Naringin protects endothelial cells from apoptosis and inflammation by regulating the Hippo-YAP Pathway

Abstract Atherosclerosis is the primary cause of several cardiovascular diseases. Oxidized low-density lipoprotein (ox-LDL)-induced apoptosis, endothelial–mesenchymal transition (EndMT), and inflammation are crucial for the progression of cardiovascular diseases, including atherosclerosis. Naringin, a major compound from tomatoes, grapefruits, and related citrus, reportedly exhibits potential protective effects during atherosclerosis development; however, its effect on ox-LDL-induced human umbilical vein endothelial cell (HUVEC) damage remains unknown. In the present study, we investigated the anti-apoptotic and anti-inflammatory activities of naringin against ox-LDL-induced endothelial cells, and the underlying mechanism. Naringin pretreatment significantly and concentration-dependently inhibited ox-LDL-induced cell injury and apoptosis. Additionally, naringin restored endothelial barrier integrity by preventing VE-cadherin disassembly and F-actin remodeling, and down-regulated pro-inflammatory factors like IL-1β, IL-6, and IL-18, in the HUVECs. We also demonstrated that naringin treatment restored ox-LDL-induced YAP (yes-associated protein) down-regulation, given the YAP-shRNA attenuated cytoprotective effect of naringin on ox-LDL-induced endothelial cell injury and apoptosis. Collectively, our data indicate that naringin reversed ox-LDL-triggered HUVEC apoptosis, EndMT, and inflammation by inhibiting the YAP pathway. Therefore, naringin may have a therapeutic effect on endothelial injury-related disorders.


Introduction
Atherosclerosis is the predominant cause of cardiovascular diseases, and is becoming a global concern, owing to its high prevalence [1]. Endothelial injury and apoptosis are the main pathological processes of atherosclerosis, known to trigger thrombosis and accelerate atherosclerotic plaque formation [2]. The arterial deposition of ox-LDL cholesterol is closely associated with atherosclerosis-related morbidity [3].
Accumulating evidence indicate the common occurrence of endothelial-mesenchymal transition (EndMT) in atherosclerosis [4]. EndMT is characterized by specific endothelial marker loss, and mesenchymal marker acquisition [5]. The loss of morphological and functional vascular endothelial cell integrity is closely related to cell inflammation and apoptosis in atherosclerosis [6,7]. Ox-LDL promotes the formation and development of atherosclerotic plaques, by inducing vascular endothelial inflammation and apoptosis [8]. Therapeutic strategies against vascular endothelial cell EndMT, inflammation, and apoptosis may lead to the remission of endothelial-related pathology [9].
Naringin is a major compound extracted from tomatoes, grapefruits, and related citrus fruits [10]. It exhibits potential protective effects against atherosclerosis development [11,12]. Pharmacological investigations have shown that naringin has anti-inflammatory, anti-oxidant, anti-apoptotic, and hepato-protective effects [13,14]. However, no reports exist on its anti-apoptotic and anti-inflammatory effects in ox-LDL-induced injury, in human umbilical vein endothelial cells (HUVECs).
The yes-associated protein 1 (YAP) is the most critical downstream effector in the Hippo signaling cascade, and is essential for physiological and pathological processes [15]. The Hippo-YAP pathway is a kinase cascade consisting of a series of protein kinases and transcription factors [16]. YAP is important for cell proliferation, endothelial cell activation, and vascular inflammation [17].
Thus, in the present study, we established an in vitro atherosclerosis model, by exposing endothelial cells to ox-LDL. The effects of naringin on ox-LDL-induced endothelial injury, apoptosis, and inflammation were investigated. We also quantitatively analyzed the mRNA expressions of YAP and inflammatory cytokines, such as IL-1β, IL-6, and IL-18. It is our hope that the present study will improve the value of naringin as a potential candidate for ox-LDL-induced endothelial injury treatment in atherosclerosis and other cardiovascular diseases.

Cell culture and treatment
HUVECs were purchased from ScienCell (San Diego, CA, U.S.A.), and maintained in ECM (San Diego, CA, U.S.A.) containing 10% heat-inactivated fetal bovine serum (Gibco, Carlsbad, CA), 1% penicillin/streptomycin solution, and 1% endothelial cell growth supplement, at 37 • C in 5% CO 2 . To evaluate the cell protective effect of naringin on ox-LDL (Yiyuan Biotechnologies, Guangzhou, China)-induced HUVECs, cells were randomly divided into three groups: the naringin-treated group where cells were exposed to naringin at 10, 50, 100 and 200 μM for 24 h; the ox-LDL-treated group where cells were exposed to ox-LDL at 20, 40, 60 and 80 μg/ml for 24 h; and the ox-LDL+ naringin-treated group where cells were pretreated with naringin for 2 h, and then incubated with ox-LDL for 24 h. Each group had a minimum of three replicated wells. Subsequently, cells were harvested and extracted for further analyses.

Cell viability assay
The cell counting Kit-8 CCK-8 (Dojindo, Kumamoto, Japan) was used to test the cell viability, following the manufacturer's instructions. Cells were plated onto 96-well plates (4 × 10 4 cells/well), each well was supplemented with 10 μl of CCK-8 solutions for 2 h at 37 • C, and the cell absorbencies measured at 450 nm, using a spectrophotometer.

Lactate dehydrogenase (LDH) release assay
To evaluate cell injury, cytosolic LDH released into culture medium was measured, as previously described [18]. After the 2 h naringin pretreatment, followed by 24 h incubation with 80 μg/ml ox-LDL, medium was collected from each well. Supernatants were obtained by centrifugation at 12,000 × g at 4 • C for 10 min. LDH release was determined using an LDH assay kit (Jiancheng Bioengineering Institute, Nanjing, China), following the manufacturer's instructions. LDH activity was determined via a colorimetric assay using an absorbance wavelength of 492 nm in a spectrophotometer (Bio-Rad Laboratories).

TUNEL staining
The TUNEL apoptosis determination kit (Beyotime, Jiangsu, China) was used to detect apoptotic cells in HUVECs, following the manufacturer's instructions. Briefly, after all treatments, cells were washed with PBS, fixed with 1% buffered formaldehyde, followed by fixation in an ethanol and acetic acid mixture. The fixed cells were stained using fluorescein-conjugated TUNEL, and the cell nuclei were stained with DAPI. The TUNEL-positive cells were observed under a fluorescence microscope (Leica DM4000, Germany). The apoptotic rate was quantified by counting TUNEL positive cells from six random fields, and the values were expressed as a percentage of the total number of cells.

Annexin-V/PI staining and flow cytometry
Apoptosis was measured by flow cytometry. HUVECs (5 × 10 5 cells) were collected by centrifugation at 1000 × g at 4 • C, for 10 min, followed by two rounds of cell pellet washing with HEPES-buffered saline. The cells were then fixed with 70% ice-cold methanol at 4 • C for 30 min. After re-suspending with the binding buffer, cells were stained with 10 μl Annexin V-fluorescein isothiocyanate (FITC), and 5 μl propidium iodide (PI) for 10 min, in the dark. The stained cells were analyzed by flow cytometry (FACSCalibur; BD Biosciences, U.S.A.). The measurements were recorded at least three times in individual experiments.

Immunofluorescent staining
HUVECs were cultured on glass slides, washed in PBS, and fixed with 4% paraformaldehyde for 30 min. The cells were then permeabilized with PBS containing 0.5% Triton X-100 for 15 min. The VE-Cadherin (Cell Signal, U.S.A., 1:1000, ab7652) or F-actin (Cell Signal, U.S.A., 1:500, ab2357) antibody was applied to the coverslip and incubated overnight at 4 • C. After three rounds of PBS washes, the fluorescent secondary antibody (Beyotime, China, 1:500, ab5468) was added and incubated for 1 h at 25 • C. Following three additional PBS washes, cell nuclei were co-stained with DAPI. Images were captured using a fluorescence microscope (Nikon, Tokyo, Japan).

Statistical analyses
Data are presented as mean + − SD. The unpaired two-tailed t test was used for comparisons between two groups. The two-way ANOVA with multiple comparisons between groups was performed to compare multiple groups. GraphPad Prism 7.0 (GraphPad, San Diego, CA, U.S.A.) was used to perform all analyses. The significance threshold was set to P values < 0.05.

Naringin prevents ox-LDL-induced endothelial injury
Initially, we tested the possible cytotoxic effects of naringin on HUVECs, and observed that at 10, 50, and 100 μM, no significant cell death was induced after 24 h of incubation, while the highest concentration; 200 μM, significantly decreased cell viability by ∼23% ( Figure 1A). In addition, 20, 40, 60, and 80 μg/l ox-LDL incubation for 24 h led to a significant dose-dependent decrease in cell viability ( Figure 1B), with only 47.5 + − 6.2% cell viability after incubation with 80 μg/l ox-LDL. Pretreatment with 10 μM naringin for 2 h had no significant influence on cell viability. However, that with 50 and 100 μM naringin significantly enhanced ox-LDL-induced HUVEC viability to 58 + − 7.3% and 79 + − 6.5%, respectively ( Figure 1C,D). We also examined the effect of naringin on LDH release. Except for the 10 μM Quantitative data show that ox-LDL increased the apoptotic rate in HUVECs, which was significantly dose-dependently reversed by naringin. Data are presented as mean+ − SD, n = 6, ### P < 0.001 vs. the control group, **P < 0.01 and ***P < 0.001 vs. the ox-LDL group.
concentration, naringin pretreatment (50 and 100 μM) significantly attenuated LDH release in HUVECs ( Figure 1E). Thus, 50 and 100 μM naringin were chosen as the experimental concentrations for studying its protective role against HUVEC injury. Our data suggests that naringin plays a protective role against ox-LDL-induced endothelial injury.

Naringin inhibits EndMT and the inflammatory response in ox-LDL-induced HUVECs
Immunofluorescent staining showed that incubating HUVECs with ox-LDL decreased the expression of the endothelial marker VE-cadherin, an effect mitigated by naringin ( Figure 3A). Western blotting consistently showed that naringin elevated VE-cadherin HUVEC levels, initially down-regulated in the presence of ox-LDL ( Figure  3C). The ox-LDL-induced change in HUVEC morphology from cobblestone-like shapes to spindle shapes was significantly reversed in naringin-pretreated cells ( Figure 3B). Moreover, Western blotting showed that F-actin protein expression, which was significantly enhanced in ox-LDL-induced HUVECs, was dose-dependently reversed in naringin-pretreated cells ( Figure 3D,E). These results suggest that naringin plays an important inhibition role in ox-LDL-induced HUVEC EndMT.

Naringin reverses ox-LDL-induced YAP attenuation in HUVECs
RT-PCR results showed that ox-LDL stimulation resulted in dose-dependent attenuated YAP mRNA levels in HU-VECs ( Figure 4A). Western blotting also showed that ox-LDL stimulation resulted in dose-dependent attenuated YAP protein levels in HUVECs ( Figure 4B). Immunofluorescent staining showed that incubating HUVECs with ox-LDL, dose-dependently decreased YAP expression ( Figure 4C,D), while Western blotting showed a dose-dependent decrease in ox-LDL-induced YAP protein down-regulation, in naringin-pretreated cells ( Figure 4E).

YAP inhibition attenuates the protective effects of naringin against ox-LDL-mediated HUVEC injury
To further investigate the role of YAP on naringin protective effects against ox-LDL-mediated HUVEC injury, we used YAP shRNA to inhibit YAP expression. HUVECs were transfected with YAP shRNA or control shRNA for 48 h, pretreated with naringin (100 μM) for 2 h, and then incubated with ox-LDL (80 μg/l) for 24 h. The results showed that YAP shRNA successfully abolished naringin-induced YAP protein up-regulation in ox-LDL-mediated HUVECs ( Figure 5A and B). Moreover, transfection with YAP shRNA reversed the effects of naringin: increased cell viability ( Figure 5C), down-regulated LDH release ( Figure 5D), and decreased cell apoptosis ( Figure 5E,F), compared with the ox-LDL+naringin group. In contrast, transfection with control shRNA had no effect on cell viability, LDH release, and cell apoptosis. These data suggest that the protective role of naringin on ox-LDL-mediated HUVEC injury was at least partially achieved by up-regulating YAP expression.

Discussion
It is commonly thought that endothelial cell injury and apoptosis are the basis and initial steps in the pathogenesis of atherosclerosis [19]. Under pathological conditions, chemical stress from harmful substances such as cytokines and oxidized lipids (such as ox-LDL), are known to be key players in endothelial cell injury [8]. ox-LDL-induced endothelial injury is considered a crucial contributor to the pathogenesis and progression of atherosclerosis [20]. Hence, inhibiting ox-LDL-induced endothelial injury may be a novel effective therapeutic approach against atherosclerosis.
In the present study, we evaluated the effect of naringin on ox-LDL-induced HUVEC injury. Previous studies have shown that naringin prevents in vitro LDL oxidation via influencing copper binding to LDL [21], probably important in preventing atherosclerosis. Reports have shown the ability of naringin to inhibit monocyte adhesion to endothelial cells induced by TNF-α [22] and high glucose [23]. Naringin also inhibits apoptosis and inflammation in LPS-induced HUVECs [24]. Research by Kim et al. indicated that naringin had no effect on ox-LDL-induced monocyte adhesion [25]. However, whether naringin affects apoptosis, EndMT, and inflammation in ox-LDL-induced HUVECs, and the underlying mechanisms, remain unclear.
Our study revealed that ox-LDL (80 μg/ml) stimulation reduced cell viability and increased LDH release in HU-VECs, which was dose-dependently reversed in cells pretreated with naringin for 2 h (50 and 100 μM). Further studies using the CCK-8 assay and flow cytometry indicated that naringin reduced apoptosis. These results suggest that naringin can inhibit ox-LDL-induced cell injury and apoptosis.
It is well known that inflammation, an important biological process, is associated with atherogenesis [26]. Our study demonstrated that naringin inhibited ox-LDL-induced release of inflammatory cytokines such as IL-1β, IL-6, and IL-18, indicating that it attenuated ox-LDL-induced inflammation. These results suggest that naringin can inhibit ox-LDL-induced HUVEC damage by suppressing inflammatory reactions. Previous studies by Bi et al. showed that naringin significantly downregulated the protein or mRNA levels of IL-1, IL-6, and TNF-α, and suppressed LPS-induced inflammation and apoptosis [24], which is consistent with our findings.
EndMT, characterized by specific endothelial marker loss and mesenchymal marker acquisition, is crucial for atherosclerosis [27]. Western blotting and immunofluorescent staining showed that the endothelial cell marker VE-cadherin was significantly attenuated in ox-LDL-induced HUVECs, a condition that was dramatically reversed by naringin. Moreover, F-actin immunofluorescent staining revealed that ox-LDL induced change in HUVEC morphology, from cobblestone-like shapes to spindle shapes, which was mitigated by naringin treatment. These results indicate that naringin modulated ox-LDL-induced EndMT in HUVECs. Vaccarin, another flavonoid glycoside isolated from vaccariae semen, was initially shown to impede ox-LDL-triggered HUVEC inflammation, EndMT, and apoptosis [28]. Our study suggests that naringin and vaccarin may have similar endothelial protective effects.
Endothelial Cell-Specific Deletion of YAP has been associated with an increase in the expression of inflammatory cytokines like IL-6, TNF-α, and IL-1β [29], which prompted us to investigate the role of YAP in naringin treatment ox-LDL-induced HUVECs. YAP/TAZ are critical in regulating several cellular behaviors, in response to various internal and external stimuli [30]. An increasing number of studies have shown that Hippo-YAP/TAZ signaling has emerged as a new blood vessel development pathway. Azad et al. reported the Hippo-YAP pathway as a critical mediator of vascular endothelial growth factor (VEGF)-induced angiogenesis, and tumor vasculogenic mimicry [31]. Our research found that naringin reversed YAP's down-regulation caused by ox-LDL, in addition, when shRNA was used to inhibit YAP expression, the cytoprotective and anti-apoptotic naringin function on ox-LDL-induced HUVECs, was inhibited. YAP negatively regulated inflammatory signaling [29] and knockdown of YAP reversed cytoprotective of naringin on ox-LDL-induced HUVECs, which suggest that YAP is a positive modulator in atherosclerosis; however, YAP knockdown attenuated the disturbed flow induction of endothelial cells proliferation and inflammation [32], which indicate that YAP is a negative regulator in atherosclerosis. A possible explanation is that different stimuli activate YAP through different mechanisms, thus play different roles in atherosclerosis. Overall, we confirmed that naringin modulated ox-LDL-induced apoptosis, EndMT, and inflammation in vitro, via the YAP pathway.
However, the present study had some limitations, given that no in vivo assay was performed to confirm the in vitro results. Hence, further in vivo experiments should be performed to demonstrate these naringin endothelial effects. In conclusion, the present study determined for the first time that naringin attenuates ox-LDL-induced HUVEC injury by suppressing cell apoptosis, EndMT, and inflammation, via inhibition of the YAP pathway.