title: Hispolon inhibition of inflammatory apoptosis through reduction of iNOS/NO production via HO-1 induction in macrophages.
abstract: BACKGROUND
  Phellinus linteus (Berkeley & Curtis), a well-known medical fungus, has long been used as a traditional medicine in Oriental countries to treat various diseases, and hispolon (HIS) is one of its bioactive components. HIS is known to possess potent antineoplastic and antiviral properties; however, its effect on inflammatory apoptosis is still undefined.
  
  
  METHODS
  RAW264.7 macrophages were incubated with HIS for 30 min followed by LPS, LTA, or PGN stimulation for 12h. The expression of indicated proteins AP-1 and NF-κB transcriptional activities was examined by Western blotting using specific antibodies. Levels of NO and ROS were examined by Griess reaction, and DCHF-DA staining via flow cytometric analysis, respectively. AP-1 and NF-κB transcriptional activities were detected by luciferase reporter assay. Knockdown of HO-1 protein expression was performed by transfection of macrophages with HO-1 siRNA. Pharmacological inhibitors including ROS scavenger NAC, JNK inhibitor SP600125, NF-κB inhibitor BAY117082 were applied for mechanism study.
  
  
  RESULTS
  HIS showed concentration-dependent inhibition of LPS, LTA, and PGN-induced iNOS protein expressions and NO production by RAW264.7 macrophages. Accordingly, HIS protected RAW264.7 cells from LPS-, LTA-, and PGN-induced apoptosis. Increased HO-1 by HIS was detected at both protein and mRNA levels along with an increase in intracellular peroxide, and this was inhibited by the translational inhibitor, cycloheximide (CHX), the transcriptional inhibitor, actinomycin D (Act D), and the reactive oxygen species scavenger, N-acetylcysteine (NAC). A mechanistic study indicated that inhibition of c-Jun N-terminal kinase (JNK) protein phosphorylation, and activator protein (AP)-1 and nuclear factor (NF)-κB activation were involved in the anti-inflammatory actions of HIS in macrophages. A structure-activity relationship analysis showed that HIS expressed the most potent effect of inhibiting iNOS and apoptosis elicited by LPS, LTA, and PGN with a significant increase in HO-1 protein in macrophages.
  
  
  CONCLUSIONS
  Evidence supporting HIS prevention of inflammatory apoptosis via blocking NO production and inducing HO-1 protein expression in macrophages is provided, and the hydroxyl at position C3 is a critical substitution for the anti-inflammatory actions of HIS.
authors.affiliation: Department of Physiology, School of Medicine, College of Medicine, Taipei Medical University, Taipei, Taiwan; Research Center for Biomedical Devices and Prototyping Production, Taipei Medical University, Taipei 11031, Taiwan.
authors.forename: Liang-Yo
authors.initials: LY
authors.lastname: Yang
authors.affiliation: Graduate Institute of Medical Sciences, College of Medicine, Taipei Medical University, Taipei, Taiwan.
authors.forename: Shing-Chuan
authors.initials: SC
authors.lastname: Shen
authors.affiliation: Department of Biochemistry and Molecular Cell Biology, School of Medicine, College of Medicine, Taipei Medical University, Taipei, Taiwan.
authors.forename: Kur-Ta
authors.initials: KT
authors.lastname: Cheng
authors.affiliation: Natsol Laboratories Pvt. Ltd., J.N. Pharma City, Visakhapatnam 531019, India.
authors.forename: Gottumukkala V
authors.initials: GV
authors.lastname: Subbaraju
authors.affiliation: Division of Nephrology, Chi Mei Medical Center, Tainan, Taiwan; Department of Food Nutrition, Chung Hwa University of Medical Technology, Tainan, Taiwan.
authors.forename: Chih-Chiang
authors.initials: CC
authors.lastname: Chien
authors.affiliation: Graduate Institute of Medical Sciences, College of Medicine, Taipei Medical University, Taipei, Taiwan; Cancer Research Center and Orthopedics Research Center, Taipei Medical University Hospital, Taipei, Taiwan. Electronic address: yc3270@tmu.edu.tw.
authors.forename: Yen-Chou
authors.initials: YC
authors.lastname: Chen
mesh_terms: D000873:Anthracenes / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects*; D002396:Catechols / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D051547:Heme Oxygenase-1; D007249:Inflammation / Q000378:metabolism*; D008070:Lipopolysaccharides / Q000037:antagonists & inhibitors; D008264:Macrophages; D016328:NF-kappa B / Q000096:biosynthesis; D009569:Nitric Oxide / Q000378:metabolism; D052247:Nitric Oxide Synthase Type II / Q000037:antagonists & inhibitors; D009570:Nitriles / Q000494:pharmacology; D034741:RNA, Small Interfering; D017382:Reactive Oxygen Species; D013329:Structure-Activity Relationship; D013450:Sulfones / Q000494:pharmacology; D013682:Teichoic Acids / Q000037:antagonists & inhibitors; D018808:Transcription Factor AP-1 / Q000096:biosynthesis
chemical_list: C434003:3-(4-methylphenylsulfonyl)-2-propenenitrile; D000873:Anthracenes; D002396:Catechols; D008070:Lipopolysaccharides; D016328:NF-kappa B; D009570:Nitriles; D034741:RNA, Small Interfering; D017382:Reactive Oxygen Species; D013450:Sulfones; D013682:Teichoic Acids; D018808:Transcription Factor AP-1; C103442:hispolon; C432165:pyrazolanthrone; D009569:Nitric Oxide; C009900:lipoteichoic acid; D052247:Nitric Oxide Synthase Type II; D051547:Heme Oxygenase-1
keywords: Apoptosis; Heme Oxygenase-1; Hispolon; Inducible Nitric Oxide Synthase