Water/ethanol extract of Cucumis sativus L. fruit attenuates lipopolysaccharide-induced inflammatory response in endothelial cells

Human endothelial SFM medium, heat inactivated FBS (Fetal Bovine Serum), antibiotic-antimycotic and Dulbecco’s phosphate buffered saline (DPBS) were purchased from Gibco-Life technologies (Carlsbad CA, USA).

RNA isolation was performed with NucleoSpin RNA kit (Macherey-Nagel GmbH & Co. KG, Düren Germany), iScript cDNA synthesis kit and iTaq Universal SYBR Green Supermix were used for cDNA synthesis and qPCR analysis (Bio-Rad Laboratories Inc., Hercules, CA, USA). All plastic supports were purchased from Falcon, Beckton-Dickinson.

Porcine Aortic Endothelial Cells (pAECs) were isolated and maintained as previously described by Bernardini and colleagues [12]. Briefly thoracic aortic traits were collected in a local slaughterhouse from adult pigs. After collection, thoracic aortic traits were washed with DPBS, ligated at the ends, and transferred to the laboratory within 1 h on ice. After ligation of all arterial side branches, aortas were cannulated with modified syringe cones and silicone tubes to set up a closed system. The vessels were repeatedly flushed with DPBS and then filled with a collagenase solution and incubated for 20 min at 37 °C. The cellular sospension were then centrifuged at 800 x g for 10 min. The cellular pellet was resuspended in 1 mL human endothelial basal growth medium (Gibco-Invitrogen, Paisley, UK) supplemented with 5% fetal bovine serum (Gibco-Invitrogen) and 1% antibiotics-antimicotics (Gibco-Invitrogen). Cell number and viability (85–90%) were determined using a Burker chamber under a phase-contrast microscope after vital staining with trypan blue dye. Cells were maintained in a logarithmic growth phase by routine passages every 2–3 days at a 1:3 split ratio. To confirm their endothelial origin, cultured cells were checked by immunocitochemistry for endothelial cell markers: CD31 and Caderine. Then cells were expanded till 20th passages. All experiments were performed with cells from the third to the eighth passage. The first seeding after thawing was always performed in T-25 tissue culture flasks (3 × 10⁵ cells/flask) and successive experiments were conducted in 24-well plates (qPCR and western blot analysis), in 96-well assay plates (cytotoxicity) and 8-well slide chamber for in vitro angiogenesis assay. Cells were cultured in Human endothelial SFM medium, added with FBS (5%) and antimicrobial/antimycotic solution (1×) in a 5% CO₂ atmosphere at 38.5 °C.

Since the non-toxicity of the extract is a fundamental pre-requisite, we first tested the cytotoxicity of the CSE in a concentration range of 0.0002–2 mg / ml. No toxicity was showed at any doses tested.

pAECs were seeded in a 96 wells plate (approximately 3 × 10³ cells/well) and exposed to increasing doses of Cucumis sativus L. (CSE) (0.02; 0.2; 2 mg/ml) in presence of lipopolysaccharide (LPS) (10 μg/ml) (E. coli 055:B5, Sigma-Aldrich Co, St Louis, MO, USA) for 24 h. Cytotoxicity was evaluated by trypan Blue exclusion dye using Countess® II FL Automated Cell Counter (Life Technologies).

pAECs were seeded in a 24 wells plate (approximately 4 × 10⁴ cells/well) and exposed to increasing doses (0.02; 0.2; 2 mg/ml) of CSE in presence of LPS (10 μg/ml) for 1, 7 and 24 h. At the end of experimental times, treated or control cells were collected and stored until gene expression analysis.

Total RNA was isolated using the NucleoSpin®RNA Kit, and high quality RNA, with A260/A280 ratio above 2.0 was used for cDNA synthesis. Total RNA (500 ng) was reverse-transcribed to cDNA using the iScript cDNA Synthesis Kit in a final volume of 20 μL. Swine primers were designed using Beacon Designer 2.07 (Premier Biosoft International, Palo Alto, CA, USA). Primer sequences, expected PCR product lengths and accession numbers in the NCBI database are shown in Table 1.

Quantitative real-time PCR was performed to evaluate gene expression profiles in CFX96 (Bio-Rad) thermal cycler using SYBR green detection system. A master mix of the following reaction components was prepared in nuclease free water to the final concentrations indicated: 0.2 μM forward primer, 0.2 μM reverse primer, 1X iTaq Universal SYBR Green Supermix. One μl of cDNA was added to 19 μl of the master mix. All samples were analyzed in duplicate. The qPCR protocol used was: 10 min at 95 °C, 40 cycles at 95 °C for 15 s and at 61 °C for 30 s, followed by a melting step from 55 °C to 95 °C (80 cycle of 0.5 °C increase/cycle).

The expression level of interest genes was calculated as fold of change using the 2^-ΔΔCT method [24].

pAECs were seeded in a 24 wells plate (approximately 4 × 10⁴ cells/well) and exposed to increasing doses (0.02; 0.2; 2 mg/ml) of CSE for 24 h. At the end of experimental time, cells were harvested and lysed in SDS solution (Tris–HCl 50 mM pH 6.8; SDS 2%; glycerol 5%). Protein Assay Kit (TP0300, Sigma) was used to determine the protein content of cellular lysates. Aliquots containing 20 μg of proteins were separated on NuPage 4–12% bis-Tris Gel (Gibco-Life-Technologies) for 50 min at 200 V. The proteins were then electrophoretically transferred onto a nitrocellulose membrane by Turbo Blot System (Bio-Rad). The blots were washed in PBS and protein transfer was checked by staining the nitro-cellulose membranes with 0.2% Ponceau Red. Non-specific binding on nitrocellulose membranes was blocked with 5% milk powder in PBS-T20 (Phosphate Buffer Saline-0.1% Tween-20) for 1 h at room temperature. The membranes were then incubated over-night at 4 °C with a 1:500 dilution of anti-HO-1 rabbit polyclonal antibody (SPA 896 StressGen Biotecnologies Corp, Victoria BC, Canada) and 1:1000 anti TLR4 mouse monoclonal antibody (NB100–56566 Novus Biologicals, Littleton, CO, USA). After several washings with PBS-T20, the membranes were incubated with the secondary biotin-conjugate antibody and then with a 1:1000 dilution of an anti-biotin horseradish peroxidase (HRP)-linked antibody.

The western blots were developed using chemiluminescent substrate (Super Signal West Pico Chemiluminescent Substrate, Pierce Biotechnology, Inc., Rockford, IL, USA) according to the manufacturer’s instructions. Chemidoc instrument using Quantity One Software (Bio-Rad) acquired the intensity of the luminescent signal of the resultant bands.

In order to normalize the HO-1 and TLR4 data on the housekeeping protein, membranes were stripped (briefly: the membranes were washed 5 min in water, then 5 min in 0.2 M NaOH and then washed again in water) and re-probed for housekeeping α-tubulin (1:500 of anti α-tubulin MA1–19162, Thermo Fisher Scientific, Rockford, IL, USA).

The relative protein content (HO-1 or TLR4/α-tubulin) was expressed as arbitrary units (AUs).

Concentration of 13 cytokines and chemokines (GM-CSF, IFN-γ, IL-1α, IL-1β, IL-1ra, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IL-18, TNF-α) was measured by quantitative multiparametric ELISA (Enzyme-linked immunosorbent assay). (Porcine Cytokine/Chemokine Magnetic Bead Panel kit, Milliplex Map Kit, EMD Millipore Corporation, Billerica MA USA), following the manufacturer’s instructions. The Luminex xMAP bead-based multiplexed immunoassay technology and MAGPIX instrument provided with xPONENT 4.2 software were used.

The experiments were carried out using 8-well slide chamber (BD Falcon Bedford, MA USA) coated with undiluted Geltrex™ LDEV-Free Reduced Growth Factor Basement Membrane Matrix. Extracellular matrix coating was carried out for 3 h in a humidified incubator, at 38.5 °C, 5% CO₂. pAECs (8 × 10⁴ cells/well) were exposed to increasing doses (0.02; 0.2; 2 mg/ml) of (CSE) in the presence of LPS (10 μg/ml) for 24 h.

At the end of experimental time, images were acquired using a digital camera installed on a Nikon epifluorescence microscope (Nikon, Yokohama, Japan) and analyzed by open software Image J 64.

Each treatment was replicated three times or six times (cytotoxicity) in three independent experiments. The data were analysed by a one-way analysis of variance (ANOVA) followed by the Tukey post hoc comparison Test. Differences of at least p < 0.05 were considered significant. Statistical analysis was carried out by using R software (http://​www.​R-project.​org) [25].

The protective effect of Cucumis sativus L. extract on LPS-induced toxicity was evaluated in pAECs. LPS treatment provoked an increased number of round and detached cells after 24 h (Fig. 1a), while CSE reduced the cellular detachment in a dose dependent manner (Fig. 1b-d). Cytotoxicity assay confirmed the ability of CSE to protect cells against LPS-induced cellular death (Fig. 1e). Moreover, we studied the expression of ZO-1, a critical component of tight junction scaffold; LPS induced the downregulation of ZO-1 gene expression, while CSE restored ZO-1 expression to control level at the intermediate and higher doses (Fig. 1f).

We studied the effect of CSE on the expression of the Toll-like receptor 4 (TLR4) that is the main receptor for LPS recognition. LPS induced a significant increase of TLR4 mRNA after 1 and 7 h of treatment, while CSE inhibited its expression at all doses studied (Fig. 2a). This inhibitory effect was confirmed at protein level by western blot analysis as shown in Fig. 2b and c. Moreover, we studied the effect of CSE on the vascular protective molecule HO-1. LPS induced HO-1 expression in pAECs, additionally CSE increased HO-1 induction at both mRNA (Fig. 3a) and protein level (Fig. 3b and c).

To assess whether CSE could influence the LPS-induced secretion of inflammatory mediators we evaluated the presence of 13 cytokines/chemokines in the culture medium of pAECs treated with LPS in the presence or absence of CSE (2 mg/ml). LPS-treated endothelial cells released significant level of Il-6, Il-8, IL-10, IL-18, GM-CSF and IFN-γ. CSE significantly influenced these cytokines setting; in particular, the presence of the extract decreased the concentration of GM-CSF, IL-8 and IL-1α with a different kinetic, whereas the concentration of IL-10, IL-18 and IFN-γ was increased at each experimental point, as shown in Table 2. The level of IL-6 showed a precocious increase (1 h) in presence of CSE but after 7 and 24 h, cells treated with plant extract produced less IL-6 than cells only treated with LPS. IL-1β, IL-1ra, IL-2, IL-4, IL-12 and TNF-α were never detected in cell culture medium (data not shown).

We examined the effect of CSE on in vitro LPS-induced pAECs angiogenesis in an extracellular matrix-based assay. Cells cultured on extracellular matrix in the presence of pro-inflammatory LPS assembled in a complete tube and network formation (Fig. 4a and e), while the in vitro angiogenesis induced by inflammatory LPS was prevented by the presence of Cucunis sativus L. extract, at any doses tested (Fig. 4b-e).