| Background and ObjectiveImpaired wound healing,one of the most challenging complications in diabetes,remain a major public health issue in China and western countries,despite the emergence of many new therapies over the past decade.Among the many influencing factors,Angiogenesis disorders is one of the important reasons that cause diabetic wounds to be difficult to heal.This may be related to the long-term chronic hyperglycemia accelerates the senescence of endothelial cells,resulting in impaired endothelial function.It has been proved that some miRNA can participate in the regulation of endothelial cell angiogenesis,and mir-30 family is rich in endothelial cells,which can promote the angiogenesis of HUVECs.Moreover,miR-30 a can regulate the senescence of BJ primary human fibroblast.But whether it is also involved in the regulation of endothelial cell senescence is unclear.A study has found that some members of miR-30 family was down-regulated to varying degrees in wound tissue of STZ-induced diabetic rats comparedwith non-diabetic rats through gene chip testing,but the role and mechanism of miR-30 a in diabetic wound healing are still unclear.Therefore,in this study,C57 BL / 6 mice and HMEC-1 cells were selected as the research objects,and miR-30 a was used as the entry point to explore whether miR-30 a can affect the healing of diabetic wounds by regulating high glucose-induced endothelial cell senescence.To explore the potential mechanism of diabetic wound healing and fund a new treatment method for the wound healing in diabetes.1: Establish a full-thickness defect model of the mouse back skin,explore whether there is a difference in the expression of miR-30 a in diabetic wound tissue and non-diabetic wound tissue,and observe the effect of miR-30 a intervention on the rate of wound healing and the senescence of wound tissue.2: Stimulate with high glucose in vitro and observe the effects of high glucose on the expression of miR-30 a,senescence-related markers like p53,p21 and β-galactosidase in HMEC-1.At the same time,the changes of HMEC-1 proliferation,migration and tube formation were observed.After that,the intervention of miR-30 a was performed and observed.3: Three databases were used to predict the possible target genes regulated by miR-30a-5p,and the possible mechanisms by which miR-30a-5p regulated endothelial cell senescence under high glucose conditions were explored through literature review and immunoblotting.Methods1.In vivo:establish a mouse model of STZ-induced diabetes,and then establish a full-thickness skin wound model on the back of normal and diabetic mice.The expression of miR-30 a in the skin tissue of the wound tissue was detected by q RT-PCR.Local multipoint injection of miR-30a-5p agomir and its control agomir NC were injected subcutaneously.Observe and calculate the speed of wound closure.The wound tissue sections on the14 th day after operation were stained with β-galactosidase and CD31.2.Simulate with high glucose in vitro,randomly divide HMEC-1 into the following groups for 48 h intervention: normal glucose group(NG:5.5mmol / L D-glucose),osmotic pressure group(OSM: 5.5mmol / L D-glucose + 24.5mmol / L mannitol),high glucose group(HG: 30 mmol / L D-glucose).The miR-30 a intervention part is divided into three groups:high glucose control group(HG: 30 mmol / L D-glucose),agomir control group(HG + agomir NC: 30 mmol / L D-glucose + agomir NC),agomir intervention group(HG + agomir: 30 mmol / L D-glucose + miR-30a-5p agomir).q RT-PCR was used to detect the expression of miR-30 a.Cell proliferation,migration,tube formation and senescence of cells were observed by Ed U assay,Scratch wound assay,Transwell assay,tube formation assay and β-galactosidase staining.The expression of p53 and p21 was detected by western blot.3.Three databases: miRNAMap,target Scan7.1,and miRDB were used to predict the possible target genes of miR-30a-5p and the genesshared by the three databases were selected.After selecting the target genes shared by the three databases for literature review,three genes related to angiogenesis and cell senescence were selected to further immunoblot analysis.Results1.The model of full-thickness skin defect on the back of mice with diabetes and non-diabetes was successfully constructed.Both mir-30a-3p and mir-30a-5p were down-regulated in the wound tissues of diabetic mice compared with non-diabetic wound tissues.At the same time,in vitro studies have found that high glucose can inhibit the expression of miR-30a-5p in HMEC-1,but has no significant effect on the expression of miR-30a-3p.And the local overexpression of miR-30a-5p in the wound can inhibit the senescence of wound tissue caused by diabetes and promote the healing of diabetic mice.2.High glucose can upregulate the expression of p21 and p53 in HMEC-1,promote the senescence of endothelial cells,and reduce their proliferation,migration and angiogenic capacity.Overexpression of miR-30a-5p can down-regulate the expression of p21 in HMEC-1,significantly improve the endothelial cell senescence induced by high glucose,and reverse the inhibition of high glucose on endothelial cell proliferation,migration and angiogenesis to a certain extent But it has no significant effect on p53.3.Using miRNAMap,Target Scan7.1 and miRDB databases to predict the possible regulation of miR-30a-5p target genes,respectively numbers:miRNAMap 514,Target Scan7.1 430,miRDB 842.And among them,there are 26 genes shared by the three databases.A literature review of these 26 genes was conducted,and three genes(CHD7,PRKRIR,ASK1)that may be related to angiogenesis and cell senescence were selected for further immunoblotting analysis.The results suggest that overexpression of miR-30a-5p under high glucose conditions,can inhibit the senescence of HMEC-1 and down-regulate the expression of ASK1,but had no significant effect on CHD7 and PRKRIR.However,whether there is a direct connection between miR-30a-5p and ASK1 still needs further study.Conclusion1.Diabetes can induce senescence of skin wound tissue in mice,inhibit the formation of wound blood vessels,and delay wound healing.The mechanism may be related to the down-regulation of miR-30a-5p expression in endothelial cells caused by high glucose,thereby up-regulating the expression of ASK1 and promoting the senescence of endothelial cells.2.miR-30a-5p can reduce the expression of ASK1 in endothelial cells,inhibit high glucose induced endothelial cell senescence,improve endothelial cell function,thereby promoting the healing of skin wounds in diabetic mice.3.miR-30a-5p regulates endothelial cell senescence and its biological behavior under high glucose conditions whether to directly target ASK1 still needs further research. |
PDF Full Text Request