Abstract

Moutan cortex radicis (MCR), the root bark of Paeonia suffruticosa, has been widely used as a traditional herb. In this study, we evaluated whether the MCR extract and two active compounds of the bark, paeoniflorin (paeo) and oxypaeoniflorin (oxypaeo), alleviate lipopolysaccharide (LPS)-induced inflammatory responses in RAW264.7 cells and whether they controlled TLR signaling pathway. RAW264.7 cells were treated with the MCR extract or two active compounds in the presence or absence of LPS. The extract and two active compounds inhibited LPS-stimulated nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) gene expression. Additionally, the extract and two active compounds suppressed inflammatory cytokine secretion and gene expression in LPS-stimulated cells. The extract and two active compounds alleviated NF-κB activation by regulating upstream genes in TLR signaling pathway. In addition, the extract and two active compounds decreased phosphorylation of ERK and p38 MAPK. These results indicate that the MCR extract, paeo and oxypaeo have anti-inflammatory effects through regulation of TLR signaling pathway in RAW264.7 cells.

1. Introduction

Inflammation is a protective response of the body and is caused by harmful stimuli ¹. In general, inflammation has beneficial effects through appropriate regulation. However, control failure caused by some stimuli induces inflammation related diseases such as cancer, atherosclerosis, rheumatoid arthritis (RA) and asthma ^{2, 3}. The appropriate regulation of pattern recognition receptors (PPRs), including trans-membrane proteins such as toll-like receptors (TLRs), c-type lectin receptors (CLRs), and NOD-like receptors (NLRs), is considered as an effective strategy for the reduction of the inflammatory responses ⁴.

Especially, TLRs are responsible for sensing various ligands such as double-stranded RNA (dsRNA), flagellin and lipoprotein ⁵. TLR4 is a member of TLR family and binds to gram-negative bacteria such as lipopolysaccharide (LPS). The activation of TLR4 by LPS stimulates the production of inflammatory cytokines, such as interleukin-1β (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor superfamily-α (TNF-α) ⁶. This delivers a signal via two different pathways, myeloid differentiation factor 88 (MyD88)-dependent and MyD88-independent pathways, within the cell ^{7, 8}. The MyD88-dependent pathway activates nuclear factor kappa B (NF-κB) through expression of the MyD88. The MyD88-independent pathway leads to transcription of interferon regulatory factor 3 (IRF3) in the nucleus through the activation of the TIR-domain containing adapter inducing interferon β (TRIF) and the TRIF-related adaptor molecule (TRAM).

Activation of mitogen-activated protein kinases (MAPKs), including p38 MAPK, extracellular signal-regulated kinase (ERK) and c-Jun amino-terminal kinase (JNK), leads to inflammatory responses in inflammation-associated diseases ^{9, 10}. MAPKs are also related to NF-κB subunit p65 transactivation ¹¹. Therefore, regulation of MAPKs plays an important role in the inhibition of inflammatory responses.

In recent studies, various phytochemicals have been demonstrated to have a beneficial effect on inflammation-associated diseases ^{1, 12}. Moutan cortex radicis (MCR) is widely used as a traditional herb in the East, and it has anti-oxidation properties and decreases periodontal diseases ^{13, 14}. Recently, several studies have shown that paeoniflorin (paeo) and oxypaeoniflorin (oxypaeo), major constituents of MCR, have neuroprotective effects and are anti-oxidants ^{15, 16}. However, the MCR extract and two active compounds, paeo and oxypaeo, have not been clearly evaluated for their anti-inflammatory effects on LPS-stimulated RAW264.6 cells. Therefore, we examined whether the MCR extract inhibits LPS-induced inflammatory responses through regulation of TLR signaling pathway. Additionally, we confirmed that paeo and oxypaeo have important roles in the MCR extract.

2. Materials and Methods

MCR was obtained from Kangwon National University (St. Gangwondaehak, Chuncheon,, South Korea). Paeoniflorin (paeo) and oxypaeoniflorin (oxypaeo) were purchased from MedChem Express (Princeton, NJ, USA). RAW264.7 cells were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). Dulbecco’s Modified Eagle’s media (DMEM), fetal bovine serum (FBS), 100 units/ml penicillin streptomycin, and 100 μg/ml streptomycin were purchased from Gibco (Grand Island, NY, USA). Lipopolysaccharide (LPS) was purchased from Sigma (St. Louis, MO, USA). Phosphate-buffered saline (PBS) was purchased from RMS Bio-solution (Seoul, Korea). Thiazolyl Blue tetrazolium bromide (MTT) was purchased from Alfa Aesar (Ward Hill, MA, US). Enzyme-linked immunosorbent assay (ELISA) kits were purchased from BioLegend (San Diego, CA, USA). TRIzol reagent was purchased from Invitrogen (Carlsbad, CA, USA). Primers were purchased from Bioneer (Daejeon, South Korea). Antibodies were purchased from Cell Signaling Technology (Boston, MA, USA) and Santa Cruz Biotechnology (Santa Cruz, CA, USA).

We used the MCR plants at six years of age for the experiment. The soil on the MCR was removed by washing with water. After air drying, it was cut into small pieces and extracted with hot water for 3 h. Next, the extract was filtrated through Whatman no. 5 filter paper and concentrated by vacuum furnace. The concentrated extract was freeze-dried at −45°C under vacuum for at least 48 h and was stored at 4°C until use.

RAW264.7 cells, a murine macrophage cell line, were cultured at 37 °C in DMEM supplemented with 10 % FBS and 1 % penicillin streptomycin. To evaluate cell viability, RAW264.7 cells (5 × 10³ cells/well) were cultured in 96-well plate. Cells were exposed to the presence or absence of the MCR extract, paeo, and oxypaeo for 24 h.

RAW264.7 cells were seeded into 12-well plates at a density of 1 × 10⁵ cells/well. RAW264.7 cells were treated with the MCR extract (0, 50, and 100 μg/mL), paeo (0, 10, and 30 μM), and oxypaeo (0, 10, and 30 μM) for 4 h. Cells were treated with LPS (1 μg/mL) and incubated for 18 h at 37 °C in a CO₂ incubator. After the various treatments, the cell culture medium (100 μL) was mixed with Griess reagent (100 μL) and incubated at room temperature for 20 min. The NO concentration was determined at 540 nm using NaNO₂ as a standard.

RAW264.7 cells were pre-treated with the three compounds for 4 h and then stimulated with or without LPS (1 μg/mL) for 18 h. Cell supernatants were thawed only once, immediately before performing the cytokine assay. We measured the cytokine levels (TNF-α, IL-1β, and IL-6) in supernatants using ELISA kits as per the manufacturer’s instructions.

Total RNAs were extracted using TRIzol reagent and subsequently used to generate cDNA using an RT-PCR system. Target gene amplification was performed using specific oligonucleotide primers in a normal PCR system. The forward and reverse sets of RT-PCR primers were designed as follows: TNF-α, forward (5′-CTACTCCTCAGAGCCCCCAG-3′) and reverse (5′-TGACCACTCTCCCTTTGCAG-3′); IL-1β, forward (5′-CAGGATGAGGACATGAGCACC-3′) and reverse (5′-CTCTGCACACTCAAACTCCAC-3′); IL-6, forward (5′-CCATCTCTCCGTCTCTCACC-3′) and reverse (5′-AGACCGCTGCCTGTCTAAAA-3′); IL-10, forward (5′-CAGTACAGCCGGGAAGACAA-3′) and reverse (5′-TCCAGCTGGTCCTTTGTTTG-3′); MyD88, forward (5′-ACTGGCCTGAGCAACTAGGA-3′) and reverse (5′-CGTGCCACTACCTGTAGCAA-3′); IRAK4, forward (5′-AGCTGCGTCACCTACCTGTT-3′) and reverse (5′-GTTTGGTGATGTTGCTGTGG-3′); were used. The PCR product was separated on 1.5 % agarose gel and visualized by UV after ethidium bromide staining.

After RAW264.7 cells were washed using 1 × PBS, cells were lysed by lysis buffer (including phosphatase inhibitor cocktail 2 and phosphatase inhibitor cocktail 3). Protein content was determined using the Bradford assay. Protein extracts were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes (Immune-Blot PVDF membrane, Bio-Rad). Membranes were immunoblotted with primary antibodies specific for iNOS, p-TBK1, TBK1, p-IRF3, IRF3, p-IKK, IKK, p-IκBα, IκBα, p-NF-κB p65, NF-κB p65, p-ERK, ERK, p-JNK, JNK, p-p38, p38 and α-tubulin at 4°C overnight. Membranes were then treated with horseradish peroxidase (HRP)-conjugated secondary antibodies (1:5,000) for 2 h. Bands were visualized using an enhanced chemiluminescence system (ECL, Thermo Fisher Scientific) and LAS image software (Fuji, New York, NY, USA).

Differences among multiple groups were determined by a one-way analysis of variance (ANOVA), followed by Duncan’s multiple range test, using the SPSS software system (SPSS for Windows, version 20; SPSS, Inc., Chicago, IL). Values marked with different letters are significantly different, p<0.05.

3. Results and Discussion

Prior to investigating the MCR extract, paeo, or oxypaeo on LPS-induced inflammation in RAW264.7 cells, we performed a cell viability assay to select the proper concentrations of the MCR extract and two compounds for further investigation. Cells were treated with increasing concentrations of the MCR extract (0, 6.3, 12.5, 25, 50, and 100 μg/mL) and two compounds (0, 10, 20, and 30 μM) for 24 h. We observed that the MCR extract and two compounds were not toxic to cells (Figure 1). Therefore, a range of concentrations of the MCR extract (0, 50, and 100 μg/mL) and two compounds (0, 10, and 30 μM) was selected for further experiments.

To determine the effect of the MCR extract, paeo, or oxypaeo on LPS-induced inflammation, cells were stimulated with LPS in the presence or absence of the MCR extract, paeo, or oxypaeo for 18 h. The production of NO is one of the markers of LPS-induced inflammation ¹⁷. As shown in Figure 2A, LPS stimulated the production of NO in RAW264.7 cells, and the MCR extract and two compounds inhibited the production of NO. Additionally, the MCR extract and two compounds suppressed the mRNA expression and protein levels of iNOS, which is a NO production-associated protein, compared to the LPS-treated cells (Figure 2B, Figure 2C). Thus, the MCR extract, paeo, and oxypaeo decreased LPS-induced NO production by regulating iNOS gene expression.

It is well known that LPS activates TLR signaling pathway and subsequently produces pro-inflammatory cytokines in macrophage cells ¹⁸. To determine the effect of the MCR extract, paeo, or oxypaeo on pro-inflammatory cytokines, cells were stimulated with LPS in the presence or absence of the MCR extract, paeo, or oxypaeo for 18 h. As shown in Figure 3A-C, it was observed that LPS increased the production of TNF-α, IL-1β, and IL-6 when compared to cells treated with the absence of LPS, while the MCR extract, paeo, or oxypaeo decreased LPS-induced production of TNF-α, IL-1β, and IL-6 compared to the corresponding control. In addition, the extract and two active compounds inhibited pro-inflammatory cytokines gene expression (Figure 3D). These results show that the MCR extract, paeo, and oxypaeo decreased secretion of LPS-induced inflammatory cytokines through regulation of pro-cytokine gene expression in cell.

We showed that the MCR extract, paeo, and oxypaeo suppress inflammatory responses of LPS–stimulated cells. To understand the molecular mechanism underlying the anti-inflammation of the MCR extract, paeo, or oxypaeo, we investigated TLR signaling pathway. TLR signaling pathway is an inflammatory response related pathway ¹⁹. In many studies, regulation of TLR signaling pathway is one of the strategic methods to alleviate inflammatory responses ^{20, 21}. We indicated a capacity of the extract and two compounds to control TLR signaling pathway. The MCR extract, paeo and oxypaeo inhibited the expression of MyD88 and IRAK4 genes, the constituents of the MyD88-dependented pathway, gene expression (Figure 4A). Additionally, the MCR extract, paeo and oxypaeo decreased LPS-stimulated TRIF, TBK1 and IRF3 gene expression, constituents of the MyD88-independent pathway (Figure 4C). Next, we studied whether the extract and two active compounds decreased LPS-induced NF-κB activation. NF-κB activation induced inflammatory responses, such as inflammatory cytokine secretion and the production of NO ²². As shown in Figure 4B, the MCR extract inhibited LPS-stimulated NF-κB activation at a low dose through regulation of the phosphorylation of IκBα. Paeo decreased NF-κB activation, whereas IKK and IκBα were not affected. Oxypaeo suppressed NF-κB activation by regulating the phosphorylation of IκBα in LPS-stimulated cells. Therefore, the MCR extract, paeo and oxypaeo had anti-inflammatory effects through targeting TLR signaling pathway.

MAPKs are related to the extracellular signals that play important roles in inflammatory responses ²³. We investigated whether the MCR extract, paeo and oxypaeo attenuate the phosphorylation of MAPKs. As shown in Figure 5, the MCR extract and paeo regulated the phosphorylation of ERK and p38 MAPK in LPS-stimulated cells. Oxypaeo only suppressed the phosphorylation of p38 MAPK, unlike extract and paeo. The phosphorylation of JNK was not affected by the extract, paeo, or oxypaeo. These results showed that the MCR extract, paeo, and oxypaeo partially affected LPS-induced phosphorylation of MAPKs in RAW264.7 cells.

4. Conclusions

Taken together, we have shown here that the MCR extract, paeo, and oxypaeo alleviate LPS-induced inflammatory responses through the regulation of TLR signaling pathway in RAW264.7 cells. In addition, the MCR extract, paeo, and oxypaeo partially suppressed the phosphorylation of MAPKs, ERK and p38 MAPK. These findings suggested that the MCR extract may function as a potential natural bioactive compound with anti-inflammatory effects. Also, paeo and oxypaeo may also function as active components from the MCR extract.

Acknowledgments

This research was partially supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF), funded by the Ministry of Education (2016R1D1A1B03932800). The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Conflicts of Interest

The authors declare no conflict of interest.

References