Lactococcus lactis-fermented spinach juice suppresses LPS-induced expression of adhesion molecules and inflammatory cytokines through the NF-κB pathway in HUVECs.

Spinach (Spinacia oleracea L.), a green leafy vegetable, is widely regarded as a functional food due to its biological activities; however, to the best of our knowledge, there are no previous studies that have investigated the protective effects of fermented spinach against endothelial dysfunction and its underlying mechanisms. Therefore, this study investigated the effects and possible mechanisms of action of fresh spinach juice (S.juice) and fermented S.juice on lipopolysaccharide (LPS)-induced inflammatory responses in human umbilical vein endothelial cells (HUVECs). The HUVECs were treated with S.juice and fermented S.juice for 18 h before LPS exposure, and the levels of cytokines and chemokines, such as monocyte chemoattractant protein-1 (MCP-1) and interleukin-6 (IL-6), were detected using enzyme-linked immunosorbent assays (ELISA). Furthermore, to examine the changes in inflammatory responses to the two treatments, immunofluorescence analysis was used to visualize the nuclear translocation of nuclear factor-κB (NF-κB). Western blot analysis was also performed to detect the differences in the expression of endothelial cell adhesion molecules, specifically vascular cell adhesion molecule 1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1). Both S.juice and fermented S.juice inhibited the LPS-induced expression of MCP-1 and IL-6, and suppressed VCAM-1 and ICAM-1. Additionally, fermented S.juice inhibited the LPS-induced activation of NF-κB and degradation of the inhibitor of NF-κB (IκBα) in an LPS dose-dependent manner. These results suggest that the anti-inflammatory effect of vitamin K2-enriched fermented S.juice is mediated by the suppression of the NF-κB pathway, suggesting its potential as a novel therapeutic candidate for inflammatory cardiovascular disease. Copyright: © Lee et al.

Introduction

Atherosclerosis is a chronic vascular inflammatory disease resulting from the buildup of cholesterol-rich fatty deposits (plaques) in the artery walls and is a major contributor to cardiovascular mortality (1). Lipopolysaccharide (LPS), the principal surface membrane component in the majority of Gram-negative bacteria, is known to cause vascular inflammation (2). The endothelial inflammatory response promotes leukocyte adhesion and increases vascular permeability by increasing the expression levels of several cell adhesion molecules, including vascular cell adhesion molecule 1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), and E-selectin, and via the release of pro-inflammatory cytokines, such as tumor necrosis factor-α (TNF-α), interleukin (IL)-6, IL-8, and monocyte chemoattractant protein-1 (MCP-1) (3-5). The activation of nuclear factor-κB (NF-κB) is a critical step in the inflammatory response, as it regulates the expression of various inflammatory mediators including cytokines and chemokines, which promotes cell adhesion and increases endothelial permeability (4,6,7). Thus, medications or nutraceuticals that downregulate the expression of inflammatory cytokines/chemokines and adhesion molecules, and inhibit the NF-κB pathway, are promising candidates for the treatment and prevention of atherosclerotic diseases. Anti-inflammatory drugs are not equally effective in all patients and are associated with adverse effects that limit their use. Phytochemicals such as flavonoids, phenylpropanoids, isothiocyanates, sulforaphanes, indole alkaloids, and sterol glucosides can inhibit the production of pro-inflammatory cytokines and subsequently their signaling pathways (8). Therefore, dietary agents with potent anti-inflammatory activity and with few or no adverse effects are promising for treating inflammatory diseases (9).

Spinach (Spinacia oleracea L.) is a green leafy vegetable that a is rich source of vitamins, minerals, phenolic compounds, and carotenoids. Its nutrient and phytochemical contents are associated with a wide range of bioactivities, including antioxidant, anti-inflammatory, hepatoprotective, anti-cancer, anti-obesity, and hypolipidemic activity (10,11). Vitamin K is naturally found in green leafy vegetables as phylloquinone (vitamin K1), while menaquinones (vitamin K2; MK-s, where s represents the number of isoprenyl side-chain units) are produced by micro-organisms including intestinal bacteria (12). Furthermore, higher concentrations of vitamin K1 have been reported in spinach compared with other foods and beverages (13). Vitamin K is a class of fat-soluble vitamins and plays important roles in the coagulation cascade, anti-inflammatory pathways, and in the regulation of serum calcium levels and bone metabolism, all of which have an effect on cardiovascular health (14). Compared with vitamin K1, vitamin K2 has more physiological benefits as it increases the regulation of transcription factors that participate in steroid hormone synthesis, and is associated with increased bone metabolism, inhibition of vascular calcification, reduction of cholesterol, and suppression of peripheral inflammation (15-17).

Lactic acid bacteria (LAB) as starter cultures, probiotics, and producers of vitamins are vital components of various food fermentation processes (18). Among LAB, Lactococcus lactis are natural producers of vitamin K2 (19). Vitamin K2 production has been observed in various bacteria that are involved in established food fermentation processes, and the exact mechanism of vitamin K2 production has been elucidated (20). However, earlier studies mainly focused on the vitamin K2 levels in fermented dairy products, with only a few studies focusing on vitamin K2 production by LAB itself (21-23). Moreover, to the best of our knowledge, there are no available reports on the fermentation of green vegetables such as spinach by L. lactis.

In this study, it was hypothesized that biotransformation of vitamin K1 to K2 in spinach juice (S.juice) during L. lactis-mediated fermentation can improve its health benefits. Therefore, the objective the present work was to determine the anti-inflammatory effects of fermented S.juice including rich vitamin K2 against the LPS-stimulated human umbilical vein endothelial cells (HUVECs) and to elucidate the potential anti-vascular inflammatory mechanism.

Materials and methods

Chemicals and reagents

Antibodies against VCAM-1 (cat. no. 13662), ICAM-1 (cat. no. 4915), and phospho-NF-κB p65 (cat. no. 3033), NF-κB p65 (cat. no. 8242), phospho-IκB-α (cat. no. 2859), IκB-α (cat. no. 9242), β-actin (cat. no. 4967) (all dilution, 1:1,000), and goat-anti-rabbit IgG-horseradish peroxidase-conjugated (HRP) secondary antibodies (dilution, 1:5,000; cat. no. 7074) were purchased from Cell Signaling Technology, Inc. Enzyme-linked immunosorbent assay (ELISA) kits for MCP-1 (Human MCP-1 ELISA Set; cat. no. 555179) and IL-6 (Human IL-6 ELISA Set; cat. no. 555220) were purchased from BD Bioscience. LPS was purchased from Sigma Chemical Co. The NE-PER Nuclear and Cytoplasmic Extraction Reagents kit (cat. no. 78833; Thermo Fisher Scientific, Inc.) were used. Triton X-100 and 10% formalin solution were obtained from Sigma-Aldrich; Merck KGaA. Additionally, 4',6-diamidino-2-phenylindole (DAPI) and Alexa Fluor dyes (488 and 555) were obtained from Thermo Fisher Scientific, Inc.

Isolation and identification of L. lactis

A strain of L. lactis (KCCM12759P) was isolated from sediment collected from the fish-farm tank at the National Institute of Fisheries Science (Pohang, Republic of Korea). The sediment sample was serially diluted, plated on De Man, Rogosa and Sharpe (MRS; Sigma-Aldrich; Merck KGaA) Agar with bromocresol purple, and incubated anaerobically at 30̊C for 24 h. For 16s rDNA gene sequencing analysis, the isolated yellow colonies were transferred to MRS broth, cultured, and DNA was subsequently extracted using the Wizard Genomic DNA Purification kit (Promega Corporation) following to the manufacturer's method. The 16s rDNA gene was amplified using 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-TACGGCTACCTTGTTACGACTT-3') primers. PCR was performed using the AccPower PCR Premix kit (Bioneer Corporation) on a Takara PCR Thermal Cycler Dice Gradient system (Takara Bio, Inc). The PCR conditions were as follows: Initial denaturation at 95˚C for 5 min, 30 cycles of denaturation at 95˚C for 30 sec, annealing at 55˚C for 30 sec and extension at 72˚C for 90 sec and final extension at 72˚C for 5 min. The PCR product was analyzed using 1% agarose gel electrophoresis, purified with a NucleoSpin Gel and PCR clean-up kit (cat. no. 740609.50; Macherey Nagel, Inc.), and sequenced using an ABI 3730 sequencer (Applied Biosystems). Homology search was performed using the Basic Local Alignment Search Tool (BLAST) program from the NCBI (https://blast.ncbi.nlm.nih.gov/Blast.cgi). The 16s rDNA of a selected isolate showed 99% similarity with L. lactis (acc. no. MG754653.1). The L. lactis used in this study was deposited at the Korean Culture Center of Microorganisms (KCCM12759P) and stored in vials of MRS broth with 50% (v/v) glycerol at -70˚C until use.

Preparation of fermented spinach juice samples

Spinach (Spinacia oleracea L.) was obtained in April 2020 from an herbal medicine cooperative situated in Gyeongsang Province, Republic of Korea. The isolated L. lactis strain (1x108 CFU/ml) was inoculated into 200 ml sterile S.juice and into MRS medium as control, and incubated anaerobically at 26˚C with agitation for 48 h. S.juice was obtained by pressing fresh spinach, lyophilized, and 5% (w/v) of S.juice was calculated as the weight of the dried product, corresponding to the volume of the juice for fermentation. After fermentation, the culture solution including the pellet and the residues of spinach were evaporated three times at 50˚C for 1 h using a rotary evaporator (N-1000; Eyela); the cell pellet was subsequently washed with 10 mM PBS (pH 7.4). Vitamin K2 (menaquinone), a fat-soluble vitamin, is produced during fermentation by bacteria such as lactic acid bacteria (20). Vitamin K2 is commonly recovered from microbial cells by liquid-liquid extraction or supercritical fluid extraction and stored at -70˚C until in experiments for vitamin K analysis (22,23).

Quantitative analysis of vitamin K in L. lactis

The vitamin K analysis was performed according to Liu et al (21) and Berenjian et al (24) with minor modifications. Briefly, the cell pellet was added to 6 ml of resuspension solution (10 mM PBS containing 1% lysozyme) and incubated at 37˚C for 1 h. Subsequently, 24 ml of extraction buffer (n-hexane-isopropanol=2:1, v/v; Sigma-Aldrich; Merck KGaA) was added, vortexed twice at 25˚C for 30 sec, and centrifugation at 3,000 x g for 10 min. Next, the lower phase was collected, an equal volume of n-hexane was added, then evaporated at 25˚C for 30 min. Subsequently, the dried pellet was dissolved by adding 2 ml isopropanol; the sample was then filtered with a 0.45 µm syringe filter (MilliporeSigma) for high-performance liquid chromatography (HPLC) analysis using an Agilent C18 column (4.6x250 mm; Agilent Technologies, Inc.), maintained at a temperature of 40˚C during the analysis. The flow rate, injection volume, and detection wavelength were 0.4 ml/min, 50 µl, and 254 nm, respectively. Reagent-grade vitamin K1 and K2 (Sigma Chemical Co.), dissolved in methanol (0.5% w/v), were used as reference standards. The mobile phase was a mixture of methanol, isopropanol, and n-hexane (25:37.5:37.5; v:v:v). The calibration curve was obtained by plotting the peak area vs. concentration. The slope, intercept, and correlation coefficients for the calibration curve were then determined. The analysis was performed in triplicate (25).

Cell culture and treatment

HUVECs were purchased from Lonza Group, Ltd. and cultured in EBM-2 basal medium (cat. no. CC-3156; Lonza Group, Ltd.) supplemented with EGM-2 SingleQuots supplement pack (cat. no. CC-4176; Lonza Group, Ltd.). HUVECs were incubated at 37˚C with 5% CO2, and the medium was replaced every 48 h.

Cytotoxicity assays

The cytotoxicity of S.juice and fermented S.juice was measured using the Cell Counting Kit-8 assay (CCK-8; Dojindo Molecular Technologies, Inc.). HUVECs were cultured in 48-well plates (1x105 cells/well) and pretreated with S.juice or fermented S.juice at 2 concentrations (200 and 400 µg/ml) for 1 h at 37˚C, followed by stimulation with LPS (10 µg/ml) for an incubation of 18 h at 37˚C. Subsequently, 400 µl of CCK-8 working solution was added to each well and incubated at 37˚C for 1.5 h. Cell viability was subsequently measured using CCK-8 solution and a microplate reader set at a detection wavelength of 450 nm (Tecan Group, Ltd.).

Enzyme-linked immunosorbent assay (ELISA)

HUVECs were seeded in 24-well plates (1x105 cells/well) and treated with LPS in the presence or absence of S.juice and fermented S.juice at 200 or 400 µg/ml for 18 h. The cell-free supernatant fractions were collected, and the levels of MCP-1 and IL-6 released into the culture supernatant were measured using the Quantikine ELISA kits according to the manufacturer's instructions.

Western blotting

The whole cell proteins were isolated using radioimmunoprecipitation assay (RIPA) lysis (cat. no. MB-030-0050; Rockland Immunochemicals Inc.) containing a 1x protease/phosphatase inhibitor cocktail (cat. no. 5872; Cell Signaling Technology, Inc.). The protein quantification was performed using the Bio-Rad Protein Assay (Bio-Rad Laboratories, Inc.). Then, 30 µg of proteins were separated by 12% SDS-PAGE and transferred to a PVDF membrane. The membrane was blocked with EveryBlot Blocking Buffer (cat. no. 12010020; Bio-Rad Laboratories, Inc.) for 1 h at room temperature, then incubated with 1:1,000 diluted primary antibodies: VCAM-1, ICAM-1, phospho-NF-κB, NF-κB, phospho-IκB-α, IκB-α, and β-actin at 4˚C overnight. Following washing with TBST, the membrane was then incubated with 1:5,000 diluted goat-anti-rabbit IgG-HRP-conjugated secondary antibody for 1 h at room temperature. The bands were detected using an enhanced chemiluminescence detection kit; Clarity Max western ECL substrate (cat.no. 1705062; Bio-Rad Laboratories, Inc.) and were analyzed using the ImageJ software (version 1.52; National Institutes of Health).

Nuclear and cytosolic fractionation

An NE-PER Nuclear and Cytoplasmic Extraction Reagent kit (Thermo Fisher Scientific, Inc.) was used to extract nuclear and cytoplasmic proteins. After treatment with S.juice and fermented S.juice, the HUVEC were homogenized in Cytoplasmic Extraction Reagent I buffer supplemented with protease inhibitor cocktail (Thermo Fisher Scientific, Inc.). After homogenization, the cells were isolated by centrifugation at 16,000 x g for 5 min at 4˚C in the presence of Cytoplasmic Extraction Reagent Ⅱ (Thermo Fisher Scientific, Inc.). The supernatant of cytoplasmic extract and left pellet were homogenized in Nuclear Extraction Reagent buffer supplemented with protease inhibitor cocktail (Thermo Fisher Scientific, Inc.). The cells were subsequently isolated by centrifugation at 16,000 x g for 10 min at 4˚C; the protein content of the supernatant (representing the nuclear extract) was quantified using Pierce™ Coomassie Plus (Bradford) Assay kit (cat. no. 23236; Thermo Fisher Scientific, Inc.).

Immunofluorescence

After treatment with S.juice and fermented S.juice, HUVECs were fixed with 10% formalin for 10 min at room temperature and immersed in 0.1% Triton X-100 (cat. no. T8787; Sigma-Aldrich; Merck KGaA) in PBS for 15 min at room temperature. Subsequently, the cells were blocked with 3% bovine serum albumin (BSA) (cat. no. 7906; Sigma-Aldrich; Merck KGaA) in PBS for 1 h at room temperature and incubated overnight at 4˚C with primary antibody (NF-κB p65; dilution, 1:200; cat. no. 8242; Cell Signaling Technology, Inc.). For fluorescence detection, the cells were incubated for 1 h at 4˚C in the dark with secondary goat anti-rabbit IgG Alexa Fluor 555 and 488 antibody (dilution 1:500; cat. no. 4413; Cell Signaling Technology, Inc.) and goat anti-rabbit IgG Alexa Fluor 488 antibody (dilution 1:200; cat. no. 4412; Cell Signaling Technology, Inc.). Next, the cells were stained with DAPI solution for the imaging of the cell nuclei through fluorescence microscopy (IX71; Olympus Corporation) at the same gain and exposure time.

Statistical analysis

All the experiments were performed at least three times and all graph bar data are presented as mean ± standard error of the mean (SEM). The mean values of different groups were compared using one-way analysis of variance (ANOVA) followed by Tukey's tests (GraphPad Prism 8; GraphPad Software, Inc.). For all data, statistical significance was defined as P<0.05.

Results

Effect of L. lactis fermentation on vitamin K contents of S.juice

Table I shows the respective vitamin K1 and vitamin K2 contents of S.juice and fermented S.juice. Single-strain L. lactis starters were used to ferment S.juice for 48 h at 26˚C. The initial LAB cell count was ~1x108 CFU/ml. After 48 h of fermentation with the L. lactis strains, the LAB cell count in fermented S.juice was 3.58x1010 CFU/ml. The vitamin K1 and K2 levels of fermented S.juice as determined using HPLC were 1.77 and 1.89 µg/ml, respectively (Table I). The vitamin K2 content of fermented S.juice was approximately 47-fold higher than in S.juice. There were no significant differences between the vitamin K1 contents of S.juice and fermented S.juice.

Table I

Vitamin K1 and K2 contents of S.juice and Lactococcus lactis-fermented S.juice.

	LAB counts (CFU/ml)
Sample	Initial	Final	Vitamin K1 content (µg/ml)	Vitamin K2 content (µg/ml)
S.juice	-	-	1.75	0.04
Fermented S.juice	1x108	3.58x1010	1.77	1.89

S.juice, spinach juice; LAB, lactic acid bacteria; CFU, colony-forming units.

Cytotoxic effects of S.juice and fermented S.juice in HUVECs

The potential cytotoxic effect of S.juice and fermented S.juice against HUVECs was evaluated using CCK-8 assays (Fig. 1). The cell viability was not affected by S.juice and fermented S.juice at either of the test concentrations (200 and 400 µg/ml), indicating no toxicity against HUVECs at these concentrations.

Figure 1

Cytotoxic effects of S.juice and Lactococcus lactis-fermented S.juice in human umbilical vein endothelial cells. The cells were treated with different concentrations of S.juice and fermented S.juice (200 and 400 µg/ml) for 1 h, followed by stimulation with LPS (10 µg/ml) for 18 h. The cell viability was determined using the Cell Counting Kit-8 assay. The results are presented as the mean ± SEM (n=3). S.juice, spinach juice; NC, negative control; LPS, lipopolysaccharide.

S.juice and fermented S.juice suppress the levels of pro-inflammatory cytokines and chemokines in LPS-stimulated HUVECs

Inflammatory cytokines and chemokines, such as MCP-1 and IL-6, play critical roles in inflammation (26). Therefore, to investigate the potential anti-inflammatory effects of LPS-stimulated HUVECs, the levels of MCP-1 and IL-6 were determined. Stimulation with LPS significantly increased the production of inflammatory cytokines such as MCP-1 and IL-6 in HUVECs (Fig. 2). However, the treatment with fermented S.juice of 400 µg/ml markedly inhibited the levels of MCP-1 and IL-6 compared with the treatment with S.juice, in a dose-dependent manner.

Figure 2

Effects of S.juice and fermented S.juice on the LPS-induced expression of (A) MCP-1 and (B) IL-6 in HUVECs. HUVECs were pretreated with S.juice and fermented S.juice (200 and 400 µg/ml) for 1 h, and subsequently treated with LPS (10 µg/ml) for 18 h. The expression levels of MCP-1 and IL-6 were determined using ELISA. The results are presented as the mean ± SEM (n=3). **P<0.01 vs. the control group. ##P<0.01, ###P<0.001 and ####P<0.001 vs. the LPS group. S.juice, spinach juice; LPS, lipopolysaccharide; MCP-1, monocyte chemoattractant protein-1; IL-6, interleukin 6; HUVECs, human umbilical vein endothelial cells.

S.juice and fermented S.juice downregulate the expression of adhesion molecules in LPS-stimulated HUVECs

Adhesion molecules such as VCAM-1 and ICAM-1 play major roles in the initial adhesion and subsequent trans-endothelial migration of leukocytes into inflamed vessels (26). In this study, the effects of S.juice and fermented S.juice on the expression VCAM-1 and ICAM-1 following LPS treatment were determined by western blotting. The expression of both VCAM-1 and ICAM-1 in LPS treatment was significantly increased compared with the control (Fig. 3A and 3B). However, the increase in the VCAM-1 and ICAM-1 levels following LPS treatment was attenuated both by S.juice and fermented S.juice in a dose-dependent manner, with the fermented S.juice exerting a greater effect.

Figure 3

Effect of S.juice and fermented S.juice on LPS-induced expression of (A) the main adhesion molecules such as (B) VCAM-1 and (C) ICAM-1 in HUVECs. HUVECs were treated with S.juice or fermented S.juice (200 and 400 µg/ml) for 1 h, followed by stimulation with LPS (10 µg/ml) for 18 h. HUVECs not treated with either of the juices were used as the control. The results for VCAM-1 and ICAM-1 were analyzed using the ImageJ software. The results are presented as the mean ± SEM (n=3). *P<0.05 and **P<0.01 vs. the control group. #P<0.05, ##P<0.01 and ###P<0.001 vs. the LPS group. S.juice, spinach juice; LPS, lipopolysaccharide; VCAM-1, vascular cell adhesion molecule 1; ICAM-1, intercellular adhesion molecule-1; HUVECs, human umbilical vein endothelial cells.

S.juice and fermented S.juice suppress the LPS-induced activation of NF-κB

NF-κB plays an important role in regulating the production of inflammatory mediators (27). Cytokines promote inflammation by promoting NF-κB phosphorylation and IκB-α degradation (28). In this study, the effects of the S.juice and fermented S.juice on NF-κB activation following LPS treatment were investigated. The levels of NF-κB activation and IκB-α degradation were greater following LPS treatment, compared with the control (Fig. 4). However, the pre-treatment with S.juice and fermented S.juice significantly inhibited the activation of NF-κB (Fig. 4A) as well as the degradation of IκB-α (Fig. 4B) induced by LPS in a concentration-dependent manner.

Figure 4

Detection of the protein levels of NF-κB p65, NF-κB p-p65, IκBα and p-IκBα using western blotting. S.juice and fermented S.juice inhibit (A) LPS-induced activation of NF-κB and (B) degradation of IκB-α. Human umbilical vein endothelial cells were pretreated with different concentrations of S.juice and fermented S.juice (200 and 400 µg/ml) for 1 h and stimulated with LPS (10 µg/ml) for 18 h. The results are presented as the mean ± SEM (n=3). **P<0.01 and ****P<0.0001 vs. the control group. ###P<0.001, and ####P<0.0001 vs. the LPS group. S.juice, spinach juice; LPS, lipopolysaccharide; nuclear factor κB, NF-κB; inhibitor of NF-κB, IκB-α; p-, phosphorylated.

S.juice and fermented S.juice suppress the NF-κB p65 signaling pathway in HUVECs

Once activated, the NF-κB p65 subunit is translocated from the cytoplasm to the nucleus and regulates the expression of target genes (28). The effect of S.juice and fermented S.juice on LPS-induced nuclear translocation of NF-κB p65 was investigated. Immunofluorescence assays demonstrated that the LPS-induced nuclear translocation of NF-κB p65 was inhibited by both S.juice and fermented S.juice (Fig. 5). Overall, this indicated that S.juice and fermented S.juice inhibited the production of inflammatory factors by suppressing the NF-κB pathway. Furthermore, fermented S.juice attenuated the LPS-induced expression of NF-κB p65 to a greater extent than S.juice.

Figure 5

Effect of S.juice and fermented S.juice on nuclear levels of NF-κB p65. (A) LPS stimulation was associated with increased nuclear translocation of NF-κB p65, which was attenuated in HUVECs following treatment with S.juice and fermented S.juice treatment. (B) Immunofluorescence staining shows the translocation of NF-κB (magnification, x200) to the nucleus in HUVECs. The results are presented as the mean ± SEM (n=3). **P<0.01 vs. the control group. ####P<0.001 vs. the LPS group. S.juice, spinach juice; nuclear factor κB, NF-κB; inhibitor of NF-κB; HUVECs, human umbilical vein endothelial cells; CON, control' LPS, lipopolysaccharide.

Discussion

The present study is, to the best of our knowledge, the first characterization of the mechanism involved in the anti-inflammatory effects of fermented S.juice in HUVECs. Although spinach has been reported to have strong protective effects against vascular inflammation, the anti-inflammatory activity of fermented spinach products remains unclear (10). Fermentation of spinach by L. lactis may synthesize into bioactive forms such as vitamin K2, MK4, MK7, MK9 and to more forms (20). First, it has been demonstrated that vitamin K2, a major bioactive component of the fermented S.juice, inhibits the endothelial inflammatory response induced by LPS (16,27). The anti-inflammatory effects include the suppression of the secretion of pro-inflammatory cytokines and chemokines and the downregulation of molecules that facilitate adhesion to endothelial cells (10). Our mechanistic analysis revealed that the anti-inflammatory effect of fermented S.juice was possibly mediated via the suppression of NF-κB activation. Thus, to the best of our knowledge, this is the first study to show that fermented S.juice attenuates HUVECs-associated inflammatory responses possibly via the inhibition of the NF-κB signaling pathway.

Spinach is a rich source of vitamins, minerals, phenolic compounds, and carotenoids that are responsible for its various bioactivities (10). The main vitamin in spinach is vitamin K1, which is used for the microbial biosynthesis of vitamin K2, including that in intestinal bacteria (12,13). An in silico study of LAB genome revealed that the genes encoding enzymes of the vitamin K2 biosynthesis pathway are different and vary depending on the strain (22,29). LAB play vital roles in food fermentation processes including vitamin K2 production (30); among the LAB, L. lactis is associated with particularly high levels of vitamin K2 production (19,31). Our results indicate that the vitamin K2 levels in S.juice increase during fermentation by L. lactis. The fat-soluble vitamin K class, including vitamin K1 and K2, regulates vascular calcification, bone metabolism, and inflammation, all of which may affect cardiovascular health (32). A previous study reported that the intake of vitamin K2, but not vitamin K1, reduced the risk of coronary heart disease mortality, all-cause mortality, as well as severe aortic calcifications (33). The typical recommended dietary intake of vitamin K in North America varies from 50 to >600 µg/day for vitamin K1, and from 5 to 600 µg/day for vitamin K2 (34). Kawashima et al (35) reported anti-atherosclerotic effects in hypercholesterolemic rabbits treated with vitamin K2 (MK-7; 1 to 10 mg/kg body weight/day) including reduced intimal thickening and ester-cholesterol deposition in the aorta, as well as a slower progression of atherosclerotic plaques. Vitamin K2 (MK-4) decreased the levels of inflammatory markers such as IL-6, MCP-1, and TNF-α in the liver (36); this might be attributed to the anti-inflammatory effects of vitamin K2. Vitamin K2 (MK-4; 0.1-10 µM) reduced the levels of IL-6, IL-1β, and TNFα in LPS-induced MG6 mouse microglia-derived cells (37). Excess vitamin K attenuated the general plasma inflammatory index (38). The overall expression of pro-inflammatory cytokines and chemokines decreased in this study; therefore, vitamin K2 might directly suppress the vascular NF-κB pathway; this is in line with a previous report (37). In the present study, the treatment levels of fermented S.juice were 200 and 400 µg/ml; 400 µg/ml fermented S.juice contained approximately 211 µg of vitamin K2 (MK-4).

Vascular inflammatory responses contribute to the pathogenesis of atherosclerosis (39), and endothelial cells play an important role in vascular inflammation (40). Pro-inflammatory stimuli such as LPS activate the endothelial inflammatory response, resulting in the secretion of pro-inflammatory cytokines that contribute to the pathogenesis of cardiovascular disease (41). Endothelial inflammatory responses are characterized by the overproduction of inflammatory mediators, including pro-inflammatory chemokines (e.g., MCP-1), cytokines (e.g., IL-6), and adhesion molecules (e.g., VCAM-1 and ICAM-1). In the present study, LPS significantly upregulated the expression of MCP-1, IL-6, VCAM-1, and ICAM-1; however, this pro-inflammatory effect of LPS was significantly inhibited by pretreatment of HUVECs with S.juice and in particular fermented S.juice.

The inhibitory effect of fermented S.juice on the expression of inflammatory mediators was similar to that of vitamin K2, an inhibitor of the NF-κB signaling pathway (37). These findings indicated that the LPS-induced secretion of MCP-1, IL-6, VCAM-1, and ICAM-1 in HUVECs was mediated via the NF-κB signaling pathway. In addition, the inhibitory effect of fermented S.juice on inflammatory mediators was likely mediated via inhibition of the NF-κB pathway.

NF-κB is involved in the LPS-induced inflammation response (42). LPS induces the phosphorylation and degradation of IκBα, as well as the phosphorylation, release, and nuclear translocation of NF-κB to activate target gene expression (43,44). To the best of our knowledge, the present study characterized for the first time the effects of S.juice and fermented S.juice on the LPS-induced activation of NF-κB.

These results are not direct evidence that the NF-κB pathway is suppressed by vitamin K2 to exert a protective effect against vascular inflammation; however, previous data that demonstrated the role of vitamin K2 in suppressing LPS-induced microglial inflammation (36), combined with our results, suggest that vitamin K2-enriched fermented S.juice may inhibit LPS-induced inflammation by suppressing the NF-κB signaling pathway.

In conclusion, this is, to the best of our knowledge, the first report of the inhibitory effect of fermented S.juice against LPS-induced inflammation via suppression of NF-κB signaling and downregulation of ICAM-1, VCAM-1, IL-6, and MCP-1 in HUVECs. Our present findings indicate that fermented S.juice might be a potential novel anti-inflammatory agent against vascular inflammation or cardiovascular disease induced by inflammation.