Sailuotong Prevents Hydrogen Peroxide (H₂O₂)-Induced Injury in EA.hy926 Cells

Sai Wang Seto, Dennis Chang, Wai Man Ko, Xian Zhou, Hosen Kiat, Alan Bensoussan, Simon M Y Lee, Maggie P M Hoi, Genevieve Z Steiner, Jianxun Liu, Sai Wang Seto, Dennis Chang, Wai Man Ko, Xian Zhou, Hosen Kiat, Alan Bensoussan, Simon M Y Lee, Maggie P M Hoi, Genevieve Z Steiner, Jianxun Liu

Abstract

Sailuotong (SLT) is a standardised three-herb formulation consisting of Panax ginseng, Ginkgo biloba, and Crocus sativus designed for the management of vascular dementia. While the latest clinical trials have demonstrated beneficial effects of SLT in vascular dementia, the underlying cellular mechanisms have not been fully explored. The aim of this study was to assess the ability and mechanisms of SLT to act against hydrogen peroxide (H₂O₂)-induced oxidative damage in cultured human vascular endothelial cells (EAhy926). SLT (1-50 µg/mL) significantly suppressed the H₂O₂-induced cell death and abolished the H₂O₂-induced reactive oxygen species (ROS) generation in a concentration-dependent manner. Similarly, H₂O₂ (0.5 mM; 24 h) caused a ~2-fold increase in lactate dehydrogenase (LDH) release from the EA.hy926 cells which were significantly suppressed by SLT (1-50 µg/mL) in a concentration-dependent manner. Incubation of SLT (50 µg/mL) increased superoxide dismutase (SOD) activity and suppressed the H₂O₂-enhanced Bax/Bcl-2 ratio and cleaved caspase-3 expression. In conclusion, our results suggest that SLT protects EA.hy916 cells against H₂O₂-mediated injury via direct reduction of intracellular ROS generation and an increase in SOD activity. These protective effects are closely associated with the inhibition of the apoptotic death cascade via the suppression of caspase-3 activation and reduction of Bax/Bcl-2 ratio, thereby indicating a potential mechanism of action for the clinical effects observed.

Keywords: Sailuotong (SLT); apoptosis; endothelial dysfunction; herbal medicine; reactive oxygen species; vascular dementia.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
(A) Representative images of the effect of Sailuotong (SLT) (50 µg/mL) on EA.hy926 cell morphology injured by H2O2 observed under an inverted/phase contract microscope; (B) Effect of Sailuotong (SLT) (0.1–50 µg/mL) on EA.hy926 cells viability injured by H2O2 (n = 3) measured by MTT assay. Data are presented as means ± S.D. *** p < 0.001 vs. control group; # p < 0.05 vs. H2O2 group; ## p < 0.01 vs. H2O2 group.
Figure 2
Figure 2
(A) Effects of SLT (1–50 µg/mL) on H2O2-induced lactate dehydrogenase (LDH) leakage in EA.hy926 cells (n = 3). Data are presented as means ± S.D. *** p < 0.001 vs. control (CLT) group; # p < 0.05 vs. H2O2 group; (B) Effects of SLT (50 µg/mL) on H2O2-inhibited superoxide dismutase (SOD) activity in EA.hy926 cells (n = 3). Data are presented as means ± S.D. ** p < 0.01 vs. control (CLT) group; # p < 0.05 vs. H2O2 group.
Figure 3
Figure 3
Effects of SLT (1–50 µg/mL) on H2O2-induced intracellular reactive oxygen species (ROS) generation in EA.hy926 cells. Gallic acid (10 µg/mL), a known potent anti-oxidant, was used as a positive control (n = 3). Data are presented as means ± S.D. *** p < 0.05 vs. control group; ### p < 0.001 vs. H2O2 group.
Figure 4
Figure 4
(A) Effects of SLT (50 µg/mL) on H2O2-upregulated Bax/Bcl-2 ratio in EA.hy926 cells (n = 3). Data are presented as means ± S.D. * p < 0.05 vs. control group; # p < 0.05 vs. H2O2 group. Images are representative of three independent experiments. The present or absent of H2O2 and SLT in the culture is indicated by + or − respectively; (B) Effects of SLT (50 µg/mL) on H2O2-upregulated cleaved caspase-3 expression in EA.hy926 cells (n = 3). Result was expressed as expression of cleaved caspase-3 protein relative to β-actin. Data are presented as means ± S.D. * p < 0.05 vs. control group; # p < 0.05 vs. H2O2 group. Images are representative of three independent experiments. The present or absent of H2O2 and SLT in the culture is indicated by + or − respectively.

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