The Mechanism And Clinical Study Of Cinnamaldehyde And DPP-4i In The Treatment Of Diabetic Ulcer

Background Diabetic ulcers(DUs)are one of the most common complications of diabetes mellitus(DM),nearly 15% of diabetic patients have DUs,14%-20% of diabetic patients sustained and developed to impaired wound that were finally resulted in nontraumatic amputation,seriously affected the quality of patients' lives.Although the standardized therapies for DUs focused on accelerating wound healing,they are usually limited to debridement and infection prevention,while lack of targeting molecular mechanisms.Therefore,searching for safe and effective targeted therapies is an urgent need for current DUs treatment.Recently,some studies have demonstrated that the Kelch-like ECH-associated protein 1,(Keap1)-nuclear factor erythroid 2-related factor(Nrf2)pathway is one of the most important endogenous antioxidant defensive system.Impairment of Keap1/ Nrf2 pathway was associated with many diseases.Previously,our study group have found that the wound edges of DUs patients were under serious oxidative stress,and that the Nrf2 pathway is compensatorily activated;intraperitoneal injection of Nrf2 agonist sulforaphane(SF)and cinnamaldehyde(CA)could promote wound healing of streptozotocin(STZ)induced diabetic mice.Moreover,some other studies have confirmed that topical addministration of Keap1 si RNA(si Keap1)in db/db diabetic mice,which could upregulate the expression of Nrf2,enhance epithelial regeneration capacity and accelerated the closure rate of diabetic wounds.In conclusion,modulation of the Nrf2 pathway in the diabetic wound could promote wound healing.In view of the fact that CA specifically activates the Nrf2 pathway,we propose that topical administration of CA can promote the healing of diabetic wound.Recent studies have found that CA is similar to SF which could dissolve in polyethylene glycol(PEG)series 200,300,400 and 600 and stably made into gel,ointment or cream.Previous studies have shown that PEG is a non-toxic,inert substance with no irritation,good water solubility and moisture retention.Therefore,we used PEG 400 mixed with CA(PEG 400 CA)to observe the effect of topical administration of CA on DUs and to further explore its possible mechanism.Research purposes 1.To observe the effect of topical administration of CA on diabetic wound healing;2.To investigate whether the effect of CA on diabetic wound healing is depend on the Nrf2 pathway.Materials and Methods Experimental materials: Eight-week old Nrf2+/+ and Nrf2-/-SKH-1 male hairless mice,which were gifted by the American professor Donna.D.Zhang of University of Arizona College of pharmacy;streptozotocin(STZ)and cinnamaldehyde(CA)were purchased from Sigma company(St.Louis MO).Experimental groups: Nrf2+/+ mice of vehicle group(Nrf2+/+ Veh group);Nrf2+/+ mice of CA treatment group(Nrf2+/+ CA group);Nrf2-/-mice of vehicle group(Nrf2-/-Veh group);the Nrf2-/-mice of CA treatment group(Nrf2-/-CA group),4 mice in each group.Experimental methods: 1.To monitor the wound healing rate at indicated time and evaluate the effect of topical addministration of CA on wound healing in Nrf2+/+ and Nrf2-/-diabetic mice;2.To analyze histological changes of the wound skin in each group by HE staining;3.To analyze the effects of topical addiministration of CA on the expression of Nrf2 and downstream target protein HO-1,and 8-oxo-d G which is one of the oxidative damanage related markers at the wound site.Research result 1.Topical application of CA significantly promoted diabetic wound healing of Nrf2+/+ mice We observed the wound healing process at indicated time point of Nrf2+/+ mice,compared with Nrf2+/+ Veh group,the wound healing rate of Nrf2+/+ CA group was significantly increased from 9th day post-surgery(P < 0.05);the newly epithelium completely formed with histological integrity of the wound of Nrf2+/+ CA group at 14 th day post-surgery,while the wounds of Nrf2+/+ Veh group were still coverd with loose granulation tissue and non peeling callus,the epithelium did not completely cover the wound.It showed that topical application of CA significantly improved wound healing in Nrf2+/+ diabetic mice.2.Topical application of CA did not affect the diabetic wound healing of Nrf2-/-mice To further observed the effect of CA on diabetic wound healing in Nrf2-/-mice.The results showed that there was no difference in the wound healing rates between the Nrf2-/-CA group and the Nrf2-/-Veh group.At the 14 th day post-surgery,there were no new epithelial covered by the histology of the wound skin,but remained the newly granulation tissue.These results suggest that topical application of CA has no significant effect on diabetic wound healing of Nrf2-/-mice.3.Topical application of CA alleviated oxidative stress damage of diabetic wound skin by activation of Nrf2 pathway Compared with the Nrf2+/+ Veh group and the Nrf2+/+ CA group,topical application of CA significantly enhanced the expression of Nrf2 and downstream target protein HO-1 at the wound sites,as well as decreased the level of 8-oxo-d G of the wound skin.It is suggested that during the wound healing process of Nrf2+/+ mice,topical application of CA activates the Nrf2 pathway and alleviates oxidative stress of the diabetic wound skin.4.Topical application of CA did not affect the oxidative stress of the diabetic wound skin in Nrf2-/-mice In Nrf2-/-Veh group and Nrf2-/-CA group,there were no expression of Nrf2,and topical application of CA did not alter the expression of HO-1 and 8-oxo-d G in the diabetic wound skin of Nrf2-/-mice.It was suggested topical administration of CA did not affect the Nrf2 pathway and oxidative stress in the diabetic wound healing of Nrf2-/-mice.In conclusion,this study found that topical administration CA promote diabetic wound healing,alleviated the oxidative stress damage at wound site depends on the Nrf2 pathway;the effect of CA PEG400 hydrogel were stable,reliable and efficacious,excluding other potential effects of CA systemic applicated,and provides the experimental basis for new therapeutic approaches for the treatment of clinical application of CA on DUs.Background There are a number of clinical anti-hyperglycemic agents for diabetes,as various complications occur frequently in diabetes,some of these anti-hyperglycemic agents may have additional effects to these complications,therefore how to choose optimal personalized hypoglycemic agents is not completely clear.For instance,liraglutide has been shown to bring cardiovascular benefits,dapagliflozin has anti-hypertensive effects,and metformin can reduce maternal weight gain without lowering infant birth weight.On the other hand,some hypoglycemic agents also cause negative side effects,such as the increased risk of heart failure hospitalization that associated with saxagliptin(Sax),elevated renal failure incidence with dapagliflozin.These results have begun to influence the selection of hypoglycemic agents,therefore an improved understanding of the additional effects of hypoglycemic agents is required to optimize individualized clinical treatment.Dipeptidyl peptidase-4 inhibitors(DPP-4i)are classic hypoglycemic drugs used worldwide and are recommended as the first-line treatment for type 2 diabetic patients(T2DM)by the American Association of Clinical Endocrinologists(AACE),due to their efficacy in glycemic control and minimum risk of hypoglycemia.Traditionally,DPP-4i is believed to reduce levels of hemoglobin A1c(Hb A1c)by prolonging the half-life of endogenous glucagon-like peptide-1(GLP-1)and glucose-dependent insulinotropic polypeptide.However,in addition to the hypoglycemic effect,several studies have indicated that DPP-4i may have additional effects on the kidney and cardiovascular system.Given the pleiotropic effects of DPP-4i,further investigations are needed to identified whether there are other additional roles during DPP-4i administration.Recent studies showed that DPP-4i was demonstrated to activate the nuclear factor E2 related factor(nuclear factor erythroid 2-related factor,Nrf2)pathway,and Nrf2 pathwayis relevant to diabetic wound healing,suggesting that DPP-4i may affect diabetic wound healing.Research purposes 1.To observe the effect of DPP-4i on diabetic wound healing;2.To explore the potential mechanism of DPP-4i on diabetic wound healing;Reseach Methods 1.To observe the effect of DPP4 i on diabetic wound healing First,the cultured human skin keratinocytes(Ha Ca T)were used to establish in vitro wound healing model to observe the effect of five common clinical DPP-4i(Saxagliptin,Sitagliptin,Alogliptin,Vilgliptin,Linagliptin)on diabetic wound healing;secondly,the mice were treated with DPP-4i(Sax)by gavage after successfully established STZ-induced diabetic mice,one week later,two 6mm diameter full-thickness skin excision wounds were conducted;then the effect of DPP-4i on diabetic wound healing were analyzed by monitoring the wound healing rate and drawing wound healing curve.In addition,HE staining was used to analyze histological differences in wound healing at 14 th day post-surgery.2.To observe whether the effect of DPP-4i on wound healing depends on the Nrf2 pathway;First,the Ha Ca T cells were cultured,and the keratinocytes were transfected with Nrf2 si RNA to block Nrf2 pathway,then the effect of DPP-4i on wound healing were compared between the control group and the Nrf2 knockdown group;secondly,the effect of DPP-4i(Sax)on wound healing of STZ induced diabetic Nrf2-/-mice were detected.3.The effect of DPP-4i on cell function during wound healing The function alterations(such as the proliferation and migration)of Ha Ca T cells and human foreskin fibroblast(HFF)were analyzed by migration assays(PDMS or transwell assay).4.The effect of DPP-4i on the functions of diabetic wound skin To compare the epithelial-mesenchymal transition(EMT)changes and SDF-1? expression by immunofluorescence and immunohistochemistry at the wound site of diabetic mice and diabetic patients.5.The expression(COL1A1,COL3A1 and FN1)and deposition of collagen at the wound site were analyzed by q RT-PCR and Masson's staining of diabetic mice.6.To observe the effect of DPP-4i(saxagliptin)on wound healing of diabetic patients The randomized controlled clinical trials(NCT02742233)comprised of 67 T2 DM with ulcers were conducted and the clinical efficacy of DPP-4i were evaluated by comparing the time of ulcer healing and the percentage of healing ulcer after 13 weeks treatment.Research results 1.DPP-4i promote diabetic wound healing were confirmed by wound healing assay in vitro.1.1 The in vitro wound healing model were performed,a clear reduction in wound area was observed upon treatment with a range of different DPP-4i(commercial human drugs Sax,Sit,Alo,Vil and Lin)at each of the time points indicated,which suggested that DPP-4i can promote wound healing.1.2 To confirm the positive wound-healing effects of DPP-4i treatment,we observed the wound closure rate in mice with STZ-induced diabetes.Consistent with in vitro results,Sax treatment significantly accelerated the diabetic wound closure rate over the course of the healing process;and the epithelial regeneration were accelerated under DPP-4i treatment by histological analysis at 14 th day post-surgery.2.DPP-4i promote diabetic wound healing not mainly through the NRF2 pathway;2.1 The NRFsi RNA were used to block the NRF2 pathway of Ha Ca T cells,and the in vitro wound healing model were established,which found that the saxagliptin treatment still promote wound healing even under the condition of blocking the NRF2 pathway,suggesting that saxagliptin promote diabetic wound healing not mainly through NRF2 pathway.2.2 The wound model of Nrf2-/-diabetic mice were successfully established,and we found that saxagliptin significantly promote diabetic wound healing at different time points,which confirmed that DPP-4i still can promote diabetic wound healing even under blocking the Nrf2 pathway.3 DPP-4i induce EMT and promote migration of keratinocytes 3.1 DPP-4i did not affect the proliferation of Ha Ca T cells and HFF cells,nor affect the migration of HFF cells,but significantly promoted the migration of Ha Ca T cells in vitro.3.2 Ha Ca T cells showed EMT-like morphological changes after DPP-4i treatment.To further detect the expression level of key EMT-associated proteins by Western blot,which showed that DPP-4i reduced E-cadherin expression while increasing the expression of Vimentin and SNAIL-1 which suggested that DPP-4i can induce EMT of keratinocytes.4.DPP-4i promotes SDF-1? production from fibroblasts indirectly driving keratinocytes EMT 4.1 To further investigate the effect of DPP-4i on the secretion function of HFF cells,we found that DPP-4i does not affect the expression of VEGF,TGF-?1,Cox-2 and HMGB1 in HFF cells,but significantly enhance the expression of SDF-1?.4.2 The followed experiments were found that SDF-1? induced EMT of keratinocytes in vitro,which suggested DPP-4i induces keratinocytes EMT by enhancing the expression of SDF-1? from fibroblast indirectly.5.DPP-4i enhanced the expression of SDF-1?,induced EMT and reduced scar formation at the wound site of diabetic mice.5.1 After the STZ induced diabetic mice wound model was established,we harvested the wound skin at different time points during wound healing.5.2 At the early stage of diabetic wound healing,DPP-4i induced EMT and enhanced SDF-1? expression at the wound site by immunofluorescence staining analysis.5.3 At the later stage of diabetic wound healing,DPP-4i reduced the expression of collagen(COL1A1,COL3A1 and FN1)and alleviate the the collagen deposition which suggested that DPP-4i can reduce scar formation.6.DPP-4i improves wound healing in diabetic patients A randomized controlled clinical study further confirmed the results above,the baseline of participants in both groups were similar.Compared to the placebo group,the median time of wound healing was reduced by 15 days(P<0.05)and the percent of healing wound were higher by 36.8%(P<0.05)after taking saxagliptin for 13 weeks,what's more,Kaplan-Meir survival analysis were used to evaluate the wound healing events(Log-rank=15.08,P=0.0001).Collectively,those results further demonstrate that DPP-4i significantly promoted wound healing independent of glycemic control.Conclusions This study revealed that DPP4 i improve diabetic wound healing by inducing EMT of keratinocytes directly and indirectly,and reducing scar formation,which may influence the strategy for clinical application of DPP-4i in the future.