Modified Autologous Blood Promotes Wound Healing In Diabetic Mice By Activating HIF-1α Pathway

Objective:To observe the effect of autologous blood treated with modified preservative solution on wound healing in diabetic mice,observe the role of HIF-1alpha signaling pathway in promoting wound healing and explore its possible mechanism.Methods:Thirty healthy male Kunming mice,aged 8 weeks,weighing 26-30 g,were fed adaptively for one week,and then injected with STZ（50 mg/kg）for 5 days to establish diabetic mice model.After successful modeling,three groups were randomly divided into three groups:blood donation group（0.6 ml per mouse heart,respectively,stored in modified preservation solution and standard preservation solution for 7 days）;experimental group（n=6）:improved group（autologous blood transfused with modified preservation solution）;standard group（n=6）（autologous blood transfused with standard preservation solution）.（1）After anesthesia,the back skin of mice was disinfected.Open skin wounds with a diameter of 2 cm were made on both sides of the spine with a perforator.Autologous blood stored in different preservation solutions was transfused on the 1st,2nd and 3rd days after the establishment of wound model.The wound sites were recorded by transparent membrane tracer on the 0^th、1^th、4^th,7^thand 14^thh days after injury,and the percentage of wound healing area was calculated.（2）The whole skin of the wound and the edge was collected and stored in liquid nitrogen immediately for testing.RT-q PCR was used to detect the expression of HIF-1α,VEGF,EGF and HSP90 in wound tissue,and immunohistochemistry was used to detect the expression of HIF-1α,VEGF and EGF in skin tissue.（3）HIF-1αwas silenced in fibroblasts by siRNA and perfused with different preservation solutions.They were divided into standard group,Standard+siRNA-HIF-1αgroup,improved group and improved+siRNA-HIF-1αgroup.The contents of HIF-1a,VEGF and MMP-2 in fibroblasts were determined by Western blot,and the levels of HIF-1α,VEGF and HSP90 were determined by RT-q PCR.（4）CCK-8 method was used to detect the activity of fibroblasts and flow cytometry was used to detect the proliferation of fibroblasts.Results:（1）Compared with the standard group,the area of back skin wound in the modified group decreased significantly on the 1th、4th,7^th and 14th day after operation（P<0.05）.（2）RT-qPCR:Compared with the standard group,the levels of HIF-1a,VEGF,EGF and HSP90 in skin tissue of the improved group were significantly higher（P<0.05）.Immunohistochemistry:Compared with the standard group,the levels of HIF-1a,VEGF and EGF in the skin tissue of the improved group increased（P<0.05）.（3）After HIF-1αsilenced in fibroblasts by siRNA,Western blot:Compared with the standard group,the expression levels of HIF-1α,VEGF and MMP-2 in the standard+siRNA-HIF-1αgroup and the improved+siRNA-HIF-1αgroup decreased（P<0.05）,and the improved group increased significantly（P<0.05）.There was no significant change in the expression of VEGF and MMP-2 protein in the improved+siRNA-HIF-1αgroup（P>0.05）.RT-qPCR:Compared with the standard group,the expression levels of HIF-1α,VEGF and HSP90in the standard+siRNA-HIF-1αgroup decreased（P<0.05）,while the expression levels of HIF-1α,VEGF and HSP90 in the improved group increased（P<0.05）,but the expression levels of HIF-1αin the improved+siRNA-HIF-1αgroup decreased,while the expression levels of VEGF and HSP90 did not change significantly（P>0.05）.（4）CCK8method:Compared with the standard group,the cell survival rate,proliferating cells and transitional cells decreased in the standard+siRNA-HIF-1αgroup and the improved group,but there was no significant difference between the improved group and the modified+siRNA HIF-1αgroup（P>0.05）.Flow cytometry:Compared with the standard group,the number of G2 or M phase cells increased in the standard+siRNA-HIF-1αgroup,but there was no significant difference in the modified+siRNA-HIF-1αgroup（P>0.05）.Conclusion:Modified autologous blood treatment can effectively promote the repair of back skin wounds in mice.Its mechanism may be related to the activation of HIF-1αsignaling pathway by modified autologous blood treatment,the regulation of fibroblast activity,the improvement of cell survival rate,cell proliferation and migration,and the promotion of wound healing in diabetic mice..