Salidroside (cat. no. 43866-25, Purity >98%), ox-LDL (cat. no. BP368) and dichloro-dihydro-fluorescein diacetate (DCFH-DA; cat. no. D6883) were obtained from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). Salidroside and ox-LDL were diluted with DMSO and PBS, respectively, and stored at −20°C. Dulbecco's modified Eagle's medium (DMEM; cat. no. 10569010), fetal bovine serum (FBS; no. 10099-141), and streptomycin/penicillin (cat. no. 15140122) were purchased from Gibco (Thermo Fisher Scientific, Inc., Waltham, MA, USA). Small interfering (si)RNA targeting SIRT1 (siSIRT1) and AMPK (siAMPK), and control siRNA (siControl) were obtained from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). The MTT assay (cat. no. C0009) and bicinchoninic acid (BCA) protein assay (cat. no. P0010) were purchased from the Beyotime Institute of Biotechnology (Beijing, China). The kits for the measurement of lactate dehydrogenase (LDH) release (cat. no. A020-2), methane dicarboxylic aldehyde (MDA) content (cat. no. A003-1), superoxide dismutase (SOD) activity (cat. no. A001-3) and glutathione peroxidase (GSH-Px) level (cat. no. A005) were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). 5, 58, 6, 68-Tetraethylbenzimidazolcarbocyanine iodide (JC-1) was supplied by BioVision (Inc., Milpitas, CA, USA; cat. no. 4999-100). The caspase-3 colorimetric assay kit was supplied by Enzo Life Sciences Inc. (Farmingdale, NY, USA; cat. no. BML-AK703-0001). The Annexin V-Fluorescein Isothiocyanate (FITC)/Propidium Iodide (PI) Apoptosis Detection kit was obtained from BD Biosciences (Franklin, Lakes, NJ, USA; cat. no. 556547). Anti-SIRT1 (cat. no. ab104833) and anti-NADPH oxidase 2 (NOX2; cat. no. ab133303) antibodies were obtained from Abcam (Cambridge, UK). Anti-phosphorylated (p)-AMPK (cat. no. 2537), AMPK (cat. no. 2793), cytochrome c (cat. no. 11940), Bax (cat. no. 774), Bcl-2 (cat. no. 15071) and GAPDH (cat. no. 2118) antibodies were obtained from Cell Signaling Technology, Inc., (Danvers, MA, USA).

HUVECs were from the American Type Culture Collection (Manassas, VA, USA; cat. no. CRL-1730) and cultured in DMEM with 10% (v/v) FBS and 1% streptomycin/penicillin (v/v) in a humidified 95% air-5% CO₂ incubator at 37°C. For ox-LDL treatment experiments, HUVECs were grown to 70-80% confluence and then switched to different concentrations of ox-LDL (10, 25, 50, 100 and 200 µg/ml) for 12, 24, or 72 h. For protection experiments, HUVECs were pretreated with salidroside (0.1, 1 and 10 µg/ml) for 1 h and incubated continuously for 24 h with ox-LDL (100 µg/ml). For mechanism research studies, the cells were pre-transfected with siSIRT1, siAMPK and siControl, and then treated with salidroside (1 µg/ml) for 1 h followed by co-incubation with ox-LDL (100 µg/ml) for 24 h.

HUVECs at the density of 4×10⁵ cells/well were seeded in a six-well plate and then transfected with siSIRT1 (50 nM), siAMPK (100 nM) or siControl (100 nM; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) using Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) in accordance with the manufacturer's protocol. The siSIRT1 sequences were 5′-CCCUCAAAGUAAGACCAGUTT-3′ (sense) and 5′-ACUGGUCUUACUUUGAGGGAA-3′ (antisense). The siAMPK sequences were 5′-GGACAGGGAAGCCUUAAAUTT-3′ (sense) and 5′-AUUUAAGGCUUCCCUGUCCTT-3′ (antisense). The negative control siRNA sequences were 5′-UUCUCCGAACGUGUCACGUTT-3′ (sense) and 5′-ACGUGACACGUUCGGAGAATT-3′ (antisense). Briefly, cells were grown to 70-90% confluence at the time of transfection. Lipofectamine^® 2000 (10 µl) was gently agitated and diluted in Opti-MEM^® I Medium (cat. no. 31985-070; Invitrogen; Thermo Fisher Scientific, Inc.) without serum (150 µl) in a separate vessel and the mixture was incubated for 5 min at room temperature. siRNA or control siControl (150 pmol) was diluted into in Opti-MEM^® I Medium without serum (150 µl). Subsequently, diluted DNA was added to diluted Lipofectamine^® 2000 Reagent (1:1 ratio). Following incubation for 5 min at room temperature, the siRNA-lipid complex (250 µl) was added to each well containing cells and medium. Following transfection for 6 h, the transfection mixture was replaced with fresh growth medium. Transfection efficiency was measured using western blot analysis. Subsequent experiments with transfected cells were performed following transfection for 24 h.

The viability of HUVECs was determined by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetra-zolium bromide (MTT) assay according to the manufacturer′s protocol. Cells at a density of 1×10⁴ cells/well were plated onto a 96-well culture plate and treated as discussed above. Following treatment for 24 h, the MTT regent (0.5 mg/ml) was supplemented added and co-incubated for 3 h at 37°C. Subsequently, DMSO (150 µl) was added to each well to fully dissolve the crystal formazan. The optical density (OD) value at 450 nm was then measured using a microplate reader (Bio-Tek Instruments, Inc., Winooski, VT, USA). Each experiment was performed three times.

The release of LDH was determined using the LDH assay kit following the manufacturer's protocol. Briefly, cells were seeded in 96-well plates at a density of 1×10⁴ cells/well and treated as discussion above for 24 h at 37°C. Then the supernatant was collected. After centrifuging at 1,000 × g for 10 min at room temperature, the supernatant (20 µl) was mixed with 2,4-dinitrophenylhydrazine (20 µl), transferred at 37°C for 15 min and then NaOH (0.4 M, 250 µl) was added into the mixture and co-incubated for a further 15 min at 37°C. The absorbance was detected at 450 nm using a microplate reader (Bio-Tek Instruments, Inc.).

HUVECs apoptosis was performed using an Annexin V-FITC/PI kit according to the manufacturer's protocol. Briefly, cells were cultured in 6-well plates at the density of 1×10⁵ cells/well and treated as discussion above. After incubation for 24 h, cells were harvested using 0.05% trypsin and washed three times with ice-cold PBS, and then re-suspended in 1X binding buffer (500 µl) included in the kit followed by mixed and incubated with 5 µl of Annexin V-FITC reagent and 5 µl of PI at room temperature for 15 min. Cell apoptosis was analyzed on a FACScan flow cytometer (BD Biosciences). The percentage of cells in quadrant 2 (Q2) and 4 (Q4) were quantitatively analyzed using FCS Express 3.0 software (De Novo Software, Glendale, CA, USA), generating the total percentage of apoptotic cells at both early and late apoptotic stage or apoptotic index. All experiments were performed in triplicate.

The intracellular ROS production was determined by a redox-sensitive fluorescent probe dichloro-dihydro-fluorescein diacetate (DCFH-DA) according to the protocol. Briefly, following treatment for 24 h, HUVECs were washed twice with ice-cold PBS and then incubated with DCFH-DA (10 µM) for 20 min at 37°C. After washing twice with PBS, the fluorescence values were detected by a F97 PRO fluorescence spectrophotometer (Shanghai Lengguang Technology Co., Ltd., Shanghai, China). In addition, following incubation with DCFH-DA (10 µM) for 20 min at 37°C, the sample was collected and centrifuged at 800 × g for 5 min at room temperature and washed twice with PBS, and then the fluorescence intensity was measured using FACScan flow cytometer at excitation and emission wavelengths of 485 and 528 nm, respectively.

The MDA content, SOD and GSH-Px activities in HUVECs were measured using colorimetric assay kits according to the manufacturer's protocols. The MDA level was measured using the thiobarbituric acid method with a maximum absorbance at 532 nm. The SOD activity was based on the combination of xanthine and xanthine oxidase and the absorbance was recorded at a wavelength of 550 nm. GSH-Px activity was determined using the enzyme-catalyzed reaction product (reduced glutathione) at a wavelength of 412 nm.

MMP was assessed using a cationic fluorescent indicator JC-1 which can selectively enter mitochondria and change color from red to green when MMP decreases. Briefly, HUVECs were washed twice with PBS and then incubated with JC-1 (200 µM) for 20 min at 37°C followed by washing with PBS to remove excess JC-1. The fluorescence was observed by a F97 PRO fluorescence spectrophotometer (Shanghai Lengguang Technology Co., Ltd.) at an excitation wavelength of 529 nm and an emission wavelength of 590 nm.

The caspase-3 activity was determined using caspase-3 colorimetric assay kit carried out as described by the protocol provided by the manufacturer. The principle of the assay is that acetyl-Asp-Glu-Val-Asp p-nitroanilide (Ac-DEVD-рNA) could be catalyzed by caspase-3 to produce a yellow substance p-nitroaniline, which reflects caspase-3 activity. In brief, HUVECs were collected and lysed with lysis buffer (provided by the kit) for 30 min on ice followed by centrifuging at 16,000 × g for 15 min at 4°C. The supernatant was then incubated with Ac-DEVD-рNA at 37°C for 2 h. Finally, the absorbance was detected at 405 nm and the relative caspase-3 activities were calculated as follows: Caspase-3 activity (%) = (OD_treatment − OD_{treatment blank})/(OD_control − OD_{control blank}) × 100%.

HUVECs were harvested and lysed in pre-cooled RIPA buffer (cat. no. P0013B; Beyotime Institute of Biotechnology) containing pheylmethanesulfonyl fluoride (0.2 mM). The cell lysate was then centrifuged at 13,400 × g for 10 min at 4°C and the total protein concentration was determined by the BCA protein assay kit according to the protocol provided by the manufacturer. Equal amounts of protein (30 µg) were separated by a 12% SDS-PAGE and then transferred to a polyvinylidene difluoride membrane. The membrane was blocked with TBS Tween-20 (TBST) buffer [20 mM Tris-HCl (pH 8.0)/150 mM NaCl/0.05% Tween-20] containing 5% non-fat dairy milk for 2 h at room temperature and then incubated overnight at 4°C with the following antibodies: Rabbit anti-SIRT1 (1:1,000), rabbit anti-phosphorylated AMPK (1:2,000), rabbit anti-AMPK (1:2,000), rabbit anti-Bax (1:1,000), rabbit anti-Bcl-2 (1:1,000), rabbit anti-NOX2 (1:1,000), rabbit anti-cytochrome c (1:1,000) or rabbit anti-GAPDH (1:1,000). After washing five times with TBST buffer, the membrane was incubated with horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (1:5,000; cat. no. 7074; Cell Signaling Technology, Inc.) at for 2 h at room temperature. The membrane was finally visualized with the BeyoECL Plus detection kit (cat. no. P0018M; Beyotime Institute of Biotechnology). Scanned images were visualized with the LI-COR Odyssey Infrared Imaging System (LI-COR Biosciences, Lincoln, NE, USA).

Results are expressed as the mean ± standard deviation from at least three separate experiments. Statistical analyses were performed using SPSS 13.0 software (SPSS, Inc., Chicago, IL, USA). The significance of differences was calculated with one-way analysis of variance followed by Tukey's test as appropriate. P<0.05 was considered to indicate a statistically significant difference.

To determine the cytotoxic potential of salidroside on HUVECs, the effects of salidroside on cell viability was evaluated. The MTT assay result demonstrated that incubation with low concentrations of salidroside (0.1, 1, 5, or 10 µM) for 24 h did not affect cell viability, whereas high concentrations of salidroside (20, 40, or 80 µM) significantly reduced cell viability (P<0.05; Fig. 1A). Therefore, three concentrations of salidroside (0.1, 1 and 10 µM) were selected for subsequent experiments. Next, in order to investigate the damaging effects of ox-LDL on HUVECs, cells were treated with ox-LDL (25, 50, 100 and 200 µg/ml) for 12, 24 or 72 h. The MTT assay results revealed that cell viability of HUVECs in response to ox-LDL (100 and 200 µg/ml) for 12, 24 and 72 h was significantly reduced (P<0.05; Fig. 1B). Notably, incubation with ox-LDL (100 µg/ml) for 24 h could reduce cell viability to 51.37±7.63%, which was selected as the next experimental condition. Subsequently, it was demonstrated that pretreatment with salidroside (0.1, 1, or 10 µM) significantly reversed ox-LDL-induced the downregulation of cell viability (P<0.05; Fig. 1C). Furthermore, the LDH release assay result revealed that ox-LDL treatment significantly increased the LDH release in HUVECs (P<0.01), while this effect was also significantly inhibited by pretreatment with salidroside (P<0.05; Fig. 1D). Flow cytometry with Annexin V-FITC/PI staining demonstrated that salidroside pretreatment significantly reversed the ox-LDL-induced upregulation of the apoptosis percentage (P<0.05; Fig. 1E and F). These results suggested that salidroside protects HUVECs from ox-LDL injury.

SIRT1 serves a vital role in promoting cell survival in the development of atherosclerosis (26). In the present study, it was demonstrated that ox-LDL (100 µg/ml) treatment significantly reduced the expression of SIRT1, while this function was significantly abolished by pretreated with salidroside (0.1, 1, or 10 µM; P<0.05; Fig. 2A). According to the Fig. 1 and this result, it was demonstrated that salidroside (10 µM) has a protective effect on ox-LDL injury and ox-LDL-induced SIRT expression downregulation. Therefore, salidroside (10 µM) was selected for subsequent experiments. To further confirm the role of SIRT1 in the cardioprotection of salidroside, HUVECs were transfected with siSIRT1 or siControl. The transfection efficiency was detected using western blotting analysis and the result demonstrated that the expression of SIRT1 in siSIRT1 transfected cells was decreased compared with in the siControl transfected cells (Fig. 2B), indicating that SIRT1 knockdown was achieved by siSIRT1 transfection. In addition, it was demonstrated that ox-LDL-induced downregulation of cell viability was exacerbated by SIRT1 knockdown and the salidroside-induced increase in the cell viability was also significantly inhibited by siSIRT1 transfection in ox-LDL-treated HUEVCs (Fig. 2C). siSIRT1 transfection also further induced the increase in LDH release and reversed salidroside-induced the decrease in the LDH release in ox-LDL-treated HUEVCs (Fig. 2D). In addition, salidroside eliminated, while siSIRT1 exacerbated, ox-LDL-induced the upregulation of apoptosis ratio (Fig. 2E and F) and caspase-3 activity (Fig. 2G). Notably, the inhibition of salidroside on apoptosis was inhibition by transfection with siSIRT1. Together, the results suggested that SIRT1 contributes to the protection of salidroside against ox-LDL injury in HUVECs.

Oxidative stress is involved in the progression of atherosclerosis, which leads to ox-LDL-induced endothelial dysfunction (8). The effects of salidroside on the oxidative stress index including ROS generation, MDA content and NOX2 expression were further investigated with ox-LDL treatment. The result demonstrated that ox-LDL treatment results in an increase in ROS green fluorescence (Fig. 3A), DCF fluorescence percentage (Fig. 3B), MDA content (Fig. 3C) and NOX2 expression (Fig. 3D and E), which were reversed by salidroside. Notably, the effects of salidroside were reversed by SIRT1 knockdown induced by siSIRT1 transfection. Furthermore, siSIRT1 transfection further aggravated ox-LDL-induced oxidative stress. The antioxidants produced endogenously including SOD and GSH-Px serve important roles in preventing oxidative damage in atherosclerosis (27). It was demonstrated that salidroside diminished the ox-LDL-induced downregulation of SOD (Fig. 3F) and GSH-Px activities (Fig. 3G). However, the functions of salidroside were inhibited by siSIRT1 transfection. Taken together, these results indicated that SIRT1 mediates the protective effect of salidroside against oxidative stress and inhibition of antioxidant defense system induced by ox-LDL.

ROS produced by mitochondrial dysfunction affecting cell survival and resulting in apoptosis and oxidative stress have been implicated in the induction of atherosclerosis (28,29). Therefore, whether salidroside effects mitochondrial function in ox-LDL-treated HUVECs and whether SIRT1 pathway is involved in this effect was investigated. JC-1 staining revealed that ox-LDL treatment resulted in the decrease in red fluorescence and the increase in green fluorescence indicating a lowering of MMP, while salidroside pretreatment resulted in the increase red fluorescence and the decrease green fluorescence, leading to increased MMP (Fig. 4A). However, the effect of salidroside was inhibited by siSIRT transfection. As an increase in cytochrome c expression and Bax/Bcl-2 ratio are associated with MMP disruption and mitochondrial dysfunction (29), the effects of salidroside on these markers of mitochondrial function were analyzed. The results of western blot analyses (Fig. 4B) demonstrated that salidroside significantly reduced the expression of cytochrome c (P<0.05; Fig. 4C) and the ratio of Bax/Bcl-2 compared with the ox-LDL treatment group, while these roles were abolished by transfection with siSIRT1 (Fig. 4D). These results suggested that salidroside improves mitochondrial dysfunction under ox-LDL injury conditions through promoting SIRT1 in HUVECs.

AMPK is a critical regulator of mitochondrial biogenesis and is reported to enhance SIRT1 (30). To further certify the role of the AMPK pathway in cardioprotection by salidroside the effects of salidroside on the AMPK pathway following ox-LDL treatment were measured. It was demonstrated that ox-LDL decreased the p-AMPK protein expression in HUVECs, however salidroside significantly upregulated the p-AMPK expression in ox-LDL-treated HUVECs (P<0.05; Fig. 5A and B), indicating that AMPK was activated by salidroside. To further investigate the roles of the AMPK pathway in the protective action of salidroside on ox-LDL injury, HUVECs were transfected with siAMPK. Western blotting analyses validation demonstrated that siAMPK transfection reduced the expression of AMPK compared with siControl transfection (Fig. 5C) following ox-LDL treatment or ox-LDL + salidroside co-treatment. In addition, it was demonstrated that transfection with siAMPK leads to the elimination of the salidroside-induced increase in cell viability (Fig. 5D) and the decrease in LDH release (Fig. 5E) in ox-LDL-treated HUVECs. Notably, it was demonstrated that siAMPK transfection further decreased the expression of SIRT1 compared with the siControl transfection and also reversed salidroside-induced the upregulation of SIRT1 expression (Fig. 5F). In conclusion, the results of the present study suggested that salidroside protects HUVECs against ox-LDL-induced injury through activating the AMPK/SIRT1 pathway.