Anti‑inflammatory effects of Nypa fruticans Wurmb via NF‑κB and MAPK signaling pathways in macrophages
- Authors:
- Published online on: November 8, 2022 https://doi.org/10.3892/etm.2022.11690
- Article Number: 755
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Copyright: © Park et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
Abstract
Introduction
Nypa fruticans (N. fruticans) Wurmb is a mangrove plant (Araceae family). It is generally distributed throughout in the tropical regions of India, the Philippines, Malaysia, and some parts of Australia (1). Traditionally, the roots, stems, and leaves of N. fruticans have been used to remedy tuberculosis, sore throat, and liver diseases (2). N. fruticans contains polyphenols, flavonoids, protocatechuic acid, chlorogenic acid, and kaempferol (3,4). N. fruticans has antioxidant, antidiabetic, and hepatoprotective activities (4-7). Plants containing phenolic compounds exhibit anti-cancer and antioxidant activities in humans (8-11). Inflammatory responses serve in the immune defense system against stimuli such as infectious agent invasion or endotoxin exposure resulting in the retrieval of normal cell function and structure (12). Macrophages are activated by numerous ligands such as Fzd1, lipopolysaccharide (LPS), and RANKL (13-15). Activated macrophages release pro-inflammatory molecules [cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), and cytokines (IL-1β, IL-6, and TNF-α)] (16). LPS propagates signaling cascades by stimulating major signaling pathways (17). The nuclear factor-kappa B (NF-κB) is found in numerous cell lines expressing cytokines and growth factors. NF-κB regulates a variety of target genes involved in cell survival, cell proliferation, and immune responses (18). Recent studies have shown that PI3K/Akt plays critical roles in various processes, including cell survival, cell cycle regulation, and NF-κB activation (19). MAPK, including ERK, JNK, and p38 mediate various cellular and biological processes related to inflammatory responses. MAPK phosphorylation activates the NF-κB signaling pathway (20). Impairment of these pathways could lead to diverse immunological diseases, including cancer and inflammation. Therefore, the MAPK, NF-κB, and PI3K/Akt signaling pathways are key targets for alleviating various inflammatory diseases. Aqueous extracts of N. fruticans have been previously reported (21). It has also been shown to have an inflammatory effect on sciatic neuropathy (22). We confirmed the effect of N. fruticans fractionated with ethyl acetate on the regulation of inflammatory factors. The present study aimed to verify the inhibitory effect of alleviating various pro-inflammatory mediators through regulation of the MAPK and Akt/IκB/NF-κB signaling pathways, including translocation of NF-κB p65.
Materials and methods
Chemicals and reagents
Dimethyl sulfoxide (DMSO), ethyl acetate, petroleum ether (PE), ethyl alcohol, and methyl alcohol were purchased from Merck. All chemical reagents were obtained from Sigma-Aldrich; Merck KGaA, unless otherwise stated. Antibodies were purchased from Cell Signaling Technology, Inc., Abcam and Santa Cruz Biotechnology, Inc.
Experimental materials extraction
Air-dried shoots of N. fruticans were purchased from Hantteus Yagcho (Seoul, Korea). The shoot extracts of N. fruticans (484.00 g) were immersed in 80% methanol (4.20 l) for 7 days using a sonicator (Hwashin Technology Co., Ltd.). The obtained extracts were then filtered by filter paper (Whatman™), evaporated under reduced pressure, and freeze-dried (N-1110S; Eyela). The powered N. fruticans was sequentially fractionated by organic solvents of increasing polarity (PE, chloroform, and ethyl acetate). The ethyl acetate fraction from N. fruticans (ENF, 0.38 g) was stored at 4˚C. ENF was diluted in DMSO and further used for experiments.
Analysis of total flavonoid contents (TFC)
100 µl of ENF (4 mg/ml) was mixed with 900 µl of distilled water, and followed by 1 ml of 10% AlCl3. The absorbance of the reaction mixture was recorded at 420 nm using Xma-3000PC (UV/visible spectrophotometer; Human Corporation) after 30 min. TFC was determined by comparing it with the reference standard curve for rutin.
Antioxidant capacity evaluation
According to the method of Bondet and Van den Berg, radical scavenging activity was determined with some modifications (23,24). The 2,2-diphenyl-1-picryl hydrazyl (DPPH) solution (300 µM) was dissolved in ethyl alcohol. The ABTS solution containing 7.4 mM of 2,2'-Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt, and potassium persulfate (2.6 mM) in distilled water was prepared for 24 h at ambient temperature. The DPPH or ABTS solution was adjusted to an absorbance value of 1.00 (515 nm or 732 nm, respectively). The sample (40 µl) was mixed with DPPH or ABTS solution (760 µl) and reacted for 30 min in the dark at ambient temperature. The data of radical scavenging activity was expressed as the decrease in the absorbance of DPPH or ABTS. The rate of decrease in the radicals was calculated using the formula as follows: [1-(AbsSample-AbsBlank)/AbsControl] x100 (%).
Here, AbsSample, AbsBlank and AbsControl are the absorbance values of the sample, ethanol or distilled water and radicals, respectively. The standard compound, L-ascorbic acid, was used at the same concentration. Then, a curve was constructed, and the equation of the curve was used to calculate the half-maximal inhibitory concentration (IC50, µg/ml).
Cell culture
Macrophages (ATCC® CRL-2278™; VA, USA) were used in this study. Cells were maintained in incubator (5% CO2, 37˚C), and cultured with DMEM [1% penicillin/streptomycin and 10% FBS (Biowest)]. Then, 0.2% prophylactic plasmocin (InvivoGen) was used to suppress the proliferation of mycoplasma.
Cell viability
To estimate the cell proliferation assay, the cytotoxic effects of ENF treatment on macrophages were assessed by the CellTiter 96® AQueous One solution (Promega). The cells were incubated in each well of 96-well plate for 24 h. The ENF (25-400 µg/ml) treatment was done at the end of 24 h and the cells were reacted with the reagent for 2 h in an incubator (37˚C, 5% CO2). The absorbance of supernatant was measured at 490 nm using a microplate reader (ELX808, BioTek).
Nitric oxide (NO) production
The cells (4.0x105 cells/well) were cultured in 6-well plates, and then treated with the appropriate concentration of ENF and 1 µg/ml of LPS (L6529, Sigma-Aldrich; Merck KGaA) for 24 h. Griess A reagent containing 1% sulfanilamide (w/v) in 5% phosphoric acid (v/v) and Griess B reagent containing 0.1% N-(1-Naphthyl) ethylenediamine dihydrochloride (w/v) in distilled water were used. The supernatant of the culture medium was collected, and Griess A/B reagent (1:1) was mixed with it in equal volumes at room temperature for 10 min. To measure the NO production, absorbance was measured using a microplate reader at 540 nm.
Western blotting
Cells were seeded in 100 π dishes (4x105 cells/well). After 24 h, the cells were treated with ENF (25, 50, and 100 µg/ml) for 2 h and then induced with LPS (1 µg/ml) for 30 min or 24 h in macrophages. The cells were washed twice with ice-cold PBS. The cells were then lysed at 4˚C using a Radio immunoprecipitation assay (RIPA) buffer [Halt™ protease and phosphatase inhibitor cocktail, EDTA solution (Thermo Fisher Scientific, Inc.)]. Following the manufacturer's protocol, a Bradford assay (Bio-Rad) was employed to determine the protein concentration. After quantification, the protein was electrophoresed on a 12% polyacrylamide gel and electro-transferred to a polyvinylidene difluoride (PVDF) membrane (Bio-Rad). A 5% Bovine serum albumin (BSA, Biosesang) in Tris-buffered saline containing 0.1% Tween-20 (TBS-T) was used as a blocking agent at room temperature for 1 h. Subsequently, the membrane was incubated with primary antibodies (diluted in 3% BSA in TBS-T) at 4˚C for overnight. The primary antibodies (1:4,000) included an iNOS (D6B6S) monoclonal antibody (#13120), a phospho-NF-κB p65 monoclonal antibody (p-p65, #3033), an IκB-α (L35A5) monoclonal antibody (#4814), a phospho-IκB-α (Ser32) (14D4) monoclonal antibody (p-IκB-α,#2859), a phospho-Akt (Ser473) (D9E) monoclonal antibody (p-Akt, #4060), a SAPK/JNK polyclonal Antibody (#9252), a phospho-SAPK/JNK (Thr183/Tyr185) polyclonal antibody (p-JNK#9251), a p44/42 MAPK (ERK) (137F5) monoclonal antibody (#4695), a phospho-p44/42 MAPK (ERK) (Thr202/Tyr204) polyclonal antibody (p-ERK, #9101), a p38 MAPK (D13E1) monoclonal antibody (#8690), and a phospho-p38 MAPK (Thr180/Tyr182) polyclonal antibody (p-p38, #9211). Antibodies were purchased from Cell Signaling Technology. The anti-COX-2 polyclonal antibody (ab15919) and anti-NF-κB p65 polyclonal antibody (ab16502) were purchased from Abcam. The membrane was then washed thrice with TBS-T for 15 min. After washing, horse radish peroxidase (HRP)-labeled secondary antibodies, including anti-mouse IgG-HRP monoclonal antibody (18-8817-33, Rockland) and anti-rabbit IgG-HRP monoclonal antibody (18-8816-33) were incubated (1:10,000) for 1 h at room temperature. Protein bands were detected using an enhanced chemiluminescence (ECL) substrate solution (Bio-Rad) and visualized using the ChemiDoc imaging system (Bio-Rad). Western blotting data were quantified using ImageJ version 1.52a (developed at the National Institutes of Health).
Reverse transcription-polymerase chain reaction (RT-PCR)
Macrophages were cultured in Dulbecco's modified Eagle's medium (DMEM) for 24 h. The following day, the cells were treated with 25, 50, and 100 µg/ml of ENF for 2 h and then induced with LPS (1 µg/ml) for 6 h. The cells were washed with PBS and lysed using 350 µl RLT buffer. Total RNA was extracted from macrophages using NucleoSpin® RNA Plus (Macherey-Nagel), as described by the manufacturer and cDNA was synthesized from 1 µg of extracted RNA using ReverTra Ace-α (Toyobo). The synthesized cDNA was used as a template, including Quick Taq® HS DyeMix (Toyobo) and primers (Table I; COX-2, iNOS, IL-1β, TNF-α, IL-6, and GAPDH). PCR was performed using a T100 Thermal Cycler (Bio-Rad) and the PCR cycles were as follows: 25-35 cycles of 30 s at 94˚C (denaturation) and 30 s at each following Tm value of primers (annealing/extension). The mRNA levels of the target genes were normalized to the housekeeping gene (GAPDH). The band density was evaluated using ImageJ software.
Quantitative PCR (qPCR)
For qPCR, the cDNA templates obtained from the RT-PCR were mixed with the Quanti Tect® SYBR-Green PCR kit (Qiagen) and all the primers. Primer sequences were designed using Primer 3 (Table I). All reactions were performed using Rotor-Gene Q series software (Qiagen) for qPCR analysis and the reaction conditions were as follows: 95˚C for 15 min (amplification), 94˚C for 15 sec (denaturation), 60˚C for 30 sec (annealing), and 72˚C for 30 sec (extension) with 40 cycles. Housekeeping gene (GAPDH) was used as a control for relative quantification of mRNA expression. The formula 2-ΔΔCq was used to analyze the mRNA expression levels, where ΔΔCq=(Cqtarget-CqGAPDH)sample-(Cqtarget-CqGAPDH)control (25).
Immunofluorescence
The cells were cultured on glass coverslips for 24 h and then treated with ENF for 2 h and later with LPS (1 µg/ml) for 30 min. After treatment, cells were fixed with 4% paraformaldehyde (Biosesang) dissolved in PBS for 15 min at ambient temperature. The cells were then washed with PBS for 3 min and blocked with 0.1% Triton X-100 at room temperature. Next, the cells were incubated with an anti-NF-κB p65 (1:1,000) and an anti-IκB-α (1:1,000) at 4˚C overnight. Subsequently, we used an anti-rabbit IgG (Alexa Fluor® 568, ab175471) and an anti-mouse IgG (Alexa Fluor® 488, ab150113) to stain the cells. The cells were incubated with DAPI (D1306, Invitrogen; Thermo Fisher Scientific, Inc.) at room temperature with light blocking. The cells were then washed at least five times with PBS. Finally, a drop of fluorescence mounting medium (S3023, Dako) was used prior to observation under a fluorescence at 400x magnification CKX53 microscope (Olympus) and images were obtained using a digital single-lens reflex camera (DS126271; Canon).
Statistical analysis
All experiments were repeated at least three times. Statistical analyses were verified using GraphPad Prism version 5.0 (GraphPad Software, Inc.) and data are presented as mean ± standard deviation. In the antioxidant capacity analysis of ENF, statistical comparisons were performed by unpaired t-test. P<0.05 was considered to indicate a statistically significant difference. In other experiments, each data point was analyzed using one-way analysis of variance. The data were analyzed using the Bonferroni post hoc test.
Results
Antioxidant capacity of ENF
The results of the DPPH and ABTS radical scavenging are shown in Fig. 1, where it demonstrated the scavenging ability of ENF against DPPH and ABTS radicals in a dose-dependent manner. The DPPH radical scavenging activity of ENF showed maximum inhibition (93.60±0.13% at 200 µg/ml). Meanwhile, the ABTS radical scavenging activity of ENF showed maximum inhibition (99.67±0.12% at 200 µg/ml). Additionally, the TFC in ENF was 85.33 mg/g. The reference standard curve of the rutin was used for normalization (y=0.001x + 0.0433, R2=0.9974). Pearson correlation coefficient statistics were analyzed to identify the relationship between total flavonoid content, ABTS radical scavenging activity, and DPPH radical scavenging activity (Fig. 2). A positive moderate correlation was found between the total flavonoid content and DPPH scavenging activity (r=0.506). There was a high positive correlation between the total flavonoid content and ABTS scavenging activity (r=0.865).
Effects of ENF on cell viability
To estimate the optimal ENF concentration not affecting the cell viability, macrophages were treated with ENF at various concentrations (25, 50, 100, 200, and 400 µg/ml). As shown in Fig. 3, concentrations ranging from 25-400 µg/ml showed no cytotoxicity as compared to that shown by the non-treated control (100%). Based on these studies (26,27), concentrations of 50 (95.83±3.73%) and 100 µg/ml (98.31±8.92%) were chosen for use in all experiments in this study.
Effects of ENF on cytokines expression
Cytokines, including TNF-α, IL-6, and IL-1β are released to amplify the inflammatory response in macrophages (28). As presented in Fig. 4, the TNF-α mRNA levels were attenuated by ENF treatment (2.38±0.20-fold at 100 µg/ml) compared with that in the LPS-induced group (5.37±0.03-fold). The IL-6 mRNA level was attenuated by ENF treatment (6.85±0.16-fold at 100 µg/ml) compared with that in the LPS-induced group (13.48±0.18-fold). In addition, the IL-1β mRNA level was attenuated by ENF treatment (3.75±0.27-fold at 100 µg/ml) compared with that in the LPS-induced group (18.66±1.91-fold).
Effects of ENF on NO production, COX-2, and iNOS
We evaluated the modulatory role of ENF treatment on NO production in LPS-induced macrophages (Fig. 5A). Nitric oxide production was significantly increased by LPS (1509.36±71.53%) as compared with the non-treated control (100%). As presented in Fig. 5A, ENF treatment potently suppressed NO production at the concentration of 50 (875.44±28.07%) and 100 µg/ml (488.89±21.22%). As shown in Fig. 5C and D, the COX-2 and iNOS expression was increased by LPS (21.26±0.09-fold and 7.87±0.35-fold, respectively) at the cellular level. The COX-2 and iNOS expression was significantly suppressed (3.43±0.22-fold and 2.05±0.04-fold, respectively) by ENF treatment at 100 µg/ml. The COX-2 and iNOS mRNA levels correlated with protein expression by RT-PCR analysis (Fig. 5E). The mRNA level of COX-2 was notably attenuated by ENF treatment at 50 (4.70±0.50-fold) and 100 µg/ml (3.29±0.40-fold) as compared to the LPS-induced group (10.87±0.92-fold). The mRNA level of iNOS was attenuated through ENF treatment at 50 (6.74±0.13-fold) and 100 µg/ml (3.88±0.18-fold) as compared to the LPS-induced group (11.92±0.07-fold). Quantitative PCR (qPCR) was utilized to normalize mRNA levels and establish the results of RT-PCR analysis. The qPCR data followed a pattern similar to that of western blotting and RT-PCR analysis.
Effects of ENF on Akt/IκB/NF-κB signaling pathway
NF-κB is a transcription factor complex, that regulates the expression of proinflammatory mediators (29). As demonstrated in Fig. 6A, ENF treatment suppressed the phosphorylation of NF-κB p65 (0.99±0.03-fold at 100 µg/ml), without affecting the total amount of NF-κB p65. The p-IκB-α was increased by LPS (1.45±0.03-fold) at the cellular level and the p-IκB-α was significantly suppressed by ENF treatment (0.34±0.02-fold at 100 µg/ml). These results showed that ENF treatment downregulated the NF-κB signaling by suppressing p-IκB-α. Located in the nucleus, NF-κB p65 phosphorylation is a major step in the activation of NF-κB (30). To understand the molecular mechanism of NF-κB p65 translocation, we investigated the nuclear fluorescence of IκB-α and NF-κB p65, which are related to the expression of the NF-κB. As presented in Fig. 7, the NF-κB p65 was translocated from the cytoplasm to the nucleus as compared with the control group. Simultaneously, the fluorescence intensity of IκB-α in the nucleus was increased by ENF treatment of live cells. The increase in Akt phosphorylation results in the release of proinflammatory mediators, including cytokines, COX-2, and iNOS (31). As demonstrated in Fig. 6A, phosphorylation of Akt was increased by LPS (3.27±0.03-fold) at the cellular level. ENF treatment significantly suppressed the phosphorylation of Akt (1.39±0.05-fold at 100 µg/ml).
Effects of ENF on MAPK signaling pathway
MAPK (JNK, ERK, and p38) signaling pathway stimulates the NF-κB signaling pathway (32). To evaluate whether ENF exerts the modulatory role on phosphorylation of the MAPK, the JNK, ERK, and p38 expression was investigated by western blotting. As demonstrated in Fig. 6D, LPS enhanced the phosphorylation of JNK (7.38±0.54-fold), ERK (17.07±1.11-fold), and p38 (9.55±0.08-fold) at the cellular level. ENF treatment significantly suppressed p-JNK (1.67±0.05-fold), p-ERK (6.22±0.37-fold), and p-p38 (6.42±0.06-fold at 100 µg/ml) without affecting the total amount of JNK, ERK, and p38. A previous study reported that blocking the phosphorylation of MAPK pathways is associated with the regulation of proinflammatory mediators (33).
Discussion
Phytochemicals (alkaloids, terpenes, saponins, phenolics, and flavonoids) are naturally occurring compounds found in plants (34,35). They exert in the protection of human health, preventing various (inflammation, colorectal cancer, liver cancer, and DNA damage) diseases (36-39). Other plants belonging to the Araceae family contain phytochemicals such as sterols, saponins, phenols, and alkaloids (34,40,41). N. fruticans contains phytochemicals, such as chlorogenic acid, kaempferol, and protocatechuic acid (4,21,42), which have antioxidant, anticancer, and anti-inflammatory effects (43-45). There are studies related to the various bioactivities of N. fruticans such as antidiabetic, antioxidant, and anti-inflammatory effects (21,46,47). ENF showed a similar level of DPPH radical scavenging activity (IC50, 3.93±0.10 µg/ml) compared to L-ascorbic acid (IC50, 2.65±0.02 µg/ml). ENF scavenged ABTS radical (IC50, 4.88±0.03 µg/ml) similar as compared to L-ascorbic acid (IC50, 2.36±0.10 µg/ml). IC50 values for DPPH and ABTS radicals of ENF were not statistically significant when compared with L-ascorbic acid (P<0.001). These results can be inferred that the total flavonoid content in ENF contributes to its antioxidant capacity. Based on research on the relationship between flavonoid content and effects of anti-inflammatory (48) and capacity of free radicals in the inflammatory processes (49), it was confirmed that the anti-inflammatory effect of ENF was related to its antioxidant capacity. LPS-induced macrophages secrete cytokines, which are pro-inflammatory mediators that trigger inflammatory response (50). The transport of cytokines, including IL-6, IL-1β, and TNF-α was reduced by ENF treatment, which could lead to regulation of the cascade. Apparently, pro-inflammatory mediators are expressed in response to inflammation via cytokines (51,52). Since excessive NO production can lead to cytotoxicity (53), cancer (54), and autoimmune diseases (55), it is supported that blocking of iNOS expression and NO production can prevent inflammatory diseases. This study showed that ENF treatment suppressed NO production in LPS-induced macrophages. The suppression of COX-2 and iNOS expression by ENF showed a similar pattern to the inhibition of NO production, demonstrating that a decrease in the expression of iNOS by ENF suppresses NO production. At the basal level, NF-κB binds to IκB-α and is localized in the cytoplasm. An external stimulus, such as LPS induces phosphorylation of IκB-α, causing IκB degradation via ubiquitination of the NF-κB dimer. The released NF-ĸB p65 translocated into the nucleus and promotes the transcription of inflammatory mediators. We investigated the molecular mechanisms of IκB-α and NF-κB p65 in macrophages following ENF treatment. We confirmed that ENF treatment inhibited IκB-α phosphorylation, thereby attenuating the NF-κB p65 translocation to the nucleus. Immunofluorescence analysis showed that ENF treatment increased the fluorescence intensity of IκB-α, thereby increasing residual NF-κB p65 in the cytoplasm. The inhibition of IκB-α phosphorylation causes NF-κB p65 to remain in the cytoplasm, and it is thought that ENF treatment regulates proinflammatory mediators by reducing the NF-κB signaling pathway. Akt is widely known as a downstream target of several cellular processes (56). The phosphorylation of Akt was inhibited by ENF treatment. Since Akt activates NF-κB by phosphorylating IκB kinase (IKK), inhibition of Akt phosphorylation through ENF treatment induces downstream NF-κB signaling pathway. Ligands activating cellular inflammatory responses induce phosphorylation of factors (JNK, ERK, and p38) in the MAPK signaling pathway in macrophages (57). ENF treatment suppressed the inflammatory cascade by alleviating the MAPK signaling pathway, which participate in NF-κB activation. In summary, ENF exhibits anti-inflammatory effects by modulating the nuclear translocation of NF-κB p65, which regulates the MAPK and Akt/IκB signaling pathways. The ethyl acetate fraction of N. fruticans can therefore be used as a functional material derived from natural products with the potential to regulate inflammatory responses.
Acknowledgements
Not applicable.
Funding
Funding: No funding was received.
Availability of data and materials
Not applicable.
Authors' contributions
HJP wrote the manuscript, performed the experiments and analyzed the data. HJP and SYH performed the experiments and interpreted the data. SOO and JBL participated in drafting the article. SSO, JBL and SMM were involved in interpretation of data. TWJ and JHP confirm the authenticity of all the raw data. JHP designed the experiments, wrote and edited the manuscript. HJP and TWJ made substantial contributions to the conception and acquisition of data. All authors read and approved the final manuscript.
Ethics approval and consent to participate
Not applicable.
Patient consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
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