3-Methyladenine – Cefaclor Chemical

Quercetin alleviates high glucose-induced damage on human umbilical vein endothelial cells by promoting autophagy

A B S T R A C T
Background: Quercetin, a ﬂavonoid antioxidant, has been found to exert therapeutic eﬀects in diabetic condi- tion. Autophagy represents a homeostatic cellular mechanism for the turnover of unfolds proteins and damaged organelles through a lysosome-dependent degradation manner. We speculated that quercetin could protect endothelial cells against high glucose-induced damage by promoting autophagic responses.
Methods: HUVECs viability was evaluated by MTT method. Griess and TBARS assays were used to monitor the levels of NO and MDA, respectively. Intracellular ROS generation was determined in DCFDA-stained cells analyzed by ﬂow cytometry. To investigate the role of quercetin in endothelial cell migratory behavior, we used a scratch test. The level of autophagy proteins LC3, Beclin-1 and P62 were measured by western blotting technique. Results: Our results showed that quercetin had the potential to increase cell survival after exposure to high glucose (P < 0.05). Total levels of oxidative stress markers were profoundly decreased and the activity of GSH was increased by quercetin (P < 0.05). High glucose suppressed HUVECs migration to the scratched area (P < 0.05). However, a signiﬁcant stimulation in cell migration was observed after exposure to quercetin (P < 0.05). Based on data, autophagy was blocked at the late stage by high glucose concentration while quer- cetin enhanced autophagic response by reducing the P62 level coincided with the induction of Beclin-1 and LC3- II to LC3-I ratio (P < 0.05). All these beneﬁcial eﬀects were reversed by 3-methyladenine as an autophagy inhibitor. Conclusion: Together, our data suggest that quercetin could protect HUVECs from high glucose induced-damage possibly by activation of the autophagy response. Introduction Diabetes mellitus, as one of the chronic metabolic disorders, is be- coming a global issue with serious outcomes to human life and health(Tabish, 2007). Epidemiological reports show an increasing number of diabetic subjects annually, which endure diabetes-associated compli- cations particularly endothelial dysfunction (Yang et al., 2010). To better realize the underlying mechanism provoking diabeticcomplications, ECs are extensively being investigated (Nachman and Jaﬀe, 2004). The most important concern of diabetic patients is char- acterized by chronic hyperglycemia-induced macro or microangiopathy (Cade, 2008). A long-term exposure of ECs to high glucose contents leads to homeostasis disturbances, originating mainly from a variety of accumulated byproducts such as ROS/RNS and advanced glycation end product (Popov, 2010). Moreover, diabetes mellitus is associated with an aberrant angiogenesis rate. In other words, excessive or defective angiogenesis under high glucose level contributes to injuries such as ocular vascular disorders while high glucose concentration impairs angiogenesis in the extremities resulting chronic nonhealing wounds such as foot ulcers (Abcouwer, 2013; Wu et al., 2007). The high content of glucose in both in vitro and in vivo models of diabetes activates nu- merous pathways of glucose metabolism and causes cellular oxidative/ nitrosative stress (Giugliano et al., 2008). Under an oxidative condition, oxidized biomolecules and damaged DNA could trigger the autophagy machinery and related signaling pathways (Ma et al., 2013). Autop- hagy, an evolutionarily conserved process, ensures cellular hemostasis by protein degradation and damaged organelles turnover during stress circumstances (Murrow and Debnath, 2013). Autophagy extremely is a context-dependent phenomenon. The autophagic response is beneﬁcial and pro-survival at the physiologic level, whiles the excessive activity of autophagy leads to cell death (Chen and Klionsky, 2011; Scarlatti et al., 2009). Among cellular stress generated by high glucose con- centration, autophagy was also induced especially at the early stages (Park and Park, 2013; Rezabakhsh et al., 2017; Surico et al., 2015). Following the onset of autophagic response, cytosolic form of micro- tubule-associated protein light chain 3(LC3)-I is conjugated to form LC3-II as a marker of the early stage of autophagy (Tanida et al., 2008). The formation of double-membrane autophagosome is initiated by PI3 kinase type III-Atg6/Beclin 1 complex activity the main characteristic ofautophagy (Kang et al., 2011). Autophagosome–lysosome fusion ismediated by sequestosome 1 also called p62 protein. The p62 protein is an ubiquitin-binding scaﬀold protein that binds directly to LC3 and transfers ubiquitinated proteins into the lysosomes. Interestingly, p62 is further degraded by the progression of autophagy. This factor is shown to be accumulated inside the target cells when autophagy impaired (Bjørkøy et al., 2009).The eﬀect of intensive therapy to prevent diabetic complications may be limited and other approaches are strongly required to prevent a progression of diabetes complications. Several kinds of drugs have been developed to manage diabetes, but most of them have several side ef- fects. Antioxidant therapy has been of great interest as a way to combat oxidative damages in diabetic patients over the past decade. In other words, antioxidant therapy could be the most promising candidate for the control or prevention of diabetic angiopathy (Skrypko, 2018). Quercetin, a natural ﬂavonoid, ubiquitously is found commonly inplants and fruits (Shanmugam et al., 2011). Besides the robust anti- oxidant, anticancer and anti-inﬂammatory properties (Bądziul et al., 2014; Milackova et al., 2015), quercetin has hypoglycemic property and could modulate the autophagy machinery. Recently, it has been shown quercetin-induced extensive autophagy and subsequent death in cancer cells mediated by the inhibition of Akt, mTOR, HIF-1α signalingpathways and proteasomal activity (Hasima and Ozpolat, 2014;Pratheeshkumar et al., 2012). Another study elucidated that quercetin rescued high glucose-induced Schwann cell damage by autophagy in- duction (Qu et al., 2014). Nevertheless, the underlying mechanism of quercetin has not been entirely clariﬁed.The aim of our study is ensuring a range of optimal autophagic activity by treatment of quercetin to protect endothelial cells and an- giogenic properties under high glucose condition.HUVECs which obtained from the National Cell of Pasteur Institute of Iran was cultured in DMEM/F12 media (Biosera, France) supple- mented with 10% fetal bovine serum (FBS, Gibco, USA), 1% Penicillin/ Streptomycin (Gibco, USA) and incubated at 37 °C in a humidiﬁed at- mosphere of 5%–7% CO2 in air. To induce normal and high glucosecondition, 5 mM (as a control group) and 30 mM D-glucose (Cat no:108337, Merck, Germany) were used, respectively. For drug prepara- tion, quercetin with purity over 95% (Cat no: Q4951; Sigma-Aldrich, Germany) was dissolved in dimethylsulfoxide to make a stock solution. The ﬁnal concentration of DMSO in the media was 0.2%. Cells at pas- sages three to six were used to subsequent analysis.Cell viability and toxicity MTT assayCell viability was assessed using MTT assay (Cat no; M5655, Sigma-Aldrich, Germany) assay. In brief, HUVECs were plated at an initial density of 1 × 104 cells/well in 96-well culture plates and subjected to an experimental procedure. After the completion of treatment, 40 μl MTT solution (5 mg/ml) was added to each well and incubated for 4 h.Then, the supernatant was discarded and 100 μl DMSO used to dissolve formazan precipitates. OD was measured at 570 nm using an ELISAmicroplate reader (Model ELx808, BioTek, USA). The viability of the control group (5 mM) was set to 100% at diﬀerent time points. (Viable cells)% = (OD of cells at diﬀerent concentrations or times/OD of re- lated control) × 100 (Shu et al., 2014).Cell toxicity was determined by measuring levels of LDH released to the supernatant using an LDH assay kit (Pars Azmun Co, Iran). Brieﬂy, 1× 104 cells were cultured in 96-well plates, allowed to attach over- night and exposed to diﬀerent concentrations of quercetin for 72 h. Thereafter, cell media were collected and centrifuged at 400 g for 10 min and the content of LDH activity was determined according to the manufacturer's recommendation. The absorbance of the samples was recorded at 432 nm using a microplate spectrophotometer system (Model ELx808, BioTek, USA). Three independent sets of experiments were performed.Assessment of oxidant and antioxidant status Nitric oxide quantiﬁcationThe level of NO was measured by Griess method (Rezabakhsh et al.,2017). In Griess reaction, nitric oxide is transformed to nitrite as a more stable metabolite. After the completion of the treatment protocol, cells were maintained in serum-free condition and the supernatant collected according to published protocols (Rezabakhsh et al., 2017). Then, the colorimetric changes were detected at 540 nm by a microplate reader (Model ELx808, BioTek, USA). The nitric oxide levels were expressed as µM. Sodium nitrite was used as a standard.To test whether that quercetin could aﬀect the production HUVECs- derived MDA, 1 × 106 cells were cultured in each well of 6-well plates. Cells were collected using enzymatic solution and protein extraction performed by using RIPA lysis buﬀer kit (Cat no: SC-24948; Santa Cruz, USA). For this propose, cells were incubated with lysis buﬀer for 20 min and centrifuged at 12,000 rpm for 20 min at 4 °C. The amount of total protein in cell lysate was measured by using a BCA kit (iNtRON Biotechnology, Korea). Lipid peroxidation in cell lysates was assessedby measuring the levels of malondialdehyde (MDA) with thiobarbituric acid reactive substance (TBARS) assay as described previously by our group (Rezabakhsh et al., 2017). The absorbance was read at 532 nm. Finally, the amount of MDA was expressed as nmol per mg protein. To evaluate GSH activity, CMAC, a speciﬁc dye-labeled GSH was used. For this purpose, cells were seeded in eight-well chamber slides at a density of 1 × 103 cells per well. After being treated, the supernatant was discarded and blue CMAC dye solution (1:1000) added to each well and slides kept for an additional 20 min at 37 °C. The wells were then washed twice with PBS. Cells were examined using up-right microscopy (Model: BX51, Olympus) and the images were processed by CellSenseSoftware Ver.1.4. Fluorescent intensity was measured at Excitation/ Emission 353⁄466 (nm) by ELISA reader spectrophotometer (Biotek, USA).To ascertain intracellular ROS content pre- and post-treatment with quercetin, ﬂow cytometric assay was performed. Cells were grown in 48-well plates over a period of 72 h. Pellets from each group containing 5× 105 cells were incubated with 10 µM DCFDA (Sigma-Aldrich) for 30 min at room temperature and washed three times with PBS. Fluorescence intensity was measured by the FACSCalibur® system (BD Bioscience). Data were analyzed by FlowJo software ver. 7.6.1.Cell migration was evaluated by conventional wound healing assay. To this end, 1 × 106 cells/well was cultured in 6-well plates and al- lowed to reach one-cell-thick monolayer. After 2 h starvation, HUVECs were exposed to quercetin under normal and high glucose condition. Then, cell monolayers were damaged by manual scratching with a sterile yellow tip. Matching wound regions per scratch line were cap- tured randomly at diﬀerent time points using an inverted microscope (Model: TCM 400; Labomed). Thereby, distances of scratch edges (µm2) were calculated by AxioVision software (Version Rel 4.8) and expressed as a percentage of closure from the original wound according to the following formula: % healing rate = (Area of the original wound − Area of the actual wound)/Area of original wound × 100.MDC is a speciﬁc marker for mature autophagy vacuoles (Merkulova et al., 2014). For visualization of MDC labeled vacuoles, HUVECs were incubated with MDC (0.05 mM, Sigma-Aldrich) solution at 37 °C for 20 min after 24 h. Then, cells were collected, washed twice with PBS, and ﬁxed in 4% paraformaldehyde. Stained cells were cap- tured with upright microscopy (Model: BX51, Olympus) and the images processed by CellSense Software Ver.1.4. The ﬂuorescence intensity was also measured by ﬂow cytometry (FACSCalibur, BD Bioscience). Data were processed with FlowJo software ver. X.0.7.HUVECs were seeded onto 8-well slide chamber at a density of 1× 103 cell/well. After the completion of treatment, cells were ﬁxed with methanol (Merck) for 10 min and blocked with 1% BSA (Sigma) and then dissolved in 0.1% Triton X-100 for 1 h. The cells were in- cubated with the anti-rabbit LC3 primary antibody (1:200) (cat no: 3868, Cell Signaling, USA) at room temperature for 1 h and washed three times with PBS. Samples were viewed and photographed with upright Microscopy (Model: BX51, Olympus) and the images were processed by CellSense Software Ver.1.4.Cells were lysed in a pre-chilled lysis buﬀer (NP-40, Sigma-Aldrich, Germany) and centrifuged at 14,000 rpm for 20 min. Supernatants were collected and the protein concentrations were determined by Nanodrop spectrophotometer (Thermo Scientiﬁc NanoDrop™ 1000). Aliquots containing 150 μg of protein were electrophoresed on 12%–15% SDS-polyacrylamide gels. Then, proteins were transferred to PVD mem-branes, which subsequently blocked by 5% skim milk and incubated with a primary antibody including LC3 I or II (cat no: 3868, Cell Signaling, USA), Beclin-1 (Cat no: 3738, Cell Signaling, USA) and P62 (Cat no: 5114, Cell Signaling, USA) overnight at 4 °C. The membranes were further incubated with HRP-conjugated anti-IgG (Cat no: 7074, Cell Signaling, USA) at room temperature for 1 h. Protein bands were detected using an enhanced chemiluminescence western blotting de- tection kit (Roche, Germany). Densitometry analysis of immunoblots was conducted with ImageJ software (NIH). Beta-actin (cat no: 8457, Cell Signaling, USA) was carried out as the housekeeping. Data are expressed as mean ± Standard Deviation (Mean ± SD) and the results of three or ﬁve independent experiments for each as- sessment were analyzed using the GraphPad InStat package version2.02 (GraphPad Software Inc.). The data analyses between groups were performed via one-way analysis of variance (ANOVA) with Tukey post hoc test. Given results at diﬀerent times were compared mean diﬀer- ences signiﬁcant at P < 0.05. In histograms, the statistical diﬀerence between the groups presented by brackets with *P < 0.05, **P < 0.01 and ***P < 0.001. Results To achieve the optimum nontoxic concentration of quercetin, we exposed cells to diﬀerent concentrations of quercetin (0, 0.1, 1, 10, 20,50, 100, 200, 400 μM) containing DMSO (below 0.2%) over 72 h. Asillustrated in Fig. 1, quercetin reduced the number of cells at con- centrations higher than 50 µM and produced a toxic eﬀect against HUVEC especially after 72 h. However, the concentrations up to 20 µM had no eﬀect either on cells viability or LDH release (Fig. 1). So, we selected the concentration of 20 µM for subsequent analyses. To de- termine the eﬀect of quercetin at 20 µM on cell viability, HUVECs were exposed to quercetin at normal and high glucose conditions for dif- ferent time points of 24, 48 and 72 hours (Fig. 2). Compared with normal glucose condition (5 mM), cell viability was signiﬁcantly de- creased (18.4% reduction) at high concentration of glucose after 72 h (P < 0.001). However, the viability of cells was increased in a normal condition containing quercetin and signiﬁcantly elevated in the high glucose condition (103.64 ± 11.52% vs. 81.58 ± 4.79%; P < 0.001).To determine antioxidant potency, GSH level was measured by a cell tracker CMAC dye kit after treatment with quercetin. As shown in immunoﬂuorescence images, cellular GSH production was reduced in high glucose condition after 72 h compared with the control group (Fig. 3a). The level of GSH was restored in quercetin group in both sets of conditions. Consistent with IF results, the changes in ﬂuorescence intensity also conﬁrmed the reduction of antioxidant potency in high glucose condition (P < 0.001) but treatment with quercetin notably increased the GSH production capacity after 72 h (P < 0.001; Fig. 3a). These data suggest that quercetin strongly recovers HUVECs defense mechanism beside oxidative stress by increasing GSH activity. Furthermore, the content of MDA, as an oxidative stress index, was in- creased both in normal- and hyper-glucose conditions after 72 h (P < 0.05). The priming of cells with quercetin remarkably declined the amount of MDA compared to the relative controls (P < 0.001; Fig. 3b). Compared with the HUVECs cultured at a normal concentration of glucose (5 mM Glu), an enhanced NO content was evident in the cells from group 30 mM glucose over time (P < 0.001; Fig. 3c). However, quercetin had potential to decrease NO levels signiﬁcantly in normaland high glucose conditions at all-time points (P < 0.001).The ﬂow cytometry showed that 30 mM glucose augmented in- tracellular ROS production time-dependently in comparison with the related control (Fig. 4a). As shown in Fig. 4b, ROS positive HUVECs reached 79 ± 10.4% after 72 h at a normal concentration of glucose (5 mM Glu), while its value was signiﬁcantly increased to98.4 ± 13.7% in the cells treated with high glucose content (P < 0.001). It is noteworthy that quercetin signiﬁcantly protected ECs by reducing the production of ROS (Fig. 4a and b). The enhanced generation of ROS was remarkably suppressed by quercetin in cells subjected to a high concentration of glucose over a period of 72 h (59.3 ± 4.8% vs. 98.4 ± 13.7%; P < 0.001) (Fig. 4a and b).In vitro scratch assay was performed to reveal the possible eﬀect of quercetin on ECs migration potency especially under high glucose condition. Data clariﬁed that high glucose content signiﬁcantly inter- rupted ECs migration compared to time-matched control (40.22% vs. 85.28%; P < 0.001; Fig. 5a and b). Notably, quercetin could not aﬀect the cell migratory function in normal glucose condition (5 mM = 85.28% vs. 5 mM + quer = 55.79%; P < 0.001; Fig. 5a andb). However, quercetin could increase the decreased cell migratory rate at end-stage of the experiment after treatment with high glucose con- dition (40.22% vs. 70.45%; P < 0.001; Fig. 5a and b).MDC-labeled autophagic vacuoles are elevated in quercetin-treated endothelial cells under high glucose condition auto phagolysosomes appeared with green punctate structures. Based on our data, a small number of MDC labeled-vacuoles were observed in the vicinity of the nucleus under high glucose condition (30 mM Glu)compared to the control HUVECs (Fig. 6a). Data also indicated that green punctate structures appeared in the cells treated with quercetin in diﬀerent conditions. 3-methyladenine (3MA), a classic inhibitor ofautophagy, signiﬁcantly reversed autophagolysosome vacuoles forma- tion (Fig. 6a). To quantify MDC-positive HUVECs, ﬂow cytometry analysis was performed (Fig. 6b). The MDC-positive cells percent was decreased in the cells from 30 mM Glu group compared to group 5 mM Glu (4.9 ± 2.4% vs. 11.3 ± 0.14%; P < 0.001; Fig. 6b). However,quercetin signiﬁcantly increased MDC-positive cells under treatment with 5 mM (10.6 ± 0.42% vs. 11.3 ± 0.14%) or 30 mM glucose(9.25 ± 0.63% vs. 4.9 ± 2.4%; P < 0.001; Fig. 6b). In presence of 3MA, the MDC-positive cells ratio signiﬁcantly was dropped (P < 0.001; Fig. 6b).To clarify whether or not, the beneﬁcial eﬀect of quercetin on cell viability is associated with autophagy pathway; we used 3MA as an autophagy inhibitor. As shown in Fig. 7a, 3MA remarkably decreased the cell survival rate compared to related control (P < 0.001; Fig. 7a). The localization of LC3 in autophagy vacuoles was determined by im- munoﬂuorescence imaging (Fig. 7b). If imaging showed the slight in- crease of LC3 accumulation in cells treated with 30 mM glucose alone while in quercetin treated-cells a signiﬁcant increase of LC3 was ob- served especially in high glucose exposed-cells. These eﬀects were re- versed after the incubation of HUVECs with 3MA (P < 0.001; Fig. 7b). We further investigated the levels of autophagy markers in quercetin-treated cells exposed to high glucose condition (Fig. 8). Western blot analysis revealed that autophagy was exclusively induced in HUVECs exposed to high glucose content in the early stage and blocked at the late phase evident by high expression of LC3II, LC3II/LC3I ratio and Beclin-1 (P < 0.001). The increased level of p62 is reversely associated with autophagy ﬂux (P < 0.001; Fig. 8). The expression of LC3II and Beclin-1 were increased in quercetin-treated cells whereas the p62 protein level was signiﬁcantly reduced under high glucose condition compared to high glucose alone (P < 0.001). 3MA alone or in combi- nation with quercetin exhibited the inhibitory eﬀect on autophagy signaling in both sets of conditions (P < 0.001; Fig. 8). The induced autophagy ﬂux by quercetin was blocked in the presence of 3MA. Discussion This study showed that treatment of HUVECs with quercetin could reverse the high glucose detrimental eﬀects on cells viability and also be able to enhance antioxidant responses. These protective eﬀects were associated with the stimulation of cell migration. The present study further depicted a putative role of quercetin on autophagy induction and revealed the underlying mechanisms involved in beneﬁcial eﬀects on ECs insuﬃciency. Nowadays, ﬂavonoids have received much at- tention as potential therapeutic agents for diabetes (Nicolle et al., 2011; van Dam et al., 2013). We recently found silibinin as a potent bioactive ﬂavonolignan of silymarin would be an appealing therapeutic option for diabetic subjects (Malekinejad et al., 2012). To seek other strong antioxidants, interest has grown in dietary ﬂavonoids, which are mainly derived from vegetables and fruits. Quercetin, a ubiquitous ﬂavonol is abundantly found in plant products, possessing antioxidative and anti- inﬂammatory properties (Formica and Regelson, 1995). Regarding the hypoglycemic eﬀect of quercetin, the therapeutic value of quercetin has been proven in diabetic rats (Kim et al., 2011). Quercetin also had beneﬁcial eﬀects on altered cell membranes integrity under diabetic conditions by restoring transmembrane potential (Margina et al., 2013). It has been proposed that quercetin could blunt exaggerated vasoconstriction, adventitial leukocyte inﬁltration, endothelial py- knosis and increased collagen deposition in vivo diabetic model (Mahmoud et al., 2013). Endothelial dysfunction is an early manifes- tation of vasculature damage developed during chronic hyperglycemia (Kaur et al., 2018). Growing body of evidence revealed that high glu- cose contents induce the production of ROS, causing endothelial dys- function and even cell apoptosis (Ho et al., 2006; Quagliaro et al., 2003). Our data added a notion that quercetin at a non-toxic con- centration (20 µM) has a cytoprotective eﬀect on HUVECs 72 h after exposure to high glucose concentration. In line with our ﬁnding, Zhao and colleagues showed that quercetin supplement in the range of 20–100 µM protected EPCs against high glucose-induced impairment (Zhao et al., 2014). The data from other studies showed high glucose- induced apoptosis was inhibited by quercetin metabolites (sulfate/ glucuronide) in a dose-dependent manner (Chao et al., 2008). In line with these statements, quercetin provokes nuclear factor erythroid like 2 (Nrf2) pathway that protects ARPE-19 cells against H2O2-induced injury (Xu et al., 2016). A series of mechanisms participate in the dia- betic-induced ECs dysfunction. Oxidative stress has been suggested as a major factor for ECs injury under hyperglycemic condition (Giacco and Brownlee, 2010). A large number of documents points that quercetin autophagy, thereby 3MA speciﬁc inhibition of PI3K has a prominent eﬀect on cell viability, which is consistent with our data. To further analyses of autophagy machinery in quercetin-treated cells, MDC im- munoﬂuorescence staining and ﬂow cytometry assessment showed that autophagic ﬂux was blocked by 30 mM glucose alone and in HUVECs received 3MA by reduction of autophagolysosomes. Western blotting showed that high concentration of glucose switched on the early stage of autophagy response by producing LC3-II and Beclin-1 in HUVECs while the elevation of p62 protein level implicated autophagy inhibi- tion at the late phase. Studies revealed that autophagy initiation was eﬃciently elicited as a compensatory response during the ﬁrst 24 h after exposure to diﬀerent stress conditions (Guo et al., 2013). Notably, quercetin promoted the autophagy ﬂux and the protective eﬀects by inducing LC3-II and Beclin-1 and consumption of p62. We also depicted could be considered as either apro-oxidantor antioxidantagentthat 3MA abolished the autophagy up-regulation triggered by quercetin(Kobori et al., 2015; Oboh et al., 2015). Of the most prominent bio- markers, MDA, ROS and NO are proposed to be deleterious factors, causing a vast range of pathological conditions (Montezano and Touyz, 2012). Considering that GSH is the main non-enzymatic anti- oxidant defense system, it should be emphasized the loss of cellular GSH seems to have an important role in cell sensitivity to insults (Ramos et al., 2011). The current experiment showed quercetin im- proves antioxidant defense system in HUVECs after exposure to high concentration of glucose. The elevated levels of cellular NO; MDA and ROS under treatment with 30 mM glucose were signiﬁcantly reduced by treating with quercetin. Hence, the protective mechanism of quercetin on high glucose-damaged ECs can be illustrated in terms of modulation of the oxidant/antioxidant imbalance, i.e., scavenging of oxygen free radicals by extract antioxidants favors recovering of GSH levels, si- multaneously decreases oxidative damage and subsequent cell death. Consistent with our ﬁndings, 100 µM of quercetin had the ability to reduce the levels of NO, MDA and intracellular ROS production and increased the GSH level in EPCs which exposed to 40 µM of glucose during 24 h (Zhao et al., 2014). There have been some contradictions in quercetin eﬀect on cell migration capacity. Our observation noted quercetin rescued the migration of ECs impaired by 30 mM glucose. It has also been found that quercetin had a stimulatory eﬀect on the EPCs migration subjected to the high level of glucose (Zhao et al., 2014). In contrast, some studies claimed quercetin at cytotoxic concentration impaired human glioblastoma and human osteosarcoma cells migration via downregulating of matrix metalloproteinase 2 and 9 (Lan et al., 2017; Liu et al., 2017). Therefore, further experiments are needed to deﬁne the precise mechanism action of quercetin. However, the lack of eﬀective antioxidant capacity against diabetic-induced cell injuries makes it necessary to explore other mechanisms governed by quercetin. Of these mechanisms, the role of autophagy is overlooked. We next explored whether quercetin could aﬀect the autophagy response in HUVECs under high glucose condition. Autophagy, as a catabolic pro- cess, plays a vital role in cellular homeostasis by acting as a house- keeper to eliminate damaged organelles (Glick et al., 2010). Autophagy is tightly regulated by some special proteins encoded by autophagy- related genes. Among these proteins, ATG8/LC3 is essential for autop- hagosome biogenesis or maturation and it also functions as an adaptor protein for selective autophagy (Lee and Lee, 2016). Based on several kinds of literature, LC3-I is converted to LC3-II which is a well-known marker of the early stage of autophagy (Tanida et al., 2008). Beclin-1, another marker of autophagy, is part of a lipid kinase complex which intervenes in the autophagy pathway. Moreover, autophagosome–-lysosome fusion is controlled indirectly by p62 protein. Our data fa- vored the notion that quercetin has the ability to maintain cell viability by inducing autophagy. To this end, we assessed the cell viability in presence of an autophagy inhibitor 3MA. The results showed that 3MA abolished the protective eﬀect of quercetin on cell viability through inhibition of the PI3K pathway. Given that PI3K pathway is being in- volved in many biological processes such as cell survival, progression, growth, migration, intracellular vesicular transport and especiallyin the high glucose condition. El-Horany et al showed that high glucose- induced inhibition of neural cell proliferation retrieved with quercetin via up-regulation of Beclin-1 and LC3, which may have neuroprotective eﬀects in diabetic peripheral neuropathy (El Horany et al., 2016). Conclusion Considering the results of the present study, it can be concluded that treatment of HUVECs with quercetin changed oxidative status, migra-tion rate and autophagy response in a set of high glucose condition. In fact, interventions directed to improve ineﬃcient autophagic ﬂux, particularly the terminal phases of autophagy and autophagic 3-Methyladenine clearance may have therapeutic potential after the onset of diabetic angiopathy.