Targeted activation of HIF-1 gene coordinated ASCs can promote the wound healing of diabetic mice

Abstract

Objective To explore the effect of combination of HIF-1α gene transfection and Adipose stem cells (ASCs) on diabetic wound healing in mice. Methods Sixty-eight SPF C57BJ/L mice were intravenously treated with 1% streptozotocin (STZ) at a dose of 60 mg/ kg versus body weight for 3 days. Animals with fasting blood glucose concentration higher than 16.7 mmol/L were employed in the following experiment. Subsequently, on the back of mice, round-thickness skin wounds with 1 cm were made. The mice were randomly divided into empty vector group (EV), a plasmid DNA construct expressing a stabilized mutant form of HIF-1α (CA5-HIF-1α) vector transfected group (CA5), EV+ saline group, CA5+saline group, EV+ASCs group, CA5+ASCs group. On the 3rd, 10rd, 17rd, 21st days after treatment, the area of the wounds was measured. The dermal blood flow was measured by Laser Doppler perfusion imaging (LDPI). The levels of HIF-1α and VEGF mRNA were measured by quantitative Real-time PCR (qRT-PCR). The blood vessel density was detected by H&E staining. The level of VEGF protein was measured by Western blot. The statistical analysis was used with the Bonferroni or Tukey post hoc variance analysis method. Results The level of HIF-1α mRNA expression treated with 125 μg CA5-HIF-1α gene was the highest among all doses (10.40±0.22) (F = 19.48, P = 0.025) so we selected 125 μg HIF-1α gene for the following experiments. By days 3 ~ 14, the wound area in CA5 group (7 828.92±294.28, 7 285.97±118.24, 4 050.41±301.97, 1 292.35±101.14) was smaller than the EV group (9 062.00±225.75, 8 534.42±189.35, 5 634.59±198.06, 2 308.15±245.36) (F = 41.37, 32.16, 27.29, 25.16, P = 0.028, 0.034, 0.038, 0.042). Besides, in the 10th and 17th days, the blood flow of mice in CA5 group (253.06±8.34, 250.59±10.13) was significantly greater than that of the EV group (158.31±9.98, 169.73±7.28) (F = 21.53, 26.08, P = 0.038, 0.032). ASCs were shown to have promotive effects on the wound healing. Therefore, we further explored the effects of combination of HIF-1α gene and ASCs therapy on skin wound healing in diabetic mice. We found that the wound area of CA5+saline group (7 656.92±177.03, 7 163.83±128.24, 4 238.23±228.36, 1 316.52±90.75), EV+ASCs group (7 593.64±192.12, 7 233.08±146.86, 4 097.58±227.91), CA5+ASCs group (7 593.64±192.12, 7 233.08±146.86, 4 097.58±227.91) were significantly smaller than the control group (6 745.25±203.16, 6 159.35±168.72, 3 682.06±257.30) (F = 39.58, 44.09, 34.67, P = 0.031, 0.028, 0.037) on 3 ~ 14 days after treatment. Besides, CA5+ASCs group showed the smallest wound area. In addition, we also found that the blood flow of CA5+saline group (262.05±9.34, 248.45±11.13), EV+ASCs group (215.33±10.75, 185.82±10.47), CA5+ASCs group (322.54±12.27, 292.49±9.57) significantly increased on day 10 compared to the control group (161.30±5.64, 134.57±8.67) (F = 29.15, 17.38, P = 0.026, 0.034). Moreover, the blood flow in combination group increased sharpest. CA5+saline group, EV+ASCs group, CA5+ASCs group showed an obvious increase in vessel density and the combined group had the highest increase in vessel density. We also found that the expression levels of VEGF mRNA and protein in CA5+saline group, EV+ASCs group, CA5+ASCs group (2.03±0.14, 2.16±0.13, 3.41±0.18) and (1.75±0.12, 1.82±0.06, 2.96±0.14) were significantly higher than those in the control group (1.05±0.02) and (1.03±0.05) (F = 34.08, 29.53, P = 0.019, 0.021). The combined group showed the highest levels of VEGF expression. Conclusion Targeted activation of HIF-1 gene coordinated ASCs can promote the wound healing of diabetic mice, and its mechanism may be related to regulation of VEGF expression. Key words: HIF-1α; Adipose stem cells; Diabetic; Mice; Wound healing; VEGF