Salidroside Ameliorated Intermittent Hypoxia-Aggravated Endothelial Barrier Disruption and Atherosclerosis via the cAMP/PKA/RhoA Signaling Pathway

FIGURE 1. Salidroside attenuated CIH-aggravated atherosclerosis in ApoE-/- mice. Male ApoE-/- mice were randomly divided into normoxia (control), CIH, and CIH + Sali groups. After experimental intervention for 7 weeks, mice were sacrificed under anesthesia (A) Representative images of aortic Oil Red O staining (B) Quantitative summary of atherosclerotic lesions in aortas (n = 4) (C) Immunofluorescence staining of VE-cadherin and CD31 and relative fluorescent intensity of VE-cadherin in the endothelium of aortic roots (5 × magnification, n = 3 sections per tissue, at least three analysis sites per slide) (D) Plaques of aortic roots were stained with ROCK (2 × magnification) (E) Quantitative summary of ROCK expression in aortic root plaques (n = 4). CIH, chronic intermittent hypoxia; Sali, salidroside; VE-cadherin, vascular endothelial cadherin; ROCK, Rho-associated protein kinase. All data are presented as mean ± SD. *p < 0.05, **p < 0.01, and ***p < 0.001 compared with CIH group.

FIGURE 2. Salidroside ameliorated IH-induced endothelial barrier dysfunction in HUVECs. The effects of IH on TEER (A) and permeability to FITC-dextran (B) were determined in HUVECs after 9, 18, 36, 72, 108 cycles of IH exposure. Effects of Sali on TEER (C) and permeability to FITC-dextran (D) were determined in HUVECs pretreated with Sali (10 µM or 100 µM) for 2 h before exposure to either normoxia or 72 cycles of IH. IH, intermittent hypoxia [one cycle includes reducing O2 for a 20-min hypoxia period (5% O2 and 5% CO2) followed by 20-min reoxygenation (21% O2 and 5% CO2)]; Sali, salidroside; TEER, transendothelial electrical resistance. All data are presented as mean ± SD. #p < 0.05 and ##p < 0.01 compared with IH group; *p < 0.05, **p < 0.01, and ***p < 0.001 compared with normoxia group.

FIGURE 3. Salidroside ameliorated the suppressive effect of IH on VE-cadherin and ZO-1 expression in HUVECs. HUVECs were pretreated with Sali (10 µM or 100 µM) for 2 h before exposure to 72 cycles of IH (A) Immunofluorographs of VE-cadherin (20 × magnification; Bar = 50 µm) and (B) ZO-1 (20 × magnification; Bar = 50 μm) (C) VE-cadherin and ZO-1 protein levels were measured by western blot analysis (D–E) Relative protein levels of VE-cadherin and ZO-1 were quantified by densitometry analysis. IH, intermittent hypoxia; Sali, salidroside; VE-cadherin, vascular endothelial cadherin; ZO-1, zonula occludens 1. All data are presented as mean ± SD. #p < 0.05, ##p < 0.01, and ###p < 0.001 compared with IH group; **p < 0.01 and ***p < 0.001 compared with normoxia group.

FIGURE 4. Salidroside attenuated IH-induced ROS overproduction in HUVECs. Intracellular ROS levels were determined in HUVECs pretreated with Sali (10 µM or 100 µM) or Tiron (10 µM) for 2 h before exposure to either normoxia or 72 cycles of IH. DCFH-DA was used as a fluorescent probe for ROS. Tiron was used as a positive control (A) Fluorographs of intracellular ROS were acquired with a fluorescence microscope (5 × magnification; Bar = 500 µm) (B) Fluorescence quantification results of intracellular ROS. IH, intermittent hypoxia; Sali, salidroside; DCFH-DA, 2ʹ,7ʹ-dichlorofluorescein diacetate; ROS, reactive oxygen species. All data are presented as mean ± SD. *p < 0.05 and ***p < 0.001 compared with IH group.

FIGURE 5. Salidroside suppressed IH-induced elevation of RhoA activity and ROCK protein expression in HUVECs through the cAMP/PKA pathway. HUVECs were pretreated with Sali (10 µM or 100 µM) or the PKA inhibitor H-89 for 2 h before exposure to 72 cycles of IH (A) Intracellular cAMP levels, as well as (B) RhoA activity and ROCK protein levels were measured (C–D) Relative levels of RhoA activity and ROCK protein expression were quantified by densitometry analysis. IH, intermittent hypoxia; Sali, salidroside; RhoA, ras homolog gene family member A; ROCK, Rho-associated protein kinase. All data are presented as mean ± SD. *p < 0.05 and ***p < 0.001 compared with IH group.

FIGURE 6. Salidroside ameliorated IH-induced ROS overproduction and barrier dysfunction in HUVECs via the cAMP/PKA/RhoA signaling pathway. Effects of drugs on ROS generation (A) and TEER (B) were determined in HUVECs pretreated with Sali (100 µM), H-89 (10 µM), Y27632 (10 µM), or Fasudil (10 µM) for 2 h before exposure to either normoxia or 72 cycles of IH. IH, intermittent hypoxia; Sali, salidroside; ROS, reactive oxygen species; TEER, transendothelial electrical resistance. All data are presented as mean ± SD of three experiments. ***p < 0.001 compared with IH group.

FIGURE 7. Schematic diagram of the potential mechanism by which salidroside protects against IH-induced ROS overproduction and barrier disruption in the endothelium. Salidroside increased intracellular cAMP levels, which subsequently activated PKA in HUVECs. Activated cAMP/PKA suppressed IH-induced elevation of RhoA and ROCK, and thereafter inhibited ROS generation. In turn, reduced ROS production inhibited RhoA/ROCK expression, breaking the vicious cycle of ROS augmentation induced by IH in the endothelium. IH, intermittent hypoxia; ROS, reactive oxygen species; RhoA, ras homolog gene family member A; ROCK, Rho-associated protein kinase; PKA, protein kinase A.