Immunostimulatory activity of Paeonia lactiflora through TLR4-dependent activation of p38, JNK, and ERK1/2 in mouse macrophages, RAW264.7 cells

ABSTRACT

Paeonia lactiflora (P. lactiflora) has been recently reported to increase the production of proinflammatory mediators and activates phagocytosis in macrophages. Thus, in this study, we tried to verify the macrophage activation of Paeoniae Radix Rubra (PRR, also known as red peony root) and elucidate its mechanism of action. PRR upregulated the production of proinflammatory mediators and activated phagocytosis in RAW264.7 cells. However, these effects were reversed by inhibition of TLR2/4. In addition, the inhibition of p38, JNK, and ERK1/2 reduced the PRR-mediated production of proinflammatory mediators, and the SPL-mediated activation of p38, JNK, and ERK1/2 was blocked by the TLR4 inhibition. These findings indicate that PRR may activate macrophages through TLR4-dependent activation of p38, JNK, and ERK1/2. These indicate that PRR has immunostimulatory activity. Thus, it is believed that PRR can be used as a functional food agent that enhances the immune system.

KEYWORDS:

• Immunostimulatory activity
• macrophage activation
• Paeonia lactiflora
• Paeoniae Radix Alba

1. Introduction

Although macrophages are innate immune cells that fight foreign pathogens at the forefront, macrophages also contribute to the activation of adaptive immune cells, T- and B-cells (Hirayama et al., 2018). Therefore, activation of macrophages can be expected to enhance both the innate immune response and the adaptive immune response at the same time (Hirayama et al., 2018). In the era of a viral pandemic such as COVID-19, the activation of macrophages is increasingly important to fight foreign pathogens such as viruses (Meidaninikjeh et al., 2021).

Paeonia lactiflora (P. lactiflora) is a medicinal plant widely used for soothing the liver, relieving pain, supplying nutrients to the blood, controlling menstruation, and preventing sweating (Wang et al., 2022). In addition, P. lactiflora has been used as a treatment for inflammatory diseases such as autoimmune diseases, rheumatoid arthritis, and systemic lupus erythematosus (He & Dai, 2011; Zhang & Wei, 2020). In fact, many scientific studies have proven the anti-inflammatory activity of peony (Chen et al., 2008; Kim & Ha, 2009; Li et al., 2007). Conversely, it was recently reported that P. lactiflora increases the production of proinflammatory mediators and activates phagocytosis in macrophages (Wang et al., 2022). This report indicates that P. lactiflora may induce macrophage activation. However, it is not known how P. lactiflora activates macrophages. Therefore, in this study, we tried to verify the macrophage activation of P. lactiflora and elucidate its mechanism of action.

2. Materials and methods

2.1. Sample preparation

P. lactiflora is divided into Paeoniae Radix Rubra (PRR, also known as red peony root) and Paeoniae Radix Alba (PRA, also known as white peony root). PRR and PRA were purchased from Humanherb (Dong-gu, Daegu). Ten grams of PRR or PRA were immersed in 200 ml of distilled water and left at 20 ^oC∼80 ^oC for 1 h∼ 24 h. Then, the extracts were centrifuged at 15,000 rpm at 4 ^oC for 10 min, and then the clear supernatant was freeze-dried. The freeze-dried extracts from PRR or PRA were dissolved in distilled water for treatment of the cells and stored at −80 ^oC.

2.2. Cell line and cell culture

Since the mouse macrophage cell line, RAW264.7 cells have been widely used to evaluate immunostimulatory activity, RAW264.7 cells were used in this study. RAW264.7 cells (American Type Culture Collection, Manassas, VA, USA) were maintained with DMEM/F-12 medium (10% fetal bovine serum, 100 U/ml of penicillin, and 100 μg/ml of streptomycin) in a CO₂ incubator (5% CO₂, 37 ^oC, Humidified atmosphere).

2.3. Measurement of cell viability

The viability of RAW264.7 cells was measured using a 3-(4,5-dimethylthiazol-2-yl)−2,5-diphenyl-tetrazolium bromide (MTT) assay. Briefly, RAW264.7 cells were treated with PRR for 24 h. Then, MTT solution (1 mg/ml, Sigma-Aldrich, St. Louis, MO, USA) was added to each well and then incubated for 4 h in an incubator (5% CO₂, 37 ^oC, Humidified atmosphere). Then, the medium of each well was aspirated, and dimethyl sulfoxide was added to each well to dissolve the purple formazan. The absorbance of dissolved purple formazan was measured at 570 nm using a UV/Visible spectrophotometer (Human Cop., Xma-3000PC, Seoul, Korea).

2.4. Treatment of chemical inhibitors

The chemical inhibitors such asTAK-242 (TLR4 inhibitor), C29 (TLR2 inhibitor), PD98059 (ERK1/2 inhibitor), SB203580 (p38 inhibitor), and SP600125(JNK inhibitor) were purchased from Sigma-Aldrich. These chemical inhibitors were treated with RAW264.7 cells 2 h before PRR treatment.

2.5. Measurement of NO production

Griess assay was performed to measure NO level in RAW264.7 cells. Briefly, RAW264.7 cells were treated with PRR or PRA for 24 h. Then, the cell culture medium and Griess solution (Sigma-Aldrich) were mixed in a 1:1 ratio and left at room temperature for 15 min. After 15 min, the absorbance of the mixture was measured at 540 nm using a UV/Visible spectrophotometer (Human Cop., Xma-3000PC).

2.6. Phagocytosis test

A Neutral red assay was performed to evaluate the phagocytosis activity of RAW264.7 cells. Briefly, RAW264.7 cells were treated with PRR for 24 h. Then, RAW264.7 cells were stained with 0.01% Neutral red (Sigma-Aldrich) for 2 h in an incubator (5% CO2, 37 oC, Humidified atmosphere). Then, lysis buffer (50% EtOH:1% acetic acid  =  1:1) for dissolving Neutral red stained to RAW264.7 cells. The absorbance was analyzed at 540 nm using a UV/Visible spectrophotometer (Human Cop., Xma-3000PC).

2.7. Reverse transcription-polymerase chain reaction (RT–PCR)

mRNA of RAW264.7 cells was isolated using a RNeasy Mini Kit. mRNA isolated from RAW264.7 cells was quantified using a GeneQuantTM 1300 (Biochrom, Cambridge, England). cDNA was made from mRNA (1 μg) using a Verso cDNA Kit. cDNA was amplified using a PCR Master Mix Kit and primers. The sequences of primers used in the amplification of the cDNA were shown in Table 1.

Table 1. The sequences of primers used in the amplification of the cDNA.

Primers	Sequences
iNOS	Forward 5’-ttgtgcatcgacctaggctggaa-3’ Reverse 5’-gacctttcgcattagcatggaagc-3’,
IL-1β	Forward 5’-ggcaggcagtatcactcatt-3’ Reverse 5’-cccaaggccacaggtattt-3’
IL-6	Forward 5′-gaggataccactcccaacagacc-3′ Reverse 5′-aagtgcatcatcgttgttcataca-3′
TNF-α	Forward 5′-tggaactggcagaagaggca-3′ Reverse 5′-tgctcctccacttggtggtt-3′
GAPDH	Forward 5’-ggactgtggtcatgagcccttcca-3’ Reverse 5’-actcacggcaaattcaacggcac-3’

2.8. Western blot analysis

Quantitative analysis of cellular proteins was carried out using a BCA kit (Thermo Fisher Scientific, Waltham, MA USA). Proteins separated using sodium dodecyl-sulfate polyacrylamide gel electrophoresis were transferred to nitrocellulose membrane (Thermo Fisher Scientific). The membranes were blocked for 1 h at room temperature and the primary antibodies such as p-p38 (#9211, Cell signalling Technology, Beverly, MA, USA), p-JNK (#9251, Cell signalling Technology), p-ERK1/2 (#4370, Cell signalling Technology), or β-actin (#5125, Cell signalling Technology) were treated to the membranes at 4 ^oC overnight. After that, Anti-rabbit IgG, HRP-linked Antibody (secondary antibody, #7074, Cell signalling Technology) was treated to membranes at room temperature for 1 h. After treating ECL Western blotting substrate on the membrane, the protein band was visualized using LI-COR C-DiGit Blot Scanner.

2.9. Statistical analysis

Statistical analyses were verified using GraphPad Prism version 5.0 (GraphPad Software, Inc.) and data are presented as mean ± standard deviation. Results with *P < 0.05 and #P < 0.05 were considered statistically significant. Each data point was analyzed using one-way analysis of variance and the data was analyzed using the Bonferroni post hoc test.

3. Results and discussion

3.1. PRR increases the production of proinflammatory mediators in RAW264.7 cells

In response to the invasion of foreign pathogens into the human body, macrophages secrete various inflammatory mediators such as nitric oxide (NO), inducible nitric oxide synthase (iNOS), interleukin-1β (IL-1β), interleukin-6 (IL-6) and tumour necrosis factor-α (TNF-α) to counteract the invading foreign pathogens. NO derived from L-arginine by the action of iNOS directly attacks foreign pathogens (Bogdan et al., 2000). In addition, NO induces the differentiation of T cells (Bogdan et al, 2000). IL-1β contributes to the survival of T cells, the activation of innate immune cells, and the antigen-presenting activity of macrophages (Seillet et al., 2014). IL-6 differentiates T and B cells to increase antibody production and activates macrophage phagocytosis (Neurath, 2014; Wang et al., 2018). TNF-α contributes to the removal of foreign pathogens by increasing the phagocytosis of macrophages (Barbara et al., 1996).

P. lactiflora is divided into Paeoniae Radix Rubra (PRR, also known as red peony root) and Paeoniae Radix Alba (PRA, also known as white peony root) (Tan et al., 2020). Therefore, we first investigated the production of proinflammatory mediators after treating RAW264.7 cells with PRR and PRA to compare macrophage activation of PRR and PRA. As shown in Figure 1, PRR increased the production of inflammatory mediators such as NO, iNOS, IL-6, IL-1β, and TNF-α higher than PRA in RAW264.7 cells. This result shows that PRR may be more suitable than PRA as an agent for activating macrophages.

Figure 1. Effect of PRR and PRA on the production of proinflammatory mediators in RAW264.7 cells. RAW264.7 cells were treated with PRR or PRA for 24 h. NO level and mRNA level were measured by Griess assay and RT-PCR, respectively. *P < 0.05 vs. control group (Cont.).

And we compared the production of proinflammatory mediators of PRR extracted by temperature (4 ^oC∼80 ^oC) and time conditions (1 h∼24 h) to establish the extraction process related to macrophage activation. As shown in Figure 2A, PRRs extracted at temperatures between 4 and 60 °C increased the production of proinflammatory mediators upwards but the production of proinflammatory mediators decreased slightly at 80 °C. As shown in Figure 2B, The PRRs extracted by time conditions at 60 °C all increased the production of pro-inflammatory mediators with no appreciable difference. Therefore, considering the industrial aspect of efficiency, we chose PRR extracted at 60°C for 1 h for further research, as shorter extraction times are advantageous. As shown in Figure 3A and B, PRR extracted for 1 h at 60 °C dose-dependently increased the production of proinflammatory mediators such as NO, iNOS, IL-6, IL-1β, and TNF-α without cytotoxicity. As shown in Figure 3C, Moreover, PRR increased macrophage phagocytosis, which is an important indicator of macrophage activation and can enhance the innate immune system. These results show that PRR may have the function of activating macrophages.

Figure 2. Effect of PRR on the production of proinflammatory mediators according to extraction conditions in RAW264.7 cells. (A) PRR was extracted at 4 ^oC∼80 ^oC for 24 h. After extraction, each PRR was treated to RAW264.7 cells for 24 h. The level of NO and mRNA was measured by Griess assay and RT-PCR, respectively. *P < 0.05 vs. control group (Cont.). (B) PRR was extracted at 60 ^oC for 1 h∼24 h. After extraction, each PRR was treated to RAW264.7 cells for 24 h. The level of NO and mRNA was measured by Griess assay and RT-PCR, respectively. *P < 0.05 vs. control group (Cont.).

Figure 3. Effect of PRR on macrophage activation in RAW264.7 cells. RAW264.7 cells were treated with PRR for 24 h. NO level (A), mRNA level (A), cell viability (B), and phagocytotic activity (C) were measured by Griess assay, RT-PCR, Neutral red assay, and MTT assay, respectively. *P < 0.05 vs. control group (PRR 0 μg/ml).

3.2. TLR2/4 are involved in the PRR-mediated production of proinflammatory mediators in RAW264.7 cells

When macrophages recognize the foreign pathogen's pathogen-associated molecular patterns (PAMPs), an inflammatory response against the foreign pathogen is initiated, which is essential for maintaining host homeostasis (Kawai & Akira, 2011). Among the pattern recognition receptors that recognize PAMPs of foreign pathogens in macrophages, Toll-like receptors (TLRs) have a unique function of recognizing an early infection and inducing a strong inflammatory response (Kawai & Akira, 2011). Indeed, it has been found that many natural products induce the TLR2/4-mediated activation of macrophages (Jiang et al., 2021; Yang et al., 2019). Thus, we investigated whether TLR2 and TLR4 are involved in the production of proinflammatory mediators and activation of phagocytosis by PRR. As shown in Figure 4A, C29 (TLR2 inhibitor) treatment decreased PRR-mediated production of NO, IL-1β, and TNF-α in RAW264.7 cells. However, the treatment of TAK-242 dramatically inhibited the PRR-mediated production of NO, iNOS, IL-6, IL-1β, and TNF-α in RAW264.7 cells. In addition, PRR-mediated activation of macrophage phagocytosis was blocked in the treatment of C29 and TAK-242 in RAW264.7 cells (Figure 4B). These results suggest that TLR4 is the major pattern recognition receptor involved in PRR-mediated macrophage activation, although both TLR2 and TLR4 are involved in PRR-mediated macrophage activation.

Figure 4. Effect of TLR2/4 on PRR-mediated production of proinflammatory mediators and phagocytosis activation in RAW264.7 cells. RAW264.7 cells were pretreated with C29 (TLR2 inhibitor, 100 μM) or TAK-242 (TLR4 inhibitor, 5 μM) for 2 h and co-treated with PRR for 24 h. NO level (A), mRNA level (A), and phagocytotic activity (B) were measured by Griess assay, RT-PCR, and Neutral red assay, respectively. *P < 0.05 vs. DMSO group without the treatment of PRR and inhibitors and #P < 0.05 vs DMSO group treated with PRR treatment without inhibitor treatment.

3.3. TLR4-dependent activation of MAPKs is involved in the PRR-mediated production of proinflammatory mediators in RAW264.7 cells

Mitogen-activated protein kinase (MAPK) signalling, including extracellular regulated kinase 1/2 (ERK1/2), p38, and c-Jun N-terminal Kinase (JNK), promotes the production of proinflammatory mediators in macrophages (Coskun et al., 2011). In fact, many natural plants with immunostimulatory activity increased the production of proinflammatory mediators through activation of MAPK signalling (Han et al., 2009; Kim et al., 2013). Thus, we investigated whether MAPK signalling is involved in the PRR-mediated production of proinflammatory mediators. As shown in Figure 5A, SB203580-induced p38 inhibition significantly attenuated the production of NO, iNOS, IL-6, IL-1β, and TNF-α in PRR-treated RAW264.7 cells. Inhibition of JNK by SP600125 decreased PRR-mediated production of NO, iNOS, and IL-1β in RAW264.7 cells. In addition, the production of IL-6, IL-1β, and TNF-α by PRR was blocked in the inhibition of ERK1/2 by PD98059. These results indicate that p38, JNK, and ERK1/2 may contribute to the PRR-mediated production of proinflammatory mediators in macrophages. Thus, we investigate whether PRR activates p38, JNK, and ERK1/2 in RAW264.7 cells. As shown in Figure 5B, the phosphorylation levels of p38, ERK1/2, and JNK were upregulated by PRR treatment in RAW264.7 cells, which indicates that PRR may activate p38, JNK, and ERK1/2 in macrophages.

Figure 5. Effect of MAPK signalling pathway on PRR-mediated production of proinflammatory mediators in RAW264.7 cells. (A) RAW264.7 cells were pretreated with PD98059 (ERK1/2 inhibitor, 40 μM), SB203580 (p38 inhibitor, 40 μM), or SP600125 (JNK inhibitor, 40 μM) for 2 h and then co-treated with PRR for 24 h. NO level and mRNA level were measured by Griess assay and RT-PCR, respectively. *P < 0.05 vs. DMSO group without the treatment of PRR and inhibitors and #P < 0.05 vs DMSO group treated with PRR treatment without inhibitor treatment. (B) RAW264.7 cells were treated with PRR for the indicated times. The protein levels were determined by Western blot analysis. Actin was used as a loading control. (C) RAW264.7 cells were pretreated with C29 (TLR2 inhibitor, 100 μM) or TAK-242 (TLR4 inhibitor, 5 μM) for 2 h and co-treated with PRR for 15 min. Protein levels were measured by Western blot analysis. Actin was used as a loading control. *P < 0.05 vs. the group without the treatment of PRR and inhibitors and #P < 0.05 vs the group treated with PRR treatment without TAK-242 treatment.

There is a report that MAPK signalling is crucial in the TLR2/4-mediated production of proinflammatory mediators in macrophages (Bai et al., 2019). Thus, since it was confirmed that the PRR-mediated production of proinflammatory mediators mainly depended on TLR4, we investigated whether TLR4 is involved in the PRR-mediated activation of p38, JNK, and EKR1/2. As shown in Figure 5C, the treatment of PRR alone dramatically phosphorylated p38, JNK, and EKR1/2 but the inhibition of TLR4 by TAK-242 downregulated the PRR-mediated phosphorylation of p38, JNK, and EKR1/2 in RAW264.7 cells. This result suggests that PRR-mediated activation of p38, JNK, and EKR1/2 may be dependent on TLR4 in macrophages.

4. Conclusion

Whenever an infectious disease such as COVID-19 occurs, interest in strengthening immunity is increasing. Although the optimal strategy to fight these infectious diseases is considered to be vaccine development, the natural enhancement of the immune system through diet has also been regarded as an important defense strategy. In this study, PRR increased macrophage-released proinflammatory mediators (NO, iNOS, IL-6, IL-1β, and TNF-α) and activated phagocytosis through TLR4-dependent activation of p38, JNK, and ERK1/2 in RAW264.7 cells. These indicate that PRR has immunostimulatory activity. Thus, it is believed that PRR can be used as a functional agent that enhances the immune system.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

This work was carried out with the support of “Cooperative Research Program for Agriculture Science and Technology Development (Project No. PJ017090052022)” Rural Development Administration, Republic of Korea.