Salidroside stimulates mitochondrial biogenesis and protects against H₂O₂-induced endothelial dysfunction

Abstract

Salidroside (SAL) is an active component of Rhodiola rosea with documented antioxidative properties. The purpose of this study is to explore the mechanism of the protective effect of SAL on hydrogen peroxide- (H2O2-) induced endothelial dysfunction. Pretreatment of the human umbilical vein endothelial cells (HUVECs) with SAL significantly reduced the cytotoxicity brought by H2O2. Functional studies on the rat aortas found that SAL rescued the endothelium-dependent relaxation and reduced superoxide anion (O2(∙-)) production induced by H2O2. Meanwhile, SAL pretreatment inhibited H2O2-induced nitric oxide (NO) production. The underlying mechanisms involve the inhibition of H2O2-induced activation of endothelial nitric oxide synthase (eNOS), adenosine monophosphate-activated protein kinase (AMPK), and Akt, as well as the redox sensitive transcription factor, NF-kappa B (NF- κ B). SAL also increased mitochondrial mass and upregulated the mitochondrial biogenesis factors, peroxisome proliferator-activated receptor gamma-coactivator-1alpha (PGC-1 α ), and mitochondrial transcription factor A (TFAM) in the endothelial cells. H2O2-induced mitochondrial dysfunction, as demonstrated by reduced mitochondrial membrane potential (Δ ψ m) and ATP production, was rescued by SAL pretreatment. Taken together, these findings implicate that SAL could protect endothelium against H2O2-induced injury via promoting mitochondrial biogenesis and function, thus preventing the overactivation of oxidative stress-related downstream signaling pathways.

Figure 1

Protective effects of SAL on H₂O₂-induced cytotoxicity in HUVECs. (a) HUVECs were exposed to various concentrations of H₂O₂ for 4 h. (b) Cells were exposed to various concentrations of SAL for 24 h. (c) HUVECs were pretreated with SAL or vehicle for 20 h and then exposed to H₂O₂ (100 μM) for 4 h. Cell viability was detected by CCK-8. Cell viability in untreated cells was assigned the value of 1. *P < 0.05, **P < 0.01 versus control; ^† P < 0.05 versus H₂O₂, n = 3–5.

Figure 2

Effect of SAL on cell free ROS detection using different assay systems. (a) OH^∙ was generated by Fenton reaction, (b) O₂ ⁻ was generated by xanthine/xanthine oxidase, and (c) H₂O₂ was exogenously added. *P < 0.05, **P < 0.01 versus blank control, n = 3.

Figure 3

Protective effects of SAL on H₂O₂-induced impairment of endothelium-dependent relaxation. (a) Rat thoracic aorta was pretreated with or without SAL for 30 min, and then H₂O₂ (100 μM) was added to incubate for another 30 min. After precontracting with 1 μM PE, ACh was added accumulatively. Complete relaxation of aorta induced by ACh was considered as 100%. *P < 0.05, **P < 0.01 versus control; ^† P < 0.05, ^†† P < 0.01 versus H₂O₂, n = 8–16. (b) HUVECs were pretreated with or without 10 μM SAL for 20 h and then loaded with DHE dye. After acquisition of basal data, H₂O₂ (100 μM) was added and the fluorescence was measured every 5 min within 30 min. The fluorescence of basal data was assigned the value of 100%. *P < 0.05, **P < 0.01 versus control; ^† P < 0.05 versus H₂O₂, n = 4. (c) HUVECs were pretreated with or without SAL for 20 h and then loaded with DAF-FM-DA dye. After acquisition of basal data, H₂O₂ (100 μM) was added and the fluorescence was measured every 5 min within 30 min. The fluorescence of basal data was assigned the value of 100%. *P < 0.05 versus control; ^† P < 0.05 versus H₂O₂, n = 4.

Figure 4

Effects of SAL on H₂O₂-induced activation of eNOS, AMPK, and Akt. After pretreatment with SAL (10 μM) for 20 h, H₂O₂ (0.1 μM or 100 μM) was added for another 4 h, and the lysates were analyzed by western blot. (a) Representative immunoblots. (b, c, d) The histogram shows quantitation of eNOS-ser1177, eNOS, AMPKa-thr172, AMPK, Akt-ser473, and Akt expression. The expressions were normalized to that obtained in untreated cells. *P < 0.05, **P < 0.01 versus control; ^† P < 0.05 versus H₂O₂, n = 4–6.

Figure 5

Effects of SAL on H₂O₂-induced activation of transcription factor NF-κB. Effects of H₂O₂ (0.1 μM) (a) and SAL (10 μM) (b) on transcription factor NF-κB activity. H₂O₂ (0.1 μM) or SAL (10 μM) was added for the indicated periods of time (0–4 h). The transcription activity in untreated cells was assigned the value of 1. *P < 0.05, **P < 0.01 versus control, n = 4. (c) Effects of SAL on H₂O₂-induced NF-κB activity. HUVECs were pretreated with SAL (1, 10 μM) for 10 min and then incubated with H₂O₂ (0.1, 100 μM) for 30 min. *P < 0.05, **P < 0.01 versus control; ^† P < 0.05, ^†† P < 0.01 versus H₂O₂, n = 4–6. (d) HUVECs were treated with TNF-α (30 ng/mL) for 30 min, and then total protein was extracted. The proteins were incubated with increasing concentrations of H₂O₂ (0.1, 1, 10, and 100 μM) for 30 min, and then the activities of NF-κB were detected as described in Section 2. The transcription activity was normalized to that obtained in untreated cells. **P < 0.01 versus control, ^†† P < 0.01 versus TNF-α, n = 3.

Figure 6

Effects of SAL on mitochondrial biogenesis. (a) Mitochondria mass was quantified using Mito Tracker Green. Scale bars = 50 μm. The fluorescence in untreated cells was assigned the value of 1. *P < 0.05 versus control, n = 4. HUVECs were treated with SAL (1, 10 μM) for 24 h, and the total protein was extracted; the lysates were analyzed by western blot. (b) Representative immunoblots. (c) Summary histograms of the relative density of PGC-1α and TFAM normalized to β-actin. The expression in untreated cells was assigned the value of 1. *P < 0.05 versus control, n = 5.

Figure 7

Effects of SAL on H₂O₂-induced mitochondrial dysfunction. HUVECs were pretreated with SAL (10 μM) for 20 h, and then H₂O₂ (100 μM) was added to incubate for another 4 h. (a) Mitochondrial membrane potential and (b) ATP content were detected as described in Section 2. ΔΨm and ATP content in untreated cells were assigned the value of 1. *P < 0.05 versus control; ^† P < 0.05 versus H₂O₂, n = 4.

Figure 8

Schematic diagram of the potential mechanisms of SAL to induce mitochondrial biogenesis. SAL inhibited transcription factors NF-κB activity, enhanced eNOS activity, and NO production, which induced mitochondrial biogenesis subsequently.