Search

Objective: To study the inhibitory effects of insulin on nuclear factor-kappa B (NF-kappaB) nuclear translocation of vascular endothelial cells induced by burn serum and its correlative mechanism., Methods: Human umbilical vein endothelial cells (HUVECs) were cultured in vitro and divided into 5 groups: blank control group (BC, ordinary culture without any stimulation), normal serum control group (NS, cultured with nutrient solution containing 20% healthy human serum), burn serum stimulation group (BS, cultured with nutrient solution containing 20% burn human serum), burn serum+insulin treatment group (BI, cultured with nutrient solution containing 20% burn human serum and 1x10(-7) mol/L insulin), inhibitor pretreatment group [IP, pretreated with 50 micromol/L protein kinase B (Akt) specific inhibitor LY-294002, then cultured with the same medium as used in BI group 30 minutes later] according to the random number table. Six hours later, the injury and apoptosis of HUVECs was respectively observed by the scanning electron microscope and determined by the flow cytometry. Meanwhile, the phosphorylation of inhibitor kappa B-alpha (p-IkappaB-alpha) and Akt (p-Akt) in cytoplasm, and the content of NF-kappaB-p65 in nucleus were determined with Western blot., Results: (1) Compared with those in BC group, HUVECs in BS group shrank obviously with irregular nuclear structure, and intercellular links jagged or vanished. Slight change was observed in HUVECs structure in NS and BI groups, with the cell ductility and nuclear structure much better than those in BS group. (2) The apoptosis rates of HUVECs in BS group [(28.5+/-2.3)%], BI group [(22.3+/-1.8)%], and IP group [(29.7+/-2.4)%] were all obviously higher than that in BC group [(15.7+/-2.2)%, F=14.288, P<0.05 or P<0.01]. There was no significant statistical difference between NS group [(17.0+/-2.5)%] and BC group in apoptosis rate (F=14.288, P>0.05). The apoptosis rate of HUVECs in BI group was obviously lower than that in BS group (F=14.288, P<0.05). (3) Compared with those in BC group, the protein expressions of p-IkappaB-alpha in cytoplasm and NF-kappaB-p65 in nucleus were up-regulated, and the protein expression of p-Akt in cytoplasm was down-regulated in BS and IP groups. The expression levels of the three proteins in NS and BI groups were close to those in BC group., Conclusions: Insulin could inhibit the IkappaB phosphorylation, and then restrict NF-kappaB nuclear translocation and improve the vascular endothelial cells function accordingly through regulating phosphatidylinositol 3 kinase/Akt pathway.

Objective: To study the effect of insulin in different concentrations on secretion function of growth factors of adipose-derived stem cells (ADSCs)., Methods: ADSCs were isolated from human abdominal adipose tissue and cultured. The immunophenotype and adipose induced-differentiation were identified, and the third generation cells were collected. The collected cells were assigned to 1 x 10(-8), 1 x 10(-7), 1 x 10(-6) mol/L insulin groups according to the concentration of added insulin. When cells grew into 70% confluence in conventional medium, ADSCs were cultured further in serum-free DMEM containing insulin in different concentrations for 3 days. ADSCs cultured in medium without insulin were used as control group. Secretion amount of vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) of ADSCs were determined by enzyme-linked immunosorbent assay. The effects of the supernatant fluid of ADSCs' nutrient solution on the proliferation and collagen synthesis of the cultured fibroblast were detected by MTT chromatometry and hydroxyproline chromatometry., Results: The secretion amounts of VEGF and HGF of ADSCs in 1 x 10(-8) and 1 x 10(-7) mol/L insulin groups [(471 +/- 41, 762 +/- 66 ng/L), (643 +/- 64, 930 +/- 67 ng/L), respectively] were significantly higher as compared with those in control group (286 +/- 47, 577 +/- 84 ng/L) (P < 0.05 or P < 0.01). No change occurred in the secretion amount of VEGF and HGF of ADSCs in 1 x l0(-6) mol/L insulin group (P > 0.05). The supernatant fluid of ADSCs' nutrient medium of 1 x 10(-8), 1 x 10(-7) mol/L insulin groups showed obvious stimulative effect on the proliferation and collagen synthesis of fibroblasts, and it was most obvious in the 1 x 10(-7) mol/L group (P < 0.05 or P < 0.01)., Conclusions: Insulin in the concentrations of 1 x 10(-8) and 1 x 10(-7) mol/L can notably promote ADSCs' function of secreting VEGF and HGF.