Acceleration Of Cutaneous Wound Healing In Diabetic Mouse By Overexpression Of Hpgds In Adipose-derived Stem Cell Seeded In Hydrogel

BackgroundDiabetic skin ulcers(DCU)refer to lower extremity infection,ulcer formation,and destruction of deep tissues due to long-term poor glycemic control in diabetic patients,which leads to neuropathy and various peripheral vascular lesions of various degrees.DCU is one of the serious complications in patients with diabetes,leading to death and disability,which cause serious life burden to the patient.Therefore,research on the treatment of diabetic skin ulcers has important clinical application value.The current treatment methods are still mainly surgical treatments,and the existing biological and physical treatment methods have many limitations,which have failed to achieve satisfactory treatment results.Previous studies have found that Hpgds is down-regulated in the wounds of diabetic mice.Hpgds is mainly expressed in hematopoietic cell lines,such as mast cells and Th2 cells,and its main role is to catalyze the production of prostaglandin D2(PGD2)in the immune system.PGD2 regulates the inflammatory response through the activation of peroxisome proliferator-activated receptor γ(PPAR-γ),which can inhibit the infiltration of iNOS,TNF-α and IL-1β and macrophages,and can regulate apoptosis of murine T cells and B cells to reduce inflammatory response.Therefore,it is speculated that the down-regulation of Hpgds expression hinders the ’resolution of inflammatory response in diabetic ulcer tissues,and the inflammatory microenvironment disorders inhibit the repair of ulcers.Therefore,the regulation of Hpgds expression in diabetic ulcer tissue may become a new target to promote its repair.In order to improve the therapeutic effect of adipose-derived stem cells(ADSC)in diabetic ulcers,ADSC was used as a vector to over-express Hpgds,which could enhance the immune regulation function of ADSC in diabetic ulcers.Due to the tissue defect,lack of blood supply,and severe inflammatory response in chronic ulcers of diabetes,the survival rate of transplantation is low,which limits the clinical application of adipose-derived mesenchymal stem cells.The RGD peptide-modified VitroGel 3D hydrogel scaffold used in this study can protect itself from the host immune system through physical separation,thereby providing an appropriate physicochemical microenvironment for ADSCHpgds adhesion and survival,and optimizing the treatment of stem cells transplantation in diabetic wound.The combination of gene and cell therapy will improve the immune and regeneration microenvironment of diabetic wounds and accelerate the healing of chronic wounds.It provides an important theoretical basis for the improvement of skin tissue engineering seed cells,and also provides an important clinical application prospect for the wound immune regulation of chronic ulcers with diabetes.Objective1.Find out the key molecules that have changed in the tissues of diabetic wounds through bioinformatics analysis,and clarify the characteristics of the immune microenvironment of diabetic wounds;2.To explore the effect of overexpressing Hpgds on biological characteristics(stem cell markers,proliferation,migration,differentiation,and apoptosis)of hADSC;3.To explore the role of RGD peptide-modified VitroGel 3D hydrogel scaffold loaded-ADSCHpgds in the healing of diabetic mice and its mechanism.Methods and Results1.Bioinformatics analysis of diabetic wounds and its immune microenvironment characteristicsMethods:Obtain high-throughput chip data of human normal skin tissue and diabetic ulcer tissue from NCBI GEO Datasets,perform Gene Ontology(GO)analysis,protein interaction network analysis,and screen for key signal molecules;Real-time PCR was used to compare the dynamic expression of Hpgds in normal mice and diabetic mice during wound healing.Compared with PBS group and control siRNA group,Hpgds siRNA was topically used to knock down Hpgds in normal mouse wounds,in order to observe the effects of Hpgds on wound healing;Compared with normal mice and diabetic mice,tissue total RNA was extracted at Oh,8h,2d,6d,10d,and 14d,and RNA-seq and ImmuCC computational models were used to detect the activity of immune-related pathways in diabetic mice wound,and the dynamic changes inflammatory factors and immune cell components.Results:21 gens were up-regulated and 22 down-regulated in diabetic wound.The GO analysis results showed that the differential genes of diabetic ulcer tissue were mainly enriched in keratinocyte differentiation,keratinization,peptide cross-linking,epidermal development,intermediate fibrous cytoskeletal tissue,glutathione derivative biosynthesis process,fungal defense,Neutrophil infiltration,glutathione metabolism,and chemokine production;the down-regulated genes are mainly enriched in the biosynthesis of glutathione derivatives and extracellular matrix components.Analysis of protein interaction network revealed three core nodes HPGDS,S100AB and KRT16 among differentially expressed proteins.During the wound healing process,the expression of Hpgds in diabetic wounds was lower than that in normal mice.The use of Hpgds siRNA to locally reduce the expression of Hpgds in normal mouse wounds can significantly affect wound healing.It has been proved that down-regulation of Hpgds expression in diabetic wounds is a key factor hindering its healing.The activation of immune-related pathways in diabetic mice was less at 8h;at 10d and 14d,the activity of immune-related pathways in wounds of diabetic mice was more active than that of normal mice.Correspondingly,at 8h,the expression of important immune-related genes in normal mouse wounds was higher than that of diabetic mice,and at 10d and 14d,it was lower than that of diabetic mice.The major immune cell components in skin wounds of diabetic and normal mice are macrophages,monocytes,CD4T cells and CD8T cells.Within two days of wound healing,the proportion of monocytes,M1 macrophages and neutrophils in diabetic mice was lower than that in normal mice;however,in the late stage of wound healing(6-14d),the proportion of monocytes,M1 macrophages,neutrophils,CD8T cells and NK cells has always been higher than that of normal mice.2.Effects of overexpressing Hpgds on biological characteristics of h ADSCMethods:Lentivirus was used to integrate mouse gene Hpgds into human ADSCs;real-time quantitative PCR was used to detect the overexpression efficiency of hADSCHpgds;ELISA kit was used to detect the expression of PGD2 in the hADSCHpgds supernatant;flow cytometry was used to analysis hADSCHpgds surface stem cell markers(CD44,CD73,CD90,CD 105);CCK8 cell proliferation assay,scratch healing experiment and Annexin V cell apoptosis assay were used to detect proliferation,migration,and apoptotic capacity,respectively;hADSCHpgds was induced to differentiate into lipid and osteoblasts,and the results of lipid and osteogenic differentiation were observed by oil red O staining and alizarin red staining respectively on the 7th and 14th day of induction.At the same time,the total NRA of differentiated cells was extracted and the expression levels of lipid-forming marker PPAR-γand osteogenic marker RUNX2 were detected by real-time quantitative PCR.Results:The expression level of Hpgds in ADSCHpgds was significantly higher,and the content of PGD2 in the culture supernatant was also significantly increased relative to ADSC.ADSCs had no significant effect on the expression of stem cell markers(CD44,CD73,CD90,CD105)after overexpression of Hpgds.ADSCs overexpression of Hpgds had no significant effect on the proliferation,migration,differentiation and apoptosis characteristics of ADSCs.3.Application RGD-VitroGel 3D hydrogel-loaded hADSCHpgds in the wound of diabetic mice and its mechanismMethods:First,the biocompatibility of hADSCHpgds and RGD-modified VitroGel 3D hydrogel was tested;hADSCHpgds was labeled with CM-dil,and the outcome of transplanted cells in the living mice was detected using IVIS imaging system;then RGD-VitroGel 3D hydrogel-loaded hADSCHpgds and hADSCPCDH were used for the treatment of diabetic mouse wounds;immunofluorescent staining and immunohistochemical methods were used to detect the expression of PCNA,CD31,and Collagen I in the wound tissue,and the tissue was detected by quantitative PCR at the same time.The expression of VEGF and Collagen I was used to analyze the effect of hADSCHpgds on the wound healing.In order to detect the immunoregulatory effect of hADSCHpgds on diabetic wounds,immunostaining was used to detect the infiltration degree of neutrophils(Ly-6g),CD8T cells and macrophages in wound tissue,and the expression of inflammatory fa.ctors IL-1β,IL-6,TNF-α,and iNOS in tissues were detected by quantitative PCR at the same time.In vitro,the regulatory effect of hADSCHpgds conditioned medium on LPS-induced RAW264.7 macrophages was examined;the difference between comparing hADSCHpgds and hADSCPCDH is that the catalytic product of Hpgdsd is PGD2,so PGD2 was added to LPS-induced RAW264.7 macrophage culture medium to detect the change of CD206,VEGF,and the inflammatory genes IL-1β,IL-6,and TNF-α expression.Results:The increasing green fluorescence area in the hydrogel was observed in the ADSCHpbds group and the ADSCPCDH group under the microscope,indicating that the cells proliferated in the hydrogel well.The IVIS imaging system was used to measure the biological fluorescence signal of living mice.The experimental results showed that the fluorescent area was limited to the wound site.The fluorescence value of the wound gradually increased within 5 days after transplantation,indicating that the transplanted cells were in a proliferative state on the wound;On the 16th day,the fluorescence signal completely disappeared,indicating that the transplanted ADSCHpgds could function in the wound environment for up to 16 days without distant metastasis.Two months after the wound was healed,wound tissue,and heart,liver,spleen,lung,and kidney tissues were dissected for histological evaluation.No significant changes in the tissue morphology were caused by ADSCHpgds transplantation.On the 17th day after transplantation,the wound healing rate of the diabetic mice in the ADSCHpgds group was significantly faster than that in control group and the ADSCPCDH group.10 days after transplantation,the area of PCNA fluorescence signal in the ADSCHpgds group was larger than that in the Control and ADSCPCDH groups;on day 13,immunohistochemical staining of wound tissue revealed that the positive rates of CD31 and Col-I cells in the ADSCHpgds group were higher than those in the Control group and ADSCPCDH.The expression of VEGF and Col-I in wound tissue of ADSCHpgds group was significantly higher than that of Control group and ADSCPCDH group tested by real-time PCR.On day 7,immunohistochemical staining revealed that the positive rate of Ly-6g and CD8T cells in the ADSCHpgds group was significantly lower than that in the control group and ADSCPCDH group.Immunofluorescence staining showed that the positive rate of f4/80+CD206+cells was higher than that of control group and ADSCPCDH group.In vitro experiments,LPS-induced RAW264.7 macrophages were cultured in ADSCHpgds and ADSCPCDH conditional media.The results showed that the expression levels of CD206 and VEGF in macrophages in the ADSCHpgds group were higher than those in the ADSCPCDH and PBS groups,while the expressions of IL-1β,IL-6,and TNF-αwere significantly reduced;The addition of PGD2 to the culture medium has consistent results with ADSCHpgds conditioned medium.Conclusion1.Down-regulated HPGDS expression in diabetic wound may lead to delayed activation of immune response in the wound,continuous activation of pro-inflammatory cells and massive accumulation of pro-inflammatory factors,which ultimately leads to delayed healing of diabetic wound surface2.Overexpression of Hpgds has no significant effect on stem cell markers,proliferation,migration,apoptosis and differentiation of hADSC;3.RGD-modified VitroGel 3D hydrogel-loaded hADSCHpgds can effectively promote the healing of diabetic mice,by inhibition of the neutrophils and CD8T cells infiltration,and proinflammatory factors IL-1β,IL-6,TNF-α and iNOS expression reduction,and polarization of M2 macrophages in the wound.