Silencing Of Topical Proline Hydroxylase Domain 2 Promotes The Healing Of Rat Diabetic Wounds By Phosphorylating AMPK

Objecive: Diabetes imposes a significant financial burden on individuals and families as well as on the national health care system.Leg ulcers occur in84% of all amputations in diabetic patients and are the leading cause of hospitalization in diabetic patients.And for diabetic ulcers impaired hypoxic response is one of the keys to their delayed healing.Prolyl hydroxylase structural domain 2(PHD2)is now widely recognized as a fine oxygen receptor,a central regulator of cellular oxygen homeostasis and a major inducer of the adaptive response to hypoxia.It has been shown that PHD2 does not only play a role as a "HIF regulator" in the oxygen-sensing signaling cascade,but also includes a number of other hypoxic protective responses that are not dependent on HIF.More critically,activation of the PHD2 signaling pathway regulates various cellular responses to hypoxic and pre-adaptive stimuli.Under hypoxic conditions,cell survival depends on the ability to efficiently maintain intracellular ATP levels in the presence of restricted mitochondrial oxidative metabolism and the absence of bioenergetic substrates.Thus,cellular energy status is a determining factor for cell survival,and maintaining cellular metabolic homeostasis during hypoxia is particularly important to promote cellular bioactivity.Adenylate-activated protein kinase(AMPK)is a major regulator of metabolic homeostasis and a cellular energy receptor.AMPK is activated under conditions of increased AMP/ATP values,including glucose deprivation,muscle contraction and hypoxia,and plays an extremely critical role in maintaining cellular energy homeostasis as well as adaptive responses.after its activation,it can play a critical role in maintaining cellular energy homeostasis by directly phosphorylating downstream proteins or After its activation,it can reduce further ATP depletion and increase cellular tolerance to hypoxia by directly phosphorylating downstream proteins or indirectly affecting the expression of some genes.Combined with the role of PHD2 in regulating the cellular response to hypoxic stimuli in hypoxia,we hypothesized that local silencing of PHD2 could promote wound healing in diabetic rats by activating the AMPK pathway.Methods.1、PHD2-shrna was lentivirally encapsulated.2、Rat dermal fibroblasts(RDFs)and rat aortic endothelial cells(RAECs)were infected with viral solution,and the success of infection was judged by fluorescence observation.3、The silencing effect of PHD2 and the AMPK phosphorylation level were judged by west-blot,PCR and other techniques.4、Detect the change of proliferation ability of the above cells by CCK8.5、Scratch assay and transwell technique to determine the migration ability of the above cells.6、The changes of cellular ATP production rate and other abilities were investigated by Real-Time ATP Rate Assay.7 、 After inhibiting cellular AMPK phosphorylation with8 u MDorsomorphin,steps 4,5 and 6 were repeated.8、20 male SD rats,randomly divided into two groups: including treatment group(sh-PHD2)(with diabetes,trauma injection of sh-PHD2 virus solution)and control group(sh-Control)(with diabetes,trauma injection of sh-Control virus solution),intraperitoneal injection of 60mg/kg streptozotocin(STZ)for type I diabetes modeling9、After successful diabetic modeling,the prototype whole skin of 2 cm in diameter was excised from the left dorsum of SD rats after anesthesia with chloral hydrate and skin preparation disinfection,and the corresponding viral solution was injected on the wound surface.10 、 Wound healing was recorded regularly and the healing rate was calculated.11 、 Wound tissue was taken at 14 days and the efficiency of PHD2 silencing and the expression of growth factors were examined by molecular biology methods.12、Histological examination was performed to assess CD31 expression and thus determine its angiogenesis.Results.1 、 PHD2 expression was reduced in RAECs after Sh-PHD2 lentivirus infection.2、The relative density of PHD2 in the sh-PHD2 group was significantly lower than that in the sh-Control group,and there was no significant change in PHD2 expression after the addition of Dorsomorphin(p<0.05),and the differences in the rest of the groups were not statistically significant.the differences in the relative density of AMPK were not statistically significant in each group,and the relative density of phoso-AMPK(p-AMPK)was significantly higher in the sh-PHD2 group(p<0.05).PHD2 group was significantly higher(p<0.05),and the differences in the remaining groups were not statistically significant(Fig.1B).3、 PHD2 silencing enhanced the proliferative capacity of R-DBF and RAECs,and the effect would disappear after AMPK phosphorylation was inhibited.4,、PHD2 silencing enhances the migration ability of R-DBF and RAECs,and this effect disappears after AMPK phosphorylation is inhibited.5、PHD2 silencing enhanced the real-time ATP rate of RAECs and RDFs,and the effect disappeared after AMPK phosphorylation was inhibited.6、The relative density of PHD2 was significantly reduced in both treatment groups on day 14(P<0.05).In contrast,the relative densities of VEGF and FGF-2 were both significantly increased(P<0.05)7、Local PHD2 silencing can promote wound repair in diabetic rats.8、Local PHD2 silencing can promote trabecular angiogenesis.Conclusion:Local proline hydroxylase structural domain 2 silencing enhances trauma healing in diabetic SD rats by activating AMPK phosphorylation thereby enhancing angiogenesis and hypoxic response in rats.This article demonstrates that the PHD2-AMPK pathway,through the exploration of RDFs and RAECs,enhances cellular metabolism thereby contributing to wound repair.