Qi Wang, Juqing Huang, Yafeng Zheng, Xuefang Guan, Chenchun Lai,Huiying Gao, Chi-Tang Ho, Bin Lin,*

a Institute of Agricultural Engineering, Fujian Academy of Agricultural Sciences, Fuzhou 350003, China

b College of Food Science, Fujian Agriculture and Forestry University, Fuzhou 350002, China

c Fujian Key Laboratory of Agricultural Product (Food) Processing, Fuzhou 350003, China

d Department of Food Science, Rutgers University, New Brunswick, NJ 08901, USA

Keywords:

Oolong tea

Selenium

Anti-inflammatory

NF-κB and MAPK pathways

A B S T R A C T

Both tea polyphenols and selenium (Se) have been suggested to exert the health benefits via the regulatory capacities of chronic inflammation, which make Se-enriched oolong tea a promising beverage as an anti-inflammatory diet.The aim of this study is to investigate the anti-inflammatory effects of Se-enriched oolong tea extract (Se-TE) and underlying mechanism in lipopolysaccharide (LPS)-induced RAW264.7 cells.Se-TE treatments (50 and 150 μg/mL) significantly suppressed the over-production of nitric oxide (NO) and prostaglandin E2 (PGE2) in LPS-stimulated macrophages via downregulating the expression of nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2).Moreover, Se-TEs also effectively inhibited the productions of inflammatory cytokines, such as tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-1β(IL-1β).Furthermore, Se-TE could block mitogen-activated protein kinase (MAPK) and nuclear factor-kappa B (NF-κB) signaling pathways through the inhibition of the phosphorylation of key proteins (IκB-α, p65, p38,ERK, and JNK) and the translocation of the p65 subunit into the nucleus.Collectively, our results indicated that Se-TE may have the potential to be used as a novel food ingredient for the development of various anti-inflammatory foods and the treatment and prevention of chronic inflammation-related diseases.

1.Introduction

Inflammation is the immune system’s response to various harmful stimuli such as pathogens, damaged cells, and chemicals,and thus a vital defense mechanism of host to protect the body from threatening invaders [1].However, when the inflammatory response fails to resolve certain persistent noxious stimuli in the body, i.e.pro-inflammatory cytokines released by adipose tissue,chronic inflammation occurs and causes silent damage systemically throughout the body [2].Increasing evidence indicates that chronic inflammation has been associated with a broad range of non-infectious diseases, including diabetes mellitus, cardiovascular disease,asthma and cancers [3].Diet is a crucial factor on the regulation of immune response.In recent decades, the health benefits of an anti-inflammatory diet has received a growing awareness and applied as healthy dietary patterns to reduce the risks of these disorders drove by prolonged inflammation [4].The most well-known antiinflammatory diets are the Mediterranean and Okinawan diets,which contains a large amount of vegetable and fruits.Expect for the beneficial macronutrients from diet, an anti-inflammatory diet can be further improved by incorporating the use of healthy beverages, herbs,spices and supplements [5].

Tea is a widely accepted healthy beverage derived from the leaves ofCamellia sinensis.The increasing tea consumption worldwide is due to its pleasant aroma, delightful flavor and more importantly its health benefits, including antioxidant, anti-inflammatory,anti-obesity, anti-hypertensive, antimicrobial, and anti-cancer properties [6].These health-promoting capacities of tea consumption have been primarily attributed to its high levels of phenolic compounds [7].In addition, the phenolic compounds of tea can exhibit its anti-inflammatory activity in suppressing the synthesis and action of many pro-inflammatory mediators [2].Based on the degree of fermentation and its polyphenols, tea can be mainly classified into 3 types: unfermented green tea, fully fermented black tea, and semifermented oolong tea.Green tea contains only monomeric catechins,i.e.(-)-epicatechin (EC), (-)-epigallocatechin (EGC), (-)-epicatechin-3-gallate (ECG) and (-)-epigallocatechin-3-gallate (EGCG).Black tea and oolong tea contain both catechins and their oxidized polymeric substances, among which thea flavins and thearubigins are the major polyphenolic compounds in black tea, whereas theasinensins are the most characteristic for oolong tea [8,9].Despite the significant difference of chemical compositions, all 3 types of tea can be used as a potent anti-inflammatory agent, which is convenient and safe in the dairy consumption [10-12].

Selenium (Se) is an essential trace element, which plays a crucial role in the antioxidant defense system and the reduction of the risks of several human diseases.Recently, increasing evidence suggests that the health benefits of Se can be related to its regulatory capacity of inflammatory response via variable and complex mechanisms [13].Tea plants have a strong ability to retain high levels of Se in its organic form, which is able to exhibit synergistic effect with the tea polyphenols in enhancing its heath functions [14,15].Therefore,Se-enriched tea has been recognized as a health-promoting beverage for its ability to provide more prominent health benefits than regular tea [14].However, unlike regular or Se-enriched green tea,whose biological compounds and their health functions have been intensively investigated [14,16-18], the anti-inflammatory potential of Se-enriched oolong tea as well as its underlying mechanism have not been adequately studied.

Based on the potential effects of tea polyphenols and Se on the inflammatory processes, this study evaluated the anti-inflammatory activity of Se-enriched oolong tea extract (Se-TE) on the inflammatory mediators in a murine macrophage (RAW264.7) cell model, which was stimulated by LPS exposure.The underlying mechanisms were then investigated through assessing the effects of Se-TE on the expression of key proteins involved in NF-κB and MAPK signaling pathways in LPS-stimulated macrophages.

2.Materials and methods

2.1 Chemicals and reagents

The tea extract (Se-TE) was prepared from a local Se-enriched oolong tea containing (1.09 ± 0.22) μg/g of Se, which was provided by Jiuhe tea plantation in Wuyishan City, Fujian China.The main chemical composition of Se-TE is as follows: total polyphenols((31.06 ± 0.67)%,m/m), caffeine ((8.96 ± 0.21)%,m/m) and organic selenium ((0.63 ± 0.05) μg/g).

Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), dimethyl sulfoxide (DMSO), penicillinstreptomycin solution and 0.25% Trypsin were purchased from Gibco (Grand Island, NY, USA).Nitric oxide (NO) assay kit was provided by JianCheng Bioengineering Institute (Nanjing, China).3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)was obtained from Sigma-Aldrich (St.Louis, MO, USA).All the primary and secondary antibodies used in Western Blotting assay were purchased from Cell Signaling Technology (Shanghai, China).Interleukin-6 (IL-6), interleukin-1β (IL-1β), prostaglandin E2 (PGE2)and tumor necrosis factor-α (TNF-α) ELISA kits were acquired from Elabscience Biotechnology Co., Ltd.(Wuhan, China).All other the chemicals used were of analytical grade.

2.2 Cell culture

The RAW264.7 murine macrophage cells were provided by Biofavor Biotech (Wuhan, China) and cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FBS and 1% penicillin-streptomycin solution (Gibco, USA) in a humidified incubator with 5% CO2at 37 °C.

2.3 Cytotoxicity assay

To evaluate the cytotoxicity of Se-TE, the viability of RAW264.7 cells was measured by the MTT assay [22].The cells (5 × 104/well)were cultured in 96-well plates and incubated at 37 °C with 5% CO2overnight.Then, the cells were treated with serial concentrations of Se-TE (25, 50, 100, 150, 250 and 500 μg/mL) and incubated for 24 h in a CO2incubator.The cells treated with culture medium along were used as a control.After the exposure period, the medium was discarded and 10 μL MTT (10 mg/mL) was added into each well and incubated at 37 °C for another 4 h.Subsequently, all of the MTT media were discarded and 100 μL DMSO was added to dissolve the MTT formazan crystals in every well.The absorbance was detected at 568 nm using a microplate reader (Multiskan MK3, Thermo Scientific, Waltham, MA, USA).Cell viability was presented as a percentage (%) of absorbance values in treated cells relative to that in control cells.The experiments were performed in triplicate.Based on the result of cell viability, low and high concentrations of Se-TE in the safe range were selected for the following experiments.

2.4 Determination of NO level

The production of NO was determined using the commercial NO assay kit according to the manufacturer’s instruction (JiangCheng,Nanjing, China).The RAW 264.7 cells (5 × 104/well) were incubated in 96-well plates at 37 °C with 5% CO2overnight and pretreated with low and high dosages of Se-TE (100 and 250 μg/mL) for 4 h prior to being stimulated with LPS (1 μg/mL) for 12 h.The blank control group was treated with DMEM without the Se-TE and LPS,while the positive group was treated with DMEM containing LPS(1 μg/mL).The cell culture supernatant was collected for the detection NO production via a chemical reaction with the Griess reagent,and the absorbance was determined at 540 nm using a microplate spectrophotometer reader with sodium nitrite as a standard.All of the experiments were performed in triplicate.

2.5 Determination of PGE2, TNF-α, IL-1β and IL-6

The levels of PGE2and the pro-inflammatory cytokines, including TNF-α, IL-1β and IL-6, were measured by using corresponding mouse immunoassay ELISA kits according to the manufacturer’s instructions.The RAW264.7 cells were treated the same as the procedure of determination of NO production.The cell-free supernatant samples were obtained after the centrifugation (1 000 ×g,20 min) at 4 °C.The absorbance of each sample was measured at 450 nm using a microplate spectrophotometer reader.

2.6 Determination of mRNA expression levels of nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2)

The mRNA expression levels ofCOX-2andiNOSin the cells were determined by qRT-PCR according to a previously reported method [22].After the RAW264.7 cells were treated by the same procedure as in the previous experiments, RNA was isolated using TRI-Reagent (Invitrogen) from the collected cells according to the manufacturer’s instructions.After the determination of the quantity and purity of the resulting RNA, 5 μg of RNA was reverse transcribed into first-stand cDNA using HiScript 1st Stand cDNA synthesis kit(Vazyme Biotech, Nanjing, China).qRT-PCR was conducted using SYBR Green Master Mix (Vazyme Biotech, Nanjing, China) on the Quant Studio 6 Flex Real-Time PCR System (ABI, CA, USA).The gene expression was normalized usingGAPDHas the reference housekeeping gene, and the relative gene expression was determined by the comparative CT (2?ΔΔCt) method.The sequences of the primers forCOX-2,iNOSandGAPDHwere listed in Table 1.

Table 1The primer sequences used for qRT-PCR.

2.7 Western blotting assay

The RAW 264.7 cells (5 × 104/well) were incubated in 6-well plates at 37 °C with 5% CO2overnight, and then were pre-treated with low and high dosages of Se-TE (100 and 250 μg/mL) for 4 h prior to being stimulated with LPS (1 μg/mL) for another 1 h.The cells were washed twice with ice-cold PBS and lysed in RIPA buffer (Beyotime,Shanghai, China) for 30 min on the ice.Afterwards, the lysates were centrifuged at 14 000 ×gfor 10 min at 4 °C, and the supernatant was collected for the measurement of protein concentration.Protein samples were dissolved in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS/PAGE) loading buffer and heated in the boiled water for 10 min.The protein samples were then resolved and separated by 5%-12% SDS/PAGE gels and then electrophoretically transferred onto polyvinylidene fluoride (PVDF) membranes, which were incubated with corresponding primary antibodies against JNK, p-JNK, P38, p-p38, ERK1/2, p-ERK1/2, p65, p-p65, IκBα,p-IκBα and GADPH overnight at 4 °C.After washing with TBST for 5 times, the PVDF membrane was reacted with the secondary antibodies (1:50 000) for another 2 h at 37 °C.Finally, an enhanced chemiluminescence (ECL) was used to detect the protein bands on PVDF membranes and scanned by an image analyzer (LAS-3000,Fujifilm, Tokyo, Japan).

2.8 Confocal microscopy

To visualize the expression and nuclear translocation of p65 in LPS-stimulated RAW264.7 cells, the described method was adapted with minor modifications [21].The RAW264.7 cells (5 × 104/well)were incubated in 24-well plates overnight and pre-treated with 250 μg/mL Se-TE for 4 h prior to being stimulated with 1 μg/mL LPS for another 12 h.Then the treated cells and the cells from control groups were collected and washed with cold PBS, and then fixed with 4% formaldehyde for 15 min at room temperature.Subsequently,cell permeabilization was performed by adding with 0.5% (V/V)Triton-X100 for 20 min and blocked with 0.5% (m/V) bovine serum albumin (BSA) dissolved in PBS for 30 min at room temperature,followed by incubation with primary antibody rabbit anti-p65(1:50, Proteintech, Wuhan, China) overnight at 4 °C.After incubation with the fluorophore (Cy3)-conjugated secondary antibody, goat antirabbit IgG (H+L) (1:100, Boster Biological Technology, Wuhan,China) at 37 °C for 1 h at the dark, the cells were washed with PBS 3 times.Following that, the cells were stained using 1 μg/mL DAPI for 5 min in the dark.Finally, the cells were observed using a confocal laser scanning microscope (Leica, Wetlzer, Germany).

2.9 Statistical analysis

All data were expressed as the means ± standard deviation (SD)from three independent experiments.Data was statistically evaluated by one-way analysis of variance (ANOVA) andP＜ 0.05 was considered as a significant difference.

3.Results

3.1 Effects of Se-TE on cell viability

To determine the safe dosages of Se-TE on the RAW264.7 cells,MTT assay was carried out to evaluate its cytotoxic effects.The cells were treated with serial concentrations (25, 50, 100, 150, 250 and 500 μg/mL) of Se-TE for 24 h.As shown in Fig.1A, at the concentrations of 25-250 μg/mL, the cell viability of Se-TE treatedcells showed no significant difference (P＞ 0.05) compared with the untreated cells in the blank control, while 500 μg/mL of Se-TE significantly decreased the cell viability to (83.15 ± 5.71)%(P＜ 0.05).Therefore, the concentration range of 25-250 μg/mL of Se-TE was proved to exhibit no cytotoxicity on the RAW264.7 cells.In the subsequent experiments, the concentrations of 50 and 150 μg/mL were selected to represent low and high dosages of Se-TE,respectively.

Fig.1 Effects of Se-TE on the cell viability in RAW246.7 cells (A).The cells were treated with Se-TE (25, 50, 100, 150, 250 and 500 μg/mL) for 24 h.Effects of Se-TE on NO (B), PGE2 (C), TNF-α (D), IL-6 (E) and IL-1β (F) production in RAW246.7 cells.The cells were pre-treated with Se-TE (0, 50 and 150 μg/mL) for 4 h,and then the groups marked with “+” were stimulated with 1 μg/mL LPS for 12 h, while the groups marked with “-” were not stimulated.Data are presented as the mean values ± SD (n = 3).Data bearing different letters are significantly different (P ＜ 0.05).

3.2 Effects of Se-TE on NO and PGE2 production

Excessively production of NO and PGE2by macrophages at the inflammatory site occurs in both acute and chronic inflammation.Therefore, to evaluate the anti-inflammatory effects of Se-TE on LPS-stimulated RAW264.7 cells, the changes of NO and PGE2production were assessed by corresponding assay kits, respectively.As shown in Figs.1B and 1C, compared to the cells in the blank control group,1 μg/mL LPS stimulation caused a significantly increased NO and PGE2concentrations by 3.77- and 8.72-folds, respectively (P＜ 0.05).Se-TE pretreatments showed significant inhibitory effects on LPS-induced NO and PGE2production (P＜ 0.05).The inhibitory rates of NO release by Se-TE pretreatments at 50 and 150 μg/mL were 26.85% and 44.63%, respectively (Fig.1B).In addition,both 50 and 150 μg/mL of Se-TEs induced a dramatically decrease in PGE2production with the inhibition rate of 29.61% and 57.64%, respectively (Fig.1C).The results demonstrated that the pretreatment with Se-TEs could simultaneously inhibit LPS-induced NO and PGE2release.

3.3 Effects of Se-TEs on the release of pro-inflammatory cytokines

To determine whether Se-TE pretreatment could inhibit the inflammatory process via suppressing the release of inflammatory cytokines, the levels of TNF-α, IL-6 and IL-1β in the culture medium were measured by ELISA kits.As shown in Figs.1D–1F, after LPS stimulation for 12 h, the release of TNF-α, IL-6 and IL-1β in the LPS-treated cells was dramatically up-regulated by 5.63-, 5.56- and 5.73-folds compared to the blank control cells (P＜ 0.05).As we expected, the LPS-stimulated production of TNF-α, IL-6 and IL-1β was significantly down-regulated by Se-TE pretreatments (P＜ 0.05).The inhibitory effects of Se-TE was dose-dependent, and high dosage(150 μg/mL) of Se-TE significantly inhibited the production of TNF-α, IL-6 and IL-1β by 56.8%, 50.2% and 56.1%, respectively.

3.4 Effects of Se-TEs on mRNA expression levels of iNOS and COX-2

To investigate the molecular mechanism of inhibitory effects of Se-TEs on the release of NO and PGE2after LPS-stimulation, the mRNA expression levels ofiNOSandCOX-2, which are responsible for the synthesis of NO and PGE2, were determined by qRT-PCR [22].As shown in Fig.2, the expression levels ofiNOSandCOX-2were sharply increased in LPS-stimulated cells (P＜ 0.05), which could explain the significantly increased release of NO and PGE2(Figs.1B, 1C).Se-TE pretreatments could significantly inhibit the mRNA expression levels ofiNOSandCOX-2in a dose-dependent way (P＜ 0.05), and thus ameliorate the over-production of NO and PGE2in LPS-stimulated cells.Taken together, the anti-inflammatory capacity of Se-TE was confirmed at the transcriptional level.

Fig.2 Effects of Se-TE on relative mRNA expression levels of iNOS (A)and COX-2 (B).RAW264.7 cells were pretreated with 0, 50 and 150 μg/mL of Se-TEs, and then the groups marked with “+” were stimulated with 1 μg/mL LPS for 12 h, while the groups marked with “-” were not stimulated.Data are presented as the mean values ± SD from three separate experiments conducted in triplicate on different days.Within each graph, means bearing different letters are significantly different (P ＜ 0.05).

3.5 Inhibitory effects of Se-TEs on LPS-induced NF-κB and MAPK activation

NF-κB and MAPK pathways are well-known signaling pathways playing important roles in the regulation of inflammation progress.Therefore, in order to clarify the potential mechanism for the anti-inflammatory function of Se-TE, the phosphorylation of key proteins involved in NF-κB and MAPK pathways were evaluated by western blotting assays.

For NF-κB signaling pathway, the expressions of NF-κB proteins, including IκB-α, phosphor-IκB-α (p-IκB-α), p65 and p-p65,were investigated.As shown in Figs.3A and 3B, LPS stimulation elevated the phosphorylation of IκB-α and p65, and thus significantly increased the relative fold of p-IκB-α/IκB-α and p-p65/p65(P＜ 0.05), which indicating the activation of NF-κB signaling pathway.The phosphorylation of IκB-α and p65 was remarkably suppressed by Se-TE treatment, especially at the concentration of 150 μg/mL.The results suggested that Se-TE treatment could block NF-κB signaling pathway via inhibiting the phosphorylation of IκB-α and p65 to exert its anti-inflammatory effects on LPS-treated RAW246.7 macrophages.

Fig.3 (Continued)

Fig.3 Effects of Se-TE on the phosphorylation of key proteins involved in NF-κB and MAPK pathways.RAW264.7 cells were pretreated with 0, 50 and 150 μg/mL of Se-TEs, and then the groups marked with “+” were stimulated with 1 μg/mL LPS for 12 h, while the groups marked with “-” were not stimulated.The relative folds of p-IκB-α/IκB-α (A), p-p65/p65 (B), p-p38/p38 (C), p-ERK/ERK (D), and p-JNK/JNK (E) were measured.Data are presented as the mean values ± SD from three separate experiments conducted in triplicate on different days.Within each graph, means bearing different letters are significantly different (P ＜ 0.05).

The expressions of MAPK proteins, including p38, p-p38, ERK,p-ERK, JNK and p-JNK were also investigated to further understand how Se-TE treatments regulate MAPKs signaling pathway.As shown in Figs.3C-3E, after LPS stimulation, MAPK signaling pathway was significantly activated via the sharply increased expressions of p-p38, p-ERK and p-JNK in RAW246.7 cells (P＜ 0.05).In addition, the productions of p-p38, p-ERK and p-JNK were found to be down-regulated in a dose-dependent manner.Taken together,our results demonstrated that Se-TE might suppress inflammation via down-regulating the phosphorylation of key proteins involved in both NF-κB and MAPK signaling pathways.

3.6 Effects of Se-TE on the phosphorylation and translocation of p65

To further confirm and visualize the phosphorylation and translocation of p65, the cells were incubated with responding antibodies, stained and observed using a confocal laser scanning microscope (Fig.4).In the blank control cells, only small amount of p65 was phosphorylated and translocated into nucleus.However,LPS stimulation significantly induced the phosphorylation and translocation of p65, which was proved by stronger red fluorescence in the nucleus of LPS-stimulated cells.Nevertheless, the phosphorylation and nuclear translocation of p65 was notably blocked by the pretreatment of Se-TE.

Fig.4 Effects of Se-TE on the expression and translocation of p65 in LPS-stimulated cells.RAW264.7 cells were pretreated with 150 μg/mL of Se-TE, and then stimulated with 1 μg/mL LPS for 12 h.The nuclear was stained by DAPI (blue fluorescence), while p65 was indicated by red fluorescence (Cy3).

4.Discussion

Both tea polyphenols and organic selenium, the main chemical compounds presented in Se-TE, have been suggested to be strongly related to the anti-inflammatory effects via variable and complex mechanisms [8,10,13].However, the synergistic effect of selenium with oolong tea polyphenols on the anti-inflammatory potential of Se-TE remains unclear.In the present study, we investigated Se-TE’s anti-inflammatory effect and explore its underlying mechanism in the cell model.The RAW246.7 macrophage cells were stimulated by exposure to LPS and thus released excessive amounts of inflammatory mediators (e.g.NO and PGE2) and pro-inflammatory cytokines (e.g.IL-6, IL-1β and TNF-α).NO, generated fromL-arginine and catalyzed by inducible iNOS [23], is a messenger molecule involved in modulating physiological and pathophysiological processes.However,over-production of NO during inflammation can lead to DNA damage and mutation in the health cells [11].Similarly, PGE2is another key inflammatory mediator produced from arachidonic acid and catalyzed by COX-1 and COX-2.During inflammation, COX-2 expression is activated by cytokines, LPS and other activators, leading to the sharply increased PGE2release, promoting further inflammation [24].Therefore, inhibiting excessive release of NO and PGE2has been considered to be an ideal strategy to deactivate the inflammation.In this study, our results showed that in LPS-stimulated macrophages,Se-TE pretreatment significantly inhibited NO and PGE2production.It was also notable that the mRNA expression levels ofiNOSandCOX-2were also downregulated by Se-TE, which well explained the underlying mechanism for the inhibitory effect of Se-TE on NO and PGE2production.

In the early stages of inflammation, LPS stimulated-macrophages release pro-inflammatory cytokines such as TNF-α, IL-1β, and IL-6,which act as messengers to promote inflammation through multiple signaling pathways [24].Increased concentrations of TNF-α are found in acute and chronic inflammatory conditions, and it has been shown to play a key role in the regulation of immune cells and inflammatory reactions [25].Dysregulation of TNF-α production causes fever,edema, apoptotic cell death, and thus has been related to various inflammatory diseases.IL-1β, belonging to IL-1 family, plays critical role in the regulation of the inflammatory response.It was found to be able to activateCOX-2expression and thus promote PGE2release [26].IL-6 is another important cytokine with multiple functions, such as inducing synthesis of acute phase proteins, stimulating antibody production, promoting differentiation or proliferation of several nonimmune cells.However, dysregulated IL-6 production can result in the onset or development of various inflammatory diseases [27].Therefore, blocking these pro-inflammatory cytokines is a useful strategy in anti-inflammatory therapy.In the present study, we provided evidence that Se-TE exerts its anti-inflammatory effects via significant inhibition of IL-1β, IL-6, and TNF-α production in LPS-stimulated RAW 264.7 cells.

NF-κB has been considered as a key pro-inflammatory signaling pathway due to its role in the expression of a number of proinflammatory genes such as cytokines, chemokines and adhesion molecules [28].Transcription factor NF-κB is composed of five subunits (p65, RelB, c-Rel, p52, and p50).Among them, the p65 subunit is considered to be the most important one, which can be phosphorylated at numerous sites [29].In its inactive form, NF-κB is presented in the cytosol by association with the inhibitor protein IκB-α.Activated by external stimuli such as LPS and TNF-α, IκB is phosphorylated and degraded, leading to NF-κB translocation to the nucleus.After nuclear translocation, phosphorylation of NF-κB subunit p65 enhances the transcription of various pro-inflammatory genes [30].Our results suggested that Se-TE pretreatment could inhibit the phosphorylation of IκB-α and p65 in LPS-stimulated RAW246.7 macrophages.Furthermore, it was confirmed that Se-TE diminished the NF-κB translocation to the nucleus according to the images of confocal laser scanning microscope.

MAPKs have been implicated as playing critical regulatory roles in regulating the expression of the pro-inflammatory cytokines genes such as IL-1β, IL-6 and TNF-α [31].Various inflammatory stimuli exert their cellular effects through phosphorylation of three major MAPK family members, namely ERK, JNK and p38 [32].Our results suggested that Se-TE pretreatment diminished the activation of MAPK pathway in the LPS-stimulated macrophages via inhibiting the phosphorylation of ERK, JNK and p38.These results collectively demonstrated that the anti-inflammatory property of Se-TE was associated with the inhibition of the NF-κB and MAPK signaling pathways, and by which suppressed the production of various proinflammatory mediators and cytokines in LPS-stimulated RAW264.7 macrophage cells.

The Se-enriched oolong tea and its extract, containing not only common bioactive compounds, i.e.polyphenols and caffeine, but also high content of organic selenium.Our results confirmed the promising anti-inflammatory effect of Se-TE.Although whether and how these bioactive compounds could synergistically act on NF-κB and MAPK pathways to modulate its function has not been completely understood,which is needed to be further investigated in our future studies.

5.Conclusion

Our results indicate that Se-TE pretreatment could suppress the inflammatory mediators, i.e.NO and PGE2production in LPS-stimulated macrophages, which might be due to the down-regulated expression ofiNOSandCOX-2.The productions of inflammatory cytokines such as TNF-α, IL-1β, and IL-6 were also significantly reduced.Moreover, Se-TE induced anti-inflammatory effects via targeting NF-κB and MAPK pathways in macrophages.Therefore,this novel Se-enriched oolong tea product may be a potential food ingredient for the development of various functional foods and the treatment of chronic inflammation-related diseases.

Declaration of competing interest

The authors declare no conflict of interest.

Acknowledgments

This work was funded by Fujian Special Research Projects for Public Scientific Research Institutions (grant number 2020R1032001).The authors greatly thankful to the colleagues who contributed to our research for their valuable assistance.