7, 8-Dihydroxyflavone Protects an Endothelial Cell Line from H2O2 Damage

7, 8-dihydroxyflavone (7, 8-DHF), a selective agonist for TrkB receptors, has been well studied for its neurotrophic functions. However, its roles outside the neural tissues have scarcely been studied as yet. In this study, we investigated the protecting roles of 7, 8-DHF in EA.hy926 cells, a human umbilic vein endothelial cell line which was exposed to hydrogen peroxide (H2O2). We found that 7, 8-DHF significantly protected the cells from being damaged by H2O2 through suppression of apoptosis, attenuation of inflammatory factor releasing and inhibition of reactive oxygen species generation. The potent biological effects of 7, 8-DHF were probably executed via its binding to TrkB receptors because the receptor specific antagonist ANA-12 significantly blocked its protecting effects. The protecting roles of 7, 8-DHF in EA.hy926 cells suggest that it will be a promising compound to be developed into a health product that definitely benefits endothelial functions and prevents cardiovascular diseases.

Introduction 7, 8-dihydroxyflavone (7,, a kind of flavonoids which are rich in fruits and vegetables, has been well demonstrated to be a selective small molecule agonist for TrkB receptors and possess strong neuroprotective function [1]. In order to explore its more applicable roles, we previously studied its effect on blood vessels and revealed that 7, 8-DHF was a potent arteryrelaxing compound [2]. Interestingly, the relaxing effect was partially dependent on the intact endothelia and mediated by endothelial NO synthesis. We also confirmed its strong antioxidant effect in PC 12 cells through PI3K/Akt pathway [3,4]. The results have invited us to hypothesize that 7, 8-DHF was probably able to affect or regulate other functions of the endothelia as well and might play a role in endothelial protection. The vascular endothelia expose itself to blood, sensing directly the changes of the components in the blood. Because of its anatomical localization lining the smooth muscle layer, it has been well known that dysfunction of vascular endothelia is the first step towards the development of vascular diseases, e.g. hypertension and atherosclerosis [5]. Since the morbidity and mortality of the diseases are high, the protection of the vascular endothelia has been a longterm focus of research. Multiple factors have been demonstrated to be capable of causing functional disorders of vascular endothelia, for instance, oxidative stress and inflammation which can lead to endothelial apoptosis, reduction of nitric oxide (NO) synthesis and increase of active substances (e.g. secretion of adhesion molecules) [6]. These pathophysiological changes of the endothelia not only elevate blood pressure but also alter the function and structure of the vascular tissues, and eventually give rise to vascular remodeling and hypertension [5,7]. When the endothelia are injured and its morphology changes, the gap between the endothelial cells will be widened and monocytes be accumulated and foamed inside the vascular walls [5]. Then, hypertension and atherosclerosis will ensue. Studies have verified that oxidative stress and inflammation through injuring the endothelia play important roles in the process of the vascular disease development and that protection of the endothelia against the damage caused by the oxidative stress and inflammation is an important approach to treating the vascular diseases [6,8,9]. Therefore, finding active substances against the oxidative stress and inflammation has become a general and effective way to fight the vascular diseases.
The vascular endothelia have been shown to express TrkB receptors [10]. Therefore, it will be interesting to know whether or not 7, 8-DHF is an active substance for the endothelia being capable of acting on the TrkB receptors and activating any biological functions, such as anti-oxidative stress or anti-inflammation. In this study, we used a human umbilic vein endothelial cell (HuVEC) line, EA.hy926, as a cell model and applied in vitro treatment with H 2 O 2 to cause cellular lesion, imitating in vivo status of oxidative stress and inflammation. Using the model, we found that 7, 8-DHF binding to TrkB receptors did prevent the cellular damage caused by H 2 O 2 through suppression of apoptosis, attenuation of inflammatory factor releasing and inhibition of reactive oxygen species generation. The possible protective mechanisms were studied as well.

MTT assay
MTT assay was used to estimate the cell proliferation with the tetrazolium dye. Cells were seeded into a 96-well plate at a density of 1x10 4 cells per well. Then the experimentally treated cells were incubated with 20 μL MTT solution (5 mg/mL in PBS) for 4 h at 37°C. The formazan crystals formed in the intact cells were dissolved in 150 μL DMSO. The absorbance was measured at 490 nm with a microplate reader (Molecular Devices, Sunnyvale, CA, USA).

DAPI staining and flow cytometry for apoptotic determination
The stained nuclei were observed under Leica DMI 4000B fluorescent microscope (Leica Microsystems, Wetzlar, Germany).
The treated EA.hy926 cells were digested with trypsin without EDTA added, collected and suspended in a binding buffer at a cell density of 1x10 6 /mL. Cells were mixed with an Annexin V-FITC/PI Apoptosis Detection Kit (Invitrogen/Life Technologies, Carlsbad, CA, USA) as instructed by the manufacturer, filtered with 200 mesh screens and passed through the flow cytometer(BD FACSCalibur, BD Biosciences, San Jose, USA) for apoptotic detection and analyses.
Cell cultures, protein extraction and western blotting for cellular proteins EA.hy926, a human umbilic vein endothelial cells (HuVECs)-derivedcell line (purchased from the Type Culture Collection of the Chinese Academy of Sciences, Shanghai, China)was cultured and passaged in DMEM media (Gibco, GrandIsland,USA) supplemented with 10% fetal bovine serum (Gibco). For total protein extraction, cells were homogenized in a lysis buffer (Beyotime Institute of Biotechnology, Shanghai, China)containing 50 mM Tris (pH 7.4), 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 2 mM sodium pyrophosphate, 25 mMβ-glycerophosphate, 1 mM EDTA, 1 mM Na 3 VO 4 , 0.5 μg/mL leupeptin, and 1 mM PMSF. PMSF was prepared immediately before use. The homogenates were centrifuged and the protein concentrations in the supernatants were determined as described [11]. SDS-PAGE for the supernatants, western blotting and chemiluminescence were performed as previously [11].
For cytoplasmic protein extraction, cells were washed in ice-cold PBS, centrifuged and resuspended in the ice-cold hypotonic buffer form a kit (Beyotime Institute of Biotechnology, Shanghai, China) and incubated on ice for 10 min, vortexed slightly and centrifuged at 15,000 g for 1 min at 4°C. The supernatant (cytoplasmic protein) was collected and stored at −80°C.

Measurement of cellular MDA and SOD activity
The commercial assay kits(Jiancheng Bioengineering Institute, Nanjing, China) were used for measurement of cellular malondialdehyde (MDA) content and cellular total superoxide dismutase(SOD) activity as described [3]. Briefly, EA.hy926 cells were homogenized and centrifuged for supernatants whose protein concentrations were then determined according to the instruction of the manufacturer before MDA or SOD assays were done. The unit for MDA content was nmol/mg protein and U/mg protein for SOD activity.

Statistical analyses
Data were expressed as means ± SEM. Student'st tests was used to compare the differences between means of two groups. One-way analysis of variance (ANOVA) was used when the means were compared among multiple groups. Statistical analyses were performed with Graph-Pad Prism 5.0 (GraphPad Software, La Jolla, CA, USA). A probability value of P < 0.05 was considered to be statistically significant.  Fig 1A). H 2 O 2 , an endogenous ROS (reactive oxygen species), is produced intracellularly and exists steadily at physiological concentrations of 0.01-0.1μM [12]. However, the threshold value for the concentration of intracellular steady state H 2 O 2 that induces apoptosis was estimated in Jurk at T cells to be around 0.7 μM [13].

7, 8-DHF via
The concentrations of H 2 O 2 used to study the redox regulation in physiological processes in experimental cell models exposed to bolus addition of H 2 O 2 were from 10 to 1000 μM [13].  (Fig 1C).
Since TrkB receptors exist on the endothelial cells [10], we hypothesized that the protecting effect of 7, 8-DHF as a TrkB receptor agonist should have been initiated from the receptors. We firstly confirmed that the proliferation-enhancing effect of 7, 8-DHF was mediated by TrkB receptors because ANA-12 [14], a TrkB receptor specific antagonist at 10 μM almost completely abrogated the cellular proliferation enhanced by 7, 8-DHF at 60 μM (Fig 1D, bars1, 3 and 10 from left)while ANA-12 alone at 10μM did not affect the proliferation(bars 1 and 9). Subsequently, using ANA-12, we demonstrated that the protecting role 7,8-DHF had played in EA.hy926 cells against H 2 O 2 was blocked by the antagonist at 5 and 10μM (Fig 1D, bars 4, 7 and 8), implying that the protecting role of 7, 8-DHF against H 2 O 2 injury was also mediated by the TrkB receptors.

7, 8-DHF inhibited H 2 O 2 -induced apoptosis in EA.hy926 cells
It is well known that H 2 O 2 reduces cellular viability through apoptosis [13]. We assumed, therefore, that 7, 8-DHF protected H 2 O 2 -attacked EA.hy926 cells by way of apoptotic suppression. The effect of 7,8-DHF on apoptosis induced by H 2 O 2 was studied with DAPI staining and flow cytometry. The DAPI staining showed that the number of apoptotic cells induced by H 2 O 2 was significantly increased compared with the control group while 7, 8-DHF evidently reduced the apoptotic number (Fig 2A). The nuclear morphology showed typical apoptotic characters: nuclear condensation and fragmentation (Fig 2A) and the size of the H 2 O 2 -treated cells was also lessened obviously (Fig 3D). Using flow cytometry to quantitatively observe the apoptotic rate of EA.hy926 cells, compared with the control group (4.01±0.43%), we found that the apoptotic rate of H 2 O 2 -treated EA.hy926 cells was significantly increased to 14.23±2.22% by about 3.5-fold. However, treatment with 7, 8-DHF lowered this rate back to 5.68± 0.52%(P<0.001, Fig 2B and 2C).
In the subsequent experiments, we examined the changes of two molecular markers of apoptosis, cytochrome C and Akt [15][16][17]. H 2 O 2 -induced apoptosis was usually accompanied by cytochrome C releasing from mitochondria and H 2 O 2 treatment inhibited phosphorylation of Akt [15][16][17]. As expected, western blotting showed that H 2 O 2 increased the protein level of cytoplasmic cytochrome C in EA.hy926 cells compared with the control group (P<0.001)  Fig 2D). Simultaneously, the phosphorylation level of Akt was found to have decreased significantly in the H 2 O 2 group while 7, 8-DHF significantly enhanced the phosphorylation of Akt in EA.hy926 cells in the presence or absence of H 2 O 2 (Fig 2E). The changes of apoptotic markers supported that H 2 O 2 initiated the apoptotic process but 7, 8-DHF prevented the process in the H 2 O 2 -treated EA.hy926 cells.

7, 8-DHF attenuated H 2 O 2 -induced inflammation in EA.hy926 cells
Another factor affecting cellular viability is cell inflammation. Using quantitative real-time PCR, the effect of 7,8-DHF was investigated on the H 2 O 2 -induced mRNA expression in EA. hy926 cells of three major inflammatory factors, IL-1β, ICAM-1 and TNFα [18]. Results showed that H 2 O 2 significantly stimulated the expression of IL-1β, ICAM-1 and TNFα It is known that NF-κB is involved in regulating the transcription of many inflammatory genes in vascular inflammation [19,20]. NF-κB is activated through IκB degradation in the process of nuclear translocation [21]. To explore the mechanism by which 7,8-DHF suppressed inflammation induced by H 2 O 2 , NF-κB nuclear translocation induced by H 2 O 2 in EA.hy926 cells was investigated. The cellular localization of the p65, a subunit of NF-κB, was examined by fluorescent immunocytochemistry. As shown in Fig 3D, in the control cells (Con), NF-κB was localized predominantly in cytoplasm. However, H 2 O 2 treatment significantly induced NF-κB to transfer from the cytoplasm into the nuclei while 7,8-DHF pretreatment significantly blocked the nuclear translocation. 7,8-DHF alone did not affect the nuclear translocation apparently. IκB is one of determinant proteins for NF-κB nuclear translocation and its degradation plays an important role to activate the process [21]. Consistently, when EA.hy926 cells were treated with H 2 O 2 and the NF-κB nuclear translocation occurred, the decreased protein level of IκB in the cytoplasm was detected by western blotting (P < 0.001, Fig 3E). Co-treatment with both 7,8-DHF and H 2 O 2 prevented the decrease of IκB protein expression. The results indicated that the expression of the inflammatory factors stimulated/suppressed by H 2 O 2 / 7,8-DHF was mediated by NF-κB and IκB factors.  Subsequently, we studied three factors related with cellular oxidative stress, HO-1 (heme oxygenase-1), MDA and SOD. HO-1 and SOD are important intracellular antioxidants [22][23][24]. MDA is a product of lipid oxidation [25]. The changes of their intracellular levels reflect the state of oxidative stress in cells. The protein expression of HO-1 and intracellular MDA level are kept in relatively low levels in resting EA.hy926 cells. However, they were significantly increased by H 2 O 2 treatment while 7, 8-DHF pretreatment prevented both the H 2 O 2 -induced increase of HO-1 protein expression and the elevated level of intracellular MDA in EA.hy926 cells (P< 0.001, Fig 4B and 4C). 7,8-DHF pretreatment could significantly enhance the cellular SOD activity of EA.hy926 cells, which was severely inhibited by H 2 O 2 (P <0.001, Fig 4D). The results indicated that 7, 8-DHF had a significant antioxidant effect and its antioxidant property was associated with the activities of cellular HO-1 and SOD.

Biological functions of 7, 8-DHF
The biological roles or functions of 7, 8-DHF are being investigated actively at present. Nonetheless, the focus of the researches has fallen on its biological effects on the neuro-regeneration and neuro-protection [26][27][28]. This is ascribed to the fact that 7, 8-DHF has been demonstrated to be a selective and hydrophobic small molecule agonist for TrkB receptors which are predominantly localized to central nervous system and play important roles in neurotrophy [1]. 7, 8-DHF is very promising to be a potent substitute for natural agonist of the receptors, brainderived neurotrophic factor(BDNF). Since the TrkB receptors are also distributed in the peripheral tissues, such as vascular endothelia [10], we thought that the roles of 7, 8-DHF in the functions of the tissues may also evident and therefore, studied its effects on the vascular contraction [2]. To be continued, its effect on the endothelial survival from oxidative-stress injury had been investigated in this project. We found that 7, 8-DHF was potent to protect the endothelial cell lineEA.hy926from injury caused by H 2 O 2 exposure. How did 7, 8-DHF protect EA.hy926 cells from the damage caused by H 2 O 2 ? H 2 O 2 is an endogenous ROS and has been long recognized as a destructive molecule whose oxidative effect on biological activities of cells has been well studied [29]. H 2 O 2 induces endothelial apoptosis by promoting the release of cytochrome C from mitochondria and starting mitochondrial apoptosis pathway, and the activated Akt can inhibit the release of cytochrome C [15][16][17]. We had investigated the antioxidant action of 7, 8-DHF against the 6-hydroxydopamine-induced cytotoxicity in PC-12 cells and found that it protected the cells through PI3K/ Akt and JNK pathways [3,4]. Since there seemed to be no TrkB receptors on PC-12, it was thought that 7, 8-DHF diffused into cytoplasm through plasma membrane and acted directly on the pathway factors to produce the antioxidant effects.
TrkB receptors have been demonstrated to be expressed on the endothelial cells [10]. Therefore, 7, 8-DHF was supposed to play an antioxidant roles in EA.hy926 cells through binding the receptors. The experiment with TrkB receptor-specific antagonist ANA-12 clarified this point of view (Fig 1D). Activation of the TrkB receptors by 7, 8-DHF triggered the signaling of PI3K/Akt pathway in neurons [1], and this pathway was also activated by 7, 8-DHF in EA. hy926 cells (Fig 2E), supporting that the TrkB receptors was activated by 7, 8-DHF, indeed.
Our previous and current experiments about the antioxidant effect of 7, 8-DHF in different types of cells (EA.hy926 and PC12) have brought forward an interesting aspect in the biological functions of the compound. TrkB receptors exist on EA.hy926 cells [30]. Therefore, it is intuitionistic to understand that the antioxidant effect of 7, 8-DHF in EA.hy926 cells is mediated via TrkB receptors. However, the receptors were not supposed to exist on PC12 cells but 7, 8-DHF played an antioxidant role in PC12 cells as well [3]. Since 7, 8-DHF is hydrophobic and membrane permeable, it is able to enter cells for its biological functions. To our limited knowledge, it remains unknown at present how 7, 8-DHF plays any biological roles inside cells. Nonetheless, it is noteworthy that 7, 8-DHF even at 5 μM effectively protected PC12 cells against 6-Hydroxydopamine (6-OHDA)-induced cell death (Fig 1B in reference 3). The toxic effect of 6-OHDA has been linked to overproduced cellular ROS such as H2O2. These results suggest that the antioxidant potency and mechanism of 7, 8-DHF probably are different among cell types. The mechanism for intracellular antioxidant influence of 7, 8-DHF requires further exploration.
In this study, we have observed that 7, 8-DHF was able to change the cellular levels of cytochrome C, HO-1, SOD activity, Akt phosphorylation, IκB degradation and NF-κB nuclear translocation. However, how these protein factors were stimulated or suppressed by 7, 8-DHF through TrkB receptors remained to be elucidated.

The significance that 7,8-DHF protects vascular endothelia
The in vitro effect of 7, 8-DHF on the damage of vascular endothelia has implied its practicability in health care. ROS is being produced constantly from cells inside the body and released into the blood. Vascular endothelia are supposed to be the forefront of oxidative defense. The oxidative stress causes cellular apoptosis and inflammation, etc. [31]. Certainly, if the oxidative stress cannot be released as soon as possible, the endothelia will be destined to be damaged.
We have evidenced that 7, 8-DHF is a potent antioxidant compound and can alleviate the oxidative state, lower the apoptotic rate and attenuate the inflammation induced by H 2 O 2 . If the concentration of 7, 8-DHF in blood serum can be elevated to some extent, we believe that it will become a promising health products for improving the health of cardiovascular system. For instance, in spontaneously hypertensive rats (an animal model for study of human essential hypertension), a large number of ROS accumulation in the vascular tissue induced inflammation of vascular endothelial cells, leading to endothelial dysfunction [32]. We hope that application of 7, 8-DHF will alleviate the inflammation of vascular endothelia in hypertensive animals or humans. Another example was that 7, 8-DHF could inhibit the secretion of inflammatory cytokines from BV2 mouse microglia induced by LPS through suppressing the NF-κB and MAPK signaling pathways [33]. Our study showed similar results. Therefore, development of 7, 8-DHF into a health product will definitely benefit endothelial function.
In conclusion, 7, 8-dihydroxyflavone (7, 8-DHF), a selective agonist for TrkB receptors with strong neurotrophic functions, also protected the EA.hy926 cells, a human umbilic vein endothelial cell line which was exposed to hydrogen peroxide (H 2 O 2 ), through suppression of apoptosis, attenuation of inflammatory factor releasing and inhibition of reactive oxygen species generation. The role in the EA.hy926 cells was mediated by its binding to TrkB receptors.