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 Table I.

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.

As shown in Fig. 1, the positive expression of Beclin-1 and c-Myc were tan color. The positive expression of Beclin-1 and c-Myc in the normal group were fewer, while those in the three burn groups were significantly increased, which showed statistically significant differences (P<0.05). According to statistical analysis of positive expression (Fig. 2), the 5-day burn group had the highest positive expression level of Beclin-1, which was obviously higher than those in the 3- and 7-day burn groups, showing statistically significant differences (P<0.05). In addition, the positive expression level of c-Myc in the 7-day burn group was the highest, which was evidently higher than those in the 3- and 5-day burn groups, showing statistically significant differences (P<0.05). This suggests that after burn, the expression levels of Beclin-1 and c-Myc in skin tissues are gradually increased with the expression level of Beclin-1 reaching the peak around the 5th day after burn and then beginning to gradually decline, and that of c-Myc was the highest around the 7th day after burn.

As shown in Fig. 3A, the expression levels of Beclin-1 protein and c-Myc protein were reduced in the normal group, while those in the three burn groups were overtly increased, and the differences were statistically significant (P<0.05). The expression levels of Beclin-1 and c-Myc proteins were subjected to statistical analysis, and the results (Fig. 3B) showed that the Beclin-1 protein expression level in the 5-day burn group was the highest, which was significantly greater than those in the 3-and 7-day burn groups, showing statistically significant differences (P<0.05). The expression level of c-Myc protein in the 7-day burn group was the highest and significantly higher than those in the 3- and 5-day burn groups. This indicates that the expression levels of Beclin-1 and c-Myc proteins in skin tissues are gradually increased after burn with the Beclin-1 protein expression level reaching the peak around the 5th day after burn and then beginning to decline, and the c-Myc protein expression level was the highest around the 7th day after burn.

According to Fig. 4, the mRNA expression levels of Beclin-1 and c-Myc were lower in the normal group, while those in the three burn groups were distinctly elevated, showing statistically significant differences (P<0.05). The mRNA expression of Beclin-1 was the highest in the 5-day burn group, which was clearly higher than those in the 3- and 7-day burn groups, and the differences were statistically significant (P<0.05). The mRNA expression of c-Myc in the 7-day burn group was the highest and obviously higher than those in the 3- and 5-day burn groups. This suggests that the expression of Beclin-1 and c-Myc mRNAs in skin tissues are gradually increased after burn with the mRNA expression level of Beclin-1 reaching the peak around the 5th day after burn and then beginning to gradually decline, and that of c-Myc was the highest around the 7th day after burn.