Abstract
OBJECTIVE: To investigate the influence and mechanism of extracellular vesicles (EVs) derived from human adipose-derived mesenchymal stem cells (hADMSCs), i.e. hADMSC-EVs on pyroptosis of human umbilical vein endothelial cells (HUVECs) induced by high glucose, with the aim of providing evidence for improving vascular function in diabetic wounds. METHODS: This study was an experimental research. The umbilical cords from 5 women aged 25 to 40 years were collected who had normal vaginal delivery at the Department of Obstetrics and Gynecology of the First Affiliated Hospital of Nanchang University from June to September in 2023, and HUVECs were isolated and successfully identified. Adipose tissue was obtained from 6 healthy women aged 25 to 35 years who underwent abdomen liposuction at the Department of Plastic Surgery of the above-mentioned hospital in the same period. After hADMSCs were isolated, hADMSC-EVs were extracted and successfully identified. The fourth passage of HUVECs were cultured in endothelial cell medium containing glucose in a molarity of 33 mmol/L and divided into phosphate buffered solution (PBS) group cultured with PBS, EV group cultured with hADMSC-EVs, and EV+LY294002 group cultured with hADMSC-EVs and the phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) signaling pathway inhibitor LY294002. Western blotting was used to detect the expressions of PI3K/Akt signaling pathway-related proteins PI3K and Akt, and pyroptosis-related proteins nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3), cysteinyl aspartate specific protease-1 (caspase-1), gasdermin D, interleukin-1β (IL-1β), and IL-18 of cells after 48 hours of culture. A cell counting kit-8 was used to test the proliferation levels of cells at 0 (immediately), 12, 24, 36, 48, 60, and 72 hours of culture. After 48 hours of culture, the cell scratch test was performed and the cell migration rates at 12 and 24 hours after scratching were calculated; the cell Transwell assay was conducted and the number of cells migrating in 24 hours was calculated; the cell tube formation experiment was performed, and the total length of tube formation and the number of branch nodes were measured and counted. The sample size was 3. RESULTS: After 48 hours of culture, the protein expressions of PI3K and Akt of cells in EV group were significantly higher than those in PBS group (P < 0.05), and the protein expressions of PI3K and Akt of cells in EV+LY294002 group were significantly lower than those in EV group (P < 0.05). After 48 hours of culture, the protein expressions of NLRP3, caspase-1, gasdermin D, IL-1β, and IL-18 of cells in EV group were 0.54±0.08, 0.96±0.11, 0.525±0.061, 1.216±0.039, and 1.317±0.023, respectively, which were significantly lower than 2.32±0.11, 1.86±0.07, 1.256±0.113, 2.589±0.084, and 2.042±0.132 in PBS group (P < 0.05); the protein expressions of NLRP3, caspase-1, gasdermin D, IL-1β, and IL-18 of cells in EV+LY294002 group were 1.16±0.05, 1.37±0.06, 0.962±0.028, 1.834±0.017, and 1.803±0.065, respectively, which were significantly higher than those in EV group (P < 0.05). At 12, 24, 36, 48, 60, and 72 hours of culture, the proliferation levels of cells in EV group were significantly higher than those in PBS group (P < 0.05), and the proliferation levels of cells in EV+LY294002 group were significantly lower than those in EV group (P < 0.05). After 48 hours of culture, the cell migration rates at 12 and 24 hours after scratching in EV group were significantly higher than those in PBS group (P < 0.05), and the cell migration rates at 12 and 24 hours after scratching in EV+LY294002 group were significantly lower than those in EV group (P < 0.05); the number of cells migrating in 24 hours in EV group was significantly greater than that in PBS group (P < 0.05), and the number of cells migrating in 24 hours in EV+LY294002 group was significantly less than that in EV group (P < 0.05). After 48 hours of culture, compared with those in PBS group, the total length of tube formation of cells in EV group was significantly prolonged (P < 0.05), and the number of branch nodes was significantly increased (P < 0.05); compared with those in EV group, the total length of tube formation in EV+LY294002 group was significantly shortened (P < 0.05), and the number of branch nodes was significantly decreased (P < 0.05). CONCLUSIONS: hADMSC-EVs can inhibit the expression of pyroptosis-related proteins in HUVECs induced by high glucose through the PI3K/Akt signaling pathway and improve the proliferation, migration, and angiogenesis capabilities of HUVECs.