Anthocyanins isolated from Hibiscus syriacus L. attenuate lipopolysaccharide-induced inflammation and endotoxic shock by inhibiting the TLR4/MD2-mediated NF-κB signaling pathway
Graphic abstract
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.
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2022, International Journal of Biological MacromoleculesAnthocyanin-enriched polyphenols from Hibiscus syriacus L. (Malvaceae) exert anti-osteoporosis effects by inhibiting GSK-3β and subsequently activating β-catenin
2021, PhytomedicineCitation Excerpt :The flower, fruit, root, and stem of H. syriacus show various pharmacological activities and have been used as an ingredient in traditional Korean medicine (Yang et al., 2020). Modern pharmacological reports have demonstrated that anthocyanin-enriched polyphenols from the petals of H. syriacus L. variety Pulsae (AHs) potently inhibited the following: melanogenesis in B16F10 melanoma cells and zebrafish larvae (Karunarathne et al., 2019); lipopolysaccharide-induced inflammation and endotoxic shock by inhibiting the Toll-like receptor 4/myeloid differentiation factor 2-mediated nuclear factor-κB signaling pathway (Karunarathne et al., 2020); oxidative stress-induced cell death by activating nuclear factor erythroid 2-related factor 2-mediated heme oxygenase-1 (Molagoda et al., 2020). However, whether AHs can promote osteoblast differentiation and osteogenesis remains unclear.