Propofol protects against high glucose-induced endothelial adhesion molecules expression in human umbilical vein endothelial cells

Abstract

Background: Hyperglycemia could induce oxidative stress, activate transcription factor nuclear factor kappa B (NF-κB), up-regulate expression of endothelial adhesion molecules, and lead to endothelial injury. Studies have indicated that propofol could attenuate oxidative stress and suppress NF-κB activation in some situations. In the present study, we examined whether and how propofol improved high glucose-induced up-regulation of endothelial adhesion molecules in human umbilical vein endothelial cells (HUVECs).

Methods: Protein expression of endothelial adhesion molecules, NF-κB, inhibitory subunit of NF-κBα (IκBα), protein kinase Cβ2 (PKCβ2), and phosphorylation of PKCβ2 (Ser(660)) were measured by Western blot. NF-κB activity was measured by electrophoretic mobility shift assay. PKC activity was measured with SignaTECT PKC assay system. Superoxide anion (O(2)(.-)) accumulation was measured with the reduction of ferricytochrome c assay. Human peripheral mononuclear cells were prepared with Histopaque-1077 solution.

Results: High glucose induced the expression of endothelial selectin (E-selectin), intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1), and increased mononuclear-endothelial adhesion. High glucose induced O(2)(.-) accumulation, PKCβ2 phosphorylation and PKC activation. Further, high glucose decreased IκBα expression in cytoplasm, increased the translocation of NF-κB from cytoplasm to nuclear, and induced NF-κB activation. Importantly, we found these high glucose-mediated effects were attenuated by propofol pretreatment. Moreover, CGP53353, a selective PKCβ2 inhibitor, decreased high glucose-induced NF-κB activation, adhesion molecules expression, and mononuclear-endothelial adhesion.

Conclusion: Propofol, via decreasing O(2)(.-) accumulation, down-regulating PKCβ2 Ser(660) phosphorylation and PKC as well as NF-κB activity, attenuated high glucose-induced endothelial adhesion molecules expression and mononuclear-endothelial adhesion.

Figure 1

Effects of glucose on adhesion molecules expression in human umbilical vein endothelial cells (HUVECs).(A) HUVECs were cultured in different concentrations of glucose (5, 10, 15, 20, 30 mM) for 4 hours. (B) HUVECs were cultured in 5 mM or 15 mM glucose for different times (1, 2, 4, 8 h). (C, D) Quantification of protein band density of adhesion molecules (*p < 0.05 vs. 5 mM glucose, n = 5). Data were expressed as mean ± SD. ■ represents E-selectin, ░ represents ICAM-1, □ represents VCAM-1.

Figure 2

Effects of propofol on adhesion molecules expression in hyperglycemic human umbilical vein endothelial cells (HUVECs).(A) HUVECs were pre-incubated with different concentrations (5, 10, 20, and 40uM) of propofol for 30 min, followed by glucose treatment. (B) Quantification of protein band density of adhesion molecules (*p < 0.05 vs. 5 mM glucose, #p < 0.05 vs. 15 mM glucose, n = 5). (C) HUVECs were pre-incubated with 10uM propofol for 30 min, followed by 5 mM glucose treatment. (D) Quantification of protein band density of adhesion molecules (*p < 0.05 vs. 5 mM glucose, #p < 0.05 vs. 15 mM glucose, n = 5). Data were expressed as mean ± SD. ■ represents E-selectin, ░ represents ICAM-1, □ represents VCAM-1.

Figure 3

Effects of propofol on 15 mM glucose-mediated nuclear factor kappa B (NF-κB) signal pathway.(A) Human umbilical vein endothelial cells were cultured in either 5 mM glucose or 15 mM glucose for 4 h with corresponding treatment. (B) Quantification of protein band density of NF-κB (p65) (*p < 0.05 vs. 5 mM glucose, #p < 0.05 vs. 15 mM glucose, n = 5). ■ represents nuclear extract, □ represents cytoplasmic extract. (C) Quantification of protein band density of IκBα (*p < 0.05 vs. 5 mM glucose, #p < 0.05 vs. 15 mM glucose, n = 5). Data were shown as mean ± SD.

Figure 4

Effects of propofol on 15 mM glucose-induced nuclear factor kappa B (NF-κB) activation. (A) Human umbilical vein endothelial cells were cultured in either 5 mM glucose or 15 mM glucose for 4 h with corresponding treatment. Shifted band was increased by 15 mM glucose, but reduced by propofol (n = 5). (B) In the supershift assay, 15 mM glucose-induced band was shifted by the pre-incubation of nuclear extracts with anti-NF-κB antibody, but was not affected by normal rabbit IgG (n = 5). (C) In the competition assay, 100-fold excess of nonradiolabeled probes were incubated with nuclear extracts 10 min before the addition of radiolabeled probes. 15 mM glucose-induced shifted band was blocked by nonradiolabeled competitors, but mutant competitors had no such effect (n = 5).

Figure 5

Effects of propofol on 15 mM glucose-induced Ser⁶⁶⁰phosphorylation of protein kinase Cβ2 (PKCβ2) and PKC activation. (A) Human umbilical vein endothelial cells (HUVECs) were cultured in either 5 mM glucose or 15 mM glucose for 4 h with corresponding treatment. (B) Quantification of protein band density of PKCβ2 or phosphorylated PKCβ2 (*p < 0.05 vs. 5 mM glucose, #p < 0.05 vs. 15 mM glucose, n = 5). ■ represents PKCβ2 phosphorylation, □ represents PKCβ2. (C) HUVECs were cultured in either 5 mM glucose or 15 mM glucose for 4 h with corresponding treatment. (*p < 0.05 vs. 5 mM glucose, #p < 0.05 vs. 15 mM glucose, n = 5). The results were shown as folds increased compared with 5 mM glucose group.

Figure 6

Effects of propofol on 15 mM glucose-induced superoxide anion (O₂^.-) accumulation. Human umbilical vein endothelial cells were cultured in either 5 mM glucose or 15 mM glucose for 4 h with corresponding treatment. (*p < 0.05 vs. 5 mM glucose, #p < 0.05 vs. 15 mM glucose, n = 5). Data were shown as mean ± SD. (a.u. = arbitrary units).

Figure 7

Effects of propofol and CGP53353 on 15 mM glucose-induced nuclear factor kappa B (NF-κB) activation.(A) Human umbilical vein endothelial cells (HUVECs) were cultured in either 5 mM glucose or 15 mM glucose for 4 h with corresponding treatment. (B) Quantification of protein band density of NF-κB (p65) (*p < 0.05 vs. 5 mM glucose, #p < 0.05 vs. 15 mM glucose, n = 5). Data were shown as mean ± SD. (C) HUVECs were cultured in either 5 mM glucose or 15 mM glucose for 4 h with corresponding treatment. Shifted band was increased by 15 mM glucose, but reduced by propofol or CGP53353 (n = 5).

Figure 8

Effects of propofol and CGP53353 on 15 mM glucose-induced adhesion molecule expression. (A) Human umbilical vein endothelial cells were cultured in either 5 mM glucose or 15 mM glucose for 4 h with corresponding treatment. (B) Quantification of protein band density of adhesion molecules (*p < 0.05 vs. 5 mM glucose, #p < 0.05 vs. 15 mM glucose, n = 5). Data were shown as mean ± SD. ■ represents E-selectin, ░ represents ICAM-1, □ represents VCAM-1.

Figure 9

15mM glucose-induced mononuclear-endothelial adhesion and its modulation by propofol and CGP53353. Compared with 5 mM glucose, 15 mM glucose greatly induced mononuclear-endothelial adhesion, which was ameliorated by propofol or CGP53353 pretreatment (*p < 0.05 vs. 5 mM glucose, #p < 0.05 vs. 15 mM glucose, n = 5). Data were shown as mean ± SD.