin tissues were detected via RT-qPCR.
Statistical Product and Service Solutions (SPSS) 20.0 software (IBM SPSS, Armonk, NY, USA) was used for statistical analysis in this study. Measurement data were expressed as mean ± standard deviation. Comparison between multiple groups was done using One-way ANOVA test followed by post hoc test (Least Significant Difference). The Chi-square test was used for enumeration data.
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Autophagy and cell proliferation in skin tissues after burn are important factors affecting wound healing. Autophagy is closely related to recycling and utilization of intracellular macromolecular substances, removal of damaged tissues and maintenance of the stability of intracellular environment (
Cell proliferation is also an important factor affecting wound healing after burn. c-Myc is an important oncogene that regulates cell division and proliferation. In addition, c-Myc is an important mediator of mitosis, which can prompt cells in the quiescent stage to rapidly transfer into the division stage, thus promoting cell division and proliferation (
In conclusion, the expression of Beclin-1 and c-Myc after burn has certain regularities, which can be used as effective targets for the treatment of burns. At the same time, corresponding intervention can be carried out for the expression features of Beclin-1 and c-Myc after burn, namely, inhibit the expression of Beclin-1 after burn to inhibit autophagy, and promote the expression of c-Myc to benefit cell proliferation, thereby promoting wound repair and healing.
Not applicable.
No funding was received.
All data generated or analyzed during this study are included in this published article.
CL analyzed and HL interpreted the data. HL performed experiments. CL wrote the manuscript. YL carried out the statistical analysis and CL edited the language. All authors have read and approved the final study.
This study was approved by the Animal Ethics Committee of Weifang People's Hospital Animal Center (Weifang, China).
Not applicable.
The authors declare that they have no competing interests.
Expression levels of Beclin-1 and c-Myc detected via immunohistochemistry.
Average optical density values of Beclin-1 and c-Myc. *P<0.05, in comparison with the normal group; #P<0.05, in comparison with the 3- and 7-day burn groups; △P<0.05, in comparison with the 3- and 5-day burn groups.
Protein expression levels of Beclin-1 and c-Myc in each group. (A) Expression levels of Beclin-1 protein and c-Myc protein detected by western blotting. (B) *P<0.05, in comparison with the normal group; #P<0.05, in comparison with the 3- and 7-day burn groups; △P<0.05, in comparison with the 3- and 5-day burn groups.
mRNA expression levels of Beclin-1 and c-Myc. *P<0.05, in comparison with the normal group; #P<0.05, in comparison with the 3- and 7-day burn groups; △P<0.05, in comparison with the 3- and 5-day burn groups.
Primer sequences.
Gene name | Primer sequences | |
---|---|---|
Upstream: | 5′-CGGAATTCTATGGAAGGGTCTAAGACGTCC-3′ | |
Downstream: | 5′-CGGGATCCTCATTTGTTATAAAATTGTGAGGACA-3′ | |
Upstream: | 5′-ATCACAGCCCTCACTCAC-3′ | |
Downstream: | 5′-ACAGATTCCACAAGGTGC-3′ | |
Upstream: | 5′-ACGGCAAGTTCAACGGCACAG-3′ | |
Downstream: | 5′-GAAGACGCCAGTAGACTCCACGAC-3′ |