Wisurumuni Arachchilage Hasitha Maduranga Karunarathne1, Kyoung Tae Lee2, Yung Hyun Choi3, Cheng-Yun Jin4, Gi-Young Kim5. 1. Department of Marine Life Sciences, Jeju National University, Jeju 63243, Republic of Korea. 2. Forest Biomaterials Research Center, National Institute of Forest Science, Jinju 52817, Republic of Korea. 3. Department of Biochemistry, College of Oriental Medicine, Dong-Eui University, Busan 47227, Republic of Korea. 4. School of Pharmaceutical Science, Institute of Drug Discovery and Development, Zhengzhou University, Henan 450001, China. 5. Department of Marine Life Sciences, Jeju National University, Jeju 63243, Republic of Korea. Electronic address: immunkim@jejunu.ac.kr.
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.
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 zebrafishendotoxic 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.
Authors: Jimena Borgo; Laura C Laurella; Florencia Martini; Cesar A N Catalán; Valeria P Sülsen Journal: Molecules Date: 2021-05-06 Impact factor: 4.411