Cynara scolymus L. (artichoke) is a species of perennial thistle, which originated from the Mediterranean, and is widely used for food as well as for medicinal purposes.¹⁾ Artichoke extract has been found to contain various components such as luteolin glycoside, chlorogenic acid, cynara saponin, and hesperetin glycoside.²⁾ Several studies have revealed the therapeutic effects of artichoke on dyspepsia, moderate hyperlipidemia, liver disease, and biliary diseases.^1,3) In addition, the antioxidant and antibacterial activity of artichoke extract has been demonstrated through various studies.^1,4-6)

Recently, in animal models of acute alcohol-induced liver damage, the protective effect of artichoke through the reduction of toll-like receptor (TLR)-4 and NF-κB expression was demonstrated.^2,7) However, the mechanism was not clear.

Inflammatory response plays an important role in protecting the host from foreign invasion.⁸⁾ Beneficial effects have been observed at the site of local injury during moderate inflammation.^9,10) On the other hand, in the case of inappropriate inflammation, nitric oxide (NO), tumor necrosis factor-α (TNF-α), interleukin 6 (IL-6) and IL-1β are overexpressed, causing acute and chronic inflammatory diseases.^11-14) These factors are closely related to the TLR4 and NF-κB pathway. Lipopolysaccharide (LPS), a cell well component of gram-negative bacteria, is a TLR4-specific protein ligand that activates TLR4-NF-κB. When TLR4 is activated, the transcription factor NF-κB is phosphorylated, translocated to the nucleus, and initiates transcription of inflammatory mediators.¹⁵⁾ Therefore, for the treatment of inflammatory diseases, the regulation of these factors is essential, and various studies related to treatment mechanisms should be conducted.

We herein investigated the effects of artichoke on the expression of inflammatory mediators such as NO, TNF-α, PGE2, IL-6 and IL-1β by ELISA and RT-PCR. T he effect of artichoke on the activation of NF-κB was examined.

Reagents and kits

Dulbecco’s modified eagle’s medium (DMEM), dulbecco’s phosphate buffered saline (DPBS) and fetal bovine serum (FBS) were obtained from Corning (New York, USA). Penicillin/Streptomycin, lipopolysaccharide (LPS), trypan blue solution, thiazolyl blue tetrazolium bromide (MTT), sulfanilamide, N-(1-Naphthyl)ethylenediamine dihydrochloride (NED), Dimethyl sulfoxide (DMSO) were purchased from SIGMA-Aldrich (St. Louis, MO, USA). ELISA kits were purchased from R&D systems (Minneapolis, MN, USA) and Elabscience (Wuhan, China). RNA isolation kit and RiboEX were purchased from GeneAll (Seoul, KOREA). AccuPower^TM HotStart PCR PreMix and AccuPower^TMCycleScript RT PreMix were obtained from Bioneer (Daejeon, Korea).

Cell culture and treatment of artichoke

RAW264.7 cells, the murine macrophage cell line, were obtained from the American Type Culture Collection (Rockville, MD, USA) and cultured in DMEM with 10% heat-inactivated FBS, penicillin (100 units/mL) and streptomycin (100 mg/mL) at 37oC in a humidified atmosphere of 5% CO₂ incubator (Sanyo, Japan). The artichoke powder (80% of Cynara scolymus L. extract, DONG IL PharmTec. Seoul, Korea) was dissolved in DMSO, and the concentration of the stock solution was 5 mg/mL. Cells were pretreated with artichoke for 1 h and then stimulated with LPS (1 mg/mL) for the indicated time. Cell supernatants and lysates were used for griess reaction, ELISA and reverse transcription polymerase chain reaction (RT-PCR) tests, respectively.

Cell viability

Cytotoxicity of artichoke was measured by MTT assay. RAW264.7 cells were plated at 5×104 cells/well (Corning, NY, USA). Cells were allowed to stabilize overnight prior to treatment with artichoke and then treated with the artichoke for 24 h. The cell supernatant was then removed and 100 mL of MTT solution was added to each well. After 4 h, the formazan crystals by MTT solution were dissolved with 100 mL of DMSO. After shaking the plate for 15 min, the optical density (OD value) was measured at 540 nm with VersaMax Microplate Reader (Molecular Devices, CA, USA).

Nitric oxide determination

The production of nitric oxide (NO) was measured by griess reaction. Cell culture supernatants were collected to observe the NO production. The cell supernatant was reacted with an equal volume of 1% sulfanilamide and 0.1% NED, and then incubated for 15 min at room temperature avoiding exposure to direct light. Optical density (OD values) was measured at 540 nm with VersaMax Microplate Reader.

Measurement of cytokine and PGE₂

The cell supernatants were used for the measurement of TNF-α, IL-6, IL-1β (DuoSet; R&D systems, Minneapolis, USA), and PGE2 (Elabscience, Wuhan, China) using ELISA method.

NF-κB activity

Cytosolic and nuclear extracts were isolated and assayed for NF-κB activity by colorimetric method system (NF-κB EZTFA Transcription Factor Assay, Upstate & Millipore, Billerica, MA, USA) according to the manufacturer’s instruction

Reverse transcription-polymerase chain reaction (RT-PCR)

Gene expression was measured by RT-PCR. RAW264.7 cells were plated into 1×106 cells/well and allowed to stabilize in an incubator overnight. Cells were pre-treated with various concentrations of artichoke for 1 h and stimulated by LPS (1 mg/mL) for 6 h. Cells were lysed to isolate messenger RNA (mRNA) with RiboEX. RNA was quantified by Colibri Microvolume Spectrometer (Titertek-Berthold, Pforzheim, Germany). Complementary DNA(cDNA) synthesis of 0.5 mg of total RNA was performed by using AccuPower^TM CycleScript RT PreMix. PCR was carried out with T100^TM Thermal Cyclers (Bio-Rad, Hercules, CA, USA) by AccuPower^TM HotStart PCR PreMix. PCR products were observed by gel electrophoresis on 2% Agarose containing Loading Gel Stain Solution instead of ethidium bromide (EtBr). Bands were visualized by Infinity-3026 (Vilber Lourmat, Collegien, France). Table 1 shows the primer sequences used in this study.

Table 1

Primer sequences used for RT-PCR in this study

Gene	Primer Sequence	Size (bp)
TNF-α	Forward: 5'-TCT TCT CAT TCC TGC TTG TG-3' Reverse: 5'-ACT TGG TGG TTT GCT ACG-3'	198
IL-6	Forward: 5'-CCT CTG GTC TTC TGG AGT ACC-3' Reverse: 5'-TGG TCC TTA GCC ACT CCT TC-3'	222
IL-1β	Forward: 5'-TCA GGC AGG CAG TAT CAC TC-3' Reverse: 5'-AGC TCA TAT GGG TCC GAC AG-3'	250
COX-2	Forward: 5'-CCC TTG GGT GTC AAA GGT AA-3' Reverse: 5'-GCC CTC GCT TAT GAT CTG TC-3'	169
iNOS	Forward: 5'-GCA GAA TGT GAC CAT CAT GG-3' Reverse: 5'-ACA ACC TTG GTG TTG AAG GC-3'	426
β-actin	Forward: 5'-CAC ACC TTC TAC AAT GAG-3' Reverse: 5'-GGT CTC AAA CAT GAT CTG-3'	117

Statistical analysis

Statistical analysis results are presented as the mean±SEM of three independent experiments. SigmaPlot 12.0 software was used for data analysis. One-way analysis of variance (ANOVA) followed by Dunnett’s test was used to assess comparisons between the groups. Values of p<0.05 and p<0.01 was considered statistically significant.

Traditionally, various herbal medicines have been widely used as agents for treating inflammatory diseases. Therapeutic and biological activity of edible plants and their bioactive compositions are potentially important for treating a variety of diseases associated with inflammation and immune dysregulation, such as autoimmune diseases, allergic diseases, type 1 diabetes, obesity, and so on. During the last decades, extracts and compounds from natural product have become a focus for the treatment of these diseases.^16-18) In the search for new therapeutic agents against inflammatory diseases, this study was designed to explore the potential anti-inflammatory effects of artichoke. We tested the effect of artichoke on the production of inflammatory mediators such as IL-1β, TNF-α, and NO in RAW264.7 cells.

The cytotoxic effect of artichoke was first evaluated and no cytotoxic effect was observed on artichoke at the concentration used (Fig. 1).

Fig. 1. Cytotoxicity of artichoke. RAW264.7 cells were treated with the artichoke extract at a dose dependent manner for 24 h, and media containing MTT were added for measurement of cell viability.

Nitric oxide (NO), synthesized by inducible nitric oxide synthase (iNOS), has been known to be an inflammatory mediator.¹⁹⁾ Therefore, we tested the effect of artichoke on LPS-induced NO production in macrophages. While treatment with LPS increased NO production in RAW264.7 cells, artichoke significantly suppressed NO production in LPS-stimulated cells in a dose-dependent manner (Fig. 2A). We next examined the effect of artichoke on the mRNA expression of iNOS in the cells activated with LPS. Result showed that artichoke suppressed mRNA expression of iNOS in a dose dependent manner (Fig. 2C). To demonstrate whether artichoke itself affects the expression of NO, cells were treated with artichoke in absence of LPS. As a result, there was no effect of NO production by artichoke (data not shown).

Fig. 2. The effect of artichoke on the expression of NO and PGE₂ in RAW264.7 cells. The cells were pre-treated with different concentrations of artichoke for 1 h and then stimulated by LPS (1 µg/mL) for 6 or 24 h for mRNA expression and protein production, respectively. (A-B) The cell supernatant was collected to measure the production of NO and PGE₂ by Griess reaction and ELISA method. (C) The cell lysate was collected and used for the mRNA expression of iNOS and COX-2 by RT-PCR. The data are presented as the mean±SEM of three independent experiments. *p<0.05, **p<0.01 compared to control groups with LPS alone.

In arachidonic acid metabolism, cyclooxygenase-2 (COX-2) produces prostaglandins, an inducible form.²⁰⁾ In particular, prostaglandin E₂ (PGE₂) is closely related to the inflammatory response and is present in high concentrations even in allergic and inflammatory diseases such as atopic dermatitis.^20,21) Therefore, the expression of PGE₂ and COX-2 mRNA was investigated. Artichoke dose-dependently suppressed the release of PGE₂ and mRNA expression of COX-2 in the macrophages stimulated with LPS (Fig. 2B and 2C). These results suggest that artichoke may be involved in the regulation of arachidonic acid metabolism.

During an inflammatory response, macrophages secrete pro-inflammatory cytokines, such as TNF-α, IL-6 and IL-1β.^22,23) We investigated the anti-inflammatory activity of the artichoke in vitro by evaluating whether it inhibited pro-inflammatory mediators. The effect of artichoke on LPS-induced TNF-α and IL-1β release in RAW 264.7 cells by ELISA was investigated. Our data showed that artichoke significantly suppressed LPS-induced IL-1β, IL-6, and TNF-α production by macrophages in a dose dependent manner. In addition, mRNA expression of these cytokines was investigated by RT-PCR. As a result, artichoke also suppressed mRNA expression of IL-1β, IL-6, and TNF-α in a dose dependent manner (Fig. 3).

Fig. 3. The effect of artichoke on the expression of pro-inflammatory cytokines. RAW264.7 cells were pre-treated with different concentrations of artichoke for 1 h and then stimulated by LPS (1 µg/mL) for 6 or 24 h for mRNA expression and protein production, respectively. (A-C) The cell supernatant was collected to measure the production of IL-1β, IL-6, and TNF-α through ELISA. (D) The cell lysate was collected and used for the mRNA expression by RT-PCR. The data are presented as the mean±SEM of three independent experiments. *p<0.05, **p<0.01 compared to control groups with LPS alone.

NF-κB, a representative transcription factor involved in inflammatory response, is phosphorylated by signaling cascade from initiation of TLR4 stimulated by LPS.¹⁵⁾ One of the NF-κB dimers, p65 (as known as RelA) is translocated from cytoplasm to nucleus when phosphorylated and then up-regulated the expression of genes mediating inflammation, such as iNOS, TNF-α, IL-1β, IL-6 and COX-2.²⁴⁾ Phosphorylation of p65 protein was inhibited by artichoke (Fig. 4).

Fig. 4. The effect of artichoke on the activation of NF-B. RAW264.7 cells were treated with or without artichoke at a dose dependent manner for 1 h, and then with LPS. After 24 h of these treatments, Cytosolic and nuclear extracts were isolated and assayed for NF-κB activity by the colorimetric method. The data are presented as the mean±SEM of three independent experiments. *p<0.05, **p<0.01 compared to control groups with LPS alone.

Another study confirmed the anti-inflammatory properties of artichokes using a carrageenan (Carr) experimental model. Their results showed that artichoke not only reduced blood biomarkers such as CRP, but also showed the effect of reducing inflammatory cell infiltration into tissues.⁶⁾ In our study, it was revealed that the inhibitory effect of inflammatory responses, including the induction of edema, was due to the regulation of the expression of inflammatory mediators.

Since the role of reactive oxygen species (ROS) on the expression of NF-kB and inflammatory factors is well known,²⁶⁾ and artichoke is also known to have an antioxidant action that inhibits the production of ROS,⁶⁾ our results suggest that inhibitory effect on NF-kB activation by artichoke is related to the inhibition of ROS production.

In conclusion, the inhibitory effect of artichoke on the inflammatory response was shown by reducing phosphorylation of NF-κB-p65 protein (phospho-p65) in RAW264.7 cells. Artichoke suppressed gene expression through an inhibitory effect on phospho-p65, reducing the expression of inflammatory mediators including NO, TNF-α, IL-6 and PGE₂. Our study showed that artichoke could be a new candidate for the treatment of inflammatory diseases. Although this study has been demonstrated about anti-inflammatory effects of artichoke in macrophages by in vitro, research on anti-inflammatory mechanisms using artichoke should be developed in other inflammatory cells in the future.

Our findings suggest that artichoke is a potent inhibitor of LPS-induced NO, TNF-α, and IL-1β production in RAW 264.7 macrophage cells. It has also been shown that this inhibitory effect of artichoke is related to NF-κB inactivation. Since NF-κB is a transcription factor that regulates the transcriptions of many inflammation-related genes, inhibition by artichoke may provide a possible approach to the prevention or treatment of serious inflammatory diseases.

This paper was supported by the Academic Research Fund of Dr. Myung Ki (MIKE) Hong in 2021

All authors declare that they have no conflict of interest.