Vascular Protective Role of Samul-Tang in HUVECs: Involvement of Nrf2/HO-1 and NO

Abstract

Samul-Tang (Si-Wu-Tang, SMT), composed of four medicinal herbs, is a well-known herbal formula treating hematological disorder or gynecologic disease. However, vascular protective effects of SMT and its molecular mechanisms on the vascular endothelium, known as the central spot of vascular inflammatory process, are not reported. The aim of this study was to investigate vascular protective effects of SMT water extract in human umbilical vein endothelial cells (HUVECs). Water extract of SMT was prepared and identified by HPLC-PDA analysis. Expression of cell adhesion molecules (CAMs) and heme oxygenase-1 (HO-1) and translocation of nuclear factor-kappa B (NF-κB) and nuclear factor-erythroid 2-related factor 2 (Nrf2) were determined by western blot. Nuclear localization of NF-κB and Nrf2 was visualized by immunofluorescence and DNA binding activity of NF-κB was measured. ROS production, HL-60 monocyte adhesion, and intracellular nitric oxide (NO) were also measured using a fluorescent indicator. SMT suppressed NF-κB translocation and activation as well as expression of CAMs, monocyte adhesion, and ROS production induced by TNF-α in HUVECs. SMT treated HUVECs showed upregulation of HO-1 and NO which are responsible for vascular protective action. Our study suggests that SMT, a traditionally used herbal formula, protects the vascular endothelium from inflammation and might be used as a promising vascular protective drug.

Figure 1

Three-dimensional chromatogram of Samul-Tang by HPLC-PDA.

Figure 2

Effects of SMT on TNF-α induced cell adhesion molecules expression in HUVECs. Cells were treated with TNF-α (50 ng/mL) for 6 h in the absence or pretreatment of SMT (10, 30, and 50 μg/mL) for 30 min. Bar represents the mean ± SEM of 3 independent experiments. ^∗ p < 0.05 versus con group. ^# p < 0.05 and ^## p < 0.01 versus TNF-α group.

Figure 3

Effects of SMT on TNF-α induced cell adhesion of HL-60 in HUVECs. HUVECs were treated with TNF-α (50 ng/mL) for 6 h in the absence or pretreatment of SMT (10, 30, and 50 μg/mL) for 30 min and then incubated with BCECF-AM labeled HL-60 cells. Adhered monocytes were captured with fluorescent microscope. Bar represents the mean ± SEM of more than 3 independent experiments. ^∗ p < 0.05 versus con group. ^# p < 0.05 versus TNF-α group.

Figure 4

Effects of SMT on TNF-α induced intracellular ROS production in HUVECs. Cells were treated with TNF-α (50 ng/mL) for 6 hours in the absence or pretreatment of SMT (10, 30, and 50 μg/mL) for 30 min and then treated with H₂DCFDA. NAC (N-acetyl-L-cysteine) was used as ROS scavenger. Bar represents the mean ± SEM of more than 3 independent experiments. ^∗ p < 0.05 versus con group. ^# p < 0.05 versus TNF-α group.

Figure 5

(a) Effects of SMT on TNF-α induced phosphorylation of IκB-α in HUVECs. (b, c) Effects of SMT on TNF-α induced NF-κB translocation in HUVECs. Cells were treated with TNF-α (50 ng/mL) for 1 hour in the absence or pretreatment of SMT (10, 30, and 50 μg/mL) for 30 min. NF-κB protein was detected by western blot and immunofluorescence. (Green: NF-κB, blue: nucleus; magnification: 400x.) Bar represents the mean ± SEM of 3 independent experiments. ^∗ p < 0.05 and ^∗∗ p < 0.01 versus con group. ^# p < 0.05 and ^## p < 0.01 versus TNF-α group.

Figure 6

Effect of SMT on TNF-α induced NF-κB activation in HUVECs. Cells were treated with TNF-α (50 ng/mL) for 1 hour in the absence or pretreatment of SMT (10, 30, and 50 μg/mL) for 30 min and nuclear extracts were prepared to perform (a) electrophoretic mobility shift assay (EMSA) and (b) luciferase promoter assay. Bar represents the mean ± SEM of 3 independent experiments. ^∗∗ p < 0.01 versus con group. ^## p < 0.01 versus TNF-α group.

Figure 7

(a, b) Effects of SMT on heme oxygenase-1 induction in HUVECs. Cells were treated with SMT as indicated without TNF-α. (c) Effects of SMT on ROS production in HUVECs. Bar represents the mean ± SEM of 3 independent experiments. ^∗ p < 0.05 and ^∗∗ p < 0.01 versus con group. ^# p < 0.05 versus respectively indicated group.

Figure 8

Effects of SMT on nuclear translocation of Nrf2 in HUVECs. Cells were incubated with SMT as indicated without TNF-α. Nrf2 was detected by (a, b) western blot and (c) immunofluorescence. (Red: Nrf2, blue: nucleus; magnification: 400x.) Bar represents the mean ± SEM of 3 independent experiments. ^∗ p < 0.05 versus con group.

Figure 9

Effect of SMT on NO (nitric oxide) synthesis in HUVECs. (a) Cells were treated SMT or ACh for 30 minutes. DAF-FM diacetate was labeled as intracellular NO indicator. (400x magnification) ACh (acetylcholine) was used as positive control. (b) Supernatant of cell cultured medium was collected after 24 h of SMT treatment and performing Griess assay. ^∗ p < 0.05 and ^∗∗ p < 0.01 versus con group.