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MIRKA LAAVOLA Immunomodulatory Properties of Wood Biochemicals (௺HFWVRQ,QÀDPPDWRU\*HQH([SUHVVLRQ DQG,QÀDPPDWRU\5HVSRQVHV,Q9LYR Tampere University Dissertations 94 7DPSHUH8QLYHUVLW\'LVVHUWDWLRQV 0,5.$/$$92/$ ,PPXQRPRGXODWRU\3URSHUWLHV RI:RRG%LRFKHPLFDOV Effects on Inflammatory Gene Expression and Inflammatory Responses In Vivo $&$'(0,&',66(57$7,21 7REHSUHVHQWHGZLWKWKHSHUPLVVLRQRI WKH)DFXOW\&RXQFLORIWKH)DFXOW\RI0HGLFLQHDQG+HDOWK7HFKQRORJ\ RIWKH7DPSHUH8QLYHUVLW\ IRUSXEOLFGLVFXVVLRQLQWKH-DUPR9LVDNRUSLDXGLWRULXP RIWKH$UYREXLOGLQJ$UYR<OS|QNDWX7DPSHUH RQ$XJXVWDWR¶FORFN $&$'(0,&',66(57$7,21 7DPSHUH8QLYHUVLW\)DFXOW\RI0HGLFLQHDQG+HDOWK7HFKQRORJ\ 7DPSHUH8QLYHUVLW\+RVSLWDO &R[D+RVSLWDOIRU-RLQW5HSODFHPHQW )LQODQG Responsible 3URIHVVRU(HYD0RLODQHQ supervisor 7DPSHUH8QLYHUVLW\ and Custos )LQODQG Supervisor(s) 'RFHQW5LLQD1LHPLQHQ 7DPSHUH8QLYHUVLW\ )LQODQG Pre-examiner(s) 3URIHVVRU6DUL0lNHOl 'RFHQW7\WWL6DUMDOD 8QLYHUVLW\RI7XUNX 8QLYHUVLW\RI2XOX )LQODQG )LQODQG Opponent(s) 3URIHVVRU5DLPR7XRPLQHQ 8QLYHUVLW\RI+HOVLQNL )LQODQG 7KHRULJLQDOLW\RIWKLVWKHVLVKDVEHHQFKHFNHGXVLQJWKH7XUQLWLQ2ULJLQDOLW\&KHFN VHUYLFH &RS\ULJKWDXWKRU &RYHUGHVLJQ5RLKX,QF ,6%1 SULQW ,6%1 SGI ,661 SULQW ,661 SGI KWWSXUQIL851,6%1 3XQD0XVWD2\±<OLRSLVWRSDLQR 7DPSHUH Contents List of Original Communications ............................................................................................ 8 Abbreviations .............................................................................................................................. 9 Abstract ...................................................................................................................................... 11 Tiivistelmä ................................................................................................................................. 13 Introduction .............................................................................................................................. 15 Review of the literature ........................................................................................................... 17 1 Inflammation ............................................................................................................... 17 1.1 Treatment of inflammation .............................................................................. 19 2 Inflammatory mediators ............................................................................................ 25 2.1 Nitric oxide ......................................................................................................... 25 2.1.1 Inducible nitric oxide synthase (iNOS) ..................................................... 27 2.1.2 Regulation of iNOS expression .................................................................. 28 2.2 Interleukin-6 ....................................................................................................... 29 2.2.1 Regulation of IL-6 expression .................................................................... 31 2.2.2 IL-6 inhibitors ............................................................................................... 32 2.3 Monocyte chemoattractant protein-1 ............................................................. 33 3 2.4 Prostanoids ......................................................................................................... 35 2.4.1 Prostaglandin E2 ............................................................................................ 36 3 Osteoarthritis ............................................................................................................... 38 3.1 Pathogenesis of osteoarthritis .......................................................................... 38 3.2 Cartilage homeostasis ........................................................................................ 39 3.2.1 Cartilage matrix components ...................................................................... 39 3.2.2 Catabolic factors ........................................................................................... 40 3.3 Drug treatment of osteoarthritis ..................................................................... 41 3.4 Drug development for osteoarthritis ............................................................. 43 4 Natural products as part of the drug discovery ..................................................... 46 4.1 Stilbenoids .......................................................................................................... 50 4.1.1 Resveratrol ..................................................................................................... 51 4.1.1.1 Anti-inflammatory effects in animal models ................................... 52 4.1.1.2 Anti-inflammatory potential in clinical studies ............................... 57 4.1.1.3 Pharmacological mechanisms ............................................................ 61 4.1.2 Pinosylvin and Monomethylpinosylvin ..................................................... 62 4.1.2.1 Immunomodulatory, antioxidant and antinociceptive properties 62 4.1.2.2 Anti-apoptotic and chemopreventive actions ................................. 63 4.2 Lignans ................................................................................................................ 64 4 4.2.1 Hydroxymatairesinol .................................................................................... 65 4.2.2 Nortrachelogenin .......................................................................................... 66 4.2.2.1 Bioactivities in vitro ............................................................................... 67 4.2.2.2 In vivo effects ......................................................................................... 68 4.3 Terpenes .............................................................................................................. 68 4.3.1 Triterpenes ..................................................................................................... 69 Aims of the study ..................................................................................................................... 71 Materials and methods ............................................................................................................. 72 5 Materials ....................................................................................................................... 72 5.1 Chemicals ............................................................................................................ 72 5.2 Antibodies ........................................................................................................... 73 6 Methods........................................................................................................................ 74 6.1 Cell cultures ........................................................................................................ 74 6.1.1 J774 murine macrophages ........................................................................... 74 6.1.2 HEK293pGL4.32NFκB Cell Line ............................................................. 74 6.1.3 Human primary chondrocytes .................................................................... 75 6.1.4 T/C28a2pGL4.32NFκB Cell Line ............................................................. 75 6.1.5 Cell viability assays ........................................................................................ 76 6.2 Nitrite assays....................................................................................................... 76 5 6.3 Preparation of cell lysates and western blotting ........................................... 76 6.4 RNA extraction and quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) ........................................................................................................ 77 6.5 Enzyme-linked immunosorbent assay and multiplex bead array ............... 80 6.6 Luciferase activity .............................................................................................. 80 6.7 Carrageenan induced inflammation in the mouse ........................................ 80 6.8 Patients and clinical studies .............................................................................. 81 6.9 Statistics ............................................................................................................... 82 Results ........................................................................................................................................ 83 7 Pine knot extract and stilbenoids have anti-inflammatory and chondroprotective properties .............................................................................................................................. 83 7.1 Pine knot extract and stilbenoids inhibit NO production and iNOS, MCP- 1 and IL-6 expression in mouse macrophages ........................................................... 83 7.2 IL-6 levels in OA patients and chondroprotective effects of stilbene derivatives ......................................................................................................................... 88 7.3 Pine knot extract and stilbenoids inhibit NF-κB mediated transcription and increase HO-1 expression .............................................................................................. 93 7.4 Stilbenes inhibit acute inflammation in vivo ................................................... 96 8 Nortrachelogenin is anti-inflammatory in