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NO pathway
European Journal of Pharmacology 868 (2020) 172885
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Full length article
Quercetin protects the vascular endothelium against iron overload damages via ROS/ADMA/DDAHⅡ/eNOS/NO pathway
T
Xuepiao Chena,1, Hongwei Lib,1, Zhiqing Wangb, Qing Zhoub, Shuping Chenb, Bin Yangb, Dong Yinc, Huan Heb,∗, Ming Hea,b a
Jiangxi Provincial Institute of Hypertension, The First Affiliated Hospital of Nanchang University, Nanchang, 330006, China Jiangxi Provincial Key Laboratory of Basic Pharmacology, Nanchang University School of Pharmaceutical Science, Nanchang, 330006, China c Jiangxi Provincial Key Laboratory of Molecular Medicine, The Second Affiliated Hospital, Nanchang University, Nanchang, 330006, China b
A R T I C LE I N FO
A B S T R A C T
Keywords: Quercetin Iron overload Vascular endothelium Mitochondrial dysfunction ROS/ADMA/DDAHⅡ/eNOS/NO pathway
The aberrant accumulation of iron causes vascular endothelium damage, which is thought to be associated with excess reactive oxygen species (ROS) generation. Quercetin (Que), as a flavonoid, has a certain ability to scavenge free radicals. Therefore, we aimed to explore the protective mechanism of Que on iron overload induced HUVECs injury focused on ROS/ADMA/DDAHⅡ/eNOS/NO pathway. In this study, HUVECs was treated with 50 μM iron dextran and 20 μM Que for 48 h. We found that Que attenuated the damages induced by iron, as evidenced by decreased ROS generation, increased DDAHⅡexpression and activity, reduced ADMA level, increased NO content and p-eNOS/eNOS ratio, and eventually caused a decrease in apoptosis. After addition of pAD/DDAHⅡ-shRNA, the effects of Que mentioned above were reversed. Meanwhile, iron overload induced mitochondrial oxidative stress, reduced mitochondrial membrane potential and increased mitochondrial permeability transition pores (mPTP) opening, which were also partially alleviated by Que. In addition, L-arginine (L-Arg), a ADMA competition substrate, ciclosporin A (CsA), a mPTP blocking agent, and edaravone (Eda), a free radical scavenger, were used as positive control reagents. The effects of Que were similar to that of L-Arg, CsA and Eda treatment. These results illustrated that Que could attenuate iron overload induced HUVECs mitochondrial dysfunction via ROS/ADMA/DDAHⅡ/eNOS/NO pathway.
1. Introduction Iron is one of the trace elements necessary for life, but it can also be harmful to the body when supply of iron exceeds the demand (Fernández-Real and Manco, 2014). Vascular endothelium are one of the target tissue that are injured by iron overload (Kraml, 2017; Vinchi et al., 2014). Iron overload impaired vascular, increased blood pressure, and ultimately leaded to cardiovascular dysfunction (Sangartit et al., 2016). In recent years, the mechanism involved in iron overload damage has been thought to be associated with oxidative stress or excessive reactive oxygen species (ROS) generation (Basu et al., 2018; Chai et al., 2015; Chan et al., 2015). Excessive ROS may cause severe mitochondrial dysfunction and eventually cell death (Akhmedov et al., 2015). Nevertheless, the potential mechanisms involved in the downstream of ROS during vascular endothelium iron overload injury remain to be elucidated. Quercetin (Que), a prominent dietary antioxidant ubiquitously
presents in vegetables, especially onions and coriander, fruits, highlighting apples and berries, wine and tea, is a type of flavonoid (Boots et al., 2008). Recently, many studies have demonstrated that intake of Que can effectively prevent various diseases such as hypertension and coronary heart disease, diabetes, neurodegenerative diseases and various tumors (Perez-Vizcaino et al., 2009; Gormaz et al., 2015; Eid and Haddad, 2017; Rauf et al., 2018). Que also exists strong antioxidant and anti-inflammatory activities (Fuentes et al., 2017; Karuppagounder et al., 2016; Kawabata et al., 2018). Que treatment has been reported to effectively prevent liver injury in iron-overload mice (Zhang et al., 2006), and also demonstrated to significantly suppress renal lipid peroxidation induced by iron overload (Eybl et al., 2008). However, Que protected against iron overload in vascular endothelium has rarely been reported, and the underlying signaling pathways of which remains to be explored. Asymmetric dimethylarginine (ADMA), an endogenous nitric oxide synthase (eNOS) inhibitor, that reduces nitric oxide (NO) production,
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Corresponding author. E-mail address: [email protected] (H. He). 1 These authors contributed equally to this work. https://doi.org/10.1016/j.ejphar.2019.172885 Received 5 August 2019; Received in revised form 17 December 2019; Accepted 18 December 2019 Available online 20 December 2019 0014-2999/ © 2019 Published by Elsevier B.V.
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and inhibits acetylcholine (Ach)-induced vasodilator in endothelial cells (Jiang et al., 2006; Memon et al., 2013). NO is synthesized by Larginine (L-Arg) under eNOS catalysis, and plays a key role in maintaining the structure and function of vascular endothelium (Balat et al., 2009). Dimethylarginine dimethylaminohydrolase (DDAH) is the main metabolic enzyme of ADMA, and has two isoforms of DDAHⅠand DDAHⅡ. DDAHⅡmainly exists in endothelial tissues (Stuhlinger et al., 2001) and is extremely sensitive to intracellular ROS, and excessive ROS can significantly inhibit DDAHⅡactivity, which leads to the accumulation of ADMA (Chen et al., 2009). At present, most studies focus on the role of ADMA/DDAHⅡ/eNOS pathway in cardiovascular injury and endothelial dysfunction (Ghebremariam et al., 2013; Zheng et al., 2015; Osorio-Yáñez et al., 2017), while the possible effects of which in endothelial cells damage caused by iron overload is rarely reported. Therefore, the purpose of this study is to explore the protective effect of Que on iron overload injury human umbilical vein endothelial cells (HUVECs) and the potential signaling pathway to prevent the mitochondrial dysfunction of HUVECs, focusing on the ROS/ADMA/ DDAHⅡ/ENOS/NO pathway, and to explore the molecular protective mechanism of Que on iron overload injury HUVECs.
viability. LDH, which is a stable cytoplasmic enzyme present in all cell types, was rapidly released into cell culture medium during the damage of plasma membrane (Zhu et al., 2013). After related treatment, the supernatant medium of HUVECs was collected to detect LDH activity using a microplate reader (Bio-rad 680) in accordance with manufacture instructions of LDH assay kit (Jiancheng, Nanjing, China).
2. Materials and methods
The intracellular ADMA content was assessed using high-performance liquid chromatography (HPLC) as previously described (Chen et al., 2008; Jiang et al., 2003). Cell lysates were firstly deproteinized with 5-sulfosalicylic acid. HPLC was carried out using an Agilent 1100 HPLC Systems (Agilent Technologies, Santa Clara, CA, USA) with Chemstation Edition Workstation and G1313A autosampler. o-Phthaldialdehyde adducts of methylated amino acids and internal standard ADMA produced by precolumn mixing were monitored using a model G1321A Fluorescence Detector set at λex = 338 nm and λem = 425 nm on an Elite C18 (5 μm, 4.6 mm × 250 mm). Samples were eluted from the column using a linear gradient containing mobile phase A consisted of 0.05 M (pH 6.8) sodium acetate-methanol-tetrahydrofuran (81:18:1 v:v:v) and mobile phase B consisted of 0.05 mM sodium acetate-methanol- tetrahydrofuran (22:77:1 v:v:v) at a flow rate of 1.0 ml/min. The intracellular NO level was reflected indirectly by the content of nitrite and nitrate using a NO assay kit (Feng et al., 2001). The samples were processed according to the manufacturer's instructions, and then the absorbance was determined at 550 nm with a spectrophotometer.
2.4. Determination of endogenous antioxidant enzymes activities and lipid peroxidation Malondialdehyde (MDA, as a lipid peroxide degradation product) level, and the activities of endogenous antioxidant enzyme (catalase: CAT, superoxide dismutase: SOD, glutathione peroxidase: GSH-Px) were estimated with spectrophotometry, respectively (Zhang et al., 2018). After the related treatment, the supernatant of cellular lysis was collected and measured according to manufacture instructions of the assay kits (Jiancheng, Nanjing, China). 2.5. Determination of ADMA and NO content
2.1. Materials Iron dextran (ID), L-Arg, ciclosporin A (CsA), and edaravone (Eda) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Adenovirus pAD/DDAHⅡ-shRNA were obtained from Gene Chem Co., Ltd (Shanghai, China). Que (purity ≥ 98%) was purchased from National Institutes for Food and Drug Control (Beijing, China). Antibodies directed against DDAHⅡ, eNOS, phospho-eNOS (Ser 1177) and cleaved caspase-3 were purchased from Cell Signaling Technology (Beverly, MA, USA). Antibodies directed against β-actin and horseradish peroxidase-conjugated IgG secondary antibodies were obtained from Zsbio (Beijing, China). 2.2. Cell culture and experimental groups HUVECs were obtained from Cell Bank of the Chinese Academy of Sciences (Shanghai, China). The cells were cultured in Ham's F-12K medium (Invitrogen) supplemented with 10% fetal bovine serum (FBS), 1% penicillin-streptomycin, 1% endothelial cell growth supplement (ECGS), and 0.1 mg/ml heparin sodium in a humidified incubator (37 °C, 5% CO2). The experiments were grouped as follows: the control group, cultured in normal conditions; the iron group, treated with 50 μM ID for 48 h; the Iron + Que group, co-treated with 50 μM ID and Que (5, 10, 20, 40, 80 μM) for 48 h; the Iron + Que + pAD/DDAHⅡ-shRNA group, pretreated with pAD/DDAHⅡ-shRNA for 2 h, and then co-incubated with 50 μM ID and 20 μM Que for 48 h; the Iron + Que + pAD/scr RNAi group, pretreated with pAD/scrRNAi for 2 h, and then co-incubated with 50 μM ID and 20 μM Que for 48 h; the Iron + L-Arg group, co-treated with 50 μM ID and 1 mM Arg for 48 h; the Iron + CsA group, co-treated with 50 μM ID and 1 μM CsA for 48 h; the Iron + Eda group, co-treated with 50 μM ID and 100 μM Eda for 48 h.
2.6. Measurement of DDAHⅡ activity DDAHⅡactivity in HUVECs was estimated by directly measuring the amount of ADMA metabolized by the enzyme as described by Lin et al. (2002). Cell lysates were divided into two groups, and 50 μM ADMA was added respectively. 30% 5-sulfurosalicylic acid was added immediately to one of the groups to inactivate DDAH, which provided a baseline of 0% DDAH activity. Additionally, the other group was incubated at 37 °C for 2 h before the addition of 30% 5-sulfurosalicylic acid. ADMA content in each group was measured by HPLC as described above. The difference in ADMA content between the two groups reflected DDAHⅡactivity. 2.7. Western blot analysis HUVECs were washed with pre-cooling PBS, scraped and lysated in a RIPA lysis buffer at 4 °C for at least 20 min, and then centrifuged at 10,800×g, 4 °C for 15 min. Total protein content was determined by a BCA protein assay kit (Thermo, USA). 30 μg protein was loaded and separated on a 12% SDS-PAGE and then transferred to polyvinylidene fluoride membranes. The membranes were blocked with 7% skim milk at 25 °C for 4 h, incubated with primary antibodies (1:1000) against βactin, DDAHⅡ, eNOS, phospho-eNOS (Ser 1177), and cleaved caspase-3 at 4 °C overnight. Subsequently, the membranes were washed with TBST for 4 × 10 min and then incubated with a horseradish peroxidase conjugated secondary antibody (1:2000) for 4 h at 25 °C. After being
2.3. Determination of cell viability and lactate dehydrogenase (LDH) activity The MTS assay (Promega, Madison, USA) was utilized to evaluate cell viability (Huang et al., 2018). HUVECs were seeded in 96-well plates at a concentration of 6000 cells/well routinely cultured for 24 h. Following related treatment, HUVECs were incubated with 20 μl MTS (5 mg/ml) in 100 μl medium at 37 °C for 1.5 h. The absorbance was measured for each well at 490 nm through a microplate reader (Bio-Rad 680, Hercules, CA, USA), which is positively correlated with cell 2
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Fig. 1. Que protects HUVECs against iron overload injury. HUVECs were treated with 50 μM ID for 48 h, to induce iron overload injury; Que treatment significantly increased cell viability and reduced LDH activity (P < 0.01) in a concentration-dependent manner. (A) and (C) Cell viability of HUVECs. (B, D) LDH activity in culture media. Values were presented as mean ± S.E.M. for 6 individual experiments. ▲▲P ﹤0.01 vs. control group, **P ﹤0.01 vs. ioron group, ##P ﹤0.01 vs. Que group, &&P﹤0.01 vs. pAD/DDAHⅡ- shRNA group.
2.10. Measurement of intracellular ROS
washed with TBST for 4 × 15 min, the membranes were saturated with enhanced chemiluminescence reagent for protein visualization. Finally, protein bands intensity were measured and analyzed with Image Jo software.
Intracellular ROS level was assessed according to a method described previously utilizing oxidation sensitive fluorescent probe DCFHDA as the substrate (Mendis et al., 2007). HUVECs were collected and washed for 2 times with serum-free media. 10 μM DCFH-DA probe (Beyotime, Shanghai, China) was added, incubated at 37 °C in darkness for 20 min with slightly reversed every 4 min. The cells were centrifuged and washed twice with PBS, and then detected by flow cytometry (Cytomics FC500, Ex = 488 nm, Em = 525 nm).
2.8. Assessment of HUVECs apoptosis Apoptosis was assessed with a Cytomics FC500 flow cytometer (Beckman Coulter, Brea, CA, USA) using an Annexin V-EGFP/PI apoptosis detection kit (BD Biosciences, San Diego, CA, USA). HUVECs were harvested and washed twice with ice-cold PBS, resuspended in 1 × Annexin V binding buffer. Cell suspension was incubated with 5 μl Annexin V-FITC at 4 °C for 15 min in darkness, co-incubated with 10 μl PI at 4 °C for 5 min in darkness, and then detected (Ex = 488 nm, Em = 578 nm) immediately. The mean fluorescence intensity of Annexin V-FITC/PI staining in the HUVECs was analyzed with CXP Analysis software.
2.11. Determination of mitochondrial membrane potential (MMP) MMP was measured using the fluorescent dye JC-1 (BestBio, Shanghai, China) (Thummasorn et al., 2011). Briefly, HUVECs were harvested and washed twice with ice-cold PBS, and the 1 ml JC-1 working solution was added to resuspend cells, incubated at 37 °C for 20 min in the darkness. Subsequently, the cell pellets were washed twice with 1 × Incubation Buffer for flow cytometry analysis (Cytomics FC500, Ex = 488 nm, Em = 530 nm). The MMP was expressed as a ratio of red to green fluorescence intensity.
2.9. TUNEL assay DNA fragmentation was detected by TUNEL assay as previously described (Bao et al., 2015). HUVECs were seeded on coverslips in 6well plates at a density of 3 × 105 cells/ml. Following the related treatment, cells were fixed in 4% paraformaldehyde for 25 min at 25 °C, then washed twice with PBS for 5 min. Triton X-100 (0.2%) was added for 8 min to permeabilize cells, and cells were rinsed again in PBS. After adding equilibration buffer for 8 min, incubated with 100 μl/slide rTdT reaction mix for 60 min at 37 °C in the dark. Added 2 × SSC for 15 min to termination reaction, washed with PBS, then cells were blocked with 0.3% H2O2 for 5 min and incubated with 100 μl HRP for 30 min. Finally, added DAB solution 100 μl for 5 min, rinsed with deionized water, stained with hematoxylin for about 1 min, then observed using an inverted fluorescence microscope (Olympus, Tokyo, Japan). Brown dots indicated the presence of apoptotic cells.
2.12. Opening of mitochondrial permeability transition pore (mPTP) mPTP openness was detected as described previously (Liu et al., 2018). The mitochondria of HUVECs were isolated by a mitochondrial/ cytosolic fractionation kit (BestBio, Shanghai, China). Afterwards, the isolated mitochondria were resuspended with 160 μl swelling buffer (KCl 120 mM, Tris-HCl 10 mM, MOPS 20 mM, KH2PO4 5 mM), measured the current absorbance at 520 nm by spectrophotometer. Then 40 μl of CaCl2 solution (200 nM) was added to induce opening of mitochondrial pore, absorbance at 520 nm was measured every 1 min until the values remaining stable. The extent of changes in absorbance was used to indicated the degree of mPTP opening. 3
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content, the effect of Que was reversed by pAD/DDAHⅡ-shRNA as well (P < 0.01, Fig. 4B). Furthermore, DDAHⅡactivity significantly reduced in the iron group (P < 0.01). However, co-treatment with Que, L-Arg, CsA and Eda enhanced DDAHⅡactivities (P < 0.01). pAD/DDAHⅡ-shRNA abolished the effect of Que (P < 0.01, Fig. 4C). Meanwhile, iron overload, like pAD/DDAHⅡ-shRNA, significantly down-regulated DDAHⅡexpression (P < 0.01); Que, L-Arg, CsA and Eda significantly up-regulate DDAHⅡexpression (P < 0.01, Fig. 4D and E). As presented in Fig. 4F, the control group had a low level of p-eNOS probably because it was not subjected to external stimulate. HUVECs with iron treatment slightly reduced the p-eNOS/eNOS ratio, but either Que, L-Arg or CsA treated could increase the ratio (P < 0.01), and also the effect of Que was reversed by pAD/DDAHⅡ-shRNA (P < 0.01).
2.13. . Statistical analysis All experimental data were expressed as Mean ± S.E.M., and tested by One-Way Analysis of Variance (One-way ANOVA) using SPSS Statistics 19.0 software. The least significant difference (LSD) test was used for further comparison between groups. P < 0.05 was considered to be statistically significant. 3. Results 3.1. Que protects HUVECs against iron overload injury Cell viability and LDH activity generally serve as indicators of cell toxicity (Huang et al., 2018). Following iron overload injury, cell viability decreased and LDH activity evaluated when compared with the control group (P < 0.01, Fig. 1A and B). After treatment with different concentrations of Que, HUVECs viability was higher than that of the iron group in a dose-dependent manner (P < 0.01). Simultaneously, LDH activity of Que-treatment group was lower than the iron group in a dose-dependent manner (P < 0.01). Based on the above results, we will select 20 μM Que as the optimal concentration in the subsequent experiments. Compared with the iron group, co-treatment with 20 μM Que, 1 mM L-Arg, or 1 μM CsA increased HUVECs viability and reduced LDH activity (P < 0.01, Fig. 1C and D). However, with the addition of the pAD/DDAHⅡ-shRNA, the protective effects of Que in HUVECs were reversed (P < 0.01), indicated that pAD/DDAHⅡ-shRNA could attenuate the protection of Que. However, cell viability and LDH activity of the pAD/scrRNAi group were not almost altered (P > 0.05), indicated that the no-load virus damages to cells can negligible. By using 20 μM Que alone, 1 mM L-Arg alone, 1 μM CsA alone, 100 μM Eda alone, and Que + pAD/DDAHⅡ-shRNA, cell viability and LDH activity did not change compared to that of the control group (P > 0.05). However, cell viability after treatment with pAD/DDAHⅡshRNA alone was lower and LDH activity was higher than that of the control group (P < 0.01, Figs. S1 and S2 of the section of Supplementary materials). These results indicate that DDAHⅡexpression play an important role in maintaining normal HUVECs function.
3.4. Que maintains mitochondrial function in HUVECs injured by iron overload Loss of plasma membrane is one of the earliest features of apoptosis (He et al., 2018). In normal cells, JC-1 can be rapidly ingested into the mitochondria to form a multimer and emits red fluorescence, but when the cells undergoing apoptosis, JC-1 exists as a monomer and emits green fluorescence. We thus use the ratio of red to green fluorescence to reflect the degree of MMP loss. In the iron group, abatement in red to green fluorescence ratio indicated a loss of MMP (P < 0.01). Nevertheless, Que, L-Arg, CsA and Eda treatment alleviated HUVECs from MMP loss after iron overload injury (P < 0.01, Fig. 5A). Furthermore, MMP was obviously reduced in the pAD/DDAHⅡ-shRNA group (P < 0.01). Increased mPTP opening is a major cause of cellular apoptosis and necrosis, and the extent of mPTP opening was determined by Ca2+induced swelling of mitochondrial (Xie et al., 2014). As shown in Fig. 5B, iron treatment resulted in mPTP opening, while co-treatment with Que, L-Arg, CsA and Eda consistently reduced the opening of mPTP (P < 0.01). Moreover, the pAD/DDAHⅡ-shRNA group showed a negative role on mPTP induction (P < 0.01). 3.5. Effect of Que on iron overload-induced HUVECs apoptosis
3.2. Que inhibits oxidative stress and excessive ROS generation in HUVECs injured by iron overload
Firstly, HUVECs were harvested for Annexin V-FITC/PI double staining and analyzed using flow cytometry (He et al., 2017). As can be seen in Fig. 6A, the ratio of apoptotic cells was significantly increased after iron treatment compared with the control group (P < 0.01). However, treatment with Que, L-Arg, CsA and Eda decreased the ratio of apoptotic cells caused by iron overload (P < 0.01). Adding pAD/ DDAHⅡ-shRNA, the positive effects of Que were weakened (P < 0.01). Furthermore, HUVECs apoptosis was determined by TUNEL staining. As shown in Fig. 7, the brown dots presented as apoptotic cells increased after iron treatment and decreased when Que, L-Arg, CsA and Eda was replenished into the cells. Moreover, cleaved caspase-3 expression showed an increase in the iron group (P < 0.01), while Que, L-Arg and CsA treatment decreased expression of cleaved caspase-3 (P < 0.01). And treatment with pAD/ DDAHⅡ-shRNA elevated the cleaved caspase-3 contrasted with the Que group (P < 0.01, Fig. 6B).
Fig. 2 showed a decrease in SOD, GSH-Px, and CAT activities of irontreated HUVECs, whereas MDA content significantly increased (P < 0.01). Que, similar to L-Arg and CsA, reversed the related effects of iron treatment (P < 0.01), and inhibited oxidative stress. After adding pAD/DDAHⅡ-shRNA, the effects of Que were basically cancelled (P < 0.01). As illustrated in Fig. 3, iron treatment caused excessive ROS generation compared with the control group, but the change could be reversed after adding Que (P < 0.01). Adding pAD/DDAHⅡ-shRNA could inhibited the effect of Que (P < 0.01). Moreover, we found that CsA (Teixeira et al., 2013), a mPTP closing agent, Eda (Masuda et al., 2016), a free radical scavenger, and L-Arg (Zheng et al., 2019), a physiological substrate for the synthesis of NO, could sharply weaken the ROS generation caused by iron overload (P < 0.01).
4. Discussion 3.3. Que regulates ADMA/DDAHⅡ/eNOS/NO pathway in HUVECs injured by iron overload
Iron is an essential trace element for all living organisms, it can also be toxic when present in excess by catalyzing the formation of ROS, thus iron intake, transport, distribution, and storage require to be precisely controlled to balance the dual features of essentiality and toxicity (Guo et al., 2016). Our results showed that HUVECs treated with 50 μM ID for 48 h, cell viability was decreased, LDH activity and cleaved caspase-3 expression and apoptotic cells significantly increased, which indicated that iron overload could damage HUVECs (Figs. 1 and
As shown in Fig. 4A, ADMA content showed a significant increased when treated with iron (P < 0.01), while Que, L-Arg, CsA and Eda attenuated the increased ADMA content induced by iron treatment (P < 0.01), and pAD/DDAHⅡ-shRNA reversed the effect of Que (P < 0.01). Conversely, iron overload markly declined NO content, and co-treatment with Que, L-Arg, CsA and Eda similarly elevated NO 4
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Fig. 2. Que inhibits oxidative stress in HUVECs injured by iron overload. 20 μM Que increased SOD (A), GSH-Px (B), and CAT (C) activities, and decreased MDA (D) content. Values were presented as mean ± S.E.M. for 6 independent experiments. ▲▲P﹤0.01 vs. control group, **P ﹤0.01 vs. iron group, P ﹤0.01 vs. Que group.
Iron (Fe2+) participates in Fenton's reaction and produces excessive reactive free radicals, iron accumulation within intravascular may lead to endothelial dysfunction and increase the risk of ischemic cardiovascular events (Vinchi et al., 2013; von Haehling et al., 2015).
7). At the same time, ROS generation in the iron group was significantly increased (Fig. 3), which was consistent with previous studies of iron overload damage mechanisms associated with ROS (Chai et al., 2015; Qiao et al., 2016).
Fig. 3. Que inhibits excessive ROS generation in HUVECs injured by iron overload. (A) Fluorescent probe DCFH-DA reflecting ROS level was detected by flow cytometry. (B) Column chart of average fluorescence intensity values. Values were presented as mean ± S.E.M. for 6 independent experiments. ▲▲P ﹤0.01 vs. control group, **P ﹤0.01 vs. iron group, ##P ﹤0.01 vs. Que group.
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Fig. 4. Que regulates ADMA/DDAHⅡ/eNOS/NO pathway in HUVECs injured by iron overload. (A) Intracellular ADMA content. (B) NO content. (C) DDAHⅡ activity in HUVECs. (D) Western blot's banding of the related proteins in HUVECs. (E) Histogram of DDAHⅡexpression in the cytoplasm. (F) Histogram of p-eNOS/eNOS expression in the cytoplasm. On (D), from left to right, lane 1: control; lane 2: iron; lane 3: Que + iron; lane 4: Que + iron + pAD/DDAHⅡ-shRNA; lane 5: LAgr + iron; lane 6: CsA + iron; lane 7: Eda + iron. Values were presented as mean ± S.E.M. for 6 independent experiments. ▲▲P ﹤0.01 vs. control group, **P ﹤0.01 vs. iron group, ##P ﹤0.01 vs. Que group.
stimulate neighbouring mitochondria to produce more ROS, a process known as ROS-induced ROS release (RIRR) (Zinkevich and Gutterman, 2011). Our results indicated that Que was likely to inhibit ROS generation (Fig. 3), keep MMP and close mPTP (Fig. 5), inhibit RIRR mechanism, thereby prevent mitochondrial dysfunction in HUVECs induced by iron overload. ADMA, a NOS inhibitor metabolized by DDAH, has been reported to associate with endothelial dysfunction (Jiang et al., 2006). Intracellular ROS can stimulate ADMA production or inhibit ADMA degradation, thereby resulting the accumulate of ADMA (Chen et al., 2009). L-Arg is a NO synthesis substrate, which can be competitive inhibited by ADMA (Siekmeier et al., 2008). Therefore, we focused on research of the downstream of ROS may be involved in the iron overload damage. Our results showed that iron overload increased ROS generation and ADMA content, declined DDAHⅡexpression and activity, p-eNOS/eNOS ratio, and NO content (Figs. 3 and 4). However, treatment with Que, the results mentioned above caused by iron overload was obliterated, but
Interestingly, consistent with the antioxidant properties of Que, we found that it could enhance SOD, GSH-Px, and CAT activities inhibited by iron overload in HUVECs, reduce MDA content (Fig. 2), and decrease ROS generation (Fig. 3). The results elucidated that Que may protect HUVECs from iron overload injury through inhibiting oxidative stress. As we all known, mitochondria are not only the position of energy metabolism, but also a vital organelle for ROS generation (Huang et al., 2007). In this study, HUVECs intracellular ROS level increased after treatment with iron (Fig. 3). However, with the utilizing of Que, a natural antioxidant, Eda (Masuda et al., 2016), a free radical scavenger, or CsA (Teixeira et al., 2013), a blocker of mPTP, no matter generation or release of ROS were blocked leading to a decrease in ROS levels (Fig. 3), a prevention in MMP loss (Fig. 5A), and a reduce in the opening of mPTP (Fig. 5B), which eventually increased cell viability (Fig. 1) and reduced apoptosis rate (Figs. 6A and 7). Previous studies have demonstrated that excessive ROS generation could weaken MMP, open mPTP, promote ROS release from mitochondria that could 6
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Fig. 5. Que maintains mitochondrial function in HUVECs injured by iron overload. (A) Fluorescent dye JC-1 reflecting MMP level was detected by flow cytometry and the ratio of red/green fluorescence. (B) The changes of absorbance at 520 nm were recorded every 1 min until 20 min and the degree of mPTP opening was reflected by the changes in absorbance (ΔOD = A5200min-A52020min). Values were presented as mean ± S.E.M. for 6 independent experiments. ▲▲P ﹤0.01 vs. control group, **P ﹤0.01 vs. iron group, ##P ﹤0.01 vs. Que group.
ADMA, and improve iron overload-induced HUVECs damage. CsA and Eda also had an effect on ADMA/DDAHⅡ/eNOS/NO pathway by preventing ROS release or inhibiting ROS generation. Many studies have shown that, as a phytochemicals with many biological activities, Que can act on multiple targets (Boots et al., 2008; Eid and Haddad, 2017). Our previous studies and others' work have
the effects of Que could be reversed by the adding of pAD/DDAHⅡshRNA (Figs. 3 and 4). These results manifested that Que was most likely to reduce intracellular ROS generation, increase DDAHⅡexpression and activity, and affect ADMA/DDAHⅡ/eNOS/NO pathway to protect HUVECs against iron overload injury. Supplement of L-Arg could effectively antagonize the reduce of NO production caused by
Fig. 6. Effect of Que on iron overload-induced HUVECs apoptosis. (A) dot plots of Annexin V-FITC/PI detected by flow cytometry and the apoptosis rate analyzed with CXP Analysis software. (B) Cleaved-caspase 3 protein expression in HUVECs. On (B), from left to right, lane 1: control; lane 2: iron; lane 3: Que + iron; lane 4: Que + iron + pAD/DDAHⅡ-shRNA; lane 5: L-Agr + iron; lane 6: CsA + iron; lane 7: Eda + iron. Values were presented as mean ± S.E.M. for 6 independent experiments. ▲▲P ﹤0.01 vs. control group, **P ﹤0.01 vs. iron group, ##P ﹤0.01 vs. Que group. 7
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Fig. 7. TUNEL assay to present the apoptotic cells iron overload-induced, and Que reduced the apoptosis. Red arrows indicate TUNEL-positive (apoptotic) HUVECs.
5. Conclusion At present, iron overload is of increasing interest as an invisible killer of human health. In this study, we found that Que could increase the activity of endogenous antioxidant enzymes, reduce oxidative stress induced by iron overload, decrease ROS generation in HUVECs, increase DDAHⅡexpression and activity, decrease ADMA content, activate eNOS, increase NO production, maintain mitochondrial function and protect vascular endothelium against iron overload injury (Fig. 8). Que is a dietary antioxidant widely existed in vegetables and fruits. Thus, it can be used as a dietary supplement for the adjuvant treatment of the related diseases. In addition, the effects of Que on ADMA metabolism and the related signaling pathways in vascular endothelium provide new ideas for the research and development of new drugs for the treatment of vascular diseases caused by iron overload injury. Declaration of competing interest The authors declared no conflict of interest.
Fig. 8. A new mechanism exhibited by Que for the protection of mitochondrial function via ADMA/DDAHⅡ/eNOS/NO pathway in HUVECs injured by iron overload. Que could increase the activity of endogenous antioxidant enzymes, reduce oxidative stress induced by iron overload, decrease ROS generation in HUVECs, increase DDAHⅡexpression and activity, decrease ADMA content, activate eNOS, increase NO production, maintain mitochondrial function and protect vascular endothelium against iron overload injury.
Acknowledgments This research was supported by grants from the Natural Science Foundation of China (№: 21467017, 81673431, 81660538, 81803534) and Jiangxi applied research and cultivation program (20181BBG78059).
found that Que could upregulate the expression of 14-3-3γ, PKCε and Bmi-1, or regulate JNK/p38, NF - κ B and ER stress pathway to play cytoprotection (Tang et al., 2013; Dong et al., 2014; Liu et al., 2016; Li et al., 2016; Cai et al., 2017; Chen et al., 2019). In the study, Que upregulate DDAHⅡexpression (Fig. 4) which played a key role, however, whether the mechanism is direct or indirect needs to be further explored. Currently, there are two types of treatment for iron overload: iron chelation therapy, which affects iron metabolism (Mobarra et al., 2016), and antioxidant therapy, which affects oxidative status (He et al., 2018). Iron chelators have been proved to be effective in the treatment of iron overload disorders (Tanner et al., 2007; Kumfu et al., 2017). Thus we inferred that iron chelating agent combined with dietary supplementation of Que to therapy iron overload disorders may produce better therapeutic effects.
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Quercetin protects the vascular endothelium against iron overload damages via ROS/ADMA/DDAHⅡ/eNOS/NO pathway
T
Xuepiao Chena,1, Hongwei Lib,1, Zhiqing Wangb, Qing Zhoub, Shuping Chenb, Bin Yangb, Dong Yinc, Huan Heb,∗, Ming Hea,b a
Jiangxi Provincial Institute of Hypertension, The First Affiliated Hospital of Nanchang University, Nanchang, 330006, China Jiangxi Provincial Key Laboratory of Basic Pharmacology, Nanchang University School of Pharmaceutical Science, Nanchang, 330006, China c Jiangxi Provincial Key Laboratory of Molecular Medicine, The Second Affiliated Hospital, Nanchang University, Nanchang, 330006, China b
A R T I C LE I N FO
A B S T R A C T
Keywords: Quercetin Iron overload Vascular endothelium Mitochondrial dysfunction ROS/ADMA/DDAHⅡ/eNOS/NO pathway
The aberrant accumulation of iron causes vascular endothelium damage, which is thought to be associated with excess reactive oxygen species (ROS) generation. Quercetin (Que), as a flavonoid, has a certain ability to scavenge free radicals. Therefore, we aimed to explore the protective mechanism of Que on iron overload induced HUVECs injury focused on ROS/ADMA/DDAHⅡ/eNOS/NO pathway. In this study, HUVECs was treated with 50 μM iron dextran and 20 μM Que for 48 h. We found that Que attenuated the damages induced by iron, as evidenced by decreased ROS generation, increased DDAHⅡexpression and activity, reduced ADMA level, increased NO content and p-eNOS/eNOS ratio, and eventually caused a decrease in apoptosis. After addition of pAD/DDAHⅡ-shRNA, the effects of Que mentioned above were reversed. Meanwhile, iron overload induced mitochondrial oxidative stress, reduced mitochondrial membrane potential and increased mitochondrial permeability transition pores (mPTP) opening, which were also partially alleviated by Que. In addition, L-arginine (L-Arg), a ADMA competition substrate, ciclosporin A (CsA), a mPTP blocking agent, and edaravone (Eda), a free radical scavenger, were used as positive control reagents. The effects of Que were similar to that of L-Arg, CsA and Eda treatment. These results illustrated that Que could attenuate iron overload induced HUVECs mitochondrial dysfunction via ROS/ADMA/DDAHⅡ/eNOS/NO pathway.
1. Introduction Iron is one of the trace elements necessary for life, but it can also be harmful to the body when supply of iron exceeds the demand (Fernández-Real and Manco, 2014). Vascular endothelium are one of the target tissue that are injured by iron overload (Kraml, 2017; Vinchi et al., 2014). Iron overload impaired vascular, increased blood pressure, and ultimately leaded to cardiovascular dysfunction (Sangartit et al., 2016). In recent years, the mechanism involved in iron overload damage has been thought to be associated with oxidative stress or excessive reactive oxygen species (ROS) generation (Basu et al., 2018; Chai et al., 2015; Chan et al., 2015). Excessive ROS may cause severe mitochondrial dysfunction and eventually cell death (Akhmedov et al., 2015). Nevertheless, the potential mechanisms involved in the downstream of ROS during vascular endothelium iron overload injury remain to be elucidated. Quercetin (Que), a prominent dietary antioxidant ubiquitously
presents in vegetables, especially onions and coriander, fruits, highlighting apples and berries, wine and tea, is a type of flavonoid (Boots et al., 2008). Recently, many studies have demonstrated that intake of Que can effectively prevent various diseases such as hypertension and coronary heart disease, diabetes, neurodegenerative diseases and various tumors (Perez-Vizcaino et al., 2009; Gormaz et al., 2015; Eid and Haddad, 2017; Rauf et al., 2018). Que also exists strong antioxidant and anti-inflammatory activities (Fuentes et al., 2017; Karuppagounder et al., 2016; Kawabata et al., 2018). Que treatment has been reported to effectively prevent liver injury in iron-overload mice (Zhang et al., 2006), and also demonstrated to significantly suppress renal lipid peroxidation induced by iron overload (Eybl et al., 2008). However, Que protected against iron overload in vascular endothelium has rarely been reported, and the underlying signaling pathways of which remains to be explored. Asymmetric dimethylarginine (ADMA), an endogenous nitric oxide synthase (eNOS) inhibitor, that reduces nitric oxide (NO) production,
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Corresponding author. E-mail address: [email protected] (H. He). 1 These authors contributed equally to this work. https://doi.org/10.1016/j.ejphar.2019.172885 Received 5 August 2019; Received in revised form 17 December 2019; Accepted 18 December 2019 Available online 20 December 2019 0014-2999/ © 2019 Published by Elsevier B.V.
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and inhibits acetylcholine (Ach)-induced vasodilator in endothelial cells (Jiang et al., 2006; Memon et al., 2013). NO is synthesized by Larginine (L-Arg) under eNOS catalysis, and plays a key role in maintaining the structure and function of vascular endothelium (Balat et al., 2009). Dimethylarginine dimethylaminohydrolase (DDAH) is the main metabolic enzyme of ADMA, and has two isoforms of DDAHⅠand DDAHⅡ. DDAHⅡmainly exists in endothelial tissues (Stuhlinger et al., 2001) and is extremely sensitive to intracellular ROS, and excessive ROS can significantly inhibit DDAHⅡactivity, which leads to the accumulation of ADMA (Chen et al., 2009). At present, most studies focus on the role of ADMA/DDAHⅡ/eNOS pathway in cardiovascular injury and endothelial dysfunction (Ghebremariam et al., 2013; Zheng et al., 2015; Osorio-Yáñez et al., 2017), while the possible effects of which in endothelial cells damage caused by iron overload is rarely reported. Therefore, the purpose of this study is to explore the protective effect of Que on iron overload injury human umbilical vein endothelial cells (HUVECs) and the potential signaling pathway to prevent the mitochondrial dysfunction of HUVECs, focusing on the ROS/ADMA/ DDAHⅡ/ENOS/NO pathway, and to explore the molecular protective mechanism of Que on iron overload injury HUVECs.
viability. LDH, which is a stable cytoplasmic enzyme present in all cell types, was rapidly released into cell culture medium during the damage of plasma membrane (Zhu et al., 2013). After related treatment, the supernatant medium of HUVECs was collected to detect LDH activity using a microplate reader (Bio-rad 680) in accordance with manufacture instructions of LDH assay kit (Jiancheng, Nanjing, China).
2. Materials and methods
The intracellular ADMA content was assessed using high-performance liquid chromatography (HPLC) as previously described (Chen et al., 2008; Jiang et al., 2003). Cell lysates were firstly deproteinized with 5-sulfosalicylic acid. HPLC was carried out using an Agilent 1100 HPLC Systems (Agilent Technologies, Santa Clara, CA, USA) with Chemstation Edition Workstation and G1313A autosampler. o-Phthaldialdehyde adducts of methylated amino acids and internal standard ADMA produced by precolumn mixing were monitored using a model G1321A Fluorescence Detector set at λex = 338 nm and λem = 425 nm on an Elite C18 (5 μm, 4.6 mm × 250 mm). Samples were eluted from the column using a linear gradient containing mobile phase A consisted of 0.05 M (pH 6.8) sodium acetate-methanol-tetrahydrofuran (81:18:1 v:v:v) and mobile phase B consisted of 0.05 mM sodium acetate-methanol- tetrahydrofuran (22:77:1 v:v:v) at a flow rate of 1.0 ml/min. The intracellular NO level was reflected indirectly by the content of nitrite and nitrate using a NO assay kit (Feng et al., 2001). The samples were processed according to the manufacturer's instructions, and then the absorbance was determined at 550 nm with a spectrophotometer.
2.4. Determination of endogenous antioxidant enzymes activities and lipid peroxidation Malondialdehyde (MDA, as a lipid peroxide degradation product) level, and the activities of endogenous antioxidant enzyme (catalase: CAT, superoxide dismutase: SOD, glutathione peroxidase: GSH-Px) were estimated with spectrophotometry, respectively (Zhang et al., 2018). After the related treatment, the supernatant of cellular lysis was collected and measured according to manufacture instructions of the assay kits (Jiancheng, Nanjing, China). 2.5. Determination of ADMA and NO content
2.1. Materials Iron dextran (ID), L-Arg, ciclosporin A (CsA), and edaravone (Eda) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Adenovirus pAD/DDAHⅡ-shRNA were obtained from Gene Chem Co., Ltd (Shanghai, China). Que (purity ≥ 98%) was purchased from National Institutes for Food and Drug Control (Beijing, China). Antibodies directed against DDAHⅡ, eNOS, phospho-eNOS (Ser 1177) and cleaved caspase-3 were purchased from Cell Signaling Technology (Beverly, MA, USA). Antibodies directed against β-actin and horseradish peroxidase-conjugated IgG secondary antibodies were obtained from Zsbio (Beijing, China). 2.2. Cell culture and experimental groups HUVECs were obtained from Cell Bank of the Chinese Academy of Sciences (Shanghai, China). The cells were cultured in Ham's F-12K medium (Invitrogen) supplemented with 10% fetal bovine serum (FBS), 1% penicillin-streptomycin, 1% endothelial cell growth supplement (ECGS), and 0.1 mg/ml heparin sodium in a humidified incubator (37 °C, 5% CO2). The experiments were grouped as follows: the control group, cultured in normal conditions; the iron group, treated with 50 μM ID for 48 h; the Iron + Que group, co-treated with 50 μM ID and Que (5, 10, 20, 40, 80 μM) for 48 h; the Iron + Que + pAD/DDAHⅡ-shRNA group, pretreated with pAD/DDAHⅡ-shRNA for 2 h, and then co-incubated with 50 μM ID and 20 μM Que for 48 h; the Iron + Que + pAD/scr RNAi group, pretreated with pAD/scrRNAi for 2 h, and then co-incubated with 50 μM ID and 20 μM Que for 48 h; the Iron + L-Arg group, co-treated with 50 μM ID and 1 mM Arg for 48 h; the Iron + CsA group, co-treated with 50 μM ID and 1 μM CsA for 48 h; the Iron + Eda group, co-treated with 50 μM ID and 100 μM Eda for 48 h.
2.6. Measurement of DDAHⅡ activity DDAHⅡactivity in HUVECs was estimated by directly measuring the amount of ADMA metabolized by the enzyme as described by Lin et al. (2002). Cell lysates were divided into two groups, and 50 μM ADMA was added respectively. 30% 5-sulfurosalicylic acid was added immediately to one of the groups to inactivate DDAH, which provided a baseline of 0% DDAH activity. Additionally, the other group was incubated at 37 °C for 2 h before the addition of 30% 5-sulfurosalicylic acid. ADMA content in each group was measured by HPLC as described above. The difference in ADMA content between the two groups reflected DDAHⅡactivity. 2.7. Western blot analysis HUVECs were washed with pre-cooling PBS, scraped and lysated in a RIPA lysis buffer at 4 °C for at least 20 min, and then centrifuged at 10,800×g, 4 °C for 15 min. Total protein content was determined by a BCA protein assay kit (Thermo, USA). 30 μg protein was loaded and separated on a 12% SDS-PAGE and then transferred to polyvinylidene fluoride membranes. The membranes were blocked with 7% skim milk at 25 °C for 4 h, incubated with primary antibodies (1:1000) against βactin, DDAHⅡ, eNOS, phospho-eNOS (Ser 1177), and cleaved caspase-3 at 4 °C overnight. Subsequently, the membranes were washed with TBST for 4 × 10 min and then incubated with a horseradish peroxidase conjugated secondary antibody (1:2000) for 4 h at 25 °C. After being
2.3. Determination of cell viability and lactate dehydrogenase (LDH) activity The MTS assay (Promega, Madison, USA) was utilized to evaluate cell viability (Huang et al., 2018). HUVECs were seeded in 96-well plates at a concentration of 6000 cells/well routinely cultured for 24 h. Following related treatment, HUVECs were incubated with 20 μl MTS (5 mg/ml) in 100 μl medium at 37 °C for 1.5 h. The absorbance was measured for each well at 490 nm through a microplate reader (Bio-Rad 680, Hercules, CA, USA), which is positively correlated with cell 2
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Fig. 1. Que protects HUVECs against iron overload injury. HUVECs were treated with 50 μM ID for 48 h, to induce iron overload injury; Que treatment significantly increased cell viability and reduced LDH activity (P < 0.01) in a concentration-dependent manner. (A) and (C) Cell viability of HUVECs. (B, D) LDH activity in culture media. Values were presented as mean ± S.E.M. for 6 individual experiments. ▲▲P ﹤0.01 vs. control group, **P ﹤0.01 vs. ioron group, ##P ﹤0.01 vs. Que group, &&P﹤0.01 vs. pAD/DDAHⅡ- shRNA group.
2.10. Measurement of intracellular ROS
washed with TBST for 4 × 15 min, the membranes were saturated with enhanced chemiluminescence reagent for protein visualization. Finally, protein bands intensity were measured and analyzed with Image Jo software.
Intracellular ROS level was assessed according to a method described previously utilizing oxidation sensitive fluorescent probe DCFHDA as the substrate (Mendis et al., 2007). HUVECs were collected and washed for 2 times with serum-free media. 10 μM DCFH-DA probe (Beyotime, Shanghai, China) was added, incubated at 37 °C in darkness for 20 min with slightly reversed every 4 min. The cells were centrifuged and washed twice with PBS, and then detected by flow cytometry (Cytomics FC500, Ex = 488 nm, Em = 525 nm).
2.8. Assessment of HUVECs apoptosis Apoptosis was assessed with a Cytomics FC500 flow cytometer (Beckman Coulter, Brea, CA, USA) using an Annexin V-EGFP/PI apoptosis detection kit (BD Biosciences, San Diego, CA, USA). HUVECs were harvested and washed twice with ice-cold PBS, resuspended in 1 × Annexin V binding buffer. Cell suspension was incubated with 5 μl Annexin V-FITC at 4 °C for 15 min in darkness, co-incubated with 10 μl PI at 4 °C for 5 min in darkness, and then detected (Ex = 488 nm, Em = 578 nm) immediately. The mean fluorescence intensity of Annexin V-FITC/PI staining in the HUVECs was analyzed with CXP Analysis software.
2.11. Determination of mitochondrial membrane potential (MMP) MMP was measured using the fluorescent dye JC-1 (BestBio, Shanghai, China) (Thummasorn et al., 2011). Briefly, HUVECs were harvested and washed twice with ice-cold PBS, and the 1 ml JC-1 working solution was added to resuspend cells, incubated at 37 °C for 20 min in the darkness. Subsequently, the cell pellets were washed twice with 1 × Incubation Buffer for flow cytometry analysis (Cytomics FC500, Ex = 488 nm, Em = 530 nm). The MMP was expressed as a ratio of red to green fluorescence intensity.
2.9. TUNEL assay DNA fragmentation was detected by TUNEL assay as previously described (Bao et al., 2015). HUVECs were seeded on coverslips in 6well plates at a density of 3 × 105 cells/ml. Following the related treatment, cells were fixed in 4% paraformaldehyde for 25 min at 25 °C, then washed twice with PBS for 5 min. Triton X-100 (0.2%) was added for 8 min to permeabilize cells, and cells were rinsed again in PBS. After adding equilibration buffer for 8 min, incubated with 100 μl/slide rTdT reaction mix for 60 min at 37 °C in the dark. Added 2 × SSC for 15 min to termination reaction, washed with PBS, then cells were blocked with 0.3% H2O2 for 5 min and incubated with 100 μl HRP for 30 min. Finally, added DAB solution 100 μl for 5 min, rinsed with deionized water, stained with hematoxylin for about 1 min, then observed using an inverted fluorescence microscope (Olympus, Tokyo, Japan). Brown dots indicated the presence of apoptotic cells.
2.12. Opening of mitochondrial permeability transition pore (mPTP) mPTP openness was detected as described previously (Liu et al., 2018). The mitochondria of HUVECs were isolated by a mitochondrial/ cytosolic fractionation kit (BestBio, Shanghai, China). Afterwards, the isolated mitochondria were resuspended with 160 μl swelling buffer (KCl 120 mM, Tris-HCl 10 mM, MOPS 20 mM, KH2PO4 5 mM), measured the current absorbance at 520 nm by spectrophotometer. Then 40 μl of CaCl2 solution (200 nM) was added to induce opening of mitochondrial pore, absorbance at 520 nm was measured every 1 min until the values remaining stable. The extent of changes in absorbance was used to indicated the degree of mPTP opening. 3
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content, the effect of Que was reversed by pAD/DDAHⅡ-shRNA as well (P < 0.01, Fig. 4B). Furthermore, DDAHⅡactivity significantly reduced in the iron group (P < 0.01). However, co-treatment with Que, L-Arg, CsA and Eda enhanced DDAHⅡactivities (P < 0.01). pAD/DDAHⅡ-shRNA abolished the effect of Que (P < 0.01, Fig. 4C). Meanwhile, iron overload, like pAD/DDAHⅡ-shRNA, significantly down-regulated DDAHⅡexpression (P < 0.01); Que, L-Arg, CsA and Eda significantly up-regulate DDAHⅡexpression (P < 0.01, Fig. 4D and E). As presented in Fig. 4F, the control group had a low level of p-eNOS probably because it was not subjected to external stimulate. HUVECs with iron treatment slightly reduced the p-eNOS/eNOS ratio, but either Que, L-Arg or CsA treated could increase the ratio (P < 0.01), and also the effect of Que was reversed by pAD/DDAHⅡ-shRNA (P < 0.01).
2.13. . Statistical analysis All experimental data were expressed as Mean ± S.E.M., and tested by One-Way Analysis of Variance (One-way ANOVA) using SPSS Statistics 19.0 software. The least significant difference (LSD) test was used for further comparison between groups. P < 0.05 was considered to be statistically significant. 3. Results 3.1. Que protects HUVECs against iron overload injury Cell viability and LDH activity generally serve as indicators of cell toxicity (Huang et al., 2018). Following iron overload injury, cell viability decreased and LDH activity evaluated when compared with the control group (P < 0.01, Fig. 1A and B). After treatment with different concentrations of Que, HUVECs viability was higher than that of the iron group in a dose-dependent manner (P < 0.01). Simultaneously, LDH activity of Que-treatment group was lower than the iron group in a dose-dependent manner (P < 0.01). Based on the above results, we will select 20 μM Que as the optimal concentration in the subsequent experiments. Compared with the iron group, co-treatment with 20 μM Que, 1 mM L-Arg, or 1 μM CsA increased HUVECs viability and reduced LDH activity (P < 0.01, Fig. 1C and D). However, with the addition of the pAD/DDAHⅡ-shRNA, the protective effects of Que in HUVECs were reversed (P < 0.01), indicated that pAD/DDAHⅡ-shRNA could attenuate the protection of Que. However, cell viability and LDH activity of the pAD/scrRNAi group were not almost altered (P > 0.05), indicated that the no-load virus damages to cells can negligible. By using 20 μM Que alone, 1 mM L-Arg alone, 1 μM CsA alone, 100 μM Eda alone, and Que + pAD/DDAHⅡ-shRNA, cell viability and LDH activity did not change compared to that of the control group (P > 0.05). However, cell viability after treatment with pAD/DDAHⅡshRNA alone was lower and LDH activity was higher than that of the control group (P < 0.01, Figs. S1 and S2 of the section of Supplementary materials). These results indicate that DDAHⅡexpression play an important role in maintaining normal HUVECs function.
3.4. Que maintains mitochondrial function in HUVECs injured by iron overload Loss of plasma membrane is one of the earliest features of apoptosis (He et al., 2018). In normal cells, JC-1 can be rapidly ingested into the mitochondria to form a multimer and emits red fluorescence, but when the cells undergoing apoptosis, JC-1 exists as a monomer and emits green fluorescence. We thus use the ratio of red to green fluorescence to reflect the degree of MMP loss. In the iron group, abatement in red to green fluorescence ratio indicated a loss of MMP (P < 0.01). Nevertheless, Que, L-Arg, CsA and Eda treatment alleviated HUVECs from MMP loss after iron overload injury (P < 0.01, Fig. 5A). Furthermore, MMP was obviously reduced in the pAD/DDAHⅡ-shRNA group (P < 0.01). Increased mPTP opening is a major cause of cellular apoptosis and necrosis, and the extent of mPTP opening was determined by Ca2+induced swelling of mitochondrial (Xie et al., 2014). As shown in Fig. 5B, iron treatment resulted in mPTP opening, while co-treatment with Que, L-Arg, CsA and Eda consistently reduced the opening of mPTP (P < 0.01). Moreover, the pAD/DDAHⅡ-shRNA group showed a negative role on mPTP induction (P < 0.01). 3.5. Effect of Que on iron overload-induced HUVECs apoptosis
3.2. Que inhibits oxidative stress and excessive ROS generation in HUVECs injured by iron overload
Firstly, HUVECs were harvested for Annexin V-FITC/PI double staining and analyzed using flow cytometry (He et al., 2017). As can be seen in Fig. 6A, the ratio of apoptotic cells was significantly increased after iron treatment compared with the control group (P < 0.01). However, treatment with Que, L-Arg, CsA and Eda decreased the ratio of apoptotic cells caused by iron overload (P < 0.01). Adding pAD/ DDAHⅡ-shRNA, the positive effects of Que were weakened (P < 0.01). Furthermore, HUVECs apoptosis was determined by TUNEL staining. As shown in Fig. 7, the brown dots presented as apoptotic cells increased after iron treatment and decreased when Que, L-Arg, CsA and Eda was replenished into the cells. Moreover, cleaved caspase-3 expression showed an increase in the iron group (P < 0.01), while Que, L-Arg and CsA treatment decreased expression of cleaved caspase-3 (P < 0.01). And treatment with pAD/ DDAHⅡ-shRNA elevated the cleaved caspase-3 contrasted with the Que group (P < 0.01, Fig. 6B).
Fig. 2 showed a decrease in SOD, GSH-Px, and CAT activities of irontreated HUVECs, whereas MDA content significantly increased (P < 0.01). Que, similar to L-Arg and CsA, reversed the related effects of iron treatment (P < 0.01), and inhibited oxidative stress. After adding pAD/DDAHⅡ-shRNA, the effects of Que were basically cancelled (P < 0.01). As illustrated in Fig. 3, iron treatment caused excessive ROS generation compared with the control group, but the change could be reversed after adding Que (P < 0.01). Adding pAD/DDAHⅡ-shRNA could inhibited the effect of Que (P < 0.01). Moreover, we found that CsA (Teixeira et al., 2013), a mPTP closing agent, Eda (Masuda et al., 2016), a free radical scavenger, and L-Arg (Zheng et al., 2019), a physiological substrate for the synthesis of NO, could sharply weaken the ROS generation caused by iron overload (P < 0.01).
4. Discussion 3.3. Que regulates ADMA/DDAHⅡ/eNOS/NO pathway in HUVECs injured by iron overload
Iron is an essential trace element for all living organisms, it can also be toxic when present in excess by catalyzing the formation of ROS, thus iron intake, transport, distribution, and storage require to be precisely controlled to balance the dual features of essentiality and toxicity (Guo et al., 2016). Our results showed that HUVECs treated with 50 μM ID for 48 h, cell viability was decreased, LDH activity and cleaved caspase-3 expression and apoptotic cells significantly increased, which indicated that iron overload could damage HUVECs (Figs. 1 and
As shown in Fig. 4A, ADMA content showed a significant increased when treated with iron (P < 0.01), while Que, L-Arg, CsA and Eda attenuated the increased ADMA content induced by iron treatment (P < 0.01), and pAD/DDAHⅡ-shRNA reversed the effect of Que (P < 0.01). Conversely, iron overload markly declined NO content, and co-treatment with Que, L-Arg, CsA and Eda similarly elevated NO 4
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Fig. 2. Que inhibits oxidative stress in HUVECs injured by iron overload. 20 μM Que increased SOD (A), GSH-Px (B), and CAT (C) activities, and decreased MDA (D) content. Values were presented as mean ± S.E.M. for 6 independent experiments. ▲▲P﹤0.01 vs. control group, **P ﹤0.01 vs. iron group, P ﹤0.01 vs. Que group.
Iron (Fe2+) participates in Fenton's reaction and produces excessive reactive free radicals, iron accumulation within intravascular may lead to endothelial dysfunction and increase the risk of ischemic cardiovascular events (Vinchi et al., 2013; von Haehling et al., 2015).
7). At the same time, ROS generation in the iron group was significantly increased (Fig. 3), which was consistent with previous studies of iron overload damage mechanisms associated with ROS (Chai et al., 2015; Qiao et al., 2016).
Fig. 3. Que inhibits excessive ROS generation in HUVECs injured by iron overload. (A) Fluorescent probe DCFH-DA reflecting ROS level was detected by flow cytometry. (B) Column chart of average fluorescence intensity values. Values were presented as mean ± S.E.M. for 6 independent experiments. ▲▲P ﹤0.01 vs. control group, **P ﹤0.01 vs. iron group, ##P ﹤0.01 vs. Que group.
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Fig. 4. Que regulates ADMA/DDAHⅡ/eNOS/NO pathway in HUVECs injured by iron overload. (A) Intracellular ADMA content. (B) NO content. (C) DDAHⅡ activity in HUVECs. (D) Western blot's banding of the related proteins in HUVECs. (E) Histogram of DDAHⅡexpression in the cytoplasm. (F) Histogram of p-eNOS/eNOS expression in the cytoplasm. On (D), from left to right, lane 1: control; lane 2: iron; lane 3: Que + iron; lane 4: Que + iron + pAD/DDAHⅡ-shRNA; lane 5: LAgr + iron; lane 6: CsA + iron; lane 7: Eda + iron. Values were presented as mean ± S.E.M. for 6 independent experiments. ▲▲P ﹤0.01 vs. control group, **P ﹤0.01 vs. iron group, ##P ﹤0.01 vs. Que group.
stimulate neighbouring mitochondria to produce more ROS, a process known as ROS-induced ROS release (RIRR) (Zinkevich and Gutterman, 2011). Our results indicated that Que was likely to inhibit ROS generation (Fig. 3), keep MMP and close mPTP (Fig. 5), inhibit RIRR mechanism, thereby prevent mitochondrial dysfunction in HUVECs induced by iron overload. ADMA, a NOS inhibitor metabolized by DDAH, has been reported to associate with endothelial dysfunction (Jiang et al., 2006). Intracellular ROS can stimulate ADMA production or inhibit ADMA degradation, thereby resulting the accumulate of ADMA (Chen et al., 2009). L-Arg is a NO synthesis substrate, which can be competitive inhibited by ADMA (Siekmeier et al., 2008). Therefore, we focused on research of the downstream of ROS may be involved in the iron overload damage. Our results showed that iron overload increased ROS generation and ADMA content, declined DDAHⅡexpression and activity, p-eNOS/eNOS ratio, and NO content (Figs. 3 and 4). However, treatment with Que, the results mentioned above caused by iron overload was obliterated, but
Interestingly, consistent with the antioxidant properties of Que, we found that it could enhance SOD, GSH-Px, and CAT activities inhibited by iron overload in HUVECs, reduce MDA content (Fig. 2), and decrease ROS generation (Fig. 3). The results elucidated that Que may protect HUVECs from iron overload injury through inhibiting oxidative stress. As we all known, mitochondria are not only the position of energy metabolism, but also a vital organelle for ROS generation (Huang et al., 2007). In this study, HUVECs intracellular ROS level increased after treatment with iron (Fig. 3). However, with the utilizing of Que, a natural antioxidant, Eda (Masuda et al., 2016), a free radical scavenger, or CsA (Teixeira et al., 2013), a blocker of mPTP, no matter generation or release of ROS were blocked leading to a decrease in ROS levels (Fig. 3), a prevention in MMP loss (Fig. 5A), and a reduce in the opening of mPTP (Fig. 5B), which eventually increased cell viability (Fig. 1) and reduced apoptosis rate (Figs. 6A and 7). Previous studies have demonstrated that excessive ROS generation could weaken MMP, open mPTP, promote ROS release from mitochondria that could 6
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Fig. 5. Que maintains mitochondrial function in HUVECs injured by iron overload. (A) Fluorescent dye JC-1 reflecting MMP level was detected by flow cytometry and the ratio of red/green fluorescence. (B) The changes of absorbance at 520 nm were recorded every 1 min until 20 min and the degree of mPTP opening was reflected by the changes in absorbance (ΔOD = A5200min-A52020min). Values were presented as mean ± S.E.M. for 6 independent experiments. ▲▲P ﹤0.01 vs. control group, **P ﹤0.01 vs. iron group, ##P ﹤0.01 vs. Que group.
ADMA, and improve iron overload-induced HUVECs damage. CsA and Eda also had an effect on ADMA/DDAHⅡ/eNOS/NO pathway by preventing ROS release or inhibiting ROS generation. Many studies have shown that, as a phytochemicals with many biological activities, Que can act on multiple targets (Boots et al., 2008; Eid and Haddad, 2017). Our previous studies and others' work have
the effects of Que could be reversed by the adding of pAD/DDAHⅡshRNA (Figs. 3 and 4). These results manifested that Que was most likely to reduce intracellular ROS generation, increase DDAHⅡexpression and activity, and affect ADMA/DDAHⅡ/eNOS/NO pathway to protect HUVECs against iron overload injury. Supplement of L-Arg could effectively antagonize the reduce of NO production caused by
Fig. 6. Effect of Que on iron overload-induced HUVECs apoptosis. (A) dot plots of Annexin V-FITC/PI detected by flow cytometry and the apoptosis rate analyzed with CXP Analysis software. (B) Cleaved-caspase 3 protein expression in HUVECs. On (B), from left to right, lane 1: control; lane 2: iron; lane 3: Que + iron; lane 4: Que + iron + pAD/DDAHⅡ-shRNA; lane 5: L-Agr + iron; lane 6: CsA + iron; lane 7: Eda + iron. Values were presented as mean ± S.E.M. for 6 independent experiments. ▲▲P ﹤0.01 vs. control group, **P ﹤0.01 vs. iron group, ##P ﹤0.01 vs. Que group. 7
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Fig. 7. TUNEL assay to present the apoptotic cells iron overload-induced, and Que reduced the apoptosis. Red arrows indicate TUNEL-positive (apoptotic) HUVECs.
5. Conclusion At present, iron overload is of increasing interest as an invisible killer of human health. In this study, we found that Que could increase the activity of endogenous antioxidant enzymes, reduce oxidative stress induced by iron overload, decrease ROS generation in HUVECs, increase DDAHⅡexpression and activity, decrease ADMA content, activate eNOS, increase NO production, maintain mitochondrial function and protect vascular endothelium against iron overload injury (Fig. 8). Que is a dietary antioxidant widely existed in vegetables and fruits. Thus, it can be used as a dietary supplement for the adjuvant treatment of the related diseases. In addition, the effects of Que on ADMA metabolism and the related signaling pathways in vascular endothelium provide new ideas for the research and development of new drugs for the treatment of vascular diseases caused by iron overload injury. Declaration of competing interest The authors declared no conflict of interest.
Fig. 8. A new mechanism exhibited by Que for the protection of mitochondrial function via ADMA/DDAHⅡ/eNOS/NO pathway in HUVECs injured by iron overload. Que could increase the activity of endogenous antioxidant enzymes, reduce oxidative stress induced by iron overload, decrease ROS generation in HUVECs, increase DDAHⅡexpression and activity, decrease ADMA content, activate eNOS, increase NO production, maintain mitochondrial function and protect vascular endothelium against iron overload injury.
Acknowledgments This research was supported by grants from the Natural Science Foundation of China (№: 21467017, 81673431, 81660538, 81803534) and Jiangxi applied research and cultivation program (20181BBG78059).
found that Que could upregulate the expression of 14-3-3γ, PKCε and Bmi-1, or regulate JNK/p38, NF - κ B and ER stress pathway to play cytoprotection (Tang et al., 2013; Dong et al., 2014; Liu et al., 2016; Li et al., 2016; Cai et al., 2017; Chen et al., 2019). In the study, Que upregulate DDAHⅡexpression (Fig. 4) which played a key role, however, whether the mechanism is direct or indirect needs to be further explored. Currently, there are two types of treatment for iron overload: iron chelation therapy, which affects iron metabolism (Mobarra et al., 2016), and antioxidant therapy, which affects oxidative status (He et al., 2018). Iron chelators have been proved to be effective in the treatment of iron overload disorders (Tanner et al., 2007; Kumfu et al., 2017). Thus we inferred that iron chelating agent combined with dietary supplementation of Que to therapy iron overload disorders may produce better therapeutic effects.
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