The Effect And Mechanism Of Insulin On Pericytes During Diabetic Wound Healing

Objection: To investage the effect and mechanism of insulin on pericytes function during diabetic wound healing Methods: The experiment were perofmed on a type 2 diabetic rats model induced by a high fat diet and intraperitoneal injection of low dose streptozotocin(STZ).Four full-thickness ischemic wounds were made on the back of the rats.Each two diagonal wounds were randomly divided into control and experiment groups.Experiment group is topically treated with neutral insulin with the dose of 0.1u/20 uLsaline,control group is treated with equal volume of normal saline.Tissues of normal,diabetic rats and wounds at day 3,day 7 and day 11 after injury were collected for immunology,immunofluorescence and transmission electron microscopy(TEM)analysis to study the vascular morphology,peiricyte quantity and apoptosis of wound blood vessels.Meanwhile,western blotting was used to detect the level of pro-apoptotic protein cleaved caspase 3 and p-Akt.Human brain vascular pericytes was cultured in normal glucose or high glucose(HG)respectively,and treated with insulin or pretreated with PI3 K inhibitor LY294002 followed with insulin treatment,the proliferation,apoptosis and expression of p-Akt of pericytes were then measured using cell counting kit and western blotting.We then utlizing a co-culture system of endothelial cells and pericytes,studied the effect of pericytes cultured in different conditions on the tube formation of endothelial cells.Results: 1.Pericyte coverage of diabetic rats skin vessels are reduced and apoptosis of PCs was increased.Compared with the control group,pericytes number of insulin group increased with reduced apoptosis,and tissues of insulin group have a higher expression of p-Akt.2.Cell viability of pericytes in HG group decreased and percentage of apoptotic cells increased,with an increasing level of Bax.Insulin can reverse the effect of HG.The above effect of insulin can be suppressed by PI3 K inhibitor.3.Capillary morphogenesis of ECs was significantly improved when cocultured with PCs.ECs mixed with pericytes cultured under high glucose conditions have poorer tube formation than in normal glucose.Insulin can reverse the effects of HG on pericytes and improve the tube formation of ECs while this can be inhibited by LY294002.Conclusion: 1.Diabetes induces apoptosis of pericytes.Insulin inhibits pericyte loss and promote angiogenesis and vascular maturation.2.Insulin inhibits the apoptois of pericyte induced by HG culture through activating PI3K-Akt signal pathway.3.PCs improve the tube formation of ECs,HG induces the impairment of PCs then suppress the tube formation of ECs.Insulin improves the functions of PCs and accelerates the tube formation of ECs by activating PI3K-Akt signal pathway.