The mechanism of nerve growth factor improving ischemic necrosis of random flap in diabetic rats
WANG Xiaowu1, ZHU Xiandong2, WANG Yongqiang1, WU Zhixuan3, WEN Zhikai2, WU Dazhou4, CHEN
Jicai4
1.Department of Hepatobiliary and Pancreatic Surgery, the First Affiliated Hospital of Wenzhou Medical University, Wenzhou 325015, China; 2.Department of Thyroid Surgery, the First Affiliated Hospital of Wenzhou Medical University, Wenzhou 325015, China; 3.Department of Breast Surgery, the First Affiliated Hospital of Wenzhou Medical University, Wenzhou 325015, China; 4.Department of Hernia and Abdominal Wall Surgery, the First Affiliated Hospital of Wenzhou Medical University, Wenzhou 325015, China
WANG Xiaowu,ZHU Xiandong,WANG Yongqiang, et al. The mechanism of nerve growth factor improving ischemic necrosis of random flap in diabetic rats[J]. JOURNAL OF WEZHOU MEDICAL UNIVERSITY, 2024, 54(2): 87-98.
Abstract：Objective: To explore the molecular mechanism of nerve growth factor (NGF) in improving the ischemic necrosis of random skin flap in diabetic rats. Methods: A Wistar rat model of type 1 diabetes was
established, who were randomly divided as the DM group (model group) and the DM+ NGF group (experimental group). A modified McFarlane flap measuring 9 cm×3 cm was designed and manufactured for skin flaptransplantation. The survival of the flaps in both groups was observed, and the survival area was measured. Ratflap tissues were collected to investigate the relationship between flap survival and angiogenesis using HE stainingand immunohistochemical staining. Western blot experiments were carried out to study the differences in CD31 and VEGF protein expression between the two groups and the changes in the AGE-RAGE/MAPK-ERK1/2 signaling pathway. Using the established high-glucose human umbilical vein endothelial cell (HUVEC) model, the cell experiment was divided into two groups: cell model group (DM group) and experimented cell group (DM+NGF group). The effects of NGF on cell proliferation, migration, and angiogenesis in HUVECs under high-glucose conditions were analyzed. This was done through CCK-8 proliferation assays, MCM2+ cell immunofluorescence assays, scratch assays, Transwell assays, and angiogenesis assays. The interaction and functional enrichment analysis of NGF with common angiogenesis-related factors such as VEGF, SDF1α, HIF-1α, PDGFA, and TGF-β1 was made using the STRING database. The effect of NGF on their expression was validated through RTqPCR and Western blot. The impact of NGF on HUVECs was further investigated through RNA-seq and Western blot experiments to explore the underlying mechanisms. Results: After successful animal modeling, it was observed that the DM+NGF group had a 10%-20% larger flap survival area compared with the DM group (P<0.01).Immunohistochemical staining of CD31 revealed a higher microvessel density in the DM+NGF group (284.2±
44.76 mm2) compared with the DM group (P<0.01), along with increased expression of CD31 and VEGF (P<0.05). Furthermore, in the flap tissue, the RAGE expression in the AGE-RAGE/MAPK-ERK1/2 signaling pathway decreased (P<0.05), while p-ERK1/2 expression increased in the DM+NGF group (P<0.05). In terms
of cellular effects, a high-glucose model with 25 mmol/L was successfully established, and NGF at 100 ng/mL was found to promote HUVEC proliferation (P<0.01). Compared with the DM group, the DM+NGF group exhibited significantly enhanced cell migration and angiogenic capabilities (P<0.01). Moreover, the DM+NGF group showed higher relative expression levels of VEGF, PDGFA, HIF-1α, Arg-1 and bFGF (P<0.05). The KEGG and GO enrichment analysis results were mainly associated with angiogenesis and the AGE-RAGE/ MAPK-ERK1/2 signaling pathway. Conclusion: NGF may promote the angiogenesis of endothelial cells in diabetic rats by affecting the signal pathway of AGE-RAGE/MAPK-ERK1/2, thus improving the ischemic necrosis of the random skin flap.
