Netrin-1 Regulates Proliferation,Migration And Repair Of Diabetic Peripheral Neurovascular Diseases

Objective: To determine the effect of diabetic peripheral neurovascular disease(DPNV)on the axon guidance factor Netrin-1 protein,and to explore the role of Netrin-1 in regulating adipose-derived stem cells(ADSCs)of their proliferation,migration and repairment of DPNV.To elucidate the specific regulatory molecular mechanism of Netrin-1 on ADSCs,which will provide theoretical basis for exploring new options for clinical prevention and treatment of DPNV.Methods:(1)Ischemic hindlimb muscle tissue and peripheral blood serum from diabetic and non-diabetic patients were obtained.The expression of Netrin-1 and inflammatory factors in diabetic patients were analyzed by immunohistochemistry,immunofluorescence,Western Blot and ELISA,as well as small vessel density,and colocalization of Netrin-1 and endothelial cells.Statistical analysis of the clinical correlation between the expression of Netrin-1 and DPNV in diabetic high glucose environment was also performed.(2)Adipose tissue was obtained from C57/BL mice,ADSCs were isolated and cultured,and a gene transfection system was successfully established to make ADSCs overexpress green fluorescent protein(GFP)and Netrin-1(N-ADSCs).CCK-8,Western Blot,flow cytometry,Transwell,etc.were performed to detect the differences in the proliferation,migration,adhesion,and differentiation into endothelial cells of N-ADSCs and ADSCs in a high glucose environment.(3)A type2 diabetes mellitus(T2DM)mice(db/db)lower extremity denervation model was constructed,N-ADSCs and ADSCs were transplantated in vivo,and blood perfusion was observed by laser Doppler,immunofluorescence and immunohistochemistry were performed to evaluate ADSCs of the survival,migration,differentiation,angiogenesis and treatment efficiency of DPNV in vivo.(4)Western Blot was performed to explore the Netrin-1-mediated signaling pathways of proliferation,migration,adhesion,differentiation,angiogenesis and apoptosis of ADSCs,ELISA was applied to detect Netrin-1-mediated paracrine secretion of ADSCs,revealing the underlying molecular mechanisms.Results:(1)The expression levels of Netrin-1 in the ischemic tissues and peripheral blood serum of diabetic patients were significantly increased,while the expression of nflammatory cytokines such as IL-6,IL-1? and MCP-1 was decreased in peripheral blood serum.The vascular density in the lower extremity muscle tissue was decreased,and Netrin-1 was found co-localized with endothelial cells;(2)Sufficient amount of ADSCs were successfully obtained from adipose tissue of C57/BL mice,the optimal time and the multiplicity of infection(MOI)were determined,and an adenovirus transfection system was established.N-ADSCs exhibit significantly improved proliferation,migration,adhesion,differentiation into endothelial cells,and anti-apoptosis ability in high glucose environments.(3)In vivo denervated T2 DM mice transplanted with N-ADSCs demonstrated significantly increased laser Doppler perfusion index and number of new small blood vessels.Immunofluorescence showed that the number of ADSCs survived and the number of differentiated endothelial cells significantly increased.(4)The results of Western Blot and ELISA demonstrated that Netrin-1 may regulate the proliferation,migration,adhesion,differentiation of ADSCs and promote angiogenesis in diabetic neurovascular diseases by up-regulating the AKT/PI3K/e NOS/P-38/NF-?B signaling pathway and promoting the paracrine of multiple factors of ADSCs to treat DPNV.Conclusion: DPNV causes a significant decrease in Netrin-1 levels in blood and ischemic tissue of the lower extremities.Overexpression of Netrin-1 by ADSCs can enhance the proliferation,migration,adhesion and differentiation of ADSCs by using a gene transfection system.Netrin-1 may up-regulate the AKT/PI3K/e NOS/P-38/NF-?B signaling pathway and promote the ADSCs paracrine of multiple factors to enhance the survival of ADSCs in vivo and promote angiogenesis,thus improving the therapeutic efficiency of ADSCs transplantation in DPNV.The results of this study provide a theoretical basis for a new option for clinical prevention and treatment of DPNV.