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Vitexin alleviates ox-LDL-mediated endothelial injury by inducing autophagy via AMPK signaling activation
Molecular Immunology 85 (2017) 214–221
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Molecular Immunology journal homepage: www.elsevier.com/locate/molimm
Vitexin alleviates ox-LDL-mediated endothelial injury by inducing autophagy via AMPK signaling activation Shaoli Zhang, Changlei Guo, Zhigang Chen ∗ , Peiyong Zhang, Jianhua Li, Yan Li Department of Cardiology, The First Affiliated Hospital of Xinxiang Medical University, Xinxiang 453100, China
a r t i c l e
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Article history: Received 28 December 2016 Received in revised form 20 February 2017 Accepted 27 February 2017 Keywords: Vitexin Endothelial dysfunction Oxidative stress Autophagy AMPK signaling
a b s t r a c t Endothelial cell injury plays a crucial role in the development and pathogenesis of cardiovascular disease. Vitexin is a natural flavonoid characterized by anti-oxidative and anti-inflammatory properties. The purpose of this study was to investigate the effects of vitexin on ox-LDL-induced endothelial dysfunction and to explore the underlying molecular mechanisms. In the present study, vitexin was found to play a protective role against ox-LDL-induced endothelial injury. Vitexin significantly promoted cell viability and inhibited apoptosis in ox-LDL-treated HUVECs. The up-regulation of cleaved Caspase-3, cleaved Caspase9 and Bax induced by ox-LDL were inhibited by treatment with vitexin; meanwhile, the down-regulation of Bcl-2 was suppressed by vitexin. Pretreatment with vitexin was found to inhibit the ox-LDL-induced overexpression of IL-1, IL-6, TNF-␣, E-selectin, ICAM1 and VCAM1. Moreover, vitexin reduced ox-LDLinduced oxidative stress by inhibiting the production of ROS and MDA, and by promoting the expression of SOD. Furthermore, we had shown that vitexin protected against the ox-LDL induced cell injury by activating autophagy. The protective effects of vitexin in ox-LDL-treated HUVECs were all reversed following treatment with the autophagy inhibitor 3-MA. In addition, we found that vitexin increased the expression of p-AMPK and decreased the expression of p-mTOR. The combination of the AMPK inhibitor Compound C plus vitexin significantly reversed the effects of vitexin in ox-LDL-treated HUVECs, such as the inhibition of autophagy, reduction in cell viability, increase in apoptosis and ROS production. In conclusion, these data suggest that vitexin ameliorates ox-LDL-mediated endothelial injury by inducing autophagy via AMPK signaling. © 2017 Elsevier Ltd. All rights reserved.
1. Introduction Vascular endothelial cells play a critical role in maintaining cardiovascular homeostasis. As previously reported, endothelial dysfunction is an early step in the development of cardiovascular diseases, including myocardial infarction, cardiac failure and atherosclerosis (AS). A variety of risk factors, such as smoking, diet, infection and air pollution, have been described to induce AS. It has been demonstrated that low-density lipoprotein is an important protein in the progression of AS (Tabas et al., 2007). Oxidized lowdensity lipoprotein (ox-LDL) can lead to the formation of foam cells and exacerbate vascular injury (Itabe, 2012; Kume and Kita, 2004). Therefore, protection of endothelial cells against ox-LDL-induced injury is a therapeutic target for cardiovascular diseases. In recent years, autophagy, as a basic catabolic process, has been attracted increasing attention. Autophagy plays a crucial role
∗ Corresponding author. E-mail address: [email protected] (Z. Chen). http://dx.doi.org/10.1016/j.molimm.2017.02.020 0161-5890/© 2017 Elsevier Ltd. All rights reserved.
in maintaining cellular energetic homeostasis by delivering damaged protein or organelles to lysosomes for degradation (Hamasaki et al., 2013; Xie and Klionsky, 2007). It has been reported that autophagy is involved in various biological processes, such as immunity (Deretic and Levine, 2009), development (Wu et al., 2013), cell death (Levine and Yuan, 2005), neurodegenerative disorders (Nixon, 2013) and cancers (Mathew et al., 2007). As previously reported, the role of autophagy in different conditions is controversial. Under basal conditions, autophagy may help to maintain cellular balance. In response to a variety of environmental stimuli, autophagy can be activated as an adaptive process. Autophagy is usually activated to play a protective role in the early stage of disease. However, the activation of autophagy may sometimes result in disease progression (Levine and Kroemer, 2008). Increasing evidence has shown that autophagy is involved in cardiovascular disease, and its role in endothelial cells has received great interest (Jiang, 2016; Schrijvers et al., 2011). Vitexin is a natural-derived flavonoid compound used as a traditional Chinese medicine in many diseases (Bhardwaj et al., 2015; He et al., 2016). It has been reported that vitexin exhibits broad anti-
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tumor, anti-oxidative and anti-inflammatory activities (An et al., 2015; Farsi et al., 2011; Kim et al., 2005; Lee et al., 2011; Tan et al., 2012). Vitexin plays a key role in cardiovascular disorders like myocardial ischemia reperfusion injury (Dong et al., 2011; Dong et al., 2013) as well as cardiac hypertrophy (Lu et al., 2013), suggesting that vitexin may be a strategy to treat cardiovascular diseases. Nevertheless, the underlying molecular mechanisms of vitexin in cardiovascular diseases are still not fully clear. Thus, an investigation into the effects of vitexin in endothelial dysfunction may contribute to understanding the role of vitexin in cardiovascular disease. In the present study, we aimed to investigate the effects of vitexin in ox-LDL-treated endothelial cells. We assessed its effects on cell viability, apoptosis, inflammatory factor production, adhesion molecule expression and ROS generation, and further investigated the underlying mechanisms. 2. Materials and methods 2.1. Materials Vitexin, 3-methyladenine (3-MA) and Compound C (C.C) were purchased from Sigma-Aldrich. Malondialdehyde (MDA) and superoxide dismutase (SOD) commercial kits were purchased from Jiancheng Bioengineering Institute. The DCFH-DA ROS Detection Kit was purchased from Beyotime Institute of Biotechnology. Anti-cleaved Caspase-3, anti-cleaved Caspase-9, anti-Bax, anti-Bcl2, anti-AMPK, anti-p-AMPK, anti-mTOR and anti-p-mTOR were purchased from Cell Signaling Technology. Anti-IL-1, anti-IL-6, anti-TNF-␣, anti-E-selectin, anti-ICAM1, anti-VCAM1 and antiGAPDH were purchased from Santa Cruz Biotechnology. Anti-Atg5, anti-Atg7, anti-Atg12, anti-LC3, anti-Beclin1and anti-p62 were purchased from Invitrogen.
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dissolving in 200 l of DMSO. The absorbance value was determined at a wavelength of 490 nm. 2.5. Apoptosis analysis HUVECs were seeded in a 6-well plate and treated according to the requirements of the different groups. Apoptosis was determined by flow cytometry. Briefly, the differently treated cells were washed in PBS and resuspended in binding buffer. Then the cells were cultured with 5 l Annexin V-FITC and 10 l PI at room temperature in the dark for 20 min. The apoptosis rates were analyzed using a FACSCalibur (Becton Dickinson) within 1 h. 2.6. Western blot analysis Cell protein extractions were prepared using lysis buffer. Protein concentration was quantified using BCA protein assay kit (Pierce, USA). Then the protein samples were separated by SDS-PAGE and transferred onto polyvinylidene difluoride (PVDF) membranes. After blocking in 5% bovine serum albumin (BSA), the membranes were immunoblotted with primary antibodies at 4 ◦ C overnight: anti-cleaved Caspase-3 (1:500), anti-cleaved Caspase-9 (1:500), anti-Bax (1:500), anti-Bcl-2 (1: 500), anti-IL-1 (1:1000), anti-IL-6 (1:1000), anti-TNF-␣ (1:1000), anti-E-selectin (1:800), anti-ICAM1(1:500), anti-VCAM1(1:500), anti-Atg5 (1:500), antiAtg7 (1:500), anti-Atg12 (1:500), anti-LC3 (1:500), anti-Beclin1 (1:1000), anti-p62 (1:1000), anti-AMPK (1:500), anti-p-AMPK (1:500), anti-mTOR (1:500), anti-p-mTOR (1:500) and anti-GAPDH (1:1000) followed by incubating with horseradish peroxidaseconjugated secondary antibodies at room temperature for 1 h. Signals were detected using ECL reagent (Millipore, Billerica MA, USA), and the relative intensities of protein bands were quantified using Image J software. 2.7. Intracellular ROS measurement
2.2. Cell culture HUVECs were purchased from Shanghai Gene Chemical Co., Ltd (Shanghai, China). They were cultured in DMEM supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin and 100 g/ml streptomycin. The cells were maintained at 37 ◦ C in a humidified chamber containing 5% CO2 . The cells were subcultured every 48 h with 0.25% trypsin digestion. Cells between passages 5–11 were used in the experiments. 2.3. Cell groups division The HUVECs were divided into six groups: control group, normal cultured HUVECs; vitexin group, cells treated with 1, 10, 20, 30, 40 and 50 M vitexin for 24 h; ox-LDL group, cells treated with 100 g/ml ox-LDL for 24 h; vitexin + ox-LDL group, cells treated with 1, 10 and 20 M vitexin for 2 h followed by treatment with 100 g/ml ox-LDL for 24 h; 3-MA + vitexin + ox-LDL group, cells treated with 5 mM 3-MA for 2 h prior to treatment of 20 M vitexin for 2 h, followed by treatment with 100 g/ml ox-LDL for 24 h; C.C + vitexin + ox-LDL group, cells treated with 5 mM C.C for 2 h prior to treatment of 20 M vitexin for 2 h, followed by treatment with 100 g/ml ox-LDL for 24 h. 2.4. Cell viability analysis HUVECs were seeded in a 96-well plate and treated according to the requirements of the different groups. Cell viability was measured by MTT assay. Briefly, the differently treated cells were treated with 100 g/ml MTT for 4 h in a 37 ◦ C incubator, following
ROS levels were determined by 2 ,7 -dichlorodihydrofluorescein diacetate probes (DCHF-DA). DCHF-DA oxidation could product the fluorescent 2 ,7 -dichlorofluorescein (DCF). In brief, cells were incubated with 10 M DCHF-DA for 20 min and then analyzed by flow cytometry. 2.8. Measurement of SOD and MDA levels The intracellular SOD and MDA levels were measured using commercially available assay kits according to the manufacturer’s guidelines. 2.9. Statistical analysis Data were analyzed by one-way ANOVA using GraphPad Prism 5.0 (GraphPad Software, San Diego, CA, USA). Data are shown as the mean ± standard error of the mean (SEM). P < 0.05 was considered to be statistically significant. 3. Results 3.1. Cytotoxic effect of vitexin in HUVECs The structure of vitexin is shown in Fig. 1A. To measure the cytotoxic effect of vitexin in HUVECs, the cells were treated with different concentrations of vitexin for 24 h and cell viability was measured by the MTT assay. As shown in Fig. 1B, the viability of cells treated with less than 20 M vitexin underwent almost no changes as compared with the control group. Cell viability was reduced after treatment with 30 M vitexin and the reduction was significant
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Fig. 1. Cytotoxic effect of vitexin in HUVECs. (A) The structure of vitexin. (B) Cells were treated with 0, 1, 10, 20, 30, 40 and 50 M vitexin for 24 h and cell viability was determined by the MTT assay. *P < 0.05 vs. control group. The experiment was performed in triplicate.
when the concentration of vitexin was more than 40 M. In subsequent experiments, the cells were treated with less than 20 M vitexin to determine its effect on endothelial injury.
3.2. Vitexin promotes cell viability and suppresses apoptosis in ox-LDL-treated HUVECs To investigate the effects of vitexin on the cell viability in ox-LDL-induced HUVECs, the cells were exposed to different concentrations (1, 10 and 20 M) of vitexin and the MTT assay was performed. As shown in Fig. 2A, ox-LDL treatment significantly reduced cell viability as compared with the control group. The ox-LDL-induced reduction in cell viability was inhibited by increasing concentrations of vitexin. We further measured apoptosis by flow cytometry. As shown in Fig. 2B and C, apoptotic cells were markedly increased in the ox-LDL group, which was dosedependently decreased by treatment with vitexin. These results suggest that vitexin exerted a dose-dependent protective effect on ox-LDL-induced endothelial injury by promoting cell viability and suppressing apoptosis. In addition, to assess detect the molecular mechanisms of apoptosis, Western blot was performed to measure the expression levels of the apoptosis-related proteins in response to 20 M vitexin. As shown in Fig. 2D and E, compared with the control group, ox-LDL treatment significantly up-regulated the expression of cleaved Caspase-3, cleaved Caspase9 and Bax, which were down-regulated following treatment with vitexin. Meanwhile, the ox-LDL-induced reduced expression of Bcl2 was significantly inhibited by 20 M vitexin. Together, these results suggest that vitexin suppresses ox-LDL-induced apoptosis by down-regulating pro-apoptosis protein and up-regulating antiapoptosis protein.
3.3. Vitexin inhibits the expression of ox-LDL-induced inflammatory factors and adhesion molecules To further investigate the protective role of vitexin in endothelial injury, we performed Western blot to measure the protein expression of inflammatory factors and adhesion molecules. As shown in Fig. 3A and B, as compared with the control group, the protein levels of IL-1, IL-6 and TNF-␣ were obviously increased following ox-LDL treatment. As compared with the ox-LDL group, the expression of IL-1, IL-6 and TNF-␣ was significantly decreased in the vitexin plus ox-LDL group. As shown in Fig. 3C and D, the adhesion molecules E-selectin, VCAM1 and ICAM1 were markedly up-regulated in the ox-LDL group as compared with the control group, while the expression was down-regulated by vitexin treatment. These results suggest that vitexin protects HUVECs against ox-LDL-induced inflammatory injury.
3.4. Vitexin suppresses ox-LDL-induced oxidative stress in HUVECs In order to investigate oxidative stress in HUVECs, the production of ROS and the intracellular levels of SOD and MDA were evaluated. As shown in Fig. 4A and B, the levels of ROS in the ox-LDL group were significantly higher than in the control group, and vitexin treatment significantly reduced ROS levels as compared with the ox-LDL group. As shown in Fig. 4C and D, compared with the control group, the levels of SOD were obviously down-regulated in the ox-LDL group, and the levels of MDA was significantly upregulated. Pretreatment with 20 M vitexin was found to increase the levels of SOD levels and to decrease MDA levels. These results indicate that vitexin may play a protective role in ox-LDL-induced endothelial injury, at least partly by suppressing oxidative stress. 3.5. Vitexin attenuates ox-LDL-induced endothelial injury by inducing autophagy To determine whether autophagy was involved in the effects of vitexin, the levels of autophagy-related proteins were measured by Western blot. As shown in Fig. 5A–C, the protein levels of Atg5, Atg7, Atg12 and Beclin1 underwent a slight increase in the oxLDL group as compared with the control group, while the level of p62 was reduced, and the expression of LC3-II was significantly increased, suggesting that autophagy may be induced in response to ox-LDL stimulation to avoid injury. Following pretreatment with vitexin, the expression of Atg5, Atg7, Atg12, LC3-II and Beclin1 showed a more significant up-regulation as compared with the oxLDL group, while the level of p62 was even more suppressed. To further explore the effects of autophagy in the protective action mediated by vitexin, we used 3-MA, a specific autophagy inhibitor, to determine the changes following ox-LDL-induced injury. Firstly, the Western blot results showed that 3-MA treatment significantly inhibited the expression of autophagy-related proteins (Fig. 5A–C). Then, we assessed cell viability and apoptosis by the MTT assay and flow cytometry, respectively. Cell viability in the vitexin plus oxLDL group was significantly elevated as compared with the ox-LDL group, which is consistent with our previous results. However, 3MA treatment counteracted the protective effect of vitexin on cell viability (Fig. 5D). The flow cytometry results also demonstrate that the inhibition of apoptosis by vitexin in ox-LDL-induced HUVECs was significantly weakened following 3-MA treatment (Fig. 5E). These results suggest that vitexin may exert a protective role in ox-LDL-induced HUVECs by inducing autophagy. To further confirm the important role of autophagy in the function of vitexin, we measured the protein expression of inflammatory factors and adhesion molecules by Western blot. As shown in Fig. 5F-H, 3MA treatment significantly enhanced the expression of IL-1, IL-6,
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Fig. 2. The effects of vitexin on cell viability and apoptosis in ox-LDL-treated HUVECs. Cells were treated with 1, 10 and 20 M vitexin for 2 h followed by treatment with ox-LDL for 24 h. (A) Cell viability was measured by the MTT assay. (B) Apoptosis was measured by flow cytometry. (C) The rate of apoptosis is indicated by histograms. (D) The expression levels of cleaved Caspase-3, cleaved Caspase-9, Bax and Bcl-2 were measured by Western blot. (E) The relative protein expression is indicated by histograms. *P < 0.05 vs. control group, #P < 0.05 vs. ox-LDL group. Each experiment was performed in triplicate.
Fig. 3. The effects of vitexin on ox-LDL-induced inflammatory and adhesion molecule expression. Cells were treated with 20 M vitexin for 2 h following by treatment with ox-LDL for 24 h. (A) The expression of IL-1, IL-6 and TNF-␣ were measured by Western blot. (B) The relative protein expression is indicated by histograms. (C) The expression of E-selectin, VCAM1 and ICAM1 were determined by Western blot. (D) The relative protein expression is indicated by histograms. *P < 0.05 vs. control group, #P < 0.05 vs. ox-LDL group. Each experiment was performed in triplicate.
TNF-␣, E-selectin, VCAM1 and ICAM1 as compared with the vitexin plus ox-LDL group. In addition, the reduction of ROS levels induced by vitexin was inhibited following 3-MA treatment (Fig. 5I). Taken together, these results demonstrate that vitexin attenuates ox-LDLinduced endothelial injury by inducing autophagy. 3.6. Vitexin plays a protective role against ox-LDL-induced endothelial injury by activating AMPK signaling It has been reported that AMPK is important in maintaining normal endothelial function, and flavonoids are considered to be AMPK activators (Gasparrini et al., 2016). So, we investigated whether the
protective role of vitexin in ox-LDL-treated HUVECs was associated with AMPK signaling. As compared with the control group, the activated p-AMPK expression level was markedly reduced, meanwhile the activated p-mTOR level was significantly enhanced in ox-LDLtreated HUVECs. When the cells were pretreated with vitexin, the p-AMPK level showed an obvious increase and the p-mTOR level was significantly suppressed (Fig. 6A and B). These results demonstrate that AMPK signaling is involved in the regulation of vitexin in ox-LDL-treated HUVECs. To further identify whether the activation of AMPK signaling was related to autophagy mediated by vitexin, after blocking AMPK activity with the AMPK inhibitor C.C, we measured the expression of autophagy-related proteins. As
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Fig. 4. The effects of vitexin on the ox-LDL-induced oxidative stress in HUVECs. Cells were treated with 20 M vitexin for 2 h following by treatment with ox-LDL for 24 h. (A) The ROS production was measured by flow cytometry using DCHF-DA probes. (B) The relative quantification of ROS is indicated by histograms. (C) The levels of SOD are indicated by histograms. (D) The levels of MDA are indicated by histograms. *P < 0.05 vs. control group, #P < 0.05 vs. ox-LDL group. Each experiment was performed in triplicate.
shown in Fig. 6A and B, C.C treatment significantly suppressed AMPK signaling by down-regulating the p-AMPK level and upregulating the p-mTOR level. We found that the vitexin-induced expression of autophagy-related proteins was dramatically inhibited following C.C treatment (Fig. 6C-E), demonstrating that AMPK signaling may be involved in the regulation of autophagy induced by vitexin. We further investigated whether blocking AMPK signaling was involved in the regulation of endothelial injury by vitexin; the results are shown in Fig. 6F-H. Consistent with our previous study, vitexin protected HUVECs against ox-LDL-induced injury by promoting cell viability, inhibiting apoptosis and reducing ROS production. However, these protective effects were reversed by C.C treatment. These results show that vitexin protects against ox-LDLinduced endothelial injury by regulating AMPK-mTOR signaling. 4. Discussion Endothelial cell dysfunction has been found to be the first cellular event in the pathogenesis of AS. Many previous studies have shown the protective effect of flavonoids in endothelial dysfunction, such as quercitrin (Zhi et al., 2016), ampelopsin (Liang et al., 2015), astragalin (Cho et al., 2014) and luteolin (Xia et al., 2014). Vitexin, as a natural-derived flavonoid, has been used in heat stress-induced oxidative injury (Bhardwaj et al., 2015), lifespan extension (Lee et al., 2015) and cancers (He et al., 2016) because of its anti-cancer, anti-oxidative and anti-inflammatory activities. With regard to the fact that endothelial dysfunction is a process including inflammation and oxidative stress, we tried to investigate the effects of vitexin on endothelial injury. Although several investigations in cancer cell lines have shown that the regulation of vitexin was involved in apoptosis and autophagy via activation of the JNK signaling pathway (He et al., 2016; Zhou et al., 2013), the functions and underlying molecular mechanisms of vitexin in different cell lines and diseases were closely associated with its concentrations in different experimental conditions. In present
study, for the first time we showed that vitexin could alleviate oxLDL-induced endothelial injury by inducing autophagy via AMPK signaling activation, contributing to understand the role of vitexin in cardiovascular disease. It has been reported that endothelial cell apoptosis can destroy the structure and function of the endothelium, accelerating the formation of foam cells. So, we first measured cell viability and apoptosis by the MTT assay and flow cytometry to assess the effects of vitexin on ox-LDL-induced HUVECs. The MTT results show that the cell viability reduction induced by ox-LDL was significantly ameliorated by treatment with vitexin in a dose-dependent manner. The flow cytometry results show that vitexin dose-dependently inhibited ox-LDL-induced apoptosis. In addition, we measured the expression of apoptosis-related proteins by Western blot assay. Caspase-9 is an initiator caspase and Caspase-3 is closely related to apoptosis. Bcl-2 is considered to be an anti-apoptotic protein while Bax is regarded as a pro-apoptosis protein (Bhola and Letai, 2016). In this study, we observed that the overexpression of cleaved Caspase-3, cleaved Caspase-9 and Bax induced by ox-LDL treatment was significantly down-regulated following treatment with vitexin. Meanwhile, the ox-LDL-induced reduction in the expression of Bcl2 was significantly inhibited by vitexin. Together, these results suggest that vitexin exerts a dose-dependent protective effect on ox-LDL-induced endothelial injury by promoting cell viability and suppressing apoptosis. Inflammation has been found to play a crucial role in the endothelial dysfunction. The expression of inflammatory cytokines, including inflammatory factors and cell adhesion molecules, can promote the adhesion and infiltration of monocytes to the vascular endotheliu, leading to the activation of macrophages; these macrophages then absorb lipoprotein, resulting in foam cell formation, which further stimulates vascular inflammation (Meliton et al., 2015; Yamagata et al., 2009). In the present study, we found that inflammatory factors such as IL-1, IL-6 and TNF-␣, were significantly increased by ox-LDL treatment; this increase was
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Fig. 5. Vitexin attenuates ox-LDL-induced endothelial injury by inducing autophagy. Cells were treated with 3-MA for 2 h and then incubated with 20 M vitexin for 2 h followed by treatment with ox-LDL for 24 h. (A) The expression of Atg5, Atg7, Atg12, LC3-II, Beclin1and p62 were measured by Western blot. (B and C) The relative protein expression is indicated by histograms. (D) Cell viability was measured by the MTT assay. (E) Apoptosis was measured by flow cytometry. The rate of apoptosis is indicated by histograms. (F) The expression of IL-1, IL-6, TNF-␣, E-selectin, VCAM1 and ICAM1 were determined by Western blot. (G-H) The relative protein expression is indicated by histograms. (I) ROS production was measured by flow cytometry. The relative quantification of ROS is indicated by histograms. & P < 0.05 vs. control group, *P < 0.05 vs. ox-LDL group, #P < 0.05 vs. vitexin + ox-LDL group. Each experiment was performed in triplicate.
inhibited by vitexin treatment. Our results show that the expression of E-selectin, VCAM1 and ICAM1 in the ox-LDL group was markedly up-regulated as compared with the control group, while the expression was down-regulated following vitexin treatment. These results suggest that vitexin protects HUVECs against ox-LDLinduced inflammatory injury. Oxidative stress has been shown to be closely associated with various pathological processes, such as apoptosis, inflammation and autophagy. Oxidative stress is usually regarded as increased bioactivity of ROS relative to antioxidant defenses (Kregel and Zhang, 2007). Elevated ROS levels play a pivotal role in many cardiovascular diseases, such as metabolic syndrome, type 2 diabetes ˜ et al., 2013; Ilkun and Boudina, 2013). SOD and dyslipidemia (Farina is a major antioxidant enzyme that counteracts oxidative damage and MDA is a marker of lipid peroxidation. In our study, we show that ox-LDL treatment caused an increase in ROS and MDA, and a decrease in SOD levels as compared with the control group. In the vitexin plus ox-LDL group, the production of ROS and MDA decreased and the SOD level increased, suggesting that vitexin protects HUVECs against oxidative damage.
Previous reports have shown that natural extracts may exert their effects through regulating autophagy. Although the function of activated autophagy is controversial, there are still many studies indicating that some flavonoid compounds protect cells against injury by inducing autophagy (Liang et al., 2015; Zhi et al., 2016). To determine whether autophagy was involved in the effects of vitexin, the levels of autophagy-related proteins were measured by Western blot. LC3II is usually regarded as a marker of autophagy (Giménez-Xavier et al., 2009). It has been reported that Atg5, Atg7 and Atg12 are crucial for the initiation of autophagy. Beclin1 is also essential for the induction of autophagy as it regulates the formation of the autophagosome. The protein levels of Atg5, Atg7, Atg12, LC3-II and Beclin1 underwent an obvious increase in the ox-LDL group, while the level of p62 was reduced, suggesting that autophagy was induced in response to ox-LDL treatment to avoid injury. The expression of Atg5, Atg7, Atg12, LC3-II and Beclin1 showed a more significant up-regulation following vitexin treatment, while the level of p62 was even lower, suggesting that vitexin treatment induced autophagy in ox-LDL-treated HUVECs. To assess the effects of autophagy in the protective action mediated by vitexin, we used 3-MA to assess endothelial injury. We
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Fig. 6. Vitexin plays a protective role in ox-LDL-induced endothelial injury by activating AMPK signaling. Cells were treated with C. C for 2 h and then incubated with 20 M vitexin for 2 h followed by treatment with ox-LDL for 24 h. (A) The expression of p-AMPK, AMPK, p-mTOR and mTOR were determined by Western blot. (B) The relative protein expression isindicated by histograms. (C) The expression of Atg5, Atg7, Atg12, LC3-II, Beclin1and p62 were measured by Western blot. (D and E) The relative protein expression is indicated by histograms. (F) Cell viability was measured by the MTT assay. (G) Apoptosis was measured by flow cytometry. The rate of apoptosis is indicated by histograms. (H) ROS production was measured by flow cytometry. The relative quantification of ROS is indicated by histograms. & P < 0.05 vs. control group, *P < 0.05 vs. ox-LDL group, #P < 0.05 vs. vitexin + ox-LDL group. Each experiment was performed in triplicate.
show that 3-MA plus vitexin significantly decreased cell viability, increased apoptosis, enhanced the expression of IL-1, IL-6, TNF␣, E-selectin, VCAM1 and ICAM1 and elevated the production of ROS as compared with vitexin treatment alone. Taken together, these results demonstrate that vitexin attenuates ox-LDL-induced endothelial injury by inducing autophagy. AMP-activated protein kinase (AMPK) is a serine/threonine protein kinase that is involved in the regulation of cellular metabolism (Mizushima et al., 2008). It has been reported that AMPK is a positive regulator of autophagy, and suppresses the mTOR pathway (Aoki et al., 2007; Kim et al., 2011). Increasing evidence indicates that mTOR inhibition stabilizes atherosclerotic plaques and slows down the progression of AS (Baetta et al., 2009; Chen et al., 2009; Mueller et al., 2008; Waksman et al., 2003). Flavonoids have been reported to activate AMPK signaling (Gasparrini et al., 2016). To determine the involvement of AMPK signaling in the protective actin of vitexin, the Western blot results show that vitexin upregulated the p-AMPK expression and down-regulated the p-mTOR level. Furthermore, after blocking AMPK activity by the AMPK inhibitor C.C, vitexin-induced autophagy was dramatically inhibited, demonstrating that AMPK signaling may be involved in the regulation of autophagy induced by vitexin. We further investigated the effects of blocking AMPK signaling on endothelial injury. The results demonstrate that the protective effects of vitexin were all reversed by C.C treatment. These results imply that vitexin plays a protective role against ox-LDL-induced endothelial injury partly by regulating AMPK-mTOR signaling. In summary, these results demonstrate that vitexin promotes cell viability, inhibits apoptosis, down-regulates inflammatory factors and adhesion molecules, and suppresses oxidative stress in ox-LDL-treated HUVECs, which may be related to the activation of
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Molecular Immunology journal homepage: www.elsevier.com/locate/molimm
Vitexin alleviates ox-LDL-mediated endothelial injury by inducing autophagy via AMPK signaling activation Shaoli Zhang, Changlei Guo, Zhigang Chen ∗ , Peiyong Zhang, Jianhua Li, Yan Li Department of Cardiology, The First Affiliated Hospital of Xinxiang Medical University, Xinxiang 453100, China
a r t i c l e
i n f o
Article history: Received 28 December 2016 Received in revised form 20 February 2017 Accepted 27 February 2017 Keywords: Vitexin Endothelial dysfunction Oxidative stress Autophagy AMPK signaling
a b s t r a c t Endothelial cell injury plays a crucial role in the development and pathogenesis of cardiovascular disease. Vitexin is a natural flavonoid characterized by anti-oxidative and anti-inflammatory properties. The purpose of this study was to investigate the effects of vitexin on ox-LDL-induced endothelial dysfunction and to explore the underlying molecular mechanisms. In the present study, vitexin was found to play a protective role against ox-LDL-induced endothelial injury. Vitexin significantly promoted cell viability and inhibited apoptosis in ox-LDL-treated HUVECs. The up-regulation of cleaved Caspase-3, cleaved Caspase9 and Bax induced by ox-LDL were inhibited by treatment with vitexin; meanwhile, the down-regulation of Bcl-2 was suppressed by vitexin. Pretreatment with vitexin was found to inhibit the ox-LDL-induced overexpression of IL-1, IL-6, TNF-␣, E-selectin, ICAM1 and VCAM1. Moreover, vitexin reduced ox-LDLinduced oxidative stress by inhibiting the production of ROS and MDA, and by promoting the expression of SOD. Furthermore, we had shown that vitexin protected against the ox-LDL induced cell injury by activating autophagy. The protective effects of vitexin in ox-LDL-treated HUVECs were all reversed following treatment with the autophagy inhibitor 3-MA. In addition, we found that vitexin increased the expression of p-AMPK and decreased the expression of p-mTOR. The combination of the AMPK inhibitor Compound C plus vitexin significantly reversed the effects of vitexin in ox-LDL-treated HUVECs, such as the inhibition of autophagy, reduction in cell viability, increase in apoptosis and ROS production. In conclusion, these data suggest that vitexin ameliorates ox-LDL-mediated endothelial injury by inducing autophagy via AMPK signaling. © 2017 Elsevier Ltd. All rights reserved.
1. Introduction Vascular endothelial cells play a critical role in maintaining cardiovascular homeostasis. As previously reported, endothelial dysfunction is an early step in the development of cardiovascular diseases, including myocardial infarction, cardiac failure and atherosclerosis (AS). A variety of risk factors, such as smoking, diet, infection and air pollution, have been described to induce AS. It has been demonstrated that low-density lipoprotein is an important protein in the progression of AS (Tabas et al., 2007). Oxidized lowdensity lipoprotein (ox-LDL) can lead to the formation of foam cells and exacerbate vascular injury (Itabe, 2012; Kume and Kita, 2004). Therefore, protection of endothelial cells against ox-LDL-induced injury is a therapeutic target for cardiovascular diseases. In recent years, autophagy, as a basic catabolic process, has been attracted increasing attention. Autophagy plays a crucial role
∗ Corresponding author. E-mail address: [email protected] (Z. Chen). http://dx.doi.org/10.1016/j.molimm.2017.02.020 0161-5890/© 2017 Elsevier Ltd. All rights reserved.
in maintaining cellular energetic homeostasis by delivering damaged protein or organelles to lysosomes for degradation (Hamasaki et al., 2013; Xie and Klionsky, 2007). It has been reported that autophagy is involved in various biological processes, such as immunity (Deretic and Levine, 2009), development (Wu et al., 2013), cell death (Levine and Yuan, 2005), neurodegenerative disorders (Nixon, 2013) and cancers (Mathew et al., 2007). As previously reported, the role of autophagy in different conditions is controversial. Under basal conditions, autophagy may help to maintain cellular balance. In response to a variety of environmental stimuli, autophagy can be activated as an adaptive process. Autophagy is usually activated to play a protective role in the early stage of disease. However, the activation of autophagy may sometimes result in disease progression (Levine and Kroemer, 2008). Increasing evidence has shown that autophagy is involved in cardiovascular disease, and its role in endothelial cells has received great interest (Jiang, 2016; Schrijvers et al., 2011). Vitexin is a natural-derived flavonoid compound used as a traditional Chinese medicine in many diseases (Bhardwaj et al., 2015; He et al., 2016). It has been reported that vitexin exhibits broad anti-
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tumor, anti-oxidative and anti-inflammatory activities (An et al., 2015; Farsi et al., 2011; Kim et al., 2005; Lee et al., 2011; Tan et al., 2012). Vitexin plays a key role in cardiovascular disorders like myocardial ischemia reperfusion injury (Dong et al., 2011; Dong et al., 2013) as well as cardiac hypertrophy (Lu et al., 2013), suggesting that vitexin may be a strategy to treat cardiovascular diseases. Nevertheless, the underlying molecular mechanisms of vitexin in cardiovascular diseases are still not fully clear. Thus, an investigation into the effects of vitexin in endothelial dysfunction may contribute to understanding the role of vitexin in cardiovascular disease. In the present study, we aimed to investigate the effects of vitexin in ox-LDL-treated endothelial cells. We assessed its effects on cell viability, apoptosis, inflammatory factor production, adhesion molecule expression and ROS generation, and further investigated the underlying mechanisms. 2. Materials and methods 2.1. Materials Vitexin, 3-methyladenine (3-MA) and Compound C (C.C) were purchased from Sigma-Aldrich. Malondialdehyde (MDA) and superoxide dismutase (SOD) commercial kits were purchased from Jiancheng Bioengineering Institute. The DCFH-DA ROS Detection Kit was purchased from Beyotime Institute of Biotechnology. Anti-cleaved Caspase-3, anti-cleaved Caspase-9, anti-Bax, anti-Bcl2, anti-AMPK, anti-p-AMPK, anti-mTOR and anti-p-mTOR were purchased from Cell Signaling Technology. Anti-IL-1, anti-IL-6, anti-TNF-␣, anti-E-selectin, anti-ICAM1, anti-VCAM1 and antiGAPDH were purchased from Santa Cruz Biotechnology. Anti-Atg5, anti-Atg7, anti-Atg12, anti-LC3, anti-Beclin1and anti-p62 were purchased from Invitrogen.
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dissolving in 200 l of DMSO. The absorbance value was determined at a wavelength of 490 nm. 2.5. Apoptosis analysis HUVECs were seeded in a 6-well plate and treated according to the requirements of the different groups. Apoptosis was determined by flow cytometry. Briefly, the differently treated cells were washed in PBS and resuspended in binding buffer. Then the cells were cultured with 5 l Annexin V-FITC and 10 l PI at room temperature in the dark for 20 min. The apoptosis rates were analyzed using a FACSCalibur (Becton Dickinson) within 1 h. 2.6. Western blot analysis Cell protein extractions were prepared using lysis buffer. Protein concentration was quantified using BCA protein assay kit (Pierce, USA). Then the protein samples were separated by SDS-PAGE and transferred onto polyvinylidene difluoride (PVDF) membranes. After blocking in 5% bovine serum albumin (BSA), the membranes were immunoblotted with primary antibodies at 4 ◦ C overnight: anti-cleaved Caspase-3 (1:500), anti-cleaved Caspase-9 (1:500), anti-Bax (1:500), anti-Bcl-2 (1: 500), anti-IL-1 (1:1000), anti-IL-6 (1:1000), anti-TNF-␣ (1:1000), anti-E-selectin (1:800), anti-ICAM1(1:500), anti-VCAM1(1:500), anti-Atg5 (1:500), antiAtg7 (1:500), anti-Atg12 (1:500), anti-LC3 (1:500), anti-Beclin1 (1:1000), anti-p62 (1:1000), anti-AMPK (1:500), anti-p-AMPK (1:500), anti-mTOR (1:500), anti-p-mTOR (1:500) and anti-GAPDH (1:1000) followed by incubating with horseradish peroxidaseconjugated secondary antibodies at room temperature for 1 h. Signals were detected using ECL reagent (Millipore, Billerica MA, USA), and the relative intensities of protein bands were quantified using Image J software. 2.7. Intracellular ROS measurement
2.2. Cell culture HUVECs were purchased from Shanghai Gene Chemical Co., Ltd (Shanghai, China). They were cultured in DMEM supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin and 100 g/ml streptomycin. The cells were maintained at 37 ◦ C in a humidified chamber containing 5% CO2 . The cells were subcultured every 48 h with 0.25% trypsin digestion. Cells between passages 5–11 were used in the experiments. 2.3. Cell groups division The HUVECs were divided into six groups: control group, normal cultured HUVECs; vitexin group, cells treated with 1, 10, 20, 30, 40 and 50 M vitexin for 24 h; ox-LDL group, cells treated with 100 g/ml ox-LDL for 24 h; vitexin + ox-LDL group, cells treated with 1, 10 and 20 M vitexin for 2 h followed by treatment with 100 g/ml ox-LDL for 24 h; 3-MA + vitexin + ox-LDL group, cells treated with 5 mM 3-MA for 2 h prior to treatment of 20 M vitexin for 2 h, followed by treatment with 100 g/ml ox-LDL for 24 h; C.C + vitexin + ox-LDL group, cells treated with 5 mM C.C for 2 h prior to treatment of 20 M vitexin for 2 h, followed by treatment with 100 g/ml ox-LDL for 24 h. 2.4. Cell viability analysis HUVECs were seeded in a 96-well plate and treated according to the requirements of the different groups. Cell viability was measured by MTT assay. Briefly, the differently treated cells were treated with 100 g/ml MTT for 4 h in a 37 ◦ C incubator, following
ROS levels were determined by 2 ,7 -dichlorodihydrofluorescein diacetate probes (DCHF-DA). DCHF-DA oxidation could product the fluorescent 2 ,7 -dichlorofluorescein (DCF). In brief, cells were incubated with 10 M DCHF-DA for 20 min and then analyzed by flow cytometry. 2.8. Measurement of SOD and MDA levels The intracellular SOD and MDA levels were measured using commercially available assay kits according to the manufacturer’s guidelines. 2.9. Statistical analysis Data were analyzed by one-way ANOVA using GraphPad Prism 5.0 (GraphPad Software, San Diego, CA, USA). Data are shown as the mean ± standard error of the mean (SEM). P < 0.05 was considered to be statistically significant. 3. Results 3.1. Cytotoxic effect of vitexin in HUVECs The structure of vitexin is shown in Fig. 1A. To measure the cytotoxic effect of vitexin in HUVECs, the cells were treated with different concentrations of vitexin for 24 h and cell viability was measured by the MTT assay. As shown in Fig. 1B, the viability of cells treated with less than 20 M vitexin underwent almost no changes as compared with the control group. Cell viability was reduced after treatment with 30 M vitexin and the reduction was significant
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Fig. 1. Cytotoxic effect of vitexin in HUVECs. (A) The structure of vitexin. (B) Cells were treated with 0, 1, 10, 20, 30, 40 and 50 M vitexin for 24 h and cell viability was determined by the MTT assay. *P < 0.05 vs. control group. The experiment was performed in triplicate.
when the concentration of vitexin was more than 40 M. In subsequent experiments, the cells were treated with less than 20 M vitexin to determine its effect on endothelial injury.
3.2. Vitexin promotes cell viability and suppresses apoptosis in ox-LDL-treated HUVECs To investigate the effects of vitexin on the cell viability in ox-LDL-induced HUVECs, the cells were exposed to different concentrations (1, 10 and 20 M) of vitexin and the MTT assay was performed. As shown in Fig. 2A, ox-LDL treatment significantly reduced cell viability as compared with the control group. The ox-LDL-induced reduction in cell viability was inhibited by increasing concentrations of vitexin. We further measured apoptosis by flow cytometry. As shown in Fig. 2B and C, apoptotic cells were markedly increased in the ox-LDL group, which was dosedependently decreased by treatment with vitexin. These results suggest that vitexin exerted a dose-dependent protective effect on ox-LDL-induced endothelial injury by promoting cell viability and suppressing apoptosis. In addition, to assess detect the molecular mechanisms of apoptosis, Western blot was performed to measure the expression levels of the apoptosis-related proteins in response to 20 M vitexin. As shown in Fig. 2D and E, compared with the control group, ox-LDL treatment significantly up-regulated the expression of cleaved Caspase-3, cleaved Caspase9 and Bax, which were down-regulated following treatment with vitexin. Meanwhile, the ox-LDL-induced reduced expression of Bcl2 was significantly inhibited by 20 M vitexin. Together, these results suggest that vitexin suppresses ox-LDL-induced apoptosis by down-regulating pro-apoptosis protein and up-regulating antiapoptosis protein.
3.3. Vitexin inhibits the expression of ox-LDL-induced inflammatory factors and adhesion molecules To further investigate the protective role of vitexin in endothelial injury, we performed Western blot to measure the protein expression of inflammatory factors and adhesion molecules. As shown in Fig. 3A and B, as compared with the control group, the protein levels of IL-1, IL-6 and TNF-␣ were obviously increased following ox-LDL treatment. As compared with the ox-LDL group, the expression of IL-1, IL-6 and TNF-␣ was significantly decreased in the vitexin plus ox-LDL group. As shown in Fig. 3C and D, the adhesion molecules E-selectin, VCAM1 and ICAM1 were markedly up-regulated in the ox-LDL group as compared with the control group, while the expression was down-regulated by vitexin treatment. These results suggest that vitexin protects HUVECs against ox-LDL-induced inflammatory injury.
3.4. Vitexin suppresses ox-LDL-induced oxidative stress in HUVECs In order to investigate oxidative stress in HUVECs, the production of ROS and the intracellular levels of SOD and MDA were evaluated. As shown in Fig. 4A and B, the levels of ROS in the ox-LDL group were significantly higher than in the control group, and vitexin treatment significantly reduced ROS levels as compared with the ox-LDL group. As shown in Fig. 4C and D, compared with the control group, the levels of SOD were obviously down-regulated in the ox-LDL group, and the levels of MDA was significantly upregulated. Pretreatment with 20 M vitexin was found to increase the levels of SOD levels and to decrease MDA levels. These results indicate that vitexin may play a protective role in ox-LDL-induced endothelial injury, at least partly by suppressing oxidative stress. 3.5. Vitexin attenuates ox-LDL-induced endothelial injury by inducing autophagy To determine whether autophagy was involved in the effects of vitexin, the levels of autophagy-related proteins were measured by Western blot. As shown in Fig. 5A–C, the protein levels of Atg5, Atg7, Atg12 and Beclin1 underwent a slight increase in the oxLDL group as compared with the control group, while the level of p62 was reduced, and the expression of LC3-II was significantly increased, suggesting that autophagy may be induced in response to ox-LDL stimulation to avoid injury. Following pretreatment with vitexin, the expression of Atg5, Atg7, Atg12, LC3-II and Beclin1 showed a more significant up-regulation as compared with the oxLDL group, while the level of p62 was even more suppressed. To further explore the effects of autophagy in the protective action mediated by vitexin, we used 3-MA, a specific autophagy inhibitor, to determine the changes following ox-LDL-induced injury. Firstly, the Western blot results showed that 3-MA treatment significantly inhibited the expression of autophagy-related proteins (Fig. 5A–C). Then, we assessed cell viability and apoptosis by the MTT assay and flow cytometry, respectively. Cell viability in the vitexin plus oxLDL group was significantly elevated as compared with the ox-LDL group, which is consistent with our previous results. However, 3MA treatment counteracted the protective effect of vitexin on cell viability (Fig. 5D). The flow cytometry results also demonstrate that the inhibition of apoptosis by vitexin in ox-LDL-induced HUVECs was significantly weakened following 3-MA treatment (Fig. 5E). These results suggest that vitexin may exert a protective role in ox-LDL-induced HUVECs by inducing autophagy. To further confirm the important role of autophagy in the function of vitexin, we measured the protein expression of inflammatory factors and adhesion molecules by Western blot. As shown in Fig. 5F-H, 3MA treatment significantly enhanced the expression of IL-1, IL-6,
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Fig. 2. The effects of vitexin on cell viability and apoptosis in ox-LDL-treated HUVECs. Cells were treated with 1, 10 and 20 M vitexin for 2 h followed by treatment with ox-LDL for 24 h. (A) Cell viability was measured by the MTT assay. (B) Apoptosis was measured by flow cytometry. (C) The rate of apoptosis is indicated by histograms. (D) The expression levels of cleaved Caspase-3, cleaved Caspase-9, Bax and Bcl-2 were measured by Western blot. (E) The relative protein expression is indicated by histograms. *P < 0.05 vs. control group, #P < 0.05 vs. ox-LDL group. Each experiment was performed in triplicate.
Fig. 3. The effects of vitexin on ox-LDL-induced inflammatory and adhesion molecule expression. Cells were treated with 20 M vitexin for 2 h following by treatment with ox-LDL for 24 h. (A) The expression of IL-1, IL-6 and TNF-␣ were measured by Western blot. (B) The relative protein expression is indicated by histograms. (C) The expression of E-selectin, VCAM1 and ICAM1 were determined by Western blot. (D) The relative protein expression is indicated by histograms. *P < 0.05 vs. control group, #P < 0.05 vs. ox-LDL group. Each experiment was performed in triplicate.
TNF-␣, E-selectin, VCAM1 and ICAM1 as compared with the vitexin plus ox-LDL group. In addition, the reduction of ROS levels induced by vitexin was inhibited following 3-MA treatment (Fig. 5I). Taken together, these results demonstrate that vitexin attenuates ox-LDLinduced endothelial injury by inducing autophagy. 3.6. Vitexin plays a protective role against ox-LDL-induced endothelial injury by activating AMPK signaling It has been reported that AMPK is important in maintaining normal endothelial function, and flavonoids are considered to be AMPK activators (Gasparrini et al., 2016). So, we investigated whether the
protective role of vitexin in ox-LDL-treated HUVECs was associated with AMPK signaling. As compared with the control group, the activated p-AMPK expression level was markedly reduced, meanwhile the activated p-mTOR level was significantly enhanced in ox-LDLtreated HUVECs. When the cells were pretreated with vitexin, the p-AMPK level showed an obvious increase and the p-mTOR level was significantly suppressed (Fig. 6A and B). These results demonstrate that AMPK signaling is involved in the regulation of vitexin in ox-LDL-treated HUVECs. To further identify whether the activation of AMPK signaling was related to autophagy mediated by vitexin, after blocking AMPK activity with the AMPK inhibitor C.C, we measured the expression of autophagy-related proteins. As
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Fig. 4. The effects of vitexin on the ox-LDL-induced oxidative stress in HUVECs. Cells were treated with 20 M vitexin for 2 h following by treatment with ox-LDL for 24 h. (A) The ROS production was measured by flow cytometry using DCHF-DA probes. (B) The relative quantification of ROS is indicated by histograms. (C) The levels of SOD are indicated by histograms. (D) The levels of MDA are indicated by histograms. *P < 0.05 vs. control group, #P < 0.05 vs. ox-LDL group. Each experiment was performed in triplicate.
shown in Fig. 6A and B, C.C treatment significantly suppressed AMPK signaling by down-regulating the p-AMPK level and upregulating the p-mTOR level. We found that the vitexin-induced expression of autophagy-related proteins was dramatically inhibited following C.C treatment (Fig. 6C-E), demonstrating that AMPK signaling may be involved in the regulation of autophagy induced by vitexin. We further investigated whether blocking AMPK signaling was involved in the regulation of endothelial injury by vitexin; the results are shown in Fig. 6F-H. Consistent with our previous study, vitexin protected HUVECs against ox-LDL-induced injury by promoting cell viability, inhibiting apoptosis and reducing ROS production. However, these protective effects were reversed by C.C treatment. These results show that vitexin protects against ox-LDLinduced endothelial injury by regulating AMPK-mTOR signaling. 4. Discussion Endothelial cell dysfunction has been found to be the first cellular event in the pathogenesis of AS. Many previous studies have shown the protective effect of flavonoids in endothelial dysfunction, such as quercitrin (Zhi et al., 2016), ampelopsin (Liang et al., 2015), astragalin (Cho et al., 2014) and luteolin (Xia et al., 2014). Vitexin, as a natural-derived flavonoid, has been used in heat stress-induced oxidative injury (Bhardwaj et al., 2015), lifespan extension (Lee et al., 2015) and cancers (He et al., 2016) because of its anti-cancer, anti-oxidative and anti-inflammatory activities. With regard to the fact that endothelial dysfunction is a process including inflammation and oxidative stress, we tried to investigate the effects of vitexin on endothelial injury. Although several investigations in cancer cell lines have shown that the regulation of vitexin was involved in apoptosis and autophagy via activation of the JNK signaling pathway (He et al., 2016; Zhou et al., 2013), the functions and underlying molecular mechanisms of vitexin in different cell lines and diseases were closely associated with its concentrations in different experimental conditions. In present
study, for the first time we showed that vitexin could alleviate oxLDL-induced endothelial injury by inducing autophagy via AMPK signaling activation, contributing to understand the role of vitexin in cardiovascular disease. It has been reported that endothelial cell apoptosis can destroy the structure and function of the endothelium, accelerating the formation of foam cells. So, we first measured cell viability and apoptosis by the MTT assay and flow cytometry to assess the effects of vitexin on ox-LDL-induced HUVECs. The MTT results show that the cell viability reduction induced by ox-LDL was significantly ameliorated by treatment with vitexin in a dose-dependent manner. The flow cytometry results show that vitexin dose-dependently inhibited ox-LDL-induced apoptosis. In addition, we measured the expression of apoptosis-related proteins by Western blot assay. Caspase-9 is an initiator caspase and Caspase-3 is closely related to apoptosis. Bcl-2 is considered to be an anti-apoptotic protein while Bax is regarded as a pro-apoptosis protein (Bhola and Letai, 2016). In this study, we observed that the overexpression of cleaved Caspase-3, cleaved Caspase-9 and Bax induced by ox-LDL treatment was significantly down-regulated following treatment with vitexin. Meanwhile, the ox-LDL-induced reduction in the expression of Bcl2 was significantly inhibited by vitexin. Together, these results suggest that vitexin exerts a dose-dependent protective effect on ox-LDL-induced endothelial injury by promoting cell viability and suppressing apoptosis. Inflammation has been found to play a crucial role in the endothelial dysfunction. The expression of inflammatory cytokines, including inflammatory factors and cell adhesion molecules, can promote the adhesion and infiltration of monocytes to the vascular endotheliu, leading to the activation of macrophages; these macrophages then absorb lipoprotein, resulting in foam cell formation, which further stimulates vascular inflammation (Meliton et al., 2015; Yamagata et al., 2009). In the present study, we found that inflammatory factors such as IL-1, IL-6 and TNF-␣, were significantly increased by ox-LDL treatment; this increase was
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Fig. 5. Vitexin attenuates ox-LDL-induced endothelial injury by inducing autophagy. Cells were treated with 3-MA for 2 h and then incubated with 20 M vitexin for 2 h followed by treatment with ox-LDL for 24 h. (A) The expression of Atg5, Atg7, Atg12, LC3-II, Beclin1and p62 were measured by Western blot. (B and C) The relative protein expression is indicated by histograms. (D) Cell viability was measured by the MTT assay. (E) Apoptosis was measured by flow cytometry. The rate of apoptosis is indicated by histograms. (F) The expression of IL-1, IL-6, TNF-␣, E-selectin, VCAM1 and ICAM1 were determined by Western blot. (G-H) The relative protein expression is indicated by histograms. (I) ROS production was measured by flow cytometry. The relative quantification of ROS is indicated by histograms. & P < 0.05 vs. control group, *P < 0.05 vs. ox-LDL group, #P < 0.05 vs. vitexin + ox-LDL group. Each experiment was performed in triplicate.
inhibited by vitexin treatment. Our results show that the expression of E-selectin, VCAM1 and ICAM1 in the ox-LDL group was markedly up-regulated as compared with the control group, while the expression was down-regulated following vitexin treatment. These results suggest that vitexin protects HUVECs against ox-LDLinduced inflammatory injury. Oxidative stress has been shown to be closely associated with various pathological processes, such as apoptosis, inflammation and autophagy. Oxidative stress is usually regarded as increased bioactivity of ROS relative to antioxidant defenses (Kregel and Zhang, 2007). Elevated ROS levels play a pivotal role in many cardiovascular diseases, such as metabolic syndrome, type 2 diabetes ˜ et al., 2013; Ilkun and Boudina, 2013). SOD and dyslipidemia (Farina is a major antioxidant enzyme that counteracts oxidative damage and MDA is a marker of lipid peroxidation. In our study, we show that ox-LDL treatment caused an increase in ROS and MDA, and a decrease in SOD levels as compared with the control group. In the vitexin plus ox-LDL group, the production of ROS and MDA decreased and the SOD level increased, suggesting that vitexin protects HUVECs against oxidative damage.
Previous reports have shown that natural extracts may exert their effects through regulating autophagy. Although the function of activated autophagy is controversial, there are still many studies indicating that some flavonoid compounds protect cells against injury by inducing autophagy (Liang et al., 2015; Zhi et al., 2016). To determine whether autophagy was involved in the effects of vitexin, the levels of autophagy-related proteins were measured by Western blot. LC3II is usually regarded as a marker of autophagy (Giménez-Xavier et al., 2009). It has been reported that Atg5, Atg7 and Atg12 are crucial for the initiation of autophagy. Beclin1 is also essential for the induction of autophagy as it regulates the formation of the autophagosome. The protein levels of Atg5, Atg7, Atg12, LC3-II and Beclin1 underwent an obvious increase in the ox-LDL group, while the level of p62 was reduced, suggesting that autophagy was induced in response to ox-LDL treatment to avoid injury. The expression of Atg5, Atg7, Atg12, LC3-II and Beclin1 showed a more significant up-regulation following vitexin treatment, while the level of p62 was even lower, suggesting that vitexin treatment induced autophagy in ox-LDL-treated HUVECs. To assess the effects of autophagy in the protective action mediated by vitexin, we used 3-MA to assess endothelial injury. We
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Fig. 6. Vitexin plays a protective role in ox-LDL-induced endothelial injury by activating AMPK signaling. Cells were treated with C. C for 2 h and then incubated with 20 M vitexin for 2 h followed by treatment with ox-LDL for 24 h. (A) The expression of p-AMPK, AMPK, p-mTOR and mTOR were determined by Western blot. (B) The relative protein expression isindicated by histograms. (C) The expression of Atg5, Atg7, Atg12, LC3-II, Beclin1and p62 were measured by Western blot. (D and E) The relative protein expression is indicated by histograms. (F) Cell viability was measured by the MTT assay. (G) Apoptosis was measured by flow cytometry. The rate of apoptosis is indicated by histograms. (H) ROS production was measured by flow cytometry. The relative quantification of ROS is indicated by histograms. & P < 0.05 vs. control group, *P < 0.05 vs. ox-LDL group, #P < 0.05 vs. vitexin + ox-LDL group. Each experiment was performed in triplicate.
show that 3-MA plus vitexin significantly decreased cell viability, increased apoptosis, enhanced the expression of IL-1, IL-6, TNF␣, E-selectin, VCAM1 and ICAM1 and elevated the production of ROS as compared with vitexin treatment alone. Taken together, these results demonstrate that vitexin attenuates ox-LDL-induced endothelial injury by inducing autophagy. AMP-activated protein kinase (AMPK) is a serine/threonine protein kinase that is involved in the regulation of cellular metabolism (Mizushima et al., 2008). It has been reported that AMPK is a positive regulator of autophagy, and suppresses the mTOR pathway (Aoki et al., 2007; Kim et al., 2011). Increasing evidence indicates that mTOR inhibition stabilizes atherosclerotic plaques and slows down the progression of AS (Baetta et al., 2009; Chen et al., 2009; Mueller et al., 2008; Waksman et al., 2003). Flavonoids have been reported to activate AMPK signaling (Gasparrini et al., 2016). To determine the involvement of AMPK signaling in the protective actin of vitexin, the Western blot results show that vitexin upregulated the p-AMPK expression and down-regulated the p-mTOR level. Furthermore, after blocking AMPK activity by the AMPK inhibitor C.C, vitexin-induced autophagy was dramatically inhibited, demonstrating that AMPK signaling may be involved in the regulation of autophagy induced by vitexin. We further investigated the effects of blocking AMPK signaling on endothelial injury. The results demonstrate that the protective effects of vitexin were all reversed by C.C treatment. These results imply that vitexin plays a protective role against ox-LDL-induced endothelial injury partly by regulating AMPK-mTOR signaling. In summary, these results demonstrate that vitexin promotes cell viability, inhibits apoptosis, down-regulates inflammatory factors and adhesion molecules, and suppresses oxidative stress in ox-LDL-treated HUVECs, which may be related to the activation of
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