1,7-Dihydroxy-3,4-Dimethoxyxanthone Inhibits Lipopolysaccharide-Induced Inflammation in RAW264.7 Macrophages by Suppress
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ORIGINAL ARTICLE
1,7-Dihydroxy-3,4-Dimethoxyxanthone Inhibits Lipopolysaccharide-Induced Inflammation in RAW264.7 Macrophages by Suppressing TLR4/NF-κB Signaling Cascades Meng-qing Tao,1 Cong-lan Ji,2 Yi-jin Wu,3 Ji-yang Dong,4 Yan Li,1 Opeyemi Joshua Olatunji,5 and Jian Zuo 1,6,7,8
Securidaca inappendiculata Hassk. is a traditional Chinese anti-rheumatic herbal medicine native to southern China. In this study, we identified a possible TLR4 inhibitor from this plant. General effects of its xanthone-rich fraction (XRF) on inflammation in vitro were investigated by immunoblotting experiments performed on lipopolysaccharides (LPS)-treated RAW264.7 cells, and the possible ligand of TLR4 within was screened out by analyzing chemical composition differences of the XRF containing cell culture medium under different inflammatory circumstances. The interaction between ligand and TLR4 was validated by cellular thermal shift assay (CETSA) and molecular docking simulation, and TLR4/ NF-κB pathway status was investigated by immunoprecipitation, ELISA, immunofluorescence, dual-luciferase reporter, and immunoblotting experiments. Treatment with XRF resulted in significant decrease in p-p65 and p-JNK, and the signal accounting for 1,7dihydroxy-3,4-dimethoxyxanthone (XAN) at 12.5 min with mass of 289.29 was greatly decreased in XRF containing medium after LPS stimulus because of enhanced interaction with increased TLR4. CETSA and molecular docking simulation demonstrated that XAN could bind to TLR4 directly on a smooth region adjacent to its contact interface with MD-2. XAN treatment inhibited the dimerization of TLR4 and transcriptional activity of NF-κB in HEK293T cells and decreased p65 accumulation in nucleus and pro-inflammatory cytokines
Abstract—
Meng-qing Tao and Cong-lan Ji contributed equally to this work. 1
Yijishan Hospital of Wannan Medical College, Wuhu, 241000, China School of Pharmacy, Anhui College of Traditional Chinese Medicine, Wuhu, 241000, China 3 The Second Affiliated Hospital of Wannan Medical College, Wuhu, 241000, Anhui, China 4 Department of Electronic Science, Xiamen University, Xiamen, 361005, China 5 Faculty of Traditional Thai Medicine, Prince of Songkla University, Hat Yai, 90110, Thailand 6 Key Laboratory of Non-coding RNA Transformation Research of Anhui Higher Education Institution, Wannan Medical College, Wuhu, 241000, China 2
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Department of Traditional Chinese Medicine, Yijishan Hospital of Wannan Medical College, No 2nd West Zheshan Road, Wuhu, 241000, China 8 To whom correspondence should be addressed at Department of Traditional Chinese Medicine, Yijishan Hospital of Wannan Medical College, No 2nd West Zheshan Road, Wuhu, 241000, China. E-mail: [email protected] Abbreviations XRF, xanthone-rich fraction; LPS, lipopolysaccharides; CETSA, cellular thermal shift assay; XAN, 1,7-dihydroxy-3,4dimethoxyxanthone; RA, rheumatoid arthritis; PRRs, pattern recognition receptors; FBS, fetal bovine serum; PBS, phosphate-buffered saline; BSA, bovine serum albumin; IP, immunoprecipitation; FLS, fi
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