title: Shikonin, a constituent of Lithospermum erythrorhizon exhibits anti-allergic effects by suppressing orphan nuclear receptor Nr4a family gene expression as a new prototype of calcineurin inhibitors in mast cells.
abstract: Over the last few decades, food allergy (FA) has become a common disease in infants in advanced countries. However, anti-allergic medicines available in the market have no effect on FA, and consequently effective drug therapies for FA are not yet available. We have already demonstrated that mucosal mast cells play an essential role in the development of FA in a murine model. Thus, we screened many constituents from medicinal herbs for the ability to inhibit rat basophilic leukemia-2H3 mast-like cell degranulation, and found that shikonin, a naphthoquinone dye from Lithospermum erythrorhizon, exhibited the most potent inhibitory effect among them. Furthermore, shikonin extremely inhibited the IgE/antigen-induced and calcium ionophore-induced upregulation of tumor necrosis factor (TNF)-α mRNA expression in mucosal-type bone marrow-derived mast cells (mBMMCs). Global gene expression analysis confirmed by real-time PCR revealed that shikonin drastically inhibited the IgE/antigen-induced and calcium ionophore-induced upregulation of mRNA expression of the nuclear orphan receptor 4a family (Nr4a1, Nr4a2 and Nr4a3) in mBMMCs, and knockdown of Nr4a1 or Nr4a2 suppressed the IgE/antigen-induced upregulation of TNF-α mRNA expression. Computational docking simulation of a small molecule for a target protein is a useful technique to elucidate the molecular mechanisms underlying the effects of drugs. Therefore, the simulation revealed that the predicted binding sites of shikonin to immunophilins (cyclophilin A and FK506 binding protein (FKBP) 12) were almost the same as the binding sites of immunosuppressants (cyclosporin A and FK506) to immunophilins. Indeed, shikonin inhibited the calcineurin activity to a similar extent as cyclosporin A that markedly suppressed the IgE/antigen-enhanced mRNA expression of TNF-α and the Nr4a family in mBMMCs. These findings suggest that shikonin suppresses mucosal mast cell activation by reducing Nr4a family gene expression through the inhibition of calcineurin activity. Therefore, shikonin has therapeutic potential for the treatment of allergic diseases as a new calcineurin inhibitor.
authors.affiliation: Division of Gastrointestinal Pathophysiology, Institute of Natural Medicine, University of Toyama, 2630 Sugitani, Toyama 930-0194, Japan.
authors.forename: Xiaoyu
authors.initials: X
authors.lastname: Wang
authors.affiliation: Division of Gastrointestinal Pathophysiology, Institute of Natural Medicine, University of Toyama, 2630 Sugitani, Toyama 930-0194, Japan.
authors.forename: Shusaku
authors.initials: S
authors.lastname: Hayashi
authors.affiliation: Research Center for Ethnomedicine, Institute of Natural Medicine, University of Toyama, 2630 Sugitani, Toyama 930-0194, Japan.
authors.forename: Masahito
authors.initials: M
authors.lastname: Umezaki
authors.affiliation: Division of Gastrointestinal Pathophysiology, Institute of Natural Medicine, University of Toyama, 2630 Sugitani, Toyama 930-0194, Japan.
authors.forename: Takeshi
authors.initials: T
authors.lastname: Yamamoto
authors.affiliation: Division of Gastrointestinal Pathophysiology, Institute of Natural Medicine, University of Toyama, 2630 Sugitani, Toyama 930-0194, Japan.
authors.forename: Natsuko
authors.initials: N
authors.lastname: Kageyama-Yahara
authors.affiliation: Department of Radiological Sciences, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, 2630 Sugitani, Toyama 930-0194, Japan.
authors.forename: Takashi
authors.initials: T
authors.lastname: Kondo
authors.affiliation: Division of Gastrointestinal Pathophysiology, Institute of Natural Medicine, University of Toyama, 2630 Sugitani, Toyama 930-0194, Japan. Electronic address: makotok@inm.u-toyama.ac.jp.
authors.forename: Makoto
authors.initials: M
authors.lastname: Kadowaki
mesh_terms: D000818:Animals; D018926:Anti-Allergic Agents / Q000737:chemistry / Q000494:pharmacology*; D065095:Calcineurin Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D015550:Cell Degranulation / Q000187:drug effects; D016572:Cyclosporine / Q000494:pharmacology; D015536:Down-Regulation; D055785:Gene Knockdown Techniques; D029684:Lithospermum / Q000737:chemistry*; D008297:Male; D008407:Mast Cells / Q000187:drug effects* / Q000502:physiology; D051379:Mice; D008807:Mice, Inbred BALB C; D062105:Molecular Docking Simulation; D009285:Naphthoquinones / Q000737:chemistry / Q000494:pharmacology*; D057105:Nuclear Receptor Subfamily 4, Group A, Member 1 / Q000235:genetics / Q000378:metabolism*; D057126:Nuclear Receptor Subfamily 4, Group A, Member 2 / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000037:antagonists & inhibitors / Q000235:genetics; D051381:Rats; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics
chemical_list: D018926:Anti-Allergic Agents; D065095:Calcineurin Inhibitors; D009285:Naphthoquinones; C539818:Nr4a1 protein, mouse; C539883:Nr4a2 protein, mouse; D057105:Nuclear Receptor Subfamily 4, Group A, Member 1; D057126:Nuclear Receptor Subfamily 4, Group A, Member 2; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha; C016101:shikonin; D016572:Cyclosporine
keywords: Calcineurin; Computational docking simulation; Mucosal mast cell; Nr4a family; Shikonin; TNF-α