Immune-enhancing effects of Hibiscus syriacus roots in RAW264.7 macrcophages

ABSTRACT

In this study, we evaluated whether extracts of the roots of Hibiscus syriacus (HSR) exert immune activation activities and elucidated its potential mechanism in macrophages. The HSR dose-dependently increased the production of immunomodulators such as nitric oxide (NO), inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), interleukin-1β (IL-1β), and tumor necrosis factor (TNF-α) activated phagocytosis in macrophages. Inhibition of toll-like receptors 4 (TLR4) reduced the production of immunomodulators induced by HSR. Inhibition of mitogen-activated protein kinase (MAPKs), nuclear factor-κB (NF-κB) and phosphoinositide-3 kinase (PI3K) signaling attenuated the production of immunomodulators induced by HSR. Based on the results of this study, HSR was thought to activate macrophages through the production of immunomodulators and phagocytosis activation through TLR4-dependent MAPKs, NF-κB and PI3K signaling pathways. Therefore, it is thought that the HSR has the potential to be used as agents for enhancing immunity.

KEYWORDS:

• Hibiscus syriacus
• immunomodulatory activity
• MAPKs
• NF-κB
• PI3K

1. Introduction

The immune system is distinguished into innate immune responses and adaptive immune responses as one of the important biological defenses against factors induced by invasion of external pathogens (Jang et al., 2016). Innate immune cells comprise populations of white blood cells such as neutrophils, eosinophils, basophils, lymphocytes (B cells, T cells and natural killer cells) and monocytes (dendritic cells and macrophages) (Lacy & Stow, 2011). The macrophages, one of the innate immune cells, are commonly used for studying immune mechanisms. The macrophages have the ability to present antigens to T cells and also function as effectors of cell-mediated immunity. Various immunostimulatory factors induced by macrophages have been known to activate T cells and B cells. These reports indicate that macrophages can contribute to the adaptive immune response as well as the innate immune response. Therefore, it has been reported that activation of macrophages related to innate immunity and adaptive immunity helps enhance the body's immune system (Duque & Descoteaux, 2014; Eo et al., 2021; Li et al., 2017; Mosser & Edwards, 2008; Son et al., 2021). Especially, the production and secretion of immunomodulators such as nitric oxide (NO), cyclooxygenase-2(COX-2), interleukin-1β (IL-1β), and tumour necrosis factor (TNF-α) inactivated RAW264.7 cells are indicators of immune function (Hayden et al., 2006; Monmai et al., 2019). In addition, the activation of RAW264.7 cells for the production of these immunomodulators has been reported to be mainly due to the activation of nuclear factor-қB (NF- қB) and mitogen-activated protein kinase (MAPKs) signalling pathways.

Hibiscus syriacus (HS) is a medicinal plant belonging to the Malvaceae family, it has been widely cultivated throughout eastern and southern Asia. The flower, fruit, root, stem, leaf and bark of HS have been widely used as traditional medicinal treatment materials in Asia (Zhang et al., 2020). The root of HS has been used as an antipyretic, anthelmintic and antifungal agent in Asia (Cheng et al., 2008; Kim et al., 2018). The bioactive compounds including flavone (saponarin), polyphenols, carotenoids, anthocyanins, fatty acid (malvalic acid, sterculic acid, lauric acid, myristic acid and palmitic acid), lignans and polysaccharides have been isolated from HS (Jang et al., 2012; Ryoo et al., 2010; Shi et al., 2014; Yang et al., 2020; Yoo et al., 1997). In recent years, many studies have shown that extract from HS had wide pharmacologic effects (Lee et al., 2015; Xu et al., 2021). However, the potential mechanism for the immune-enhancing activity of the roots of HS (HSR) is still insufficient. In the present study, the immune-enhancing effects of HSR on the RAW264.7 cells were determined, and the associated TLR4-dependent activation of MAPKs, NF-κB and PI3K signalling pathway was investigated.

2. Materials and methods

2.1. Material

Griess reagent, 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide, (Methylthiazolyldiphenyl-tetrazolium bromide, MTT), 2-(2-Amino-3-methoxyphenyl)-4H-1-benzopyran-4-one (PD98059, ERK1/2 inhibitor), 4-(4-Fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)-1H-imidazole (SB203580, p38 inhibitor), 1,9-Pyrazoloanthrone (SP600125, JNK inhibitor), (E)-3-(4-Methylphenylsulfonyl)-2-propenenitrile (BAY 11–7082, IKK inhibitor) and TAK-242 (Toll-like receptor 4 inhibitor, TLR4 inhibitor) were purchased from Sigma Aldrich (St. Louis, MO, USA). C29 (TLR2 inhibitor) was purchased from Bio Vision, Inc. (Milpitas, CA, USA).

2.2. Sample preparation

Hibiscus syriacus roots (HSR) were collected after botanical identification in an experimental site (37°15′04″N, 126°57′17″E) of the National Institute of Forest Science, Suwon, Korea. 20 g of powdered HSR was immersed in 400 ml of distilled water and then extracted for 48 h under stirring at 150 rpm at 4°C. After 48 h, the extracts were centrifuged for 10 min at 15,000 rpm, and then the supernatant was taken and lyophilized. The lyophilized water extracts from HSR were stored at -80°C until use.

2.3. Cell culture and treatment

The mouse macrophage cell line, RAW264.7 cells were purchased American Type Culture Collection (ATCC, Virginia, USA) and grown at 37°C in DMEM medium supplemented with 10% fetal bovine serum (FBS), penicillin (100 units/mL) and streptomycin (100 ug/mL) in a humidified atmosphere of 5% CO₂. The sample was dissolved in dimethyl sulfide (DMSO) and treated in cells. DMSO was used as a vehicle and the final DMSO concentration did not exceed 0.1% (v/v).

2.4. Cell viability assay

Cell viability was performed by MTT assay. Briefly, RAW264.7 cells were seeded at a density of 1 × 10⁶ cells/well in 12-well plate and incubated for 24 h. The RAW264.7 cells were treated with HSR at the indicated concentrations for 24 h. Then, the RAW264.7 cells were incubated with 200 μl of MTT solution (1 mg/ml) for an additional 2 h. The resulting crystals were dissolved in DMSO. The formation of formazan was measured by reading absorbance at a wavelength of 570 nm using UV/Visible (Perkin Elmer, Norwalk, CT, USA).

2.5. Measurement of nitric oxide production

RAW264.7 cells were incubated 12-well plate for overnight. The RAW264.7 cells were pretreated with HSR at the indicated concentrations for 24 h. NO level was evaluated by Griess assay. Briefly, 50 μl of the RAW264.7 cells culture supernatants were mixed with 50 μl of Griess reagent (Sigma Aldrich, St. Louis, MO, USA) and followed by reaction for 10 min at the room temperature. After 10 min, absorbance values were determined using a UV/Visible spectrophotometer (Perkin Elmer, Norwalk, CT, USA) at 540 nm.

2.6. Reverse transcriptase-polymerase chain reaction (RT–PCR)

After treatment of HSR, total RNA was extracted from RAW264.7 cells using a RNeasy Mini Kit (Qiagen, Valencia, CA, USA) and total RNA (1 μg) was synthesized using a Verso cDNA kit (Thermo Scientific, Pittsburgh, PA, USA) according to the manufacturer’s protocol. PCR was performed using PCR Master Mix Kit (Promega, Madison, WI, USA) and mouse primers for iNOS, COX-2, IL-1β, TNF-α and GAPDH were as follows; iNOS: forward 5′-GTGCTGCCTCTGGT CTTGCAAGC-3′ and reverse 5′-AGGGGCAGGCTGGGAATTCG-3′, COX-2: forward 5′-GGAG AGACTATCAAGATAGTGATC-3′ and reverse 5′-ATGGTCAGTAGACTTTTACAGCTC-3′, IL-1β: rorward 5′-GAAGCTGTGGCAGCTACCTATGTCT-3′ and reverse 5′-CTCTGCTTGTGAGGTGCTG ATGTAC-3′, TNF-: forward 5′- TACTGAACTTCGGGGTGATTGGTCC-3′ and reverse 5′-CAGCC TTGTCCCTTGAAGAGAACC-3′, GAPDH: forward 5′-CAGGAGCGAGACCCCACTAACAT-3′ and reverse 5′-GTCAGATCCACGACGGACACATT-3′. The PCR results were visualized using agarose gel electrophoresis. The density of mRNA bands was calculated using the Chemi Doc MP Imaging system (Bio-rad, CA, USA).

2.7. Statistical analysis

All the data are shown as mean ± SD (standard deviation). Statistical analysis was determined by Student’s t-test. Differences with *P or #P < 0.05 were considered statistically significant.

3. Results and discussion

3.1. HSR induces macrophage activation in RAW264.7 cells

NO produced by macrophages is an important signalling agent that protects against tumour cells or infected microorganisms in the immune system. Cytokine production during innate immunity activates local cellular responses to infections or injuries while mobilizes many defense mechanisms throughout the body (Bogdan, 2001; Coleman, 2001; Marshall et al., 2018). Thus, Production of NO in macrophages such as RAW264.7 cells and secretion of cytokines are indicators of immune function. To evaluate the effect of HSR on the production of immunomodulators such as NO, iNOS, COX-2, IL-1β, and TNF-α in RAW264.7 cells were treated with HSR for 24 h. As shown in Figure 1(A), there was no cytotoxicity against RAW264.7 cells at 12.5, 25 and 50 μg/ml concentrations of HSR. As shown in Figure 1(B), HSR promoted NO secretion in macrophages in a dose-dependent manner. HSR dose-dependently activated mRNA expression of iNOS, COX-2, IL-1β and TNF-α in RAW264.7 cells (Figure 1C). These results suggest that HSR may induce macrophage activation.

Figure 1. Effect of HSR on macrophage activation in RAW264.7 cells. RAW264.7 cells were treated with HSR for 24 h. (A) NO level, (B) cell viability and (C) mRNA level were measured by the Griess assay, MTT assay and RT-PCR, respectively. *P < 0.05 compared to the cells without the treatment.

3.2. The production of immunomodulators by HSR is dependent on TLR4 in RAW264.7 cells

Toll-like receptors (TLR) are superior characteristic pattern recognition receptors that can recognize and respond to various pathogen-related or risk-related molecular patterns during infection. TLR signalling in RAW264.7 macrophages generates in the intracellular signalling pathways through the recruitment of various adaptor and signalling proteins, and results in the activation of effector mechanisms and pathways that are significant for host defense to intracellular bacteria (Jo et al., 2019; Wang et al., 2020). We investigated that effects of TLR2 or TLR4 on the induction of immunomodulators such as NO, iNOS, IL-1β, IL-6 and TNF-α in HSR-treated RAW264.7 cells. As shown in Figure 2(A and B), the treatment of anti-TLR4 attenuated HSR-induced NO production, but not the treatment of anti-TLR2 in RAW264.7 cells. In addition, the mRNA expressions of iNOS, COX-2, IL-1β and TNF-α induced by HSR were blocked in RAW264.7 cells treated with anti-TLR4 (Figure 2C). These results indicate that TLR4 may be a major receptor involved in the production of immunomodulators by HSR. Thus, the effect of HSR on the expression of TLR4 was investigated.

Figure 2. Effect of TLR2/TLR4 on HSR-mediated production of immunomodulators in RAW264.7 cells. RAW264.7 cells were pretreated with C29 (TLR2 inhibitor, 10 μM) or TAK-242 (TLR4 inhibitor, 10 μM) for 2 h and co-treated with HSR (50 μg/ml) for 24 h. (A) NO level (C29), (B) NO level (TAK-242), and (C) mRNA level (TAK-242) were measured by Griess assay (A and B) and RT-PCR (C), respectively. *P < 0.05 compared to that for cells without HSR treatment.

3.3. MAPKs signalling contributes to the production of immunomodulators by HSR in RAW264.7 cells

The MAPKs, comprised of extracellular signal-regulated kinase (ERK), p38, and c-Jun NH2-terminal kinase (JNK), play prominent roles in the innate and adaptive immune systems. Active MAPKs phosphorylates transcription factors and other targets to control gene transcription and immune response (Kim & Choi, 2010; Huang et al., 2009). To determine the mechanism by which HSR activates cytokines and up-regulates NO production, we checked MAPKs signalling pathway. Thus, the effects of MAPKs signalling inhibition on HSR-induced NO production were investigated in RAW264.7 cells. As shown in Figures 3(A and B), the inhibition of ERK1/2 by PD98059 or p38 by SB203580 did not affect NO production by HSR. However, JNK inhibition by SP600125 dramatically inhibited NO production by HSR (Figure 3C). Because NO production by HSR was dramatically suppressed by the inhibition of JNK, the mRNA expression of the immunomodulators such as iNOS, COX-2, IL-1β and TNF-α was investigated in RAW264.7 cells according to JNK inhibition (Figure 3D). These results suggest that HSR may be involved in the production of immunomodulators through JNK activation.

Figure 3. Effect of MAPK signalling pathway on HSR-mediated production of immunomodulators in RAW264.7 cells. RAW264.7 cells were pretreated with (A) PD98059 (ERK1/2 inhibitor, 20 μM), (B) SB203580 (p38 inhibitor, 20 μM) or (C) SP600125 (JNK inhibitor, 20 μM) for 2 h and then co-treated with HSR (50 μg/ml) for 24 h. NO level was measured by the Griess assay. (D) RAW264.7 cells were pretreated with SP600125 (JNK inhibitor, 20 μM) for 2 h and then co-treated with HSR (50 μg/ml) for 24 h. mRNA level was measured by the RT-PCR. *P < 0.05 compared to that for cells without HSR treatment.

3.4. NF-κB signalling contributes to the production of immunomodulators by HSR in RAW264.7 cells

The NF-κB signalling pathway is a protein family that is involved in inflammatory response regulation, immune modulation, apoptosis, cell proliferation and epithelial differentiation, which forms the central axis of the intracellular signalling system (Chae, 2005). The NF-κB signalling pathway is one of the best understood immune-related pathways. NF-κB signalling is activated by numerous discrete stimuli and is not only master regulator of the inflammatory response to pathogens and cancerous cells, but also a key regulator of autoimmune diseases. NF-κB signalling is crucial for a number of important immunological transcriptional programmes, including inflammatory responses to microorganisms and viruses by innate immune cells, development and activation of adaptive immune cells, and the development of secondary lymphoid organs (Dorrington & Fraser, 2019). NO production by HSR was slightly attenuated in RAW264.7 cells pretreated with BAY 11–7082 (NF-κB inhibitor) (Figure 4A). As shown in Figure 4(B), inhibition of NF-κB blocked the mRNA expression of iNOS, COX-2, IL-1β and TNF-α by HSR. These results suggest that HSR may be involved in the production of immunomodulators through NF-κB activation.

Figure 4. Effect of NF-κB signalling pathway on HSR-mediated production of immunomodulators in RAW264.7 cells. (A and B) RAW264.7 cells were pretreated with BAY 11–7082 (NF-κB inhibitor, 20 μM) for 2 h and then co-treated with HSR (50 μg/ml) for 24 h. (A) NO level was measured by the Griess assay and (B) RT-PCR, respectively. *P < 0.05 compared to that for cells without HSR treatment.

3.5. PI3K signalling contributes to the production of immunomodulators by HSR in RAW264.7 cells

The members of the phosphoinositide-3 kinase (PI3K) family control several cellular responses including cell growth, proliferation, differentiation, motility, survival and the trafficking of intracellular organelles in many different types of cells. The PI3K signalling pathway regulates the activities of a wide range of downstream molecular effectors, which in turn synergizes to mediate multiple cell behaviours and characteristics under both normal and pathological conditions. The PI3K signalling pathway regulates several cell processes underlying immune response to pathogens or malignant cells. In particular PI3K has important functions in the immune system (Dituri et al., 2011; Koyasu, 2003). To determine the mechanism by which HSR activates cytokines and up-regulates NO production, we checked PI3K signalling pathway. The NO production by HSR was slightly attenuated in RAW264.7 cells pretreated with LY294002 (PI3K inhibitor) (Figure 5A). As shown in Figure 5(B), inhibition of PI3K blocked the mRNA expression of iNOS, COX-2, IL-1β and TNF-α by HSR. These results suggest that HSR may be involved in the production of immunomodulators through PI3K activation.

Figure 5. Effect of PI3K signalling pathway on HSR-mediated production of immunomodulators in RAW264.7 cells. (A and B) RAW264.7 cells were pretreated with LY294002 (PI3K inhibitor, 20 μM) for 2 h and then co-treated with HSR (50 μg/ml) for 24 h. (A) NO level was measured by the Griess assay and (B) RT-PCR, respectively. *P < 0.05 compared to that for cells without HSR treatment.

4. Conclusion

In this study, we investigated whether MAPKs, NF-κB and PI3K signalling result in the production of immunomodulators such as iNOS, COX-2, IL-1β and TNF-α by HSR in RAW264.7 macrophages. In view of the above results, the inhibitions of JNK by SP600125, NF-κB signalling by BAY 11–7082 reduced the production of IL-1β by HSR when compared to cells treated with HSR alone. PI3K signalling by LY294002 reduced the production of iNOS and COX-2 by HSR when compared to cells treated with HSR alone. However, inhibitions of ERK1/2 by PD98059 and p38 by SB203580, there was no change in the production of NO by HSR. Through these results, JNK, NF-κB and PI3K are thought to be an important signalling involved in the production of immunomodulators by HSR in macrophages. In conclusion, we investigated the immune-enhancing activity of HSR through macrophage activation (Figure 6). The HSR induces NO production through stimulation of TLR4, which activates MAPKs, NF-κB and PI3K signalling pathways to promote the production of immunomodulators.

Figure 6. Scheme of the pathways of HSR-mediated activation of RAW264.7 macrophages. HSR increases the production of immunomodulators through the activation of MAPKs, NF-κB and PI3K pathways via the stimulation of TLR4 in RAW264.7 macrophages.

Acknowledgements

This study was supported by the National Institute of Forest Science, Korea (project number FG0403-2018-01-2022).

Disclosure statement

No potential conflict of interest was reported by the authors.