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Table of Contents
ORIGINAL ARTICLE
Year : 2021  |  Volume : 64  |  Issue : 1  |  Page : 32-42

The Anti-inflammatory Effects of the Bioactive Compounds Isolated from Alpinia officinarum Hance Mediated by the Suppression of NF-kappaB and MAPK Signaling


1 PhD Program of Biotechnology and Industry, College of Biotechnology and Bioresources, Da-Yeh University, Changhua; Department of Life-and-Death Studies, Nanhua University, Chiayi, Taiwan
2 Department of Food Science and Biotechnology, Da-Yeh University, Changhua, Taiwan
3 Department of Biomedical Sciences, Chung Shan Medical University; Department of Medical Research, Chung Shan Medical University Hospital, Taichung, Taiwan
4 Department of Food Science and Biotechnology, National Chung Hsing University, Taichung, Taiwan
5 Functional Neurosurgery Division, Neurological Institute, Taichung Veterans General Hospital, Taichung, Taiwan

Date of Submission15-Oct-2020
Date of Decision20-Dec-2020
Date of Acceptance20-Jan-2021
Date of Web Publication25-Feb-2021

Correspondence Address:
Dr. Ming-Shiun Tsai
Department of Food Science and Biotechnology, Da-Yeh University, No. 168, University Road, Dacun, Changhua 51591
Taiwan
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/CJP.CJP_81_20

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  Abstract 


This study was designed to evaluate the anti-inflammatory effects of Alpinia officinarum Hance extract (AOE) and identify its main active ingredients. AOE was obtained using a 95% ethanol extraction method. Lipopolysaccharide (LPS) were used to induce an inflammatory response in RAW264.7 cells. The results showed that AOE exerts anti-inflammatory effects via inhibition of prostaglandin E2 secretion and cyclooxygenase -2 (COX-2) production. We further analyzed the components of AOE using high-performance liquid chromatography and found that AOE is comprised of several bioactive flavonoids including quercetin (Q), kaempferol (K), galangin (G), and curcumin (C). These four flavonoids effectively inhibited nitric oxide (NO), interleukin (IL)-1β, IL-6, and tumor necrosis factor-α production. Moreover, they reduced COX-2 and inducible NO synthase expressions via regulation of nuclear factor kappa-light-chain-enhancer of activated B cells and c-Jun N-terminal kinase signaling pathways. Furthermore, we compared and contrasted the anti-inflammatory effects and mechanisms of these four flavonoids at the same dose in the LPS-induced cell inflammation model. The results showed that C is the most effective inhibitor of LPS-induced NO production. However, only Q and K effectively attenuated LPS-induced extracellular signal-regulated kinase and p38 elevations. In conclusion, AOE and its major bioactive compounds exert anti-inflammatory effects on LPS-induced inflammation. As A. officinarum Hance is much cheaper than any of its four flavonoids, especially G, we suggest using AOE as an anti-inflammatory agent.

Keywords: Alpinia officinarum Hance extract, anti-inflammatory effects, curcumin, galangin, kaempferol, quercetin


How to cite this article:
Li CY, Cheng SE, Wang SH, Wu JY, Hsieh CW, Tsou HK, Tsai MS. The Anti-inflammatory Effects of the Bioactive Compounds Isolated from Alpinia officinarum Hance Mediated by the Suppression of NF-kappaB and MAPK Signaling. Chin J Physiol 2021;64:32-42

How to cite this URL:
Li CY, Cheng SE, Wang SH, Wu JY, Hsieh CW, Tsou HK, Tsai MS. The Anti-inflammatory Effects of the Bioactive Compounds Isolated from Alpinia officinarum Hance Mediated by the Suppression of NF-kappaB and MAPK Signaling. Chin J Physiol [serial online] 2021 [cited 2021 Apr 21];64:32-42. Available from: https://www.cjphysiology.org/text.asp?2021/64/1/32/310129

The authors Chia-Yu Li, Szu-En Cheng, and Sue-Hong Wang contributed equally to this work.





  Introduction Top


Inflammation is generally considered the first line of defense against pathogens and is associated with the elimination of damaged or infected cells to promote tissue repair and recovery.[1] Inflammation can be divided into two types: acute inflammation which occurs in response to tissue injury and is a short-term process lasting several hours and chronic inflammation which can last for months or, in some cases, years. Inflammation is the body's response to injury. However, excessive inflammation can damage the body and even trigger immune cells to attack healthy cells, resulting in autoimmune diseases such as multiple sclerosis. In addition, chronic inflammation mediates severe chronic diseases, including cancer, diabetes, cardiovascular diseases, allergies, and chronic inflammatory diseases, some of which are significant causes of death worldwide.[2],[3] This means that the development of novel anti-inflammatory compounds is critical to improving global health.

Alpinia officinarum Hance (AO) is an herbaceous plant of the Zingiberaceae family that is mainly distributed throughout tropical and subtropical Asia. AO is used widely in traditional medicine and nutritional supplements. For example, in traditional Chinese medicine, AO has been used to relieve stomach aches and cold symptoms, invigorate the circulatory system, treat vomiting, and reduce swelling.[4] Several studies have revealed various biological functions of AO.[5] Methanol extracts of AO have been shown to inhibit MCF-7 cells by blocking cell cycle progression and inducing cell apoptosis.[6] These extracts also exhibit remarkable antitumor activity in mouse skin models.[7] Moreover, ethanol extracts of AO have been reported to possess anti-inflammatory[8] and anticancer activities in PC-3 prostate cancer cells,[9] in addition to anti-obesity and hypolipidemic effects[10] and antibacterial activity against Bacillus cereus, Staphylococcus aureus, Pseudomonas aeruginosa, and  Escherichia More Details coli.[11] The n-hexane extract of AO effectively inhibits the growth of THP-1 and leukemia cells.[12] Dichloromethane AO extracts exert cytotoxic effects on COR L23 cells, a non-small cell lung cancer cell line.[13] Acetone extracts of AO inhibit nitric oxide (NO) production in lipopolysaccharide (LPS)-activated murine macrophages,[14] while water extracts of AO display anti-inflammatory and anticancer activities.[5] Taken together, various extracts of AO possess anti-inflammatory, antioxidant, anticancer, and antibacterial effects. Previous studies have shown that the bioactive compounds in AO extracts include pinocembrin, galangin (G), quercetin (Q), kaempferol (K), hexahydrocurcumin, and 3-O-methylgalangin.[5] The aim of this study was to explore the anti-inflammatory effects of AO extract (AOE) and its underlying mechanisms of action.

Our results demonstrated that AOE effectively decreases inflammation in vitro. Further analysis of the active ingredients of AOE suggests that these effects are mediated by Q, K, G, and curcumin (C). In addition, AOE and its four major compounds inhibit LPS-induced NO, prostaglandin E2 (PGE2), interleukin (IL)-1β, IL-6, tumor necrosis factor-α (TNF-α), cyclooxygenase-2 (COX-2), and inducible nitric oxide synthase (iNOS) that are regulated by nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and mitogen-activated protein kinase (MAPK) signaling. Therefore, AOE and its four bioactive compounds are potential anti-inflammatory agents.


  Materials and Methods Top


Chemicals, reagents, and antibodies

AO rhizomes were purchased from Hua-Chang Co., Ltd. (Tainan, Taiwan). Q, K, G, C, LPS, dimethyl sulfoxide (DMSO), and Griess reagent were obtained from Sigma-Aldrich (St. Louis, MO, USA). Antibodies included anti-iNOS (Santa Cruz, CA, USA), anti-COX-2 (Abcam, Cambridge, MA, USA), anti-NF-κB p65 (Franklin Lakes, NJ, USA), anti-β-actin (Merck Millipore, Darmstadt, Germany), and anti-lamin B1 (Abcam). Goat polyclonal secondary antibodies to rabbit immunoglobulin G (IgG)-heavy and light chain were purchased from Abcam, and goat anti-mouse IgG horseradish peroxidase (HRP) was purchased from Jackson ImmunoResearch Laboratories (West Grove, PA, USA).

Alpinia officinarum Hance extract preparation

AO rhizomes were dehydrated in an incubator at 40°C and then crushed using an electric mill. Subsequently, 95% ethanol in a solid-liquid ratio of 1:10 was used to develop extract from the powder at room temperature for 60 min. The resulting extract was filtered using an ADVDNTEC NO. 131 membrane (Toyo Roshi Kaisha, Ltd., Japan), concentrated, and freeze-dried. The powder (AOE, 50 mg) was dissolved in 1 mL DMSO to prepare a 50 mg/mL stock solution that was stored at −20°C. The components of AOE were determined by high performance liquid chromatography (HPLC) which was outsourced to Vercotech Inc. (Taipei, Taiwan). HPLC was performed using Daisogel C8 column (5 μm, 4.6 mm × 150 mm) and Thermo Fisher Scientific UltiMate 3000 HPLC system with 360 nm ultraviolet detector. Mobile phase A: 10 mM ammonium acetate in H2 O; mobile phase B: 10 mM ammonium acetate in H2 O/acetonitrile (1:9); mobile phase C: isopropyl alcohol/tetrahydrofuran (3:7). Gradient: from 90% in mobile phase A and 10% in mobile phase B to 95% in mobile phase B and 5% in mobile phase C in 25 min at a flow rate of 1 mL/min.

Cell culture and treatment

Murine macrophage RAW264.7 cells were purchased from the Bioresource Collection and Research Center (Hsinchu, Taiwan). These cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated fetal bovine serum, penicillin, streptomycin, and L-glutamine (Biological Industries, Kibbutz Beit Haemek, Israel) at 37°C in a 5% CO2 humidified incubator. Cells were seeded onto plates or dishes, cultured for 24 h, refreshed with new medium, and then exposed to LPS in the presence or absence of AOE or flavonoids. After 24 h, the cells or cultured media were collected and used for experimental analyses.

Cell viability assay

Cell viability was evaluated on 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. RAW 264.7 cells were seeded (5 × 105 cells/well) onto a 12-well plate, cultured for 24 h, and then treated with 1 μg/mL LPS and varying concentrations of AOE or flavonoids for 24 h. The medium was then removed and 500 μL of the MTT solution (500 μg/mL, Amresco, Solon, OH, USA) was added to each well, followed by incubation for 4 h. After removal of the solution, 500 μL of DMSO was added to dissolve the purple precipitates. The absorbance was measured at a wavelength of 570 nm using an ELISA reader (LT-4500, Labtech International Ltd., Heathfield, UK). Cell viability was calculated based on the following formula: Cell viability (%) = absorbance of the sample/absorbance of the control × 100%.

Nitric oxide measurement

The amount of NO in the culture media was determined on Griess reagent assay. RAW264.7 cells were seeded onto 12-well plates (5 × 105 cells/well) and incubated for 24 h. Cells were then treated with or without 1 μg/mL LPS and varying concentrations of AOE or flavonoids for 24 h. Subsequently, 100 μL of the supernatant from each well was transferred onto a 96-well plate. Each well was supplemented with 50 μL of 1% sulfanilamide in 5% phosphoric acid and 50 μL of 0.1% N-(1-Naphthyl)ethylenediamine dihydrochloride. The absorbance of the solution was measured at 550 nm using an ELISA reader and the concentrations of nitrite were calculated using a standard calibration curve based on different concentrations of sodium nitrite.

Prostaglandin E2, interleukin-1β, interleukin-6, and tumor necrosis factor-α measurements

The levels of PGE2 and various pro-inflammatory cytokines (IL-1β, IL-6, and TNF-α) in the culture media were determined using mouse PGE2 ELISA kit (Cayman Chemical Company, MI, USA), IL-1β ELISA kit (eBioscience Inc., CA, USA), IL-6 ELISA kit (eBioscience Inc., CA, USA), and TNF-α ELISA kit (eBioscience Inc., CA, USA), according to the manufacturers' instructions. Cells (5 × 105 cells/well) were cultured on 12-well plates for 24 h and then treated with or without 1 μg/mL LPS and varying concentrations of AOE or flavonoids. After 24 h, media were harvested and the concentrations of PGE2, IL-1β, IL-6, and TNF-α were calculated using standard calibration curves.

Western blots

After determining protein concentrations using the bicinchoninic acid protein assay kit (Thermo Fisher Scientific, USA), an equal amount (40 μg) of proteins from each sample were separated using 8% or 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes (FluoroTrans, PALL, Driesch, Germany). Immunoblots were incubated in 5% skim milk in 0.1% Tween 20 in Tris-buffered saline (TBS-T) at room temperature for 1 h and then overnight with the appropriate primary antibodies at 4°C. The membranes were washed three times with TBS-T and incubated with the HRP-conjugated secondary antibody at room temperature for 1 h. Blots were then washed another three times in TBS-T, developed using enhanced chemiluminescence (ECL, Bionovas, Toronto, Canada) for signal detection, and visualized on an ImageQuant LAS 4000 digital imaging system (GE LAS-4000, GE Healthcare Life Sciences, NJ, USA). Protein expression levels were determined using ImageJ software 1.47t (National Institutes of Health, Bethesda, MD, USA). Protein levels = ([intensity of treatment/intensity of internal control] of the protein of interest)/([intensity of treatment/intensity of internal control] for β-actin or lamin B1).

Nuclear extraction

Treated cells were washed three times in cold phosphate-buffered saline and resuspended in a hypotonic buffer (10 mM HEPES [pH 7.9], 1.5 mM MgCl2, 10 mM KCl, 0.2 mM phenylmethanesulfonyl fluoride [PMSF], 0.5 mM dithiothreitol (DTT), and 10 μg/mL aprotinin) and proteinase inhibitor mixture (PIM, Roche, Mannheim, Germany) and then incubated on ice for 15 min. Cells were subsequently lysed using 0.5% NP-40 and vortexed vigorously for 10 s. After centrifugation at 12,000 rpm and 4°C for 5 min, the pellet was collected and washed with hypotonic buffer. The pellet was mixed with nuclear extraction buffer (20 mM HEPES [pH 7.5], 400 mM NaCl, 1 mM EDTA, 1 mM PMSF, and 1 mM DTT) and PIM and then incubated on ice for 40 min. After centrifugation at 12,000 rpm and 4°C for 20 min, the supernatant was collected and protein expression of NF-κB p65 was analyzed on Western blot as described above.

Mitogen-activated protein kinase family detection

The concentrations of phosphorylated p38, extracellular signal-regulated kinase (ERK), and c-Jun N-terminal kinase (JNK) in cell lysates were determined using Activation Multispecies InstantOne™ ELISA kits (Invitrogen, Thermo Fisher Scientific), according to the manufacturer's protocol. Each well of a 96-well plate was supplemented with 50 μL of cell lysis mix (negative control) and 50 μL of positive control cell lysate (positive control) or 20 μL of cell lysate sample. After incubation on a microplate shaker at room temperature for 1 h, wells were washed three times in wash buffer. After adding 50 μL of the detecting antibody cocktail to each well, samples were incubated on a microplate shaker at room temperature for 1 h. Wells were washed three times in wash buffer and then 100 μL of the detection reagent was added. This was followed by incubation of the plates for another 10–30 min with shaking. Stop solution was added to the wells, and the absorbance of the samples was recorded at 450 nm using an ELISA reader.

Statistical analysis

Results are expressed as the mean ± standard deviation from at least three independent experiments. Comparisons between groups were performed using one-way ANOVA or Student's t-test. The results were considered statistically significant when P < 0.05.


  Results Top


Effects of Alpinia officinarum Hance extract on cell viability and nitric oxide production in lipopolysaccharide-treated macrophages

MTT assay was used to evaluate the effects of AOE treatments on LPS-stimulated RAW264.7 macrophages. The cells were treated with 1 μg/mL LPS and various concentrations (0, 25, 37.5, 50, and 100 μg/mL) of AOE for 24 h, and the results are summarized in [Figure 1]a. The addition of 1 μg/mL LPS resulted in a significant decrease in cell viability compared to the no-treatment control (NC). However, there was a significant increase in cell viability following treatment with AOE at concentrations of 25 and 37.5 μg/mL, suggesting that AOE treatment attenuates LPS-induced inhibition of cell proliferation. We then performed Griess assay to evaluate the effects of AOE treatments on the production of LPS-induced NO. Treatment with AOE inhibited LPS-induced NO production in a dose-dependent manner in RAW264.7 macrophages [Figure 1]b. AOE at 25, 37.5, 50, and 100 μg/mL reduced NO production in RAW264.7 cells to 35.88 ± 4.09%, 24.10 ± 3.11%, 2.94 ± 0.95%, and 1.20 ± 0.11% that in LPS-only treated cells, respectively. AOE treatment showed dosage-dependent increases in the NO inhibitions. Since treatments with AOE from 50 to 100 μg/mL showed significant inhibitions of cell proliferations compared to the NC [Figure 1]a, that AOE at concentrations of 50 and 100 mg/mL attenuated NO productions induced by LPS may result from these inhibitions. From these results, we identified 37.5 μg/mL AOE as the optimal concentration for our subsequent experiments due to significant increase in cell viability and inhibition of NO production.
Figure 1: Alpinia officinarum Hance extract inhibited the production of nitric oxide in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages. (a) Cells were treated with or without 1 μg/mL LPS and 0–100 μg/mL of Alpinia officinarum Hance extract for 24 h and cell viability was determined on 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Results are expressed as percentages of respective control, no-treatment control (NC) values (100%). (b) Cells were incubated with 1 μg/mL LPS and 0–100 μg/mL of Alpinia officinarum Hance extract for 24 h and nitric oxide production rates in media were evaluated on Griess reagent assay. Results are shown as percentages of the LPS only values (100%). #P < 0.05 compared to NC; *P < 0.05 compared to LPS.

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Effects of Alpinia officinarum Hance extract on lipopolysaccharide-induced prostaglandin E2 production and cyclooxygenase-2 expression

To identify the potential molecular mechanisms underlying the effects of AOE, we evaluated PGE2 and COX-2 expressions using ELISA and Western blotting, respectively. RAW 264.7 macrophages were treated with or without 1 μg/mL LPS and 37.5 μg/mL AOE for 24 h. Cultured media were collected for analyzing PGE2 levels. AOE suppressed PGE2 secretion in the culture medium with the concentration of PGE2 decreasing from 2.64 ± 0.23 ng/mL after LPS induction (LPS) to 0.08 ± 0.01 ng/mL (L + A) [Figure 2]a. In addition, Western blot demonstrated an increase in COX-2 expression in response to LPS stimulation and a reduction in response to AOE treatment (L + A) [Figure 2]b. Moreover, AOE treatment inhibited LPS-induced PGE2 secretion and COX-2 expression. However, AOE treatment alone did not significantly affect PGE2 secretion or COX-2 expression in RAW264.7 macrophages.
Figure 2: Effect of Alpinia officinarum Hance extract on prostaglandin E2 production and cyclooxygenase-2 expression in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages. Cells were treated with or without 1 μg/mL LPS and 37.5 μg/mL Alpinia officinarum Hance extract and then incubated for 24 h. Prostaglandin E2 content in media was evaluated by ELISA (a) and cyclooxygenase-2 expression levels were evaluated by Western blot (b, upper panel). The expression level of each protein was then quantified using ImageJ software. After normalization with β-actin, relative cyclooxygenase-2 expression levels are shown (b, lower panel). NC: no-treatment control, LPS: treated with LPS only, AOE: treated with AOE only, L + A: treated with LPS and AOE. #P < 0.05 compared to NC; *P < 0.05 compared to LPS.

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High performance liquid chromatography analysis of Alpinia officinarum Hance extract

To identify the components within AOE, we performed HPLC analysis. On profiles of HPLC analysis [Figure 3]a, at least four major components of AOE, Q (retention time [RT]: 11.085 min), K (RT: 12.742 min), G (RT: 15.958 min), and C (RT: 16.585 min), were identified. G (~92%) was the most abundant compound. The chemical structures and molecular weights of these four flavonoids are shown in [Figure 3]b. G, K, and Q possess similar chemical structures, except that Q has one more hydroxyl group than K and K has one more hydroxyl group than G. Given that all four compounds are known to exert many biological effects, we evaluated the anti-inflammatory potential of each of these flavonoids in LPS-stimulated RAW 264.7 macrophages.
Figure 3: High performance liquid chromatography profiles for Alpinia officinarum Hance extract and four flavonoid standards (a) and chemical structures and molecular weights of four flavonoid standards (b). High-performance liquid chromatography profiles for Alpinia officinarum Hance extract (green line) and four reference standards of quercetin (brown line), kaempferol (pink line), galangin (blue line), and curcumin (black line).

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Four flavonoids increase cell viability and reduce nitric oxide production in lipopolysaccharide-treated macrophages

Cells were cultured with or without 1 μg/mL LPS and 0, 5, 2.5, and 12.5 μg/mL Q, K, G, and C for 24 h. Cell viability was evaluated on MTT assay. The results [Figure 4]a showed that LPS stimulation induces a decline in RAW264.7 viability and treatment with any of the flavonoids at 2.5 and 12.5 μg/mL significantly reversed this inhibition. These results suggested that these flavonoids attenuate LPS-induced decreases in cell viability. In addition, results of Griess reagent assays revealed that while LPS stimulation significantly increases the levels of NO in the culture media, the addition of any of these flavonoids suppresses this response [Figure 4]b. When these four flavonoids were added at a concentration of 12.5 μg/mL, we observed reductions in NO production in culture media to 2.4 ± 0.15% (Q), 13.30 ± 1.41% (K), 6.0 ± 0.71% (G), and 2.1 ± 0.1% (C) that of LPS treatment alone. IC50 values of inhibition of LPS-stimulated NO production for Q, K, G, and C were 6.16 μg/mL (20.38 μM), 7.04 μg/mL (24.60 μM), 7.01 μg/mL (25.94 μM), and 7.47 μg/mL (20.28 μM), respectively. C showed better inhibition than Q, followed by K and G. These results indicated that AOE containing Q, K, G, and C relieves inflammation in LPS-stimulated RAW264.7 macrophages.
Figure 4: Four different flavonoids inhibited nitric oxide production in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages. (a) Cells were treated with or without 1 μg/mL LPS and 0–12.5 μg/mL quercetin, kaempferol, galangin, or curcumin as indicated for 24 h and cell viability was evaluated on 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Results are expressed as percentages of no-treatment control (NC) values (100%). (b) Nitric oxide production rates in the media were evaluated on Griess reagent assay. Results are shown as percentages of the LPS only values (100%). #P < 0.05 compared to NC; *P < 0.05 compared LPS only.

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Flavonoid treatments inhibit production of lipopolysaccharide-induced pro-inflammatory cytokines

To further explore the molecular mechanisms of these flavonoids in LPS-stimulated macrophages, the effects of 12.5 μg/mL Q, K, G, and C on the LPS-stimulated productions of IL-1β, IL-6, and TNF-α were investigated using ELISA [Figure 5]a, [Figure 5]b, [Figure 5]c. Following LPS stimulation, cells secreted 1.27 ± 0.05 ng/mL IL-1β, 3.31 ± 0.03 ng/mL IL-6, and 0.72 ± 0.01 ng/mL TNF-α, which were significant increases when compared with NC group. However, simultaneous treatments with Q, K, G, and C led to decreases in IL-1β to 0.25 ± 0.05, 0.26 ± 0.04, 0.27 ± 0.08, and 0.26 ± 0.06 ng/mL, respectively; IL-6 to 1.64 ± 0.08, 1.51 ± 0.12, 1.66 ± 0.06, and 1.65 ± 0.17 ng/mL, respectively; and TNF-α to 0.34 ± 0.03, 0.33 ± 0.05, 0.33 ± 0.02, and 0.34 ± 0.02 ng/mL, respectively. These concentrations were significantly reduced when compared with LPS group. In addition, we analyzed the protein expressions of COX-2 and iNOS on Western blots [Figure 5]d. These two proteins mediated the syntheses of PGE2 and NO, respectively. After normalization by β-actin, both COX-2 and iNOS levels significantly increased after LPS stimulation but significantly decreased in response to Q, K, G, and C treatments [Figure 5]e, [Figure 5]f. This indicated that the anti-inflammatory effects of Q, K, G, and C are due to downregulations of IL-1β, IL-6, TNF-α, iNOS, and COX-2 levels in LPS-stimulated macrophages.
Figure 5: Effects of treatments with four different flavonoids on lipopolysaccharide (LPS)-induced pro-inflammatory cytokines and cyclooxygenase-2 and inducible nitric oxide synthase protein expressions. RAW264.7 cells were treated with or without 1 μg/mL LPS and 12.5 μg/mL quercetin (Q), kaempferol (K), galangin (G), or curcumin (C) for 24 h. (a-c) Levels of interleukin-1β (a), interleukin-6 (b), and tumor necrosis factor-α (c) in media were determined by ELISA. The cyclooxygenase-2 and inducible nitric oxide synthase expression levels were evaluated by Western blots (d). The expression level of each protein was quantified using ImageJ software. After normalization by β-actin, relative cyclooxygenase-2 (e) and inducible nitric oxide synthase (f) expression levels are shown. L + Q: treated with LPS and Q, L + K: treated with LPS and K, L + G: treated with LPS and G, L + C: treated with LPS and C. #P < 0.05 compared to NC *P < 0.05 compared to LPS.

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Flavonoid treatments suppress lipopolysaccharide-induced inflammation through nuclear factor-kappaB and mitogen-activated protein kinase signaling pathways

To decipher the signaling pathway(s) involved in Q, K, G, and C suppression of LPS-induced inflammation, we determined whether these flavonoids regulate the NF-κB signaling pathway. The protein expression levels of NF-κB p65 were determined after nuclear protein extraction on Western blotting. As shown in [Figure 6]a, all flavonoids at a dose of 12.5 μg/mL reduced expression levels of NF-κB p65 induced by LPS in the nucleus. Following this, we evaluated the effects of these flavonoids on the MAPK signaling pathway, including ERK, JNK, and p38, by Activation Multispecies InstantOne™ ELISA kits [Figure 6]b, [Figure 6]c, [Figure 6]d. LPS treatment significantly increased phosphorylated ERK, JNK, and p38 (286.80 ± 10.56%, 336.10 ± 10.71%, and 140.60 ± 12.97%, respectively) expressions compared to NC group in RAW264.7 cells. Phosphorylated ERK levels significantly decreased only when simultaneously treated with either 12.5 μg/mL Q (186.85 ± 10.64%) or K (263.05 ± 9.55%), while phosphorylated JNK levels were all significantly reduced following simultaneous treatments with Q (195.90 ± 17.82%), K (189.30 ± 12.73%), G (163.85 ± 8.78%), and C (180.90 ± 11.18%). Phosphorylated p38 expression levels also significantly decreased in cells simultaneously treated with only Q (124.3 ± 6.36%) or K (107.05 ± 11.81%). Therefore, NF-κB and MAPK signaling pathways are targets for Q, K, G, and C mediated anti-inflammatory responses in LPS-stimulated RAW264.7 cells.
Figure 6: Effects of treatments with four different flavonoids on nuclear translocation of nuclear factor-kappaB (NF-κB) and mitogen-activated protein kinase (MAPK) expression levels in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages. RAW264.7 cells were treated with or without 1 μg/mL LPS and 12.5 μg/mL Q, K, G, or C for 24 h. (a) Cells were collected, followed by extraction of nuclear fraction. NF-κB p65 expressions were evaluated by Western blotting (upper panel). The expression level of each protein was quantified using ImageJ software. After normalization by lamin B1, relative NF-κB expression levels are shown as folds of no-treatment control (NC) values (1-fold) (lower panel). The total cell lysates were analyzed for phosphorylated extracellular signal-regulated kinase (ERK) (b), c-Jun N-terminal kinase (JNK) (c), and p38 (d) levels using ELISA. The relative amount of each phosphorylated MAPK was expressed as percentages of NC values (100%). #P < 0.05 compared to NC; *P < 0.05 compared to LPS. (e) Schematic diagram describes the potential anti-inflammatory mechanisms of AOE and four different flavonoids in LPS-stimulated macrophages.

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In summary, the major components of AOE, Q, K, G, and C, exhibited significant activities including reductions in IL-1β, IL-6, TNF-α, iNOS, and COX-2, via regulation of NF-κB nuclear translocation and MAPK signaling [Figure 6]e. From these results and the high proportion of G (~ 92%) in AOE, we suggest that G is an ideal candidate for the development of novel anti-inflammatory therapeutics. However, purifying G is very expensive. As AO is much cheaper than pure flavonoids, AOE can be directly used as an anti-inflammatory agent.


  Discussion Top


Inflammation is one of the initiating factors in the development of chronic diseases, and several studies have described the use of LPS-induced RAW264.7 cells and animal models in the evaluation of potential anti-inflammatory therapeutic interventions.[15] AOE has been shown to inhibit tetradecanoylphorbol-13-acetate-induced inflammatory ear edema[7] and alleviate Freund's adjuvant-induced arthritis in rats by decreasing NO production, making it a candidate for the development of therapeutic or preventive interventions for acute and chronic arthritis.[16] Furthermore, AOE exerted antiulcer effects in a rat gastric injury model via the mediation of COX and non-COX responses.[17] These reports have demonstrated that AOE exerts an anti-inflammatory effect on arthritis and gastric injury in vivo. However, there is no precise data on the anti-inflammatory effects and mechanisms of AOE in vitro. Our results demonstrated that AOE represses LPS-induced inflammation in RAW264.7 cells when administered at a dose of 25–37.5 μg/mL. Moreover, AOE effectively reduces NO production, PEG2 secretion, and COX-2 expression in LPS-stimulated RAW264.7 cells.

After demonstration of the anti-inflammatory effects of AOE, we analyzed AOE composition by HPLC. G, Q, K, and C are the four major constituents of AOE with G accounting for about 92% of the extract. Previous studies have found that methanol extracts of AO contain various bioactive compounds including chrysin, pinocembrin, tectochrysin, apigenin, G, 3-O-methylgalangin, acacetin, K, kaempferide, Q, isorhamnetin, and rutin.[18] Ethanol extracts of AO have been found to include pinocembrin, G, kaempferide, and 3-O-methylgalangin.[19] In addition, acetate AO extracts have been shown to include G, kaempferide, pinobanksin, 3-phenylpropanoic acid, and zingerone.[14],[20] Interestingly, G is a common constituent in all these extracts while Q and K are detected in ethanol and methanol AO extracts. C was not detected in any of the previous solvent extracts of AO and comprised <5% of our AOE, indicating that C is not the main active compound in AOE.

We then deciphered the anti-inflammatory mechanisms of G, Q, K, and C. Our results suggested that all four compounds exhibit anti-inflammatory effects, consistent with the findings of previous studies. G has been reported to reduce propacetamol-induced acute liver injury[21] and liver fibrosis,[22],[23] as well as to relieve arthritis and bronchitis via the regulation of NF-κB expression.[24],[25] G has also been shown to have therapeutic potential in stroke and neurodegenerative diseases[26],[27] and to inhibit the expression of matrix metalloproteinase-9 in human fibrosarcoma cells HT-1080 to reduce tumor metastasis.[28] Our previous report has shown that G ameliorates cisplatin-induced nephrotoxicity through inhibition of ERK and NF-κB signaling.[29] Q has been reported to have anti-inflammatory,[30] anti-fibrosis,[31] antioxidant,[32] and antidiabetic activities[33], and has been evaluated as a treatment for neurodegenerative and cardiovascular diseases.[34],[35] K has been shown to alleviate inflammation by regulating NF-κB[36] and inhibiting COX-2 expression in nephritis.[37] It also induces cell apoptosis by activating MEK-MAPK signaling in A549 cells.[38] When K is combined with 5-fluorouracil, it represses colon cancer cell growth by mediating PI3K/Akt signaling.[39] Our previous report has shown that K protects against propacetamol-induced acute liver injury through attenuation of ERK and JNK-mediated inflammation and apoptosis.[40] C has been shown to exert anti-inflammatory[41],[42],[43] and anticancer activities in lung, breast, gastric, and prostate cancers,[44] as well as to possess antioxidant effects.[45],[46] Recently, C has been widely used in dietary and nutritional supplements designed to prevent chronic diseases. Therefore, Q, K, G, and C are all solid candidates for novel interventions in inflammation-induced diseases.

NF-κB activates various members of the MAPK pathway, including JNK, ERK, and p38,[47] and stimulates the transcriptions of IL-6, IL-1β, TNF-α, COX-2, PEG2, and iNOS.[48],[49] Here, we demonstrated that all four compounds (G, Q, K, and C) significantly reduce NF-κB nuclear translocation and JNK phosphorylation, but only Q and K reduced ERK and p38 phosphorylations. Reductions of LPS-induced NF-κB nuclear translocation and MAPK activations lead to decrease IL-6, IL-1β, TNF-α, COX-2, and iNOS levels and inhibitions of PEG2 and NO productions. These compounds showed similar anti-inflammatory mechanisms to those reported in previous studies, including NF-κB-mediated inhibitions of MAPK and Akt/PI3K, downregulations of IL-6, IL-1β, and TNF-α, and reductions of COX-2 and iNOS expressions. G exhibited similar anti-inflammatory effects to Q, K, and C and compared favorably with other AOE compounds. However, as purifying G is very expensive, we suggest direct use of AOE as an anti-inflammatory agent.


  Conclusion Top


Our results confirmed the anti-inflammatory activities of AOE in LPS-induced RAW264.7 cells and demonstrated that AOE is composed of about 92% G and approximately 5% Q, K, and C. In addition, G was the main active compound in our AOE and exerted anti-inflammatory effects via the downregulation of IL-1β, IL-6, TNF-α, COX-2, and iNOS expressions via regulation of NF-κB and JNK signaling. This means that AOE and G can be used as therapeutic agents for the treatment of inflammation-related diseases.

Acknowledgments

We would like to thank Ms. Cheryl Robbins for manuscript editing.

Financial support and sponsorship

This study was supported by grants from the Nanhua University, Taiwan (grant number C107000379), and the Taichung Veterans General Hospital/Da-Yeh University Joint Research Program (TCVGH-DYU1088303).

Conflicts of interest

There are no conflicts of interest.



 
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