Hispolon inhibition of inflammatory apoptosis through reduction of iNOS/NO production via HO-1 induction in macrophages Liang-Yo Yang a,b,1 , Shing-Chuan Shen c,1 , Kur-Ta Cheng d , Gottumukkala V. Subbaraju e , Chih-Chiang Chien f,g , Yen-Chou Chen c,h,n a Department of Physiology, School of Medicine, College of Medicine, Taipei Medical University, Taipei, Taiwan b Research Center for Biomedical Devices and Prototyping Production, Taipei Medical University, Taipei 11031, Taiwan c Graduate Institute of Medical Sciences, College of Medicine, Taipei Medical University, Taipei, Taiwan d Department of Biochemistry and Molecular Cell Biology, School of Medicine, College of Medicine, Taipei Medical University, Taipei, Taiwan e Natsol Laboratories Pvt. Ltd., J.N. Pharma City, Visakhapatnam 531019, India f Division of Nephrology, Chi Mei Medical Center, Tainan, Taiwan g Department of Food Nutrition, Chung Hwa University of Medical Technology, Tainan, Taiwan h Cancer Research Center and Orthopedics Research Center, Taipei Medical University Hospital, Taipei, Taiwan article info Article history: Received 24 April 2014 Received in revised form 25 July 2014 Accepted 25 July 2014 Available online 14 August 2014 Keywords: Hispolon Heme Oxygenase-1 Inducible Nitric Oxide Synthase Apoptosis abstract Ethnopharmacological relevance: 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. Materials and methods: RAW264.7 macrophages were incubated with HIS for 30 min followed by LPS, LTA, or PGN stimulation for 12 h. 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 expres- sions 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. & 2014 Elsevier Ireland Ltd. All rights reserved. Contents lists available at ScienceDirect journal homepage: www.elsevier.com/locate/jep Journal of Ethnopharmacology http://dx.doi.org/10.1016/j.jep.2014.07.054 0378-8741/& 2014 Elsevier Ireland Ltd. All rights reserved. Abbreviations: HIS, hispolon; HIS-ME, hispolon Methyl ether; M-HIS, methoxyhispolon; D-HIS, dehydroxyhispolon; H-HIS, hydroxyhispolon; LPS, lipopolysaccharide; LTA, lipoteichoic acid; PGN, peptidoglycan; NO, nitric oxide; Act D, actinomycin D; AP-1, activator protein-1; COX, cyclooxygenase; BCIP, 5-bromo-4-chloro-3-indolyl phosphate; CH, cyclohexenone; CHX, cycloheximide; CO, carbon monoxide; HO-1, heme oxygenase 1; iNOS, inducible nitric oxide synthase; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyl tetrazolium bromide; NAC, N-acetylcysteine; NBT, nitroblue tetrazolium; SP, SP600125; ROS, reactive oxygen species; siRNA, small interfering RNA; SnPP, tin protoporphyrin; ERKs, extracellular signal-regulated kinases; JNKs, c-Jun N-terminal kinases (JNKs); SAR, structure-activity relationship; PARP, poly ADP ribose polymerase; α–TUB, α-tubulin; NAME, N-nitro-L-arginine methyl ester n Corresponding author at: Graduate Institute of Medical Sciences, Taipei Medical University, Taipei, Taiwan. Tel.: þ886 2 27361661x3421; fax: þ886 2 2378620. E-mail address: yc3270@tmu.edu.tw (Y.-C. Chen). 1 Dr. Yang and Dr. Shen contributed equally to the present work. Journal of Ethnopharmacology 156 (2014) 61–72