Promotion Mechanism Of Tetrahedral Framework Nucleic Acids On The Healing Of Diabetic Skin Ulcers:an Experimental Study

BackgroundDiabetic skin ulcers are one of the most common and complex complications of diabetes.With the number of diabetes rising,its incidence in the world has increased year by year.Due to the delayed wound healing,diabetic skin ulcers are susceptible to infection and tissue necrosis.These have made diabetic skin ulcers to become a major problem in the field of wound repair.At present,it is considered that the local cellular dysfunction,insufficient vascularization,impaired inflammatory response and inadequate growth factors are the crucial factors leading to the delayed healing of diabetic wounds,which tend to develop into chronic ulcers.Although a number of treatments have achieved certain therapeutic effect,they require higher costs,longer cycles and more complex procedures.Tetrahedral framework nucleic acids(t FNAs)are novel type of DNA nanomaterials with tetrahedral space structure.Due to the excellent mechanical,chemical and biological properties,t FNAs have become a hot topic in the field of biomedicine in recent years.By regulating the biological behaviors of various cells,t FNAs play an active and effective role in tissue regeneration and repair,and can be used as an ideal material for wound treatment.In view of the factors of delayed diabetic wound healing,this study will use the advantages of t FNAs to design experiments which explore the potential possibility of t FNAs as a treatment for diabetic skin ulcers.ObjectiveThe aim of this study is to prepare t FNAs,and to explore the effects of t FNAs on diabetic skin ulcers both in vitro and in vivo.What’s more,the specific pathway and possible molecular mechanism would be further studied.Methods1.Preparation and characterization of t FNAs1.1 t FNAs were synthesized by using four specific ss DNA.1.2 The molecular weight,spatial shape,particle size and ZETA potential of t FNAs were detected by PAGE,AFM,TEM,nanoparticle size and potential analysis,respectively.2.Effect of t FNAs on some wound healing related cells in high glucose environmentThe cells were divided into blank group(normal environment),control group(high glucose environment)and experimental group(high glucose environment + t FNAs).2.1 The proliferation of HaCaT cells,HDF and HUVECs in each group was detected by CCK-8 method.2.2 The migration of Ha Ca T cells,HDF and HUVECs in each group was detected by cell scratch test.2.3 The secretions of bFGF,TGF-β1 and VEGF-A in each group were detected by ELISA.2.4 The gene expression of b FGF,TGF-β1 and VEGF-A in each group was detected by RT-q PCR.3.Effect of t FNAs on wound healing of diabetic skin ulcersAnimals were divided into control group(drug delivery: normal saline)and experimental group(drug delivery: t FNAs).3.1 The skin ulcers models of diabetic mice were made.The method of drug-delivery was injection around the wound.The wound healing rates were recorded and compared.3.2 HE and Masson staining were used to compare the quality of wound healing in each group.3.3 Immunohistochemistry and immunofluorescence staining were used to compare the angiogenesis of wounds in each group.3.4 The protein expression of bFGF,TGF-β1 and VEGF-A in each group was detected by ELISA.3.5 The protein expression of Wnt signaling pathway(β-Catenin,c-Myc and Cyclin D1)in each group was detected by WB.Results1.Preparation and characterization of t FNAst FNAs were successfully prepared.The t FNAs had a tetrahedral structure.The molecular weight was about 200 bp.The particle size was about 18 nm.ZETA potential was slightly negative.2.Effect of t FNAs on some wound healing related cells in high glucose environment2.1 The proliferation and migration of HaCaT cells and HUVECs in control group were less than those in blank group,while the proliferation of HDF was less.The proliferation and migration of Ha Ca T cells,HDF and HUVECs in experimental group were more than those in control group.2.2 The gene expression and protein secretions of b FGF,TGF-β1 and VEGF-A in control group were lower than those in blank group.The gene expression and protein secretion of b FGF,TGF-β1 and VEGF-A in experimental group were significantly higher than those in control group.3.Effect of t FNAs on wound healing of diabetic skin ulcers3.1 Compared with control group,the wound healing rate of experimental group was significantly accelerated.3.2 Compared with control group,the regenerative epidermal thickness,deposited collagen percentage and the number of new vessels in experimental group were increased.3.3 On the 14 th day after surgery,compared with normal skin,only the expression level of b FGF was slightly increased in the wounds of control group,while the expression levels of TGF-β1 and VEGF-A had no statistical significance.On the 21 st day after surgery,there were no significant differences in the expression of b FGF,TGF-β1and VEGF-A in control group.The expression levels of b FGF,TGF-β1 and VEGF-A in experimental group were significantly higher than those in control group on the 14 th and21st day after surgery.3.4 On the 14 th and 21 st day after surgery,the expression levels of β-catenin,c-Myc and Cyclin D1 in the wounds of control group were not significantly different from those of normal skin.The expression levels of β-catenin,c-Myc and Cyclin D1 in experimental group were significantly higher than those in control group.ConclusionIn the light of all the research on this subject,the conclusions are as follows:1.t FNAs were successfully prepared.The t FNAs had a tetrahedral structure.The molecular weight was about 200 bp.The particle size was about 18 nm.ZETA potential was slightly negative.Besides,the preparation process was simple and efficient.2.t FNAs could facilitate the proliferation and migration of HaCaT cells,HDF and HUVECs,and promote the gene expression and protein secretion of b FGF,TGF-β1 and VEGF-A in high glucose environment.3.t FNAs could significantly accelerate the wound healing rate of diabetic mice.It could facilitate diabetic cutaneous wound healing by promoting epithelialization,vascularization,collagen synthesis and the expression of growth factors via the Wnt signaling pathway.4.t FNAs can possibly be used as a new treatment for diabetic skin ulcers.