| ObjectiveDiabetes Mellitus(DM)is a metabolic disease with a high incidence worldwide and characterized by chronic hyperglycemia due to defective insulin secretion or insulin dysfunction.Diabetic foot is one of the most serious chronic complications of diabetes and an important factor of disability and death in diabetic patients.Diabetic foot has drawn great attention from the medical community and the society because of its characteristics of unhealed wound,high disability rate and high treatment cost.It has become an important research topic to seek effective methods and drugs to treat diabetic foot wound healing.Neferine(Nef)is a dibenzyl hydroquinone alkaloid extracted from the green germ of lotus seed.Nef has many pharmacological effects,such as anti-tumor,anti-depression,anti-oxidation and anti-inflammation.Previous studies have shown that Nef play important roles in the treatment of diabetes.However,whether Nef can be used in the treatment of diabetic foot remains unknown.Recent studies have shown that Long non-coding RNA(LncRNA)is involved in the wound healing process of diabetes.In addition,many drugs can exert their pharmacological effects by changing the expression of LncRNA.However,whether Nef regulates wound healing by changing LncRNA remains to be studied.The study was aimed to prove whether Nef improved wound healing in diabetic rats.Firstly,the animal model of diabetic wound healing was established to observe whether Nef treatment could improve wound tissue healing indexes,reduce inflammation and peroxidation,and increase the expression of healing related proteins.In vitro high-glucose(HG)induced fibroblast model was used to investigate the effects of Nef on fibroblasts proliferation,migration and the expression of healing related proteins.Furthermore,the study was aimed to explore the possible mechanism of Nef promoting wound healing in diabetes.Firstly,the expression of long-chain intergene non-protein coding RNA 1118(LINC01118)was detected in Nef and HG induced fibroblasts.Second,the study clarified the effect of knockdown LINC01118 on the fibroblast proliferation,migration and the expression of healing related proteins.Then the study revealed the effect of Nef on the expression of LINC01118,nuclear transcription factor SP1 and the miR--766-3p/RIPK1 axis.Methods1.Study on the effect of Nef improving wound healing in diabetic ratsThe wound healing model of diabetic rats was established to explore the effect of Nef.The wound area of each group was measured and recorded on day 1,7 and 14 after modeling,and the wound healing ratio was calculated.Blood glucose,lipid and insulin levels were measured on day 14.In addition,the levels of oxidative stress(MDA and SOD)and inflammatory cytokines(TNF-α,IL-6 and IL-1β)were measured in the wound tissues.Moreover,the content of Collagen was detected.The protein expression levels of VEGF,Collage-1 and α-SMA in the wound tissues were detected by western blot.The wound healing was observed by HE staining.HG was used to establish in vitro cultured rat fibroblasts.MTT,flow cytometry,wound healing,ELISA,RT-qPCR,western blot,and immunofluorescence assays were used to detect the effect of Nef on fibroblasts proliferation,apoptosis,migration,inflammatory response and the levels of VEGF,Collage-1 and α-SMA.2.Study on the mechanism of Nef promoting wound healing in diabetic rats.The expression of LncRNA in serum and wound tissue of Nef treated diabetic rats was determined by RT-qPCR.The expression of LINC01118 in fibroblasts treated with Nef and induced by HG was detected by RT-qPCR.MTT,flow cytometry,wound healing,ELISA,RT-qPCR,western blot,and immunofluorescence assays were used to detect the effect of knockdown LINC01118 on fibroblasts proliferation,apoptosis,migration,inflammatory response and the protein levels of VEGF,Collage-1 and α-SMA.The potential downstream miRNAs of LINC01118 and the potential downstream target gene of miR-766-3p were screened through bioinformatics analysis.The expression of miRNAs,target genes and transcription factor SP1 in HG induced fibroblasts was detected by RT-qPCR.The relationship between LINC01118 and miR-766-3p,miR-766-3p and RIPK1,LINC01118 and SP1 were verified by Dual luciferase reporter assay.RT-qPCR was used to detect the expression of LINC01118,miR-766-3p,RIPK1 and SP1 in the wound tissue and serum of Nef treated diabetic rats,as well as in fibroblasts induced by HG.The effect of LINC01118/miR-766-3p/RIPK1 axis on diabetic wound healing was evaluated by a functional compensatory experiment through joint knockdown of LINC01118,RIPK1 and miR-766-3p inhibitor.Results1.Nef promotes wound healing in diabetic rats.Animal model studies showed that Nef accelerated the wound healing of diabetic rats,inhibited lipid peroxidation and inflammatory reaction,and increased the content of Collagen and the expression of VEGF,Collage-1 and α-SMA.The results of cell experiments showed that Nef significantly inhibited fibroblasts apoptosis,promoted cell viability and migration,inhibited the release of inflammatory factors,and increased the expression of VEGF,Collagen-1 andα-SMA.2.Mechanism of Nef promoting wound healing in diabetic rats.The results indicated that Nef could significantly inhibit the expression of LINC01118 in diabetic animal wound tissue and HG-induced fibroblasts.Further experiments suggested that knockdown of LINC01118 reduced fibroblast apoptosis,promoted cell viability and migration,inhibited the release of inflammatory factors,and increased the expression of VEGF,Collagen-1 and α-SMA.In addition,Nef inhibited transcription factor SP1 to down-regulate the expression of LINC01118,and LINC01118 targeted miR-766-3p to regulate the expression of RIPK1.LINC01118 inhibited diabetic wound healing through miR-766-3p/RIPK1 axis.ConclusionNef can significantly promoted diabetic rats wound healing,and promoted fibroblasts viability and migration,inhibited the release of inflammatory factors,and increased the expression of VEGF,Collagen-1 and α-SMA.Nef may exert its pharmacological effect by inhibiting the expression of transcription factor SP1,downregulating LINC01118,and regulating the effect of miR-766-3p/RIPK1 axis. |
PDF Full Text Request