Study On The Effect And Mechanism Of Promoting Skin Wound Healing By Intermittent Fasting

The treatment of skin trauma in diabetes and large area burn is still a difficult problem in clinic.Diabetic skin wound healing cycle is long,and easy to be infected;Burn wounds heal by osmosis,which takes longer than normal wounds.Intermittent fasting has been shown to improve health and enhance cellular protection.Objective:1.To explore the effect of intermittent fasting on skin healing of diabetic mice and scald mice..2.To explore the effects of intermittent fasting on the activity,proliferation,migration and pro-angiogenesis of skin related cells in vitro.3.To screen target genes of skin-associated cell activity by intermittent fasting.4.To verify that vasoactive factors can promote skin healing through intermittent fasting.Methods:1.C57BL/6 mice were used as research objects to explore the effects of fasting on wound healing in diabetic skin trauma model mice and burn model mice.Two kinds of skin lesions model mice were divided into three groups: control group,fasting group 1 and fasting group 2.(1)Control group: Never fasting before and after Skin lesions modeling.(2)Fasting group 1: 24 hours fasting on the seventh day before defect modeling.Normal feeding for 72 hours and then 24 hours fasting again.followed by normal feeding for 48 hours.Then the skin model lesions were maked.No longer starve after skin lesions are molded.(3)Fasting group 2: Normal feed before skin lesion modeling,the day of skin lesion modeling was recorded as day 0.After successful modeling,the conventional feed for 48 hours.and then fasting 24 hours.The conventional feed 72 hours,then fasting again 24 hours.Normal feed until the 12 th day.Three groups at the same time to build wound and then at the day of0,4,6,8,12 take photos to observe,statistic and analysis of the rate of wound healing.Using the histological analysis,immune dyeing,photo counting method to calculate and statistic wound re-epithelial change rate,scar formation rate,collagen deposition rate,skin cells around the increment rate,the number of regenerated vessels of skin at the end of animal experiment.Then compare with the difference among three groups.2.Human microvascular endothelial cells(HMECs)were taken as the research object,and the same cell line was proliferated to a enough number,and the cells were subcultured at 1 : 4,and grouped as follows:The first group was cultured in the medium containing 10% fetal bovine serum at conventional concentration for 24 hours(N-24h).The second group: 0 serum medium for fasting culture for 24 hours(F-24h);The third group: 0 serum medium for 24 hours and then cultured with 10% fetal bovine serum medium(F-24h+N-24h)for 24 hours.The fourth group: medium containing 10% fetal bovine serum at conventional concentration 48 hours(N-48h).The first and second groups 24 hours later,and the third and fourth groups 48 hours later compared with the activity,the proliferation,the migration and angiogenesis of human microvascular endothelial cells.3.Cultivate HMECs,when the cell density reaches about 90%,1 :4 vials of cells were cultured at different concentrations and at different times to obtain(N-24 h,F-24 h,F-24h+N-24 h,N-48h)4 vials of cells.Reverse transcriptional sequencing was performed to detect the comparison of the m RNA content of vasculogenic factors in each group of cells,and WB method was used to verify the performance of SMOC1 and SCG2 in the hypothesized cells.4.Immunofluorescence staining was used to detect target gene content in skin wounds of diabetic mice and burned mice.After silencing the expression of target gene in HMECs with si RNA transfection kit,the activity,proliferation,migration and vasogenic ability of HMECs in serum after fasting were observed in four groups of HMECs cells(N-24 h,F-24 h,F-24h+N-24 h,N-48h).Results:1.Either before or after wound injury,fasting can accelerate the wound healing of diabetic mice and burned mice.Compared with the control group,the mice in the fasting group had better epidermal and dermal regeneration,less scarring,and higher levels of angiogenesis.The cuticle and dermis of the fasting group 1 were faster than those of the fasting group 2,the wound healed better,the distance between scars was shorter,and there was more subcutaneous angiogenesis around the wound.2.In vitro,cell experiments showed that serum starvation alone did not increase the activity,proliferation and migration of human microvascular endothelial cells,and the ability to form vascular like,but decreased.However,culture with normal serum concentration after cell serum feeding not only activated endothelial cells,but also further enhanced the proliferation,migration and vasculogenic ability of endothelial cells.3.Sequencing results: Pairs were compared according to the time sequence of HMECs cell intervention culture.First,the expression of1437 genes was up-regulated and 853 genes were down-regulated in the group(N-24h)and the group(F-24h).Second,the expression of 318 genes increased and 416 genes decreased in the(F-24h)and(F-24h+N-24h)groups.Finally,76 genes were up-regulated and 131 genes downregulated in the(F-24h+N-24h)group and the(N-48h)group.SMOC1 and SCG2 had the highest and second highest up-regulated expression levels after 24 h of fasting.The protein levels of SMOC1 and SCG2 in HMECs were not significantly affected or even decreased by 24 h of fasting,while the expressions of these two proteins were significantly increased by refeeding after fasting.4.Immunofluorescence staining confirmed that m RNA expressions of SMOC1 and SCG2 were increased in diabetic skin lesions and burn wounds after fasting treatment.When SMOC1 or SCG2 expression was down-regulated,the angiogenic effect of HMECs induced by fasting and refeeding was significantly reduced.Conclusions:SMOC1 and SCG2 can be activated by fasting,and the combination of fasting and refeeding,can enhance the proliferation,migration and angiogenesis of activated SMOC1 and SCG2 endothelial cells,promoting the formation of neovascularization and accelerating wound healing.