Objective: To explore the regulatory mechanism of
E2F1 transcription factor on M2 macrophages in full-thickness skin defect
wounds of mice. Methods: E2F1 gene knockout heterozygotes C57BL/6 mice and wild-type C57BL/6 mice were introduced and self-reproduced. Two weeks after birth, E2F1 gene knockout homozygotes mice and wild-type mice were identified by polymerase chain reaction (PCR). Twelve identified 6-8 weeks old male E2F1 gene knockout homozygotes C57BL/6 mice and wild-type C57BL/6 mice were selected respectively according to the random number table and set as E2F1 gene knockout group and wild-type group. A full-thickness skin defect
wound was made on the back of each mouse. On post injury day (PID) 2 and 7, 6 mice in each group were selected according to the random number table and sacrificed, and the
wound tissue was excised. The expression of CD68 and CD206 double positive M2 macrophages was observed by immunofluorescence method, and the percentage of CD206 positive cells was calculated. The
protein expression of CD206 was detected by Western blotting. The
mRNA expression of
arginase 1 was detected by real-time fluorescent quantitative reverse transcription PCR (RT-PCR).
Wound tissue specimens of the two groups on PID 7 were obtained, and the
protein and
mRNA expressions of
peroxisome proliferator-activated receptor gamma (
PPAR-γ) were detected by Western blotting and real-time fluorescent quantitative RT-PCR respectively. The above-mentioned experiments were repeated four times. Three specimens of
wound tissue of mice in wild-type group on PID 7 were obtained to detect the relationship between E2F1 and
PPAR-γ by co-immunoprecipitation and Western blotting, and this experiment was repeated two times. Data were processed with unpaired t test. Results: The size of PCR products of E2F1 gene knockout homozygotes C57BL/6 mice and wild-type C57BL/6 mice were 227 and 172 bp respectively, which were the same as those of the designed
DNA fragments. On PID 2 and 7, the number of CD68 and CD206 double positive M2 macrophages in the
wound tissue of mice in E2F1 gene knockout group was more than that of wild-type group, and the percentages of CD206 positive cells in the
wound tissue of mice in E2F1 gene knockout group were (0.234±0.032)% and (0.584±0.023)% respectively, which were significantly higher than (0.129±0.017)% and (0.282±0.071)% of wild-type group (t=3.29, 3.54, P<0.05). On PID 2 and 7, the
protein expression of CD206 in the
wound tissue of mice in E2F1 gene knockout group were 1.00±0.23 and 1.63±0.26 respectively, which were significantly higher than 0.43±0.06 and 0.97±0.08 of wild-type group (t=2.41, 2.45, P<0.05). On PID 2 and 7, the
mRNA expressions of
arginase 1 in the
wound tissue of mice in E2F1 gene knockout group were 0.482±0.105 and 0.195±0.031 respectively, which were significantly higher than 0.163±0.026 and 0.108±0.017 of wild-type group (t=3.04, 2.86, P<0.05). On PID 7, the
protein and
mRNA expressions of
PPAR-γ in the
wound tissue of mice in E2F1 gene knockout group were 0.61±0.12 and 0.51±0.13 respectively, which were significantly higher than 0.20±0.04 and 0.20±0.04 of wild-type group (t=3.36, 2.86, P<0.05). On PID 7, detection of the
wound tissue of mice in wild-type group showed that
PPAR-γ had unidirectional effect on E2F1. Conclusions:
E2F1 transcription factor affects the polarization of M2 macrophages by inhibiting the expression of
PPAR-γ, thereby inhibiting the healing process of full-thickness skin defect
wounds in mice.