Effect Of Exogenous High Mobility Group Box 1 On Angiogenesis In Early Stage Of Burn

Objective: To investigate the effect of exogenous high mobility group box 1 protein(HMGB1)on angiogenesis in the ischemic region of scalded skin on the back of rats.Methods: Thirty-six male SD rats were randomly divided into two groups,the scald group and the HMGB1 group,with18 rats in each group.All rats were anesthetized with intraperitoneal injection of 20 g/L pentobarbital 3 mg/kg,and 8% sodium sulfide aqueous solution was used for back anesthesia for 24 hours,and then anesthetized again with the same method as before.The customized copper scalding model was heated at100° C.in boiling water.3-After 5 minutes,it was placed on the back of the rat's back and completely fitted with the back skin tissue of the rat.After waiting for 20 s,it was removed to prepare four 10*20mm III° scald areas and three 5*20mm ischemic areas.Comb-burn wound model.Immediately after scald,rats in the HMGB1 group were injected with 400 ng of HMGB1 in 0.1ml phosphate buffer(PBS)subcutaneously into the ischemic region of the wound.The same part of the wound was injected subcutaneously with the same amount of PBS.After treatment,the rats of the two groups were harvested at 24,48,and 72 h(6 rats in each group at each time point)and stored in neutral formaldehyde solution and RNA at room temperature.Hematoxylin-eosin staining(HE staining)method was used to test the changes of histopathological changes in ischemic area to verify the successful preparation of burn wound ischemic area model ? The expression of vascular endothelial growth factor(VEGF)on the 24 th,48th and 72 th hour after ischemia was detected by Biotin-Streptavidin-Peroxidase(SP)Immunohistochemistry.And 48 th,72th hour platelet-endothelial cell adhesion molecule(CD31)protein expression,compared the two groups of ischemic tissue 48 th,72h microvessel density;real-time fluorescent quantitative PCR was used to compare the two groups of ischemia The expression of VEGF and CD31 mRNA in 24 h,48 h and 72 h in regional tissues.The obtained data were analyzed by factorial analysis of variance,between groups by t-test and Bonferroni correction,P <0.05 considered statistically significant.Results(1)The results of HE staining showed that the epidermis in the ischemic region of the scald wounds in rats were intact,the structure of dermis was partially disordered,the structure of the skin appendages was incomplete,and the model of ischemic area of scald wounds was successfully established.(2)The results of immunohistochemical staining showed that,The content of VEGF protein in HMGB1 group(0.100 ±0.007,0.318 ± 0.034 and 0.572 ± 0.067,respectively)at 24 h,48h and 72 h after SCI in scalp was higher than that of scald group(0.053 ± 0.001,0.122 ± 0.008,0.247 ± 0.080 respectively)(t = 4.437-7.496,P <0.05 or P <0.01).The levels of CD31 protein in HMGB1 group at 48 and 72 h were 0.039 ± 0.008 and 0.058 ±0.001,respectively,(0.013 ± 0.005 and 0.030 ± 0.004,respectively)(t = 10.257 and 15.055 respectively,P <0.01).(3)The results of microvessel density showed that the number of microvessels in HMGB1 group at 48 and 72 h after scalding were 2.333 ± 0.408,5.000 ± 0.408)were more than those in the scald group(1.667 ± 0.408,2.333 ± 0.408,respectively)(t = 3.536,4,P <0.05respectively);(4)Real-In the back of the rat skin tissue The expression of VEGF mRNA in HMGB1 group at the 24 th,48th,and 72 th hour after injury was 4.38 ± 1.12,2.37 ± 0.49 and 2.37 ± 0.56,respectively,which were significantly higher than those in the scald group(1.08 ± 0.54 and 1.04 ±0.35)(t = 3.356 ~ 4.406,P <0.05).The expression of CD31 mRNA in the ischemic area of HMGB1 group at 24 and 48 h were 1.01 ± 0.13 and 1.01± 0.21,respectively(2.13 ± 0.53,2.92 ± 1.08,respectively)(t = 3.466 and 4.113 respectively,P <0.05).The expression of CD31 mRNA in HMGB1 group at 72h(1.24 ± 0.98)was significantly higher than that in simple scald group 1.23 ±0.26),the difference was not statistically significant(t value was 0.01,P value>0.05).Conclusions 1,exogenous HMGB1 in scalded wounds in rats early Blood region has the role of promoting neovascularization;2,exogenous HMGB1 promote angiogenesis may depend on the role of promoting VEGF and CD31 expression.