Elsevier

Phytomedicine

Volume 76, September 2020, 153237
Phytomedicine

Anthocyanins isolated from Hibiscus syriacus L. attenuate lipopolysaccharide-induced inflammation and endotoxic shock by inhibiting the TLR4/MD2-mediated NF-κB signaling pathway

https://doi.org/10.1016/j.phymed.2020.153237Get rights and content

Highlights

  • PS decreases the expression of iNOS and COX-2 concomitant with the production of NO and PGE2.

  • PS attenuates LPS-induced TNF-α, IL-6, and IL-12 expression.

  • PS inhibits the MyD88-IRAK4-NF-κB signal cascade.

  • PS hindered the binding between LPS and TLR4/MD2 complex.

  • PS decreases LPS-induced mortality and abnormality in zebrafish larvae.

Abstract

Background

Hibiscus syriacus L. has been used as a medicinal plant in many Asian countries. However, anti-inflammatory activity of H. syriacus L. remains unknown.

Purpose

This study was aimed to investigating the anti-inflammatory effect of anthocyanin fractions from the H. syriacus L. variety Pulsae (PS) on the lipopolysaccharide (LPS)-induced inflammation and endotoxic shock.

Study design and methods

MTT assay and flow cytometry analysis were performed to determine cytotoxicity of PS. RT-PCR, western blotting, and ELISA were conducted to evaluate the expression of proinflammatory mediators and cytokines. Molecular docking study predicted the binding scores and sites of PS to TLR4/MD2 complex. Immunohistochemical assay was conducted to evaluate the binding capability of PS to TLR4/MD2 and nuclear translocation of NF-κB p65. A zebrafish endotoxic shock model was used to evaluate anti-inflammatory activity of PS in vivo.

Results

PS suppressed LPS-induced nitric oxide and prostaglandin E2 secretion concomitant with the downregulation of inducible nitric oxide synthase and cyclooxygenase-2 expression. Furthermore, PS inhibited the production of proinflammatory cytokines such as TNF-α, IL-6, and IL-12 in LPS-stimulated RAW 264.7 macrophages. Additionally, molecular docking data showed that PS mostly fit into the hydrophobic pocket of MD2 and bound to TLR4. In particular, apigenin-7-O-glucoside powerfully bound to MD2 and TLR4 via hydrogen bonding. Additionally, immunohistochemistry assay revealed that PS inhibited LPS-induced TLR4 dimerization or expression on the cell surface, which consequently decreased MyD88 recruitment and IRAK4 phosphorylation, resulting in the inhibition of NF-κB activity. PS also attenuated LPS-mediated mortality and abnormality in zebrafish larvae and diminished the recruitment of neutrophils and macrophages at the inflammatory site accompanied by the low levels of proinflammatory mediators and cytokines.

Conclusion

PS might be a novel immunomodulator for the effective treatment of LPS-mediated inflammatory diseases.

Introduction

Inflammation is a highly regulated self-limiting process to identify and destroy invading pathogens and restore normal tissue structure and function (Grivennikov et al., 2010). However, an excessive inflammatory response has been recognized as the principle cause of acute and chronic inflammation, including vascular disease, rheumatoid arthritis, inflammatory bowel disease, and cancer (Horiguchi et al., 2018). Macrophages are the differentiated tissue cells originating from blood monocytes and play a role in the initiation and propagation of inflammatory responses by releasing proinflammatory mediators such as nitric oxide (NO) and prostaglandin E2 (PGE2) as well as cytokines such as tumor necrosis factor‑α (TNF‑α), interleukin‑6 (IL‑6), and IL‑12 (Chung et al., 2009). Therefore, the inhibition of inflammatory mediators and cytokines has been highlighted as a promising strategy for anti-inflammatory drugs. In this regard, plant extracts and polyphenolic compounds have been playing an advantageous role in the prevention of inflammatory diseases (Yahfoufi et al., 2018).

Lipopolysaccharide (LPS) is a major component of the outer membrane of gram‑negative bacteria and strongly triggers macrophages to produce proinflammatory mediators and cytokines (Peri et al., 2015). Toll-like receptor 4 (TLR4) is a major LPS receptor on the surface of macrophages, dendritic cells, and monocytes, and mediates innate immunity (Park and Lee, 2013). Five lipid chains of LPS directly bind to the hydrophobic pocket of myeloid differentiation protein 2 (MD2), and a remaining lipid chain is exposed to MD2 surface, which is associated with TLR4 (Billod et al., 2016). Once bound to LPS, the homodimer TLR4 forms a complex with MD2 on the cell surface, which recruits central adaptor proteins such as myeloid differentiation factor 88 (MyD88) linking to interleukin-1 receptor-associated kinase-4 (IRAK-4), resulting in the induction of inflammatory response by activating the nuclear factor‑κB (NF‑κB) signaling pathway (Kim et al., 2007). Recently, lipid A derivatives with four lipid chains such as lipid IVa and eritoran have been shown to target the hydrophobic pocket of MD2, which destabilize TLR4 dimerization as TLR4/MD2 antagonists (Matsushima et al., 2015). Therefore, several MD2 antagonists and TLR4 signaling inhibitors are used in preclinical and clinical trials to treat inflammatory diseases such as sepsis, diabetes, rheumatoid arthritis, and atherosclerosis (Hennessy et al., 2010; Xu et al., 2018).

The root bark of Hibiscus syriacus L. has been used as an alternative medicine in Asia for the treatment of tinea, eczema, scabies, and dysentery (Akdis et al., 2016). Previous studies have shown that the root bark extracts of H. syriacus L. provided anti-cancer (Shi et al., 2014), neutrophil elastase inhibitory (Ryoo et al., 2010), anti-oxidant (Lee et al., 1999), and anti-depressant activities (Kim et al., 2018). Moreover, the flower extracts of H. syriacus L. showed potent anti-oxidant (Jin et al., 2019; Molagoda et al., 2020) and wound-healing activity on the injury of the skin (Yoon et al., 2017). Recently, we also reported that the anthocyanin extract from the petals of H. syriacus L. variety Pulsae (PS) potently inhibited melanogenesis in B16F10 melanoma cells and zebrafish larvae (Karunarathne et al., 2019). Nevertheless, whether PS modulates LPS-induced inflammation and endotoxic shock is not yet known. Therefore, to our knowledge, we evaluated the anti-inflammatory activity of PS in LPS-stimulated inflammation and endotoxic shock.

In this study, we found that PS significantly downregulated LPS-induced proinflammatory mediators such as NO and PGE2, as well as cytokines such as TNF-α, IL-6, and IL-12 by suppressing the binding between TLR4/MD2 complex and LPS, causing the inhibition of NF-κB signaling pathway. In addition, PS diminished LPS-mediated mortality and endotoxic shock in zebrafish larvae.

Section snippets

Plant material and PS preparation

H. syriacus L. was cultivated in the Hibiscus clonal archive of the Korea Forest Research Institute, Suwon, Republic of Korea (N 37° 15′ 5.56″, E 126° 57′ 16.11″) and identified by Dr. H.-Y. Kwon (National Institute of Forest Science, Suwon, Gyeonggi-do, Republic of Korea). Voucher specimens were deposited in the Korea Forest Service (NF-H8-F). According to our previous study, PS contains 17 anthocyanins; cyanidin-3-O-galactoside (1COG), cyanidin-3-O-glucoside (2COG), orientin-7-O-glucoside

High concentration of PS decreases the viability of RAW 264.7 macrophages

To evaluate the effect of PS on the viability of RAW 264.7 macrophages, we treated the cells with the indicated concentrations of PS (0-1000 µg/ml) for 24 h. No significant changes in morphology were noted with all concentrations of PS tested (Fig. 1A). However, PS only at 1000 µg/ml showed a slight reduction (86.0 ± 3.4%) of cell viability compared to that of the untreated group (Fig. 1B). Flow cytometry analysis was also performed to confirm whether PS influenced cell viability and total cell

Discussion

Anthocyanins are more attractive than other small compounds because of their powerful anti-diabetic, anti-cancer, anti-inflammatory, anti-obesity, anti-microbial, and anti-oxidant activities (Khoo et al., 2017; Ullah et al., 2019). Previously, Riaz et al. revealed that Hibiscus sabdariffa has been used for nutritional food and therapeutic use because of the presence of affluent bioactive constituents such as flavonoids (Riaz and Chopra, 2018). Similarly, flavonoids from Hibiscus deflersii and

Conflict of interest

The authors declare that they have no conflicts of interest concerning this article.

Acknowledgments

This research was supported by Basic Science Research Program (2018R1D1A1B07045460) and RIBS of Jeju National University (2019R1A6A1A10072987) through the National Research Foundation of Korea (NRF) funded by the Ministry of Education.

References (49)

  • G. Riaz et al.

    A review on phytochemistry and therapeutic uses of Hibiscus sabdariffa L

    Biomed. Pharmacother.

    (2018)
  • L.S. Shi et al.

    Cytotoxic effect of triterpenoids from the root bark of Hibiscus syriacus

    Fitoterapia

    (2014)
  • N.A. Siddiqui et al.

    Concurrent analysis of bioactive triterpenes oleanolic acid and beta-amyrin in antioxidant active fractions of Hibiscus calyphyllus, Hibiscus deflersii and Hibiscus micranthus grown in Saudi Arabia by applying validated HPTLC method

    Saudi Pharm. J.

    (2018)
  • F. Spiller et al.

    Targeting nitric oxide as a key modulator of sepsis, arthritis and pain

    Nitric Oxide

    (2019)
  • T. Tanaka et al.

    Chapter 27 - Anti-interleukin-6 receptor antibody therapy against autoimmune inflammatory diseases

  • A. Thompson et al.

    Emerging IL-12 family cytokines in the fight against fungal infections

    Cytokine

    (2018)
  • L. Wang et al.

    ΔNp63a modulates phosphorylation of p38 MAP kinase in regulation of cell cycle progression and cell growth

    Biochem. Biophys. Res. Commun.

    (2019)
  • Y. Xu et al.

    Discovery of novel small molecule TLR4 inhibitors as potent anti-inflammatory agents

    Eur. J. Med. Chem.

    (2018)
  • J.M. Billod et al.

    Computational approaches to toll-like receptor 4 modulation

    Molecules

    (2016)
  • J.M. Cavaillon et al.

    Monocytes/macrophages and sepsis

    Crit. Care Med.

    (2005)
  • M.K. Evans et al.

    XIAP regulation by MNK links MAPK and NFκB signaling to determine an aggressive breast cancer phenotype

    Cancer Res.

    (2018)
  • S. Gargiulo et al.

    Relation between TLR4/NF-kappaB signaling pathway activation by 27-hydroxycholesterol and 4-hydroxynonenal, and atherosclerotic plaque instability

    Aging Cell

    (2015)
  • E.A. Harvie et al.

    Neutrophils in host defense: new insights from zebrafish

    J. Leuk. Biol.

    (2015)
  • E.J. Hennessy et al.

    Targeting toll-like receptors: emerging therapeutics?

    Nat. Rev. Drug Discov.

    (2010)
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