天然物由来物質における抗炎症作用機序の解明とラットモデルを用いたその活用に関する研究

Title 天然物由来物質における抗炎症作用機序の解明とラットモ_{デルを用いたその活用に関する研究( 本文(Fulltext) )} Author(s) 米澤, 豊 Report No.(Doctoral Degree) 博士(獣医学) 甲第519号 Issue Date 2019-03-13 Type 博士論文 Version ETD URL http://hdl.handle.net/20.500.12099/77950 ※この資料の著作権は、各資料の著者・学協会・出版社等に帰属します。

ኳ↛≀⏤᮶≀㉁࡟࠾ࡅࡿᢠ⅖⑕స⏝ᶵᗎࡢゎ᫂

࡜ࣛࢵࢺࣔࢹࣝࢆ⏝࠸ࡓࡑࡢά⏝࡟㛵ࡍࡿ◊✲

 ᖺ





ᒱ㜧኱Ꮫ኱Ꮫ㝔㐃ྜ⋇་Ꮫ◊✲⛉

ᖏᗈ␆⏘኱Ꮫ









⡿⃝ ㇏



ኳ

ኳ↛≀⏤᮶≀㉁࡟࠾ࡅࡿᢠ⅖⑕స⏝ᶵ

ᗎࡢゎ᫂࡜ࣛࢵࢺࣔࢹࣝࢆ⏝࠸ࡓࡑࡢ

ά⏝࡟㛵ࡍࡿ◊✲

┠ḟ ᗎㄽ --- 1 ➨1 ❶ ࣐࢘ࢫ࣐ࢡࣟࣇ࢓࣮ࢪᵝ⣽⬊ᰴ RAW264.7 ࢆ⏝࠸ࡓ lipopolysaccharide ่ ⃭ヨ㦂࡟࠾ࡅࡿhydroxytyrosol ࡢస⏝ࡢゎᯒ --- 3 ㅖゝ --- 4 ᮦᩱཬࡧ᪉ἲ --- 6 ⤖ᯝ --- 10 ⪃ᐹ --- 12 ᑠᣓ --- 15 ᅗ⾲ --- 16 ➨2 ❶ ⣽⬊ෆࢩࢢࢼࣝᅉᏊࡢ inhibitor ࢆ⏝࠸ࡓ hydroxytyrosol ࡢస⏝ᶵᗎࡢゎᯒ --- 23 ⥴ゝ --- 24 ᮦᩱཬࡧ᪉ἲ --- 26 ⤖ᯝ --- 28 ⪃ᐹ --- 30 ᑠᣓ --- 33 ᅗ⾲ --- 34 ➨3 ❶ ࣛࢵࢺ࢝ࣛࢤࢽࣥ⫥ᾋ⭘ࣔࢹࣝࢆ⏝࠸ࡓ hydroxytyrosol ࡢᢠ⅖⑕స⏝ࡢホ ౯ --- 39 ⥴ゝ --- 40

ᮦᩱཬࡧ᪉ἲ --- 41 ⤖ᯝ --- 47 ⪃ᐹ --- 49 ᑠᣓ --- 51 ᅗ⾲ --- 52 ➨4 ❶ Hydroxytyrosol ࡢ⤒ཱྀᢞ୚࡟ࡼࡿ࢔ࢫࣆࣜࣥㄏⓎ⫶₽⒆࡬ࡢᙳ㡪 --- 59 ⥴ゝ --- 60 ᮦᩱཬࡧ᪉ἲ --- 62 ⤖ᯝ --- 65 ⪃ᐹ --- 66 ᑠᣓ --- 68 ᅗ⾲ --- 69 ⥲ᣓ --- 74 ㅰ㎡ --- 77 ᘬ⏝ᩥ⊩ --- 78

␎ྕ୍ぴ (࢔ࣝࣇ࢓࣋ࢵࢺ㡰)

AP-1 activator protein 1, ࢔ࢡࢳ࣮࣋ࢱ࣮ࢱࣥࣃࢡ㉁1

COX-2 cyclooxygenase-2, ࢩࢡࣟ࢜࢟ࢩࢤࢼ࣮ࢮ-2

ERK1/2 p44/42 mitogen-activated protein kinase, ⣽⬊እࢩࢢࢼࣝㄪ⠇࢟ࢼ࣮ࢮ

GAPDH glyceraldehyde-3-phosphate dehydrogenase, ࢢࣜࢭࣝ࢔ࣝࢹࣄࢻ-3-ࣜ

ࣥ㓟ࢹࣄࢻࣟࢤࢼ࣮ࢮ

HT hydroxytyrosol, ࣄࢻࣟ࢟ࢩࢳࣟࢰ࣮ࣝ

IL interleukin, ࢖ࣥࢱ࣮ࣟ࢖࢟ࣥ

iNOS inducible NO synthase, ㄏᑟᆺNO ྜᡂ㓝⣲

JNK c-jun N-terminal kinase, c-Jun N ᮎ➃࢟ࢼ࣮ࢮ

LDA low-dose aspirin, ప⏝㔞࢔ࢫࣆࣜࣥ

LDH lactate dehydrogenase, ங㓟⬺Ỉ⣲㓝⣲

LPS lipopolysaccharide, ࣏ࣜከ⢾

MAPK mitogen-activated protein kinase, ศ⿣ಁ㐍ᅉᏊάᛶ໬ࢱࣥࣃࢡ㉁࢟ࢼ

࣮ࢮ

MC methylcellulose, ࣓ࢳࣝࢭ࣮ࣝࣟࢫ

NF-κB nuclear factor-kappa B, ᰾ෆᅉᏊκB

NO nitric oxide, ୍㓟໬❅⣲

NSAIDs nonsteroidal anti-inflammatory drugs, 㠀ࢫࢸࣟ࢖ࢻᛶᢠ⅖⑕⸆

PG prostaglandin, ࣉࣟࢫࢱࢢࣛࣥࢪࣥ

PVDF polyvinylidene difluoride, ࣏ࣜࣇࢵ໬ࣅࢽࣜࢹࣥ

RRT-PCR real-time RT-PCR, ࣜ࢔ࣝࢱ࢖࣒RT-PCR

TBS tris-buffered saline, ࢺࣜࢫ⦆⾪⏕⌮㣗ሷỈ

TBS-T TBS with Tween 20

TLR toll-like receptor, Toll ᵝཷᐜయ

1 ᗎㄽ ⅖⑕ࡣឤᰁࡸእയ࡞࡝࡛⤌⧊ࡀᦆയࡋࡓ࡜ࡁ࡟యෆ࡛⏕ࡌࡿ⏕య㜵ᚚ཯ᛂ࡛ ࠶ࡿࠋලయⓗ࡟ࡣ㸪≀⌮ⓗ่⃭ (ᶵᲔⓗእയ㸪⇕㸪ᨺᑕ⥺㸪ග⥺㸪␗≀) ࡸ໬Ꮫ ⓗ่⃭ (ᙉ㓟㸪ᙉ࢔ࣝ࢝ࣜ)㸪⑓ཎᚤ⏕≀ࡢឤᰁ㸪⮬ᕫᢠయ࡞࡝ࡢච␿ᢠཎࡀཎᅉ ࡜࡞ࡾ㸪ᒁᡤⓗ࡞Ⓨ㉥㸪⭘⬽㸪⑊③㸪Ⓨ⇕࡜࠸ࡗࡓ4 ୺ᚩࡀᘬࡁ㉳ࡇࡉࢀࡿࠋ⅖ ⑕཯ᛂ࡟ࡼࡿ③ࡳࡣ⏕య࡟࡜ࡗ࡚ࡢ༴㝤ಙྕࢆ▱ࡽࡏࡿᚲせ୙ྍḞ࡞ࡶࡢࡢ㸯 ࡘ࡛ࡣ࠶ࡿࡀ㸪㐣๫࡟Ⓨ⌧ࡋࡓሙྜ㸪⅖⑕ࡣ៏ᛶ໬࠶ࡿ࠸ࡣ㑄ᘏࡋ㸪⏕య⤌⧊ࡢ ᦆയࡸᶵ⬟పୗ࡞࡝ேయ࡟࡜ࡗ࡚ᝏᙳ㡪ࢆ࿊ࡍࡿࡇ࡜࡜࡞ࡿࠋᮏ≧ែࢆ៏ᛶ⅖⑕ ࡜࿧ࡪ (1)ࠋ ㏆ᖺ㸪ࡀࢇࡸ⏕ά⩦័⑓࡞࡝ࡢ៏ᛶ⑌ᝈࡢ⑓ែᙧᡂ࡟㸪៏ᛶ⅖⑕ࡀ῝ࡃ㛵ࢃࡗ ࡚࠸ࡿࡇ࡜ࡀὀ┠ࡉࢀ࡚࠸ࡿ (2)ࠋ៏ᛶ⅖⑕࡛ࡣ㸪㓟໬ࢫࢺࣞࢫ࡞࡝ࡢ่⃭ࢆཷ ࡅࡓ⮚ჾࡢ⣽⬊࠿ࡽᨺฟࡉࢀࡿ⮬ᕫ⏤᮶ࡢᡂศࡀ㸪࣐ࢡࣟࣇ࢓࣮ࢪ࡟Ⓨ⌧ࡍࡿ Toll ᵝཷᐜయ (TLR) (3) ࡟ࡼࡾㄆ㆑ࡉࢀ㸪ࢧ࢖ࢺ࢝࢖ࣥ㸪ࣉࣟࢫࢱࢢࣛࣥࢪࣥ (PG) E2㸪୍㓟໬❅⣲ (NO) ࢆࡣࡌࡵ࡜ࡋࡓᵝࠎ࡞⅖⑕ᛶ࣓ࢹ࢕࢚࣮ࢱ࣮ࡀ⏘⏕ࡉ ࢀࡓ⤖ᯝ㸪ᜏᖖⓗ࡞⅖⑕཯ᛂࡀㄏᑟࡉࢀࡿ (2)ࠋࡇࡢࡼ࠺࡟㸪࣐ࢡࣟࣇ࢓࣮ࢪ࡜ ៏ᛶ⅖⑕࡜ࡢ㛵㐃ࡀὀ┠ࡉࢀ࡚࠾ࡾ㸪࣐ࢡࣟࣇ࢓࣮ࢪ࡟ࡼࡿᵝࠎ࡞࣓ࢹ࢕࢚࣮ࢱ ࣮ࡢ⏘⏕ࡢண㜵࡟ᙺ❧ࡘ᪂つ໬ྜ≀ࡢ᥈⣴ࡀ㐍ࡵࡽࢀ࡚࠸ࡿࠋ ኳ↛≀⏤᮶≀㉁࡛࠶ࡿ hydroxytyrosol (HT) ࡣ࣮࢜ࣜࣈࡢⴥࡸᐇ࡟㇏ᐩ࡟ྵࡲ

ࢀࡿపศᏊࣇ࢙ࣀ࣮ࣝ໬ྜ≀ (Fig. 1) ࡛࠶ࡾ㸪in vitro ᐇ㦂࡟࠾࠸࡚ᢠ⅖⑕ (4–7)㸪

ᢠ㓟໬ (8, 9)㸪ᢠ࢘࢖ࣝࢫ (10) ཬࡧᢠ⳦ (11) స⏝ࢆ♧ࡍࡇ࡜ࡀ᫂ࡽ࠿࡜࡞ࡗ࡚

2 (LPS) ่࡛⃭ࡋࡓ࣐࢘ࢫࡢ⭡⭍࣐ࢡࣟࣇ࢓࣮ࢪ࡟ HT ࢆస⏝ࡉࡏࡓ࡜ࡇࢁ㸪ㄏᑟ ᆺNO ྜᡂ㓝⣲ (iNOS) 㑇ఏᏊⓎ⌧ࡢపୗ㸪࢖ࣥࢱ࣮ࣟ࢖࢟ࣥ-10 (IL-10) 㑇ఏᏊ Ⓨ⌧ࡢୖ᪼㸪NO ᨺฟࡢపୗ➼㸪ᢠ⅖⑕ᛶస⏝࡟㛵ࡍࡿኚ໬ࡀ☜ㄆࡉࢀࡓࠋ୍᪉ ࡛㸪⅖⑕ᛶ཯ᛂ࡟㛵୚ࡍࡿ࡜⪃࠼ࡽࢀࡿnuclear factor-kappa B (NF-κB)㸪⭘⒆ቯṚ ᅉᏊ-α (TNF-α)㸪ࢩࢡࣟ࢜࢟ࢩࢤࢼ࣮ࢮ-2 (COX-2) ࡟ࡘ࠸࡚ࡣ HT ࡟ࡼࡿⓎ⌧ᢚ ไస⏝ࡣㄆࡵࡽࢀ࡞࠿ࡗࡓࠋࡇࢀࡽࡢ⤖ᯝ࠿ࡽ㸪HT ࡀ♧ࡋࡓᢠ⅖⑕స⏝ࡣ NF-κB㸪 TNF-α㸪COX-2 ࢆ௓ࡋࡓࢩࢢࢼࣝఏ㐩⤒㊰࡛ࡣ↓ࡃ㸪ูࡢࢩࢢࢼࣝ⤒㊰ࢆ௓ࡋ࡚ ⏕ࡌࡓྍ⬟ᛶࡀ⪃࠼ࡽࢀ㸪HT ࡢస⏝࣓࢝ࢽࢬ࣒ࢆ≉ᐃࡍࡿࡓࡵ࡟᭦࡞ࡿ᳨ウࡀ ᚲせ࡜⪃࠼ࡽࢀࡓ (12)ࠋ ࡑࡇ࡛㸪ᮏ◊✲࡛ࡣHT ࡢᢠ⅖⑕స⏝࡟㛵ࡋ࡚㸪ࡑࡢస⏝ᶵᗎࢆ᫂ࡽ࠿࡟ࡍࡿ ࡜࡜ࡶ࡟㸪ື≀ࣔࢹࣝ࡟࠾ࡅࡿᢠ⅖⑕స⏝ࢆ᫂ࡽ࠿࡟ࡍࡿࡇ࡜ࢆ┠ⓗ࡜ࡋࡓࠋ ➨1 ❶࡛ࡣ㸪࣐࢘ࢫ࣐ࢡࣟࣇ࢓࣮ࢪᵝ⣽⬊ᰴ RAW264.7 ࢆ⏝࠸࡚㸪LPS ㄏⓎᛶ ⅖⑕࡟࠾ࡅࡿHT ࡢᢠ⅖⑕స⏝ࢆホ౯ࡋࡓࠋ➨ 2 ❶࡛ࡣ㸪⣽⬊ෆࢩࢢࢼࣝᅉᏊࡢ inhibitor ࢆ⏝࠸࡚㸪HT ࡢస⏝࣓࢝ࢽࢬ࣒ࢆゎᯒࡋࡓࠋ➨ 3 ❶࡛ࡣ㸪࢝ࣛࢤࢽࣥㄏ Ⓨ⫥ᾋ⭘ࣔࢹࣝࢆ⏝࠸࡚㸪HT ࡢᢠ⅖⑕స⏝ࢆ᳨ウࡋࡓࠋຍ࠼࡚➨ 4 ❶࡛ࡣ㸪HT ࡢ⤒ཱྀᢞ୚࡟ࡼࡿ࢔ࢫࣆࣜࣥㄏⓎ⫶₽⒆࡬ࡢస⏝࡟ࡘ࠸᳨࡚ウࡋࡓࠋ

3

➨1 ❶

࣐࢘ࢫ࣐ࢡࣟࣇ࢓࣮ࢪᵝ⣽⬊ᰴRAW264.7 ࢆ⏝࠸ࡓ lipopolysaccharide ่⃭ヨ㦂࡟

4 ㅖゝ ⅖⑕ࡣ㸪⑓ཎయ౵ධ㸪໬Ꮫ≀㉁่⃭㸪᪂㝞௦ㅰ␗ᖖ࡟ࡼࡿ⤌⧊⣽⬊ࡢ␗ᖖኚ໬ ➼࡟ࡼࡾ㸪⏕యࡀෆእ࠿ࡽ᭷ᐖ࡞่⃭ࢆཷࡅࡓ࡜ࡁ࡟⏕ࡌࡿ㜵ᚚ཯ᛂ࡛࠶ࡿࠋ⅖ ⑕ࡢᶵ⬟ࡣ㸪⣽⬊യᐖࡢึᮇཎᅉࢆ᤼㝖ࡋ㸪⅖⑕ࣉࣟࢭࢫ࡟ࡼࡗ࡚ᦆയࡋࡓቯṚ ⣽⬊ཬࡧ⤌⧊ࢆ㝖ཤࡋ㸪⤌⧊ಟ᚟ࢆ㛤ጞࡍࡿࡇ࡜࡛࠶ࡿࠋࡋ࠿ࡋ㸪㐣๫࡞⅖⑕ࡣ㸪 㛵⠇⅖㸪ႍᜥ㸪ከⓎᛶ◳໬⑕ཬࡧ࢔ࢸ࣮࣒ࣟᛶື⬦◳໬⑕ࢆྵࡴ៏ᛶ⅖⑕ࢆᘬࡁ ㉳ࡇࡍ (1)ࠋ Ⓨ⇕ࡸⓎ㉥࡞࡝ࡢ⅖⑕ᛂ⟅ࡢ㛤ጞ࡟㔜せ࡞ᙺ๭ࢆᯝࡓࡍࡢࡣ㸪ࠕ⮬↛ච␿ࠖ࡜ ࿧ࡤࢀࡿ⏕య࡟ࡼࡿ␗≀ࡢ≉␗ⓗㄆ㆑ᶵᵓ࡛࠶ࡿࠋ࣐ࢡࣟࣇ࢓࣮ࢪࡣ⮬↛ච␿࡟ 㛵ࢃࡿከᶵ⬟ᛶࡢⓑ⾑⌫࡛㸪⏕య࡟౵ධࡋ࡚ࡁࡓ␗≀ࢆㄆ㆑࣭᤼㝖ࡍࡿࢭࣥࢧ࣮ ࡜ࡋ࡚ാࡃࠋ࣐ࢡࣟࣇ࢓࣮ࢪࡢάᛶ໬ࡣ㸪⅖⑕཯ᛂ࡟࠾࠸࡚㔜せ࡞ᙺ๭ࢆᯝࡓࡍࠋ ࢢ࣒ࣛ㝜ᛶ⣽⳦ࡢ⣽⬊ቨࡢᡂศ࡛࠶ࡿ࣏ࣜከ⢾ (LPS) ࡣ㸪࣐ࢡࣟࣇ࢓࣮ࢪ࡟ࡼ ࡾㄆ㆑ࡉࢀ㸪⅖⑕཯ᛂࢆᘬࡁ㉳ࡇࡍࠋ࣐ࢡࣟࣇ࢓࣮ࢪ࡟ࡼࡿ LPS ࡢㄆ㆑ࡣ㸪⣽ ⬊⾲㠃ཪࡣෆ㒊ࡢ TLR (3) ࡬ࡢ LPS ࡢ⤖ྜ࡟ࡼࡗ࡚ᘬࡁ㉳ࡇࡉࢀࡿ (13)ࠋLPS ࡀ⤖ྜࡋࡓTLR ࡣ㸪⣽⬊ෆࢩࢢࢼࣝఏ㐩⤒㊰ࢆ௓ࡋ࡚㌿෗ᅉᏊࢆάᛶ໬ࡋ㸪NO㸪 PGE2࡞࡝ࡢ⏕⌮άᛶ≀㉁ࡸ㸪⅖⑕ᛶࢧ࢖ࢺ࢝࢖࡛ࣥ࠶ࡿTNF-α㸪IL-1β ➼ࡢ⅖⑕ ᛶ࣓ࢹ࢕࢚࣮ࢱ࣮ࡢⓎ⌧ㄏᑟࢆಁࡍ (14, 15)ࠋ NO ࡣ㸪NOS ࡟ࡼࡗ࡚ L-࢔ࣝࢠࢽࣥ࠿ࡽྜᡂࡉࢀࡿࠋiNOS ࡣ㸪⅖⑕ㄏⓎ่⃭ ࢆཷࡅࡓ㝿࡟Ⓨ⌧ࡍࡿࠋ୍ᗘⓎ⌧ࡍࡿ࡜㸪iNOS ࡣ኱㔞ࡢ NO ࢆ⏕ᡂࡋ㸪㐣๫࡟ ⏕ᡂࡋࡓNO ࡣ㸪༢⊂࠶ࡿ࠸ࡣࢫ࣮ࣃ࣮࢜࢟ࢧ࢖ࢻ࡜཯ᛂࡋ࡚ DNA 㞀ᐖᛶࢆ᭷ ࡍࡿ࣌ࣝ࢜࢟ࢩள◪㓟ࢆ⏘⏕ࡋ㸪⣽⬊ࡸ⤌⧊࡟㞀ᐖࢆ୚࠼㸪⅖⑕⑕≧ࢆቑᝏࡍࡿࠋ

5 (16, 17)ࠋ ࣮࢜ࣜࣈἜ୰࡟ྵࡲࢀࡿᑠࡉ࡞ࣇ࢙ࣀ࣮ࣝศᏊ࡛࠶ࡿHT ࡣ௚ࡢኳ↛≀⏤᮶≀ ㉁࡜ẚ㍑ࡋ࡚ᙉຊ࡞ᢠ㓟໬άᛶࢆⓎ᥹ࡋ㸪ᢠ⅖⑕㸪ᢠ⾑ᰦ㸪ᢠ⭘⒆ཬࡧᢠ⳦స⏝ ࢆᣢࡘࡇ࡜ࡀ▱ࡽࢀࡿ (4–12, 18)ࠋ   Ṋ⏣ࡽ (12) ࡣ௨๓ࡢ◊✲࡟࠾࠸࡚㸪HT ࡣ㸪LPS ࡛ฎ⌮ࡋࡓ࣐࢘ࢫ⭡⭍࣐ࢡࣟ ࣇ࢓࣮ࢪ࡟࠾ࡅࡿiNOS 㑇ఏᏊⓎ⌧ཬࡧ NO ⏘⏕ࢆᢚไࡍࡿࡇ࡜ࢆ᫂ࡽ࠿࡟ࡋࡓࠋ ࡉࡽ࡟㸪HT ࡣ NF-κB ࢩࢢࢼࣝఏ㐩⤒㊰㜼ᐖ࡜ࡣ⊂❧ࡋࡓ࣓࢝ࢽࢬ࣒ࢆ௓ࡋ࡚ NO ⏘⏕ࢆᢚไࡍࡿྍ⬟ᛶࡀ࠶ࡿࡇ࡜ࢆሗ࿌ࡋࡓࠋ୍᪉㸪Zhang ࡽ (19) ࡣ㸪HT ࡣࣄ ࢺ༢⌫THP-1 ⣽⬊࡟࠾࠸࡚ LPS ࡢᏑᅾୗ࡛ NF-κB ࡢ᰾࡬ࡢ⛣⾜ࢆᢚไࡋࡓࡇ࡜ ࢆሗ࿌ࡋ࡚࠸ࡿࠋࡇࢀࡽࡢ2 ࡘࡢ◊✲࡛ࡣ㸪⏝࠸ࡓ⣽⬊⣔ࡣ␗࡞ࡗ࡚࠸ࡓࡀ㸪⏝ ࠸ࡓHT ࡢ⃰ᗘࡣྠᵝ࡛࠶ࡗࡓ (80 μM ཬࡧ 100 μM)ࠋྠᵝࡢ HT ⃰ᗘ࡛␗࡞ࡿ⤖ ᯝࡀㄆࡵࡽࢀࡓཎᅉ࡜ࡋ࡚㸪100 μM ⛬ᗘࡢ HT ࡣ NF-κB ࢩࢢࢼࣝఏ㐩⤒㊰ࢆ௓ ࡋ࡚㉳ࡇࡿ⅖⑕཯ᛂࢆᖖ࡟ᢚไࡍࡿࢃࡅ࡛ࡣ࡞࠸ࡇ࡜㸪ࡲࡓࡣ㸪࣐ࢡࣟࣇ࢓࣮ࢪ ⣔ࡢᵝࠎ࡞⣽⬊⣔ࡣ㸪LPS ่⃭࡟ᑐࡋ࡚␗࡞ࡿᛂ⟅ࢆ♧ࡍࡇ࡜ࡀ⪃࠼ࡽࢀࡓࠋ LPS ࡣ NF-κB ࢩࢢࢼࣝఏ㐩⤒㊰࡜ࡣ␗࡞ࡿ⤒㊰ࡶㄏᑟࡍࡿࡇ࡜ࡀሗ࿌ࡉࢀ࡚࠸ ࡿࠋ≉࡟㸪p44/42 mitogen-activated protein kinase (ERK1/2) ࡣ㸪༢⌫ཬࡧ࣐ࢡࣟࣇ

࢓࣮ࢪ࡟࠾࠸࡚LPS ࡟ࡼࡗ࡚ᙉࡃㄏᑟࡉࢀࡿࡇ࡜࠿ࡽ (20, 21)㸪HT ࡣࡇࡢࢩࢢ ࢼࣝఏ㐩⤒㊰ࢆᢚไࡍࡿࡇ࡜࡟ࡼࡗ࡚ᢠ⅖⑕ຠᯝࢆⓎ᥹ࡍࡿྍ⬟ᛶࡀ⪃࠼ࡽࢀ ࡿࠋ ᮏ◊✲࡛ࡣ㸪࣐࢘ࢫ࣐ࢡࣟࣇ࢓࣮ࢪᵝ⣽⬊ᰴRAW264.7 ࢆ⏝࠸࡚㸪LPS ㄏⓎᛶ ⅖⑕࡟࠾ࡅࡿHT ࡢᢠ⅖⑕స⏝ཬࡧࡑࡢ࣓࢝ࢽࢬ࣒ࢆ᫂ࡽ࠿࡟ࡍࡿࡇ࡜ࢆ┠ⓗ࡜ ࡋࡓࠋ

6 ᮦᩱཬࡧ᪉ἲ ⿕㦂≀㉁➼ HT ࡣᮾி໬ᡂᕤᴗᰴᘧ఍♫ (ᮾி㸪᪥ᮏ) ࡢ⣧ᗘ >98.0%ࡢ〇ရࢆ⏝࠸ࡓࠋHT ࢆ⵨␃Ỉ (ᰴᘧ఍♫኱ሯ〇⸆ᕤሙ㸪ᮾி㸪᪥ᮏ) ࢆ⏝࠸࡚ 100 mg/mL ࡢ⃰ᗘ࡟࡞ ࡿࡼ࠺⁐ゎࡋ㸪–80 °C ࡛ಖᏑࡋࡓࠋ LPS ࡣ࿴ග⣧⸆ᕤᴗᰴᘧ఍♫ (኱㜰㸪᪥ᮏ) ࡢ〇ရࢆ⏝࠸ࡓࠋLPS ࢆ⏕⌮㣗ሷ Ỉ (ᰴᘧ఍♫኱ሯ〇⸆ᕤሙ) ࢆ⏝࠸࡚ 1 mg/mL ࡢ⃰ᗘ࡟࡞ࡿࡼ࠺⁐ゎࡋ㸪–80 °C ࡛ಖᏑࡋࡓࠋ HT ཬࡧ LPS ࡣ㸪10%࢘ࢩ⫾௘⾑Ύࢆྵࡴࢲࣝ࣋ࢵࢥኚἲ࢖࣮ࢢࣝᇵᆅ (࿴ග ⣧⸆ᕤᴗᰴᘧ఍♫) (௨ୗ㸪ᇵ㣴ᾮ) ࡛ẁ㝵ᕼ㔘ࡋᐇ㦂࡟⏝࠸ࡓࠋ ᐇ㦂᪉ἲ 1. HT ࡢ RAW 264.7 ࡟ᑐࡍࡿ⣽⬊യᐖᛶࡢ᳨ド

 ࣐࢘ࢫ࣐ࢡࣟࣇ࢓࣮ࢪᵝ⣽⬊ᰴRAW264.7 (DS Pharma Biomedical, Co., Ltd., ኱

㜰㸪᪥ᮏ) ࡣᇵ㣴ᾮࢆ⏝࠸࡚⣽⬊ᇵ㣴⏝ 10 cm ࢩ࣮࡛ࣕࣞᇵ㣴ࡋࡓࠋᇵ㣴᮲௳ࡣ 37 Υ㸪5.0% CO2⃰ᗘ࡜ࡋ㸪CO2࢖࣮ࣥ࢟ࣗ࣋ࢱ࣮ෆ࡛ᇵ㣴ࡋࡓࠋ⥅௦ࡢ⣽⬊⃰ ᗘࡣ 0.5–1.05/well ࡜ࡋࡓࠋᇵ㣴ᾮࡢ஺᥮ࡣ⣽⬊ࡢ⥅௦࡜ྠ᫬Ⅼ࡛⾜࠸㸪㢖ᗘࡣ 3 ࠥ4 ᪥࡟ 1 ᅇ࡜ࡋࡓࠋᐇ㦂࡟ࡣ᭱኱࡛⥅௦ 12 ௦┠ࡲ࡛ࡢ⣽⬊ࢆ౑⏝ࡋࡓࠋ ⥅௦⣽⬊ࡀ༑ศ࡞ᩘ࡟࡞ࡗࡓ᫬Ⅼ࡛⣽⬊ࢆᅇ཰ࡋ㸪⣽⬊ᇵ㣴⏝ᖹᗏ 24-well ࣉ ࣮ࣞࢺ࡟2×106/well ࡢ⣽⬊ࢆ᧛✀ࡋ࡚ 2 ᫬㛫ᇵ㣴ࡋࡓᚋ㸪᥋╔ࡋ࡞࠿ࡗࡓ⣽⬊ࢆ 㝖࠸ࡓࠋࡑࡢᚋ㸪HT ࢆࡑࢀࡒࢀ 0–50 μg/mL ࡢ⃰ᗘ࡛ྵ᭷ࡉࡏࡓᇵ㣴ᾮ࡟஺᥮ࡋ㸪

7

CO2࢖࣮ࣥ࢟ࣗ࣋ࢱ࣮ෆ࡛24 ᫬㛫ᇵ㣴ࡋࡓࠋᇵ㣴ᚋ㸪ᇵ㣴ୖΎࢆᅇ཰ࡋ㸪⏕໬Ꮫ

⮬ືศᯒ⿦⨨ (LABOSPECT006㸪᪥❧ࣁ࢖ࢸࢡࣀࣟࢪ࣮ࢬ㸪ᮾி㸪᪥ᮏ) ࢆ⏝࠸

࡚㸪ୖΎ୰࡟ྵࡲࢀࡿlactate dehydrogenase (LDH) ࢆ ᐃࡋࡓࠋ

2. Real-time RT-PCR (RRT-PCR) ࡟ࡼࡿ㑇ఏᏊⓎ⌧ࡢゎᯒ

⣽⬊ᇵ㣴⏝ᖹᗏ12-well ࣉ࣮ࣞࢺ࡟ 2×106/well ࡢ RAW264.7 ⣽⬊ࢆຍ࠼࡚ 2 ᫬

㛫ᇵ㣴ࡋࡓᚋ㸪᥋╔ࡋ࡞࠿ࡗࡓ⣽⬊ࢆ㝖࠸ࡓࠋࡑࡢᚋ㸪1) ᇵ㣴ᾮࡢࡳ㸪2) LPS 0.25

μg/mL㸪3) LPS 0.25 μg/mL + HT (1.56–12.5 μg/mL) ࢆࡑࢀࡒࢀῧຍࡋࡓ well ࢆタ

ࡅCO2࢖࣮ࣥ࢟ࣗ࣋ࢱ࣮ෆ࡛ᇵ㣴ࡋࡓࠋ

ᇵ㣴 3 ཬࡧ 24 ᫬㛫ᚋ࡟⣽⬊ࢆᅇ཰ࡋ㸪RNeasy® Mini Kit (QIAGEN, Venlo,

Netherlands) ࢆ౑⏝ࡋ㸪࢟ࢵࢺࡢྲྀᢅㄝ᫂᭩࡟ᚑࡗ࡚⣽⬊ࡢ total RNA ࢆᢳฟࡋࡓࠋ

cDNA ྜᡂ࡟ࡣ㸪QuantiTectTM Reverse Transcription Kit (QIAGEN) ࢆ౑⏝ࡋ㸪1 ࢧ

ࣥࣉࣝ࠶ࡓࡾ1 μg ࡢ total RNA ࢆ⏝࠸࡚㸪௨ୗࡢ᮲௳࡛㏫㌿෗ࡋࡓ (42 °C 15 ศ

㛫㸪95 °C 3 ศ㛫)ࠋReal-time RT-PCR (RRT-PCR) ࡣ㸪cDNA ཬࡧ QuantiFast SYBR®

Green PCR (QIAGEN) ཬࡧ AriaMx (Agilent Technologies, Santa Clara, CA, USA) ࢆ

⏝࠸࡚⾜ࡗࡓࠋ⏝࠸ࡓࣉࣛ࢖࣐࣮ (QuantiTectTM Primer Assays, QIAGEN) ࢆ Table

1 ࡟♧ࡋࡓࠋPCR ཯ᛂࡣ 95 °C ࡛ 5 ศ㛫཯ᛂࡉࡏࡓᚋ㸪95 °C ࡛ 10 ⛊㛫㸪60 °C ࡛ 30 ⛊㛫ࡢ 40 ࢧ࢖ࢡ࡛ࣝ⾜ࡗࡓࠋ㜈್ࢧ࢖ࢡࣝ (Ct) ࡣ㸪⺯ගᙉᗘࡀ㜈್ࢆ㉸࠼࡚

ୖ᪼ࡍࡿࡢ࡟ᚲせ࡞ࢧ࢖ࢡࣝᩘ࡜ࡋ࡚ᐃ⩏ࡋࡓࠋᚓࡽࢀࡓ Ct ್ࢆᇶ࡟ෆᅾᛶࢥ

ࣥࢺ࣮࡛ࣟࣝ࠶ࡿglyceraldehyde-3-phosphate dehydrogenase (GAPDH) ࡟ᑐࡍࡿྛ

⩌㛫ࡢ┦ᑐⓗ࡞㑇ఏᏊⓎ⌧㔞ࢆ⟬ฟࡋࡓࠋ⤖ᯝࡣ㸪MIQE ࢞࢖ࢻࣛ࢖ࣥ (22) ࡟ ᚑࡗ࡚㸪㑇ఏᏊⓎ⌧ࡢ┦ᑐ್࡜ࡋ࡚⾲ࡋࡓࠋ

8

3. ᇵ㣴ୖΎ୰ࡢ NO㸪PGE2㸪TNF-α㸪IL-1β ࡢ⃰ᗘཬࡧ⣽⬊୰ࡢ COX-2 ⃰ᗘ ᐃ

2 ࡜ྠᵝࡢᐇ㦂ࢆ⾜࠸㸪ᇵ㣴 24 ᫬㛫ᚋ࡟⣽⬊ୖΎࢆᅇ཰ࡋࡓࠋ⣽⬊ୖΎ୰ࡢ

NO㸪PGE2㸪TNF-α ཬࡧ IL-1Eࡢ⃰ᗘࢆ㸪ࡑࢀࡒࢀ Nitric Oxide (total), detection kit

(Enzo Life Sciences, Farmingdale, NY, USA)㸪Prostaglandin E2 ELISA Kit (Abcam,

Cambridge, UK)㸪Mouse TNF alpha SimpleStep ELISA® Kit (Abcam) ཬࡧ IL-1 beta

Mouse SimpleStep ELISA® Kit (Abcam) ࢆ⏝࠸࡚ ᐃࡋࡓࠋࡉࡽ࡟㸪⣽⬊୰ࡢ

COX-2 ⃰ᗘࢆ Mouse COX2 SimpleStep ELISA® Kit (Abcam) ࡛ ᐃࡋࡓࠋPGE2㸪

TNF-α㸪IL-1Eཬࡧ COX-2 ࡟㛵ࡋ࡚ࡣ ELISA ἲ࡛ ᐃࡋ㸪NO ࡟㛵ࡋ࡚ࡣ Griess ἲ (23) ࡛ ᐃࡋࡓࠋ

4. Western blotting ࡟ࡼࡿ⣽⬊ෆࢩࢢࢼࣝᅉᏊࡢⓎ⌧ゎᯒ

2 ࡜ྠᵝࡢᐇ㦂ࢆ⾜࠸㸪 ⣽⬊ࢆ 15 ศᚋ࡟ᅇ཰ࡋ࡚ࣜࣥ㓟໬ NF-κB (pNF-κB) ཬ

ࡧࣜࣥ㓟໬ERK1/2 (pERK1/2) ࡢⓎ⌧ࢆ㸪ࡲࡓ 6 ᫬㛫ᚋ࡟ᅇ཰ࡋ࡚ iNOS ࡢⓎ⌧

ࢆ㸪ࡑࢀࡒࢀWestern Blotting ἲ࡟ࡼࡾゎᯒࡋࡓࠋ

Mini-PROTEAN TGX Precast Gels (Bio-Rad, Hercules, CA, USA) ࢆ ⏝ ࠸ ࡚ SDS-PAGE ࡟ࡼࡾࢱࣥࣃࢡ㉁ࢆศ㞳ࡋࡓᚋ࡟㸪Trans-Blot Turbo Transfer Pack (Bio-Rad) ࢆ⏝࠸࡚ polyvinylidene difluoride (PVDF) ࣓ࣥࣈࣞࣥ࡟㌿෗ࡋࡓࠋ㌿෗ ᚋࡢ࣓ࣥࣈࣞࣥࡣ㸪0.3%ࢫ࣒࣑࢟ࣝࢡྵ᭷ Tris Buffered Saline with Tween 20 (TBS-T) (࿴ග⣧⸆ᕤᴗᰴᘧ఍♫) ࡟ࡼࡾ 4 °C ࡛ 24 ᫬㛫ࣈࣟࢵ࢟ࣥࢢࢆ⾜ࡗࡓࠋ

ࣈࣟࢵ࢟ࣥࢢᚋ㸪࢘ࢧࢠ⏤᮶୍ḟᢠయ㸸ᢠ NF-κB㸪ᢠ pNF-κB㸪ᢠ ERK1/2㸪ᢠ

pERK1/2 ཬࡧᢠ iNOS (Cell Signaling Technology, Danvers, MA, USA) ࢆ 0.3%ࢫ࢟

9

3 ᅇὙίࡋࡓࠋ஧ḟᢠయ࡟ࡣ Anti-rabbit IgG, HRP-linked Antibody (Cell Signaling Technology) ࢆ 0.3%ࢫ࣒࣑࢟ࣝࢡྵ᭷ TBS-T ࡛ 1,000 ಸᕼ㔘ࡋ࡚⏝࠸㸪ᐊ ࡛ 1

᫬㛫཯ᛂࡉࡏࡓࠋTBS-T ࡛ 3 ᅇὙίᚋ㸪SuperSignalTM West Pico (Thermo Scientific,

Waltham, MA, USA) ࢆ⏝࠸࡚࣓ࣥࣈࣞࣥ࡟⤖ྜࡋࡓᢠཎᢠయ」ྜయࢆ᳨ฟࡋ㸪 LAS-3000 (ᐩኈࣇ࢖࣒ࣝᰴᘧ఍♫㸪ᮾி㸪᪥ᮏ) ࡛ศᯒࡋࡓࠋෆ㒊ࢥࣥࢺ࣮ࣟࣝ

࡜ࡋ࡚㸪anti-β-Actin HRP-DirecT (Medical & Biological Laboratories, Co., Ltd., ឡ▱㸪

᪥ᮏ) ࢆ⏝࠸࡚ β-࢔ࢡࢳࣥࢆ᳨ฟࡋࡓࠋࡉࡽ࡟, ᚓࡽࢀࡓࣂࣥࢻࡢ⃰ᗘࢆ Quantity

One version 4.5 (Bio-Rad) ࢆ⏝࠸࡚ ᐃࡋ㸪┠ⓗࢱࣥࣃࢡ㉁ࡢ⃰ᗘࢆ β-࢔ࢡࢳࣥࡢ ⃰ᗘ࡛๭ࡿࡇ࡜࡟ࡼࡾ┦ᑐⓗ࡞ࢱࣥࣃࢡ㉁Ⓨ⌧㔞ࢆ⟬ฟࡋࡓࠋ

⤫ィฎ⌮

⤖ᯝࡣ㸪ᖹᆒ ± ᶆ‽ㄗᕪ (S.E.) ࡜ࡋ࡚♧ࡋࡓࠋ᭷ពᕪ᳨ᐃ࡟ࡣ㸪࢘࢕ࣜ࢔࣒

ࢬࡢከ㔜ẚ㍑ヨ㦂ࢆ⏝࠸ࡓࠋ᭷ពỈ‽ࡣ୧ഃ 5%࡜ࡋࡓࠋ⤫ィゎᯒ࡟ࡣ⤫ィࢯࣇ

ࢺ࢙࢘࢔࡛࠶ࡿSAS (SAS Institute Inc., Cary, NC, USA) ཬࡧ EXSUS (ᰴᘧ఍♫

10 ⤖ᯝ 1. RAW264.7 ⣽⬊࡟࠾ࡅࡿ HT ࡢ⣽⬊യᐖᛶ RAW264.7 ⣽⬊࡟࠾ࡅࡿ HT (0–50 μg/mL) ࡢ⣽⬊യᐖᛶࢆホ౯ࡋࡓࠋ࠸ࡎࢀࡢ ⃰ᗘ࡟࠾࠸࡚ࡶ㸪⣽⬊യᐖᛶࡢᣦᶆ࡛࠶ࡿ LDH ࡢቑຍࡣㄆࡵࡽࢀ࡞࠿ࡗࡓࠋࡋ ࠿ࡋ㸪25 μg/mL ௨ୖࡢ⃰ᗘ࡟࠾࠸࡚ LDH ࡣࢃࡎ࠿࡟ῶᑡࡋࡓ (Fig. 2)ࠋཎᅉࡣ୙ ࡛᫂࠶ࡗࡓࡀ㸪ఱࡽ࠿ࡢᙳ㡪ࡀ࠶ࡿ࡜⪃࠼㸪ࡑࡢᚋࡢᐇ㦂ࡣ0–12.5 μg/mL ࡢ⃰ᗘ ࡛ᐇ᪋ࡋࡓࠋ 2. RAW264.7 ⣽⬊࡟࠾ࡅࡿ⅖⑕㛵㐃㑇ఏᏊࡢⓎ⌧ ḟ࡟㸪LPS (0.25 μg/mL) ่࡛⃭ࡋࡓ RAW264.7 ⣽⬊࡟࠾ࡅࡿ HT ࡢຠᯝࢆホ౯ ࡋࡓࠋ௨๓ࡢ◊✲ (12) ࡢᐇ㦂᮲௳࡟ຍ࠼㸪ࡼࡾ᪩ᮇ࡛ࡢ㑇ఏᏊⓎ⌧ኚືࢆㄪ࡭ ࡿࡓࡵ࡟㸪RAW264.7 ⣽⬊ࢆ LPS (0.25 μg/mL) ཬࡧ HT (0–12.5 μg/mL) ࡛ 3 ᫬㛫 ཬࡧ24 ᫬㛫ฎ⌮ࡋࡓࠋ 3 ᫬㛫ฎ⌮࡟࠾࠸࡚㸪LPS (0.25 μg/mL) ༢⊂࡛ࡣ㸪ࢥࣥࢺ࣮ࣟࣝ࡜ẚ㍑ࡋ࡚㸪 iNOS㸪COX-2㸪TNF-α㸪IL-1β ཬࡧ IL-6 ࡢ㑇ఏᏊⓎ⌧ࡀ᭷ព࡟ቑຍࡋࡓࠋNF-κB ཬࡧIL-10 㑇ఏᏊⓎ⌧ࡶቑຍࡋࡓࡀ㸪⤫ィᏛⓗ࡟᭷ព࡛ࡣ࡞࠿ࡗࡓࠋHT ࡣ㸪LPS ࡀㄏⓎࡋࡓTNF-α ཬࡧ IL-1β ࡢ㑇ఏᏊⓎ⌧ቑຍࢆ㸪1.56–12.5 μg/mL ࡢ⃰ᗘ࡛᭷ព ࡟ῶᑡࡉࡏࡓ (Fig. 3)ࠋ 24 ᫬㛫ฎ⌮࡟࠾࠸࡚㸪LPS (0.25 μg/mL) ༢⊂࡛ࡣ㸪ࢥࣥࢺ࣮ࣟࣝ࡜ẚ㍑ࡋ࡚㸪 iNOS㸪COX-2㸪IL-1β ཬࡧ IL-6 ࡢ㑇ఏᏊⓎ⌧ࡀ᭷ព࡟ቑຍࡋࡓࠋᑐ↷ⓗ࡟㸪TNF-α, NF-κB ཬࡧ IL-10 㑇ఏᏊⓎ⌧ࡣ LPS ࡟ࡼࡗ࡚ኚືࡋ࡞࠿ࡗࡓࠋHT ࡣ㸪LPS ࡀㄏ

11

ⓎࡋࡓiNOS㸪COX-2㸪ཬࡧ IL-1β ࡢ㑇ఏᏊⓎ⌧ቑຍࢆ㸪ࡑࢀࡒࢀ 12.5 μg/mL㸪1.56–

12.5 μg/mL㸪ཬࡧ 1.56–12.5 μg/mL ࡢ⃰ᗘ࡛᭷ព࡟ῶᑡࡉࡏࡓ (Fig. 4)ࠋ

3. RAW264.7 ⣽⬊ࡢᇵ㣴ୖΎ୰ࡢ NO㸪PGE2㸪TNF-α㸪IL-1β ࡢ⃰ᗘཬࡧ⣽⬊୰ࡢ

COX-2 ⃰ᗘ

LPS (0.25 μg/mL) Ꮡᅾୗ࡛ᇵ㣴ࡋࡓ RAW264.7 ⣽⬊ࡼࡾᅇ཰ࡋࡓ⣽⬊ୖΎ࡛ࡣ㸪

NO㸪PGE2㸪TNF-α ཬࡧ IL-1β ࡢ⏘⏕ࡀ᭷ព࡟ቑຍࡋ࡚࠸ࡓࠋLPS ࡟ࡼࡿ NO㸪PGE2

ཬࡧIL-1β ࡢ⏘⏕ࡣ㸪ࡑࢀࡒࢀ 3.13–12.5 μg/mL㸪1.56–12.5 μg/mL ཬࡧ 3.13–12.5 μg/mL ࡢ HT ࡛⃰ᗘ౫Ꮡⓗ࡟ᢚไࡉࢀࡓࠋᑐ↷ⓗ࡟㸪LPS ࡟ࡼࡿ TNF-α ࡢ⏘⏕ࡣ㸪 HT ࡟ࡼࡗ࡚ᢚไࡉࢀ࡞࠿ࡗࡓࠋ⣽⬊୰ࡢ COX-2 ⃰ᗘࡣ㸪LPS ࡟ࡼࡾ᭷ព࡟ቑຍ ࡋࡓࠋLPS ࡟ࡼࡿ COX-2 ⏘⏕ࡣ HT ࡟ࡼࡾᢚไࡉࢀࡓࡀ㸪⤫ィᏛⓗ࡟᭷ព࡛ࡣ࡞ ࠿ࡗࡓ (Fig. 5)ࠋ 4. RAW264.7 ⣽⬊࡟࠾ࡅࡿ NF-κB 㛵㐃ࢱࣥࣃࢡ㉁ࡢⓎ⌧

Western blotting ἲࢆ⏝࠸࡚㸪LPS ่⃭ୗࡢ RAW264.7 ⣽⬊ࢆ HT Ꮡᅾࡲࡓࡣ㠀

Ꮡᅾୗ࡛15 ศ㛫ᇵ㣴ࡋࡓᚋࡢ pNF-κB ཬࡧ pERK1/2 ࡢⓎ⌧㸪࡞ࡽࡧ࡟ 6 ᫬㛫ᇵ

㣴ࡋࡓ⣽⬊࡟࠾ࡅࡿiNOS ࡢⓎ⌧ࢆホ౯ࡋࡓࠋLPS (0.25 μg/mL) ࡢῧຍ࡟ࡼࡾ㸪

pNF-κB㸪pERK1/2 ཬࡧ iNOS ࡢⓎ⌧ࡣ࠸ࡎࢀࡶቑຍࡋࡓࠋLPS ࡀㄏⓎࡋࡓ pNF-κB

ཬࡧiNOS Ⓨ⌧ࡢୖ᪼ࡣ㸪12.5 μg/mL ࡢ⃰ᗘࡢ HT ࡢῧຍ࡟ࡼࡾᢚไࡉࢀࡓࠋᑐ

12 ⪃ᐹ ᮏ◊✲࡛ࡣ㸪in vitro ⅖⑕ࣔࢹࣝ࡜ࡋ࡚ከ⏝ࡉࢀ࡚࠸ࡿ࣐࢘ࢫ࣐ࢡࣟࣇ࢓࣮ࢪᵝ ⣽⬊ᰴRAW264.7 ࢆ⏝࠸㸪HT ࡢ LPS ࡟ࡼࡿ⅖⑕ᛶࢧ࢖ࢺ࢝࢖ࣥཬࡧ࣓ࢹ࢕࢚࣮ ࢱ࣮ࡢㄏᑟ࡟ᑐࡍࡿస⏝࡟ࡘ࠸᳨࡚ドࡋࡓࠋ HT ࡣ RAW264.7 ⣽⬊࡟࠾ࡅࡿ LPS ㄏⓎᛶࡢ NF-κB άᛶ໬㸪TNF-α 㑇ఏᏊⓎ⌧㸪

IL-1β ⏘⏕㸪COX-2-PGE2ཬࡧiNOS-NO ⣔ࢆᢚไࡋࡓࠋTLR4 ࡣ㸪LPS ࡟ࡼࡗ࡚ά

ᛶ໬ࡉࢀ㸪NF-κB ࢩࢢࢼࣝఏ㐩ཬࡧ⅖⑕ᛶࢧ࢖ࢺ࢝࢖ࣥ⏘⏕ࢆㄏⓎࡋ㸪⮬↛ච␿

⣔ࢆάᛶ໬ࡍࡿ (24, 25)ࠋIL-1β ཬࡧ TNF-α ࡣ㸪ឤᰁ㸪യᐖཬࡧච␿่⃭࡟ᑐࡍ

ࡿᛴᛶᮇᛂ⟅࡜ࡋ࡚㎿㏿࡟⩻ヂࡉࢀ㸪ᨺฟࡉࢀࡿ (26–28)ࠋiNOS ཬࡧ COX-2 ࡣ㸪

IL-1β㸪TNF-α ཬࡧ௚ࡢࢧ࢖ࢺ࢝࢖ࣥ࡟ࡼࡗ࡚ㄏᑟࡉࢀࡿࡓࡵ (29–31)㸪⅖⑕ᛂ⟅

ࣉࣟࢭࢫࡢᚋ༙࡟ᨺฟࡉࢀࡿࠋᮏ◊✲࡛ࡣ㸪HT ࡣึᮇẁ㝵࡛ TNF-α ཬࡧ IL-1β

ࡢⓎ⌧ࢆᢚไࡋࡓ (Fig. 3)ࠋࡋࡓࡀࡗ࡚㸪TNF-α ཬࡧ IL-1β ࡢᢚไࡀ㸪COX-2-PGE2

ཬࡧiNOS-NO ⣔ࢆ஧ḟⓗ࡟ᢚไࡋࡓྍ⬟ᛶࡀ⪃࠼ࡽࢀࡓࠋ Western blotting ゎᯒ࡛ࡣ㸪HT ࡀ 12.5 μg/mL ࡢ⃰ᗘ࡛ LPS ่⃭࡟ࡼࡿ pNF-κB ࡢⓎ⌧ࢆᢚไࡋࡓࡀ㸪6.25 μg/mL ᮍ‶ࡢ⃰ᗘ࡛ࡣᢚไࡋ࡞࠿ࡗࡓࠋZhang ࡽ (19) ࡣTHP-1 ⣽⬊࡟࠾ࡅࡿ NF-κB ࡢ᰾࡬ࡢ⛣⾜ࡣ㸪100 μM ࡢ HT ࡟ࡼࡗ࡚ᢚไࡉࢀ ࡓࡀ㸪50 μM ࡢ HT ࡛ࡣᢚไࡉࢀ࡞࠿ࡗࡓ࡜ሗ࿌ࡋ࡚࠸ࡿࠋ୍᪉㸪௒ᅇࡢᐇ㦂࡟ ࠾࠸࡚㸪ప⃰ᗘࡢHT ࡣ NF-κB ࢩࢢࢼࣝఏ㐩ࢆᢚไࡋ࡞࠿ࡗࡓࡀ㸪TNF-α ཬࡧ IL-1β ࡢ㑇ఏᏊⓎ⌧ཬࡧ iNOS-NO ⣔ࢆᢚไࡋࡓࠋࡇࢀࡽࡢ⤖ᯝࡣ㸪HT ࡣప⃰ᗘ࡛ࡣ NF-κB ࢩࢢࢼࣝ࡟౫Ꮡࡋ࡞࠸⤒㊰࡛ LPS ㄏⓎᛶࡢ⅖⑕ᛂ⟅ࢆᢚไࡍࡿࡇ࡜ࢆ♧ࡋ ࡚࠸ࡿࠋ

13

pERK1/2 ࡣ LPS ࡟ࡼࡗ࡚ᙉࡃㄏᑟࡉࢀࡿࡇ࡜ࡀሗ࿌ࡉࢀ࡚࠸ࡿ (20, 21)ࠋ௒ᅇ ࡢᐇ㦂࡟࠾࠸࡚ HT ࡣ㸪pNF-κB ཬࡧ iNOS ࡟ᑐࡍࡿᢚไຠᯝ࡜ࡣᑐ↷ⓗ࡟㸪࠸ࡎ

ࢀࡢ⃰ᗘ࡛ࡶpERK1/2 ࡢⓎ⌧ࢆᢚไࡋ࡞࠿ࡗࡓࠋChan ࡽ (32) ࡣ㸪RAW264.7 ⣽

⬊࡟࠾ࡅࡿLPS ㄏⓎ iNOS ⏘⏕ࡀ㸪ERK1/2 ⤒㊰࡟ຍ࠼࡚㸪c-Jun N ᮎ➃࢟ࢼ࣮ࢮ

(c-jun N-terminal kinase : JNK) ཬࡧ p38 ศ⿣ಁ㐍ᅉᏊάᛶ໬ࢱࣥࣃࢡ㉁࢟ࢼ࣮ࢮ (p38) ⤒㊰࡟ࡼࡗ࡚ㄪ⠇ࡉࢀࡿ࡜ሗ࿌ࡋ࡚࠾ࡾ㸪HT ࡢస⏝ᶵᗎࢆᢸ࠺ࢩࢢࢼࣝఏ 㐩⤒㊰ࢆ᫂ࡽ࠿࡟ࡍࡿࡓࡵ࡟ࡣ᭦࡞ࡿ◊✲ࡀᚲせ࡜⪃࠼ࡽࢀࡓࠋ HT ࡀ iNOS 㑇ఏᏊⓎ⌧ࢆῶᑡࡉࡏ㸪NO ⏘⏕ࢆᢚไࡍࡿ࡜࠸࠺⤖ᯝࡣ㸪࣐࢘ࢫ ⭡⭍࣐ࢡࣟࣇ࢓࣮ࢪࢆ⏝࠸ࡓṊ⏣ࡽࡢ⤖ᯝ (12) ࡜୍⮴ࡍࡿࡶࡢ࡛࠶ࡗࡓࠋࡋ࠿ ࡋ㸪Ṋ⏣ࡽࡢ◊✲࡛ࡣ㸪COX-2 㑇ఏᏊࡢⓎ⌧ࡣ 3.13–12.5 μg/mL ࡢ HT ࡟ࡼࡗ࡚ ᢚไࡉࢀ࡞࠿ࡗࡓࠋ࣐࢘ࢫ⭡⭍࣐ࢡࣟࣇ࢓࣮ࢪࢆ⏝࠸ࡓ㐣ཤࡢูࡢ◊✲࡟࠾࠸࡚ ࡶ㸪Ṋ⏣ࡽࡢᐇ㦂⤖ᯝ࡜ྠᵝ࡟㸪HT ࡣ LPS ㄏⓎᛶࡢ COX-2 ࢱࣥࣃࢡ㉁Ⓨ⌧ቑຍ ࢆᢚไࡋ࡞࠿ࡗࡓ (33)ࠋ ⯆࿡῝࠸ࡇ࡜࡟㸪Maiuri ࡽ (5) ཬࡧ Zhang ࡽ (19) ࡣ㸪 HT ࡣ LPS ㄏⓎᛶࡢ COX-2 㑇ఏᏊⓎ⌧࡜ PGE2⏘⏕ࡢ୧᪉ࡢㄏᑟࢆᢚไࡍࡿࡇ࡜ ࢆሗ࿌ࡋ࡚࠸ࡿࠋࡇࢀࡽ2 ࡘࡢ◊✲࡛ࡣ㸪࣐࢘ࢫ࣐ࢡࣟࣇ࢓࣮ࢪᵝᰴ໬⣽⬊ J774 ཬࡧࣄࢺ༢⌫THP-1 ⣽⬊ࡀ⏝࠸ࡽࢀ࡚࠸ࡿࠋ࣐࢘ࢫ⭡⭍࣐ࢡࣟࣇ࢓࣮ࢪ࡛ࡣ㸪ᇵ 㣴⣽⬊ᰴ࡜ࡣ␗࡞ࡾ㸪LPS ࡞࡝ࡢච␿㈿ά๣࡟ࡼࡿάᛶ໬㐣⛬࡟࠾࠸࡚ᮍ▱ࡢ⏕ ≀ᏛⓗᅉᏊࡢᙳ㡪ࢆཷࡅࡿྍ⬟ᛶࡀ⪃࠼ࡽࢀ࡚࠸ࡿ (33)ࠋin vivo ࡛ࡣከࡃࡢ␗࡞ ࡿࢱ࢖ࣉࡢ࣐ࢡࣟࣇ࢓࣮ࢪࡀᏑᅾࡍࡿࡢ࡛㸪༢୍⣽⬊ᆺ࠿ࡽᚓࡽࢀࡓᐇ㦂⤖ᯝࡣ in vivo ࡟࠾ࡅࡿ⌧㇟ࢆ᏶඲࡟཯ᫎࡋ࡞࠸ࡇ࡜࡟ὀពࡍࡿࡇ࡜ࡀ㔜せ࡛࠶ࡿ࡜⪃࠼ ࡽࢀࡓࠋ ᮏ◊✲࡛ࡣ㸪HT ࡣ LPS ㄏⓎᛶࡢ⅖⑕ᛂ⟅ࢆᢚไࡍࡿࡇ࡜ࢆ♧ࡋ㸪ᢠ⅖⑕⸆࡜

14

ࡋ࡚ࡢ㛤Ⓨࡢྍ⬟ᛶࡀぢฟࡉࢀࡓࠋ᪤Ꮡࡢᢠ⅖⑕⸆࡛࠶ࡿ㠀ࢫࢸࣟ࢖ࢻᛶᢠ⅖⑕ ⸆ (nonsteroidal anti-inflammatory drugs㸹NSAIDs) ࡢ๪స⏝࡟ࡣ㸪⫶ࡸᑠ⭠࡟ᑐࡍ ࡿ⢓⭷ᦆയ (34) ࡸ⭈㞀ᐖ (35) ࡀ▱ࡽࢀ࡚࠸ࡿࠋHT ࡣࡆࡗṑ㢮ࢆ⏝࠸ࡓᏳ඲ᛶ

ヨ㦂࡛ẘᛶࢆ♧ࡉࡎ (36)㸪ࡍ࡛࡟㣗ရࡸᰤ㣴⿵ຓ㣗ရ࡛ᗈࡃᕷ㈍ࡉࢀ࡚࠸ࡿ (37)ࠋ

HT ࡢᢠ⅖⑕ຠᯝࢆ᏶඲࡟⌮ゎࡍࡿࡓࡵ࡟ࡣࡉࡽ࡞ࡿ◊✲ࡀᚲせ࡛࠶ࡿࡀ㸪HT ࡀ ື≀ࣔࢹࣝ࡟࠾࠸࡚᭷ᐖ࡞⅖⑕ᛂ⟅ࢆຠᯝⓗ࡟ᢚไࡍࡿ࠿࡝࠺࠿ࢆヨ㦂ࡍࡿ౯ ್ࡀ࠶ࡿ࡜⪃࠼ࡽࢀࡿࠋ

15 ᑠᣓ ᭷ᐖ࡞่⃭ࢆཷࡅࡓ࡜ࡁ࡟⏕ࡌࡿ㜵ᚚ཯ᛂ࡛࠶ࡿ⅖⑕ࡢไᚚࡣ㸪⑓ែࡢ㌿ᖐ࡜ ᝈ⪅ࡢQOL ࡢ୧㠃࡛㔜せ࡛࠶ࡿࠋࡋ࠿ࡋ࡞ࡀࡽ㸪NSAIDs ࡟௦⾲ࡉࢀࡿࡼ࠺࡟ከ ࡃࡢᢠ⅖⑕ᛶࡢ἞⒪ἲ࡟ࡣ๪స⏝ࡀ࠶ࡾ㸪᪂ࡋ࠸Ᏻ඲࡞἞⒪ࡀồࡵࡽࢀ࡚࠸ࡿࠋ 㐣ཤࡢ◊✲࡛ࡣ㸪LPS ่࡛⃭ࡋࡓ࣐࢘ࢫ⭡⭍࣐ࢡࣟࣇ࢓࣮ࢪ࡟࠾࠸࡚㸪HT ࡣ 12.5 μg/mL ࡢ⃰ᗘ࡛ iNOS 㑇ఏᏊⓎ⌧ཬࡧ NO ⏘⏕ࢆᢚไࡋࡓࠋ୍᪉࡛㸪NF-κB 㑇ఏᏊⓎ⌧ࡣኚ໬ࡋ࡞࠿ࡗࡓࠋᮏ◊✲࡛ࡣ㸪࣐࢘ࢫ࣐ࢡࣟࣇ࢓࣮ࢪᵝ⣽⬊ᰴ RAW264.7 ࢆ⏝࠸࡚㸪✀ࠎࡢ⃰ᗘࡢ HT ࡢᢠ⅖⑕ຠᯝࢆホ౯ࡋࡓࠋHT ࡣ 12.5 μg/mL ࡢ⃰ᗘ࡛ࡣNF-κB ࢩࢢࢼࣝఏ㐩⤒㊰ࢆᢚไࡍࡿࡇ࡜࡛㸪TNF-α 㑇ఏᏊⓎ⌧㸪IL-1β ⏘⏕㸪COX-2-PGE2ཬࡧiNOS-NO ⣔ࢆᢚไࡋ㸪12.5 μg/mL ࡼࡾప࠸⃰ᗘ࡛ࡣูࡢ ࢩࢢࢼࣝఏ㐩⤒㊰࡟㛵୚ࡍࡿࡇ࡜࡛⅖⑕཯ᛂࢆㄪ⠇ࡍࡿྍ⬟ᛶࡀ⪃࠼ࡽࢀࡓࠋ୍ ᪉㸪HT ࡣ LPS ㄏⓎᛶࡢ pERK1/2 Ⓨ⌧ࢆᢚไࡋ࡞࠿ࡗࡓࠋHT ࡢᢠ⅖⑕ຠᯝࢆ᏶ ඲࡟⌮ゎࡍࡿࡓࡵ࡟ࡣࡉࡽ࡞ࡿ◊✲ࡀᚲせ࡛࠶ࡿࡀ㸪ᮏ◊✲࡛ࡣ㸪HT ࡀ LPS ่ ⃭RAW264.7 ⣽⬊࡟ᑐࡋ࡚ᢠ⅖⑕ຠᯝࢆ᭷ࡍࡿࡇ࡜ࡀ᫂ࡽ࠿࡜࡞ࡗࡓࠋ HT ࡣ᪤࡟ᰤ㣴⿵ຓ㣗ရ࡜ࡋ࡚฼⏝ྍ⬟࡛࠶ࡾ㸪ẘᛶࡢᙳ㡪ࡣሗ࿌ࡉࢀ࡚࠸࡞ ࠸ࠋᚑࡗ࡚㸪HT ࡣ᪂つᢠ⅖⑕⸆࡟࡞ࡾ࠺ࡿ࡜⪃࠼ࡽࢀࡓࠋ᪂つᢠ⅖⑕⸆࡜ࡋ࡚ ࡢ㛤Ⓨࢆぢᤣ࠼㸪HT ࡢస⏝࣓࢝ࢽࢬ࣒ࢆ᫂ࡽ࠿࡟ࡍ࡭ࡃ㸪⣽⬊ෆࢩࢢࢼࣝᅉᏊ ࡢinhibitor ࢆ⏝࠸ࡓ᳨ウࢆ㐍ࡵࡿࡇ࡜࡟ࡋࡓࠋ

16

ᅗ⾲

Table 1. Primers sets (QuantiTect TM Primer Assays, QIAGEN) used in real-time RT-PCR

Gene name Assay name

iNOS (nitric oxide synthase 2, inducible)

COX-2 (prostaglandin-endoperoxide synthase 2) TNF-α (tumor necrosis factor alpha)

NF-κB

(nuclear factor of kappa light polypeptide gene enhancer in B-cells)

IL-1β (interleukin 1 beta) IL-6 (interleukin 6) IL-10 (interleukin 10)

Gapdh (glyceraldehyde-3-phosphate dehydrogenase)

Mm_LOC673161_1_SG Mm_Ptgs2_1_SG Mm_Tnf_1_SG Mm_Nfkb1_1_SG Mm_Il1b_2_SG Mm_Il6_1_SG Mm_Il10_1_SG Mm_Gapdh_3_SG

17

18

Fig. 2. Analysis of the cytotoxicity of HT against RAW264.7 cells cultured with HT (0–50 μg/mL) for 24 h.

Cytotoxicity was evaluated as the LDH concentration in the culture supernatant. Mean ± S.E. (n = 3).

19

Fig. 3. Expression of the iNOS, COX-2, TNF-α, NF-κB, IL-1β, IL-6 and IL-10 genes in cultures of RAW264.7 cells treated with LPS (0.25 μg/mL) and HT (0–12.5 μg/mL) for 3 h.

Relative mRNA expression levels of iNOS, COX-2, TNF-α, NF-κB, IL-1β, IL-6 and IL-10 were evaluated by real-time RT-PCR.

20

Fig. 4. Expression of the iNOS, COX-2, TNF-α, NF-κB, IL-1β, IL-6 and IL-10 genes in cultures of RAW264.7 cells treated with LPS (0.25 μg/mL) and HT (0–12.5 μg/mL) for 24 h.

Relative mRNA expression levels of iNOS, COX-2, TNF-α, NF-κB, IL-1β, IL-6 and IL-10 were evaluated by real-time RT-PCR.

Mean ± S.E. *P < 0.05, **P < 0.01, ***P < 0.001 (Williams’ multiple comparison test) (n = 4).

21

Fig. 5. Concentration of NO, PGE2, TNF-α and IL-1β in the culture supernatant

and COX-2 in the cell extract of RAW264.7 cells cultured with LPS (0.25 μg/mL) and HT (0–12.5 μg/mL) for 24 h.

NO concentration was measured by Griess method. Concentration of PGE2, TNF-α,

IL-1β and COX-2 was measured by ELISA.

Mean ± S.E. *P < 0.05, **P < 0.01, ***P < 0.001 (Williams’ multiple comparison test) (n = 3).

22

Fig. 6. Expression of pNF-κB (A), pERK1/2 (B), and iNOS (C) in RAW264.7 cells cultured with LPS (0.25 μg/mL) and HT (0–12.5 μg/mL).

Western blotting analysis was used to determine the levels of these proteins in cells treated for 15 min (A, B) or 6 h (C). Representative results of duplicate experiments are

shown. The graph shows the relative  protein  expression  levels measured by

23

➨2 ❶

24 ⥴ゝ ྂ᮶ࡼࡾࣇࣛࣥࢫ➼ࡢᆅ୰ᾏἢᓊ࡟ఫࡴேࠎࡣẚ㍑ⓗ㧗⬡⫫㣗ࢆᦤྲྀࡍࡿ࡟ ࡶ㛵ࢃࡽࡎ㸪㛗ᑑ࡛㸪ᚰ⮚⑓ࡸ≉ᐃࡢࡀࢇ࡞࡝㸪⏕ά⩦័⑓࡜ࡉࢀࡿ⑓ẼࡢⓎ⑕ ⋡ࡀ๻ⓗ࡟ప࠸ࡇ࡜ࡀ▱ࡽࢀ࡚࠸ࡓ (38–41)ࠋ࢔࣓ࣜ࢝ࡢ⏕⌮Ꮫ⪅ Ancel Keys ࡣ㸪 ࡇࡢ஦ᐇࡀࠕ㣗஦ࡢࢫࢱ࢖ࣝࡢ㐪࠸ࠖ࡜ࡢ௬ㄝࡢඖ࡛◊✲ࢆ⾜࠸㸪࣮࢜ࣜࣈ࢜࢖ ࣝࢆጞࡵ࡜ࡍࡿ Mediterranean food ࡟ື⬦◳໬ࢆ㜵ࡄᢠ㓟໬స⏝ࡀ࠶ࡿࡇ࡜ࢆ᫂ ࡽ࠿࡟ࡋࡓ (42)ࠋࡑࡢᚋࡢ◊✲࡟࠾࠸࡚㸪࣮࢜ࣜࣈ࢜࢖ࣝ୰࡟ぢࡽࢀࡿᑠࡉ࡞ࣇ ࢙ࣀ࣮ࣝศᏊ࡛࠶ࡿHT ࡣᙉຊ࡞ᢠ㓟໬άᛶࢆⓎ᥹ࡋ㸪ᢠ⅖⑕㸪ᢠ⾑ᰦ㸪ᢠ⭘⒆ ཬࡧᢠ⳦స⏝➼ࢆᣢࡘࡇ࡜ࡀ᫂ࡽ࠿࡜࡞ࡗࡓ (4–12, 18)ࠋ ➨1 ❶࡟࠾࠸࡚㸪HT ࡀ㸪࣐࢘ࢫ࣐ࢡࣟࣇ࢓࣮ࢪᵝ⣽⬊ᰴ RAW264.7 ࡟࠾࠸࡚ LPS ่⃭࡛ㄏᑟࡉࢀࡿ NO ཬࡧ PGE2⏘⏕ࢆᢚไࡍࡿࡇ࡜ࢆ᫂ࡽ࠿࡟ࡋࡓࠋࡉࡽ ࡟㸪HT ࡣ 12.5 μg/mL ௨ୖࡢ⃰ᗘ࡛ࡣ NF-κB ࢩࢢࢼࣝఏ㐩⤒㊰ࢆᢚไࡍࡿࡇ࡜࡛㸪 iNOS㸪COX-2 ཬࡧ IL-1β 㑇ఏᏊࡢⓎ⌧ࢆᢚไࡋ㸪12.5 μg/mL ࡼࡾప࠸⃰ᗘ࡛ࡣู ࡢࢩࢢࢼࣝఏ㐩⤒㊰࡟㛵୚ࡍࡿࡇ࡜࡛⅖⑕཯ᛂࢆㄪ⠇ࡍࡿྍ⬟ᛶࢆぢฟࡋࡓࠋ HT ࡀప⃰ᗘ࡛స⏝ࡋ࡚࠸ࡿࢩࢢࢼࣝఏ㐩⤒㊰࡜ࡋ࡚㸪✀ࠎࡢࢧ࢖ࢺ࢝࢖ࣥࡸ 㓟໬ࢫࢺࣞࢫ➼࡟ࡼࡾάᛶ໬ࡉࢀࡿศ⿣ಁ㐍ᅉᏊάᛶ໬ࢱࣥࣃࢡ㉁࢟ࢼ࣮ࢮ (mitogen-activated protein kinase: MAPK) (43, 44) ࡀ㛵୚ࡍࡿ⤒㊰ࡶ⪃࠼ࡽࢀࡿࠋ MAPK ࡣ㸪ERK1/2㸪p38㸪JNK ࡢ 3 ࡘ࡟኱ูࡉࢀ㸪ࡑࢀࡒࢀࡢୖὶᇦ࡟࠾࠸࡚≉ ␗ⓗ࡞࢝ࢫࢣ࣮ࢻࢆᙧᡂࡋ࡚࠸ࡿ (45)ࠋERK1/2 ࡣቑṪᅉᏊཷᐜయ࡞࡝ࡢୗὶ࡛

25

⑕ᛶࢧ࢖ࢺ࢝࢖ࣥ࡞࡝࡛άᛶ໬ࡉࢀ࢔࣏ࢺ࣮ࢩࢫࢩࢢࢼࣝఏ㐩࡟㛵୚ࡋ࡚࠸ࡿࠋ HT ࡢᢠ⅖⑕స⏝ࡢᶵᗎࡢゎ᫂ࡣ㸪๪స⏝ࡢᑡ࡞࠸᪂つᢠ⅖⑕⸆ࡢ㛤Ⓨ࡟⧅ࡀ

ࡿྍ⬟ᛶࡀ࠶ࡿ࡜⪃࠼㸪⣽⬊ෆࢩࢢࢼࣝᅉᏊࡢ inhibitor ⏝࠸ࡓᐇ㦂ࢆ⾜࠸㸪HT

26

ᮦᩱཬࡧ᪉ἲ

⿕㦂≀㉁➼

HT ཬࡧ LPS ࡢ⏤᮶ཬࡧㄪ〇᪉ἲࡣ➨ 1 ❶࡜ྠᵝ࡛࠶ࡿࠋ

⣽⬊ෆࢩࢢࢼࣝᅉᏊࡢinhibitor ࡜ࡋ࡚㸪࿴ග⣧⸆ᕤᴗᰴᘧ఍♫ࡢ U0126 (ERK1/2

⤒㊰㜼ᐖ) (46)㸪SB203580 (p38 㜼ᐖ) (47) ཬࡧ SP600125 (JNK 㜼ᐖ) (48) ࢆ⏝࠸ࡓࠋ Celastrol (NF-κB ⤒㊰㜼ᐖ) (49) ࡣᮾி໬ᡂᕤᴗᰴᘧ఍♫ࡢ〇ရࢆ⏝࠸ࡓࠋྛ inhibitor ࢆ dimethylsulfoxide (ྠோ໬Ꮫ◊✲ᡤ㸪኱㜰㸪᪥ᮏ) ࢆ⏝࠸࡚ 1 mg/mL ࡢ ⃰ᗘ࡟࡞ࡿࡼ࠺⁐ゎࡋ㸪–80 °C ࡛ಖᏑࡋࡓࠋ ᐇ㦂᪉ἲ 1. ⣽⬊ୖΎ୰ࡢ NO㸪PGE2ཬࡧ TNF-α ࡢ⃰ᗘ ᐃ  ➨1 ❶࡜ྠᵝࡢ᪉ἲ࡛ RAW264.7 ⣽⬊ࢆᇵ㣴ࡋࡓࠋ⮳㐺⣽⬊ᩘ࡟ቑṪᚋ㸪ᢚไ స⏝ࡀ⏕ࡌࡿ⃰ᗘ࡛࠶ࡿ 10 μM ࡢ U0126㸪SB203580㸪SP600125 ཬࡧ celastrol (46-49) ࢆࡑࢀࡒࢀຍ࠼࡚ࡉࡽ࡟ 2 ᫬㛫ᇵ㣴ࡋࡓࠋࡑࡢᚋ㸪1) ᇵ㣴ᾮࡢࡳ㸪2) LPS 0.25 μg/mL㸪3) LPS 0.25 μg/mL㸩HT (1.56–12.5 μg/mL) ࢆࡑࢀࡒࢀῧຍࡋࡓ well ࢆタࡅ CO2࢖࣮ࣥ࢟ࣗ࣋ࢱ࣮ෆ࡛ᇵ㣴ࡋࡓࠋᇵ㣴 24 ᫬㛫ᚋ࡟⣽⬊ୖΎࢆᅇ཰ࡋ

ࡓࠋ⣽⬊ୖΎ୰ࡢNO㸪PGE2ཬࡧTNF-α ⃰ᗘࡢ ᐃࢆ㸪Nitric Oxide (total) detection

kit (Enzo Life Sciences)㸪Prostaglandin E2 ELISA Kit (Abcam) ཬࡧ Mouse TNF alpha

SimpleStep ELISA® Kit (Abcam) ࢆ⏝࠸࡚ᐇ᪋ࡋࡓࠋ

2. Western blotting ࡟ࡼࡿ⣽⬊ෆࢩࢢࢼࣝᅉᏊࡢⓎ⌧ゎᯒ

27

࡚㸪➨1 ❶࡜ྠᵝࡢ᪉ἲ࡛ pNF-κB ཬࡧ pERK1/2 ࡢⓎ⌧ࢆゎᯒࡋࡓࠋ୍ḟᢠయ࡟

ࡣᢠNF-κB㸪ᢠ pNF-κB㸪ᢠ ERK1/2㸪ᢠ pERK1/2㸪ᢠ p38㸪ᢠ pp38㸪ᢠ JNK ཬ

ࡧᢠpJNK (Cell Signaling Technology) ࢆ 0.3%ࢫ࣒࣑࢟ࣝࢡྵ᭷ TBS-T ࡛ 1,000 ಸ

ᕼ㔘ࡋ࡚⏝࠸ࡓࠋ

⤫ィฎ⌮

⤖ᯝࡣ㸪ᖹᆒ ± ᶆ‽ㄗᕪ (S.E.) ࡜ࡋ࡚♧ࡋࡓࠋ᭷ពᕪ᳨ᐃ࡟ࡘ࠸࡚ࡣ㸪࢘࢕

ࣜ࢔࣒ࢬࡢከ㔜ẚ㍑ヨ㦂ࢆ⏝࠸ࡓࠋ᭷ពỈ‽ࡣ୧ഃ5%࡜ࡋࡓࠋ⤫ィゎᯒ࡟ࡣ SAS

28

⤖ᯝ

1. RAW264.7 ⣽⬊ࡢᇵ㣴ᾮ୰ࡢ NO㸪TNF-α㸪ཬࡧ PGE2⃰ᗘ

NO ࡟㛵ࡋ࡚㸪U0126 (ERK1/2 ⤒㊰㜼ᐖ)㸪SB203580 (p38 㜼ᐖ) ཬࡧ SP600125 (JNK 㜼ᐖ) ࡛ฎ⌮ࡋࡓ RAW264.7 ⣽⬊࡟࠾࠸࡚ࡣ㸪LPS ㄏⓎᛶࡢ NO ⏘⏕ࡣ inhibitor ฎ⌮ࡋ࡞࠿ࡗࡓ⣽⬊ (Fig. 5) ࡜ྠ⛬ᗘ࡟ㄆࡵࡽࢀࡓࠋࡲࡓ㸪ࡇࢀࡽ LPS ㄏⓎᛶࡢNO ࡢ⏘⏕ࡣ HT ࡟ࡼࡗ࡚⃰ᗘ౫Ꮡⓗ࡟ᢚไࡉࢀࡓࠋCelastrol (NF-κB ⤒ ㊰㜼ᐖ) ࡛ฎ⌮ࡋࡓ⣽⬊࡟ࡘ࠸࡚ࡣ㸪NO ࡢ⏘⏕ࡣㄆࡵࡽࢀ࡞࠿ࡗࡓ (Fig. 7)ࠋ TNF-α ࡟㛵ࡋ࡚㸪U0126㸪SB203580 ཬࡧ SP600125 ࡛ฎ⌮ࡋࡓ RAW264.7 ⣽ ⬊࡟࠾࠸࡚ࡣ㸪LPS ㄏⓎᛶࡢ TNF-α ⏘⏕ࡣ inhibitor ฎ⌮ࡋ࡞࠿ࡗࡓ⣽⬊ (Fig. 5) ࡜ẚ㍑ࡋ࡚1/5ࠥ1/10 ⛬ᗘ࡟ᢚไࡉࢀࡓࠋ࡞࠾㸪LPS ㄏⓎᛶࡢ TNF-α ⏘⏕ࡣ U0126 ࡛ฎ⌮ࡋࡓ⣽⬊࡟࠾࠸࡚ࡣ㸪HT (1.56–3.13 μg/mL) ࡟ࡼࡗ࡚⃰ᗘ౫Ꮡⓗ࡟ᢚไࡉ ࢀࡓࠋSB203580 ཬࡧ SP600125 ࡛ฎ⌮ࡋࡓ⣽⬊࡟࠾࠸࡚ࡣ HT ῧຍ࡟ࡼࡗ࡚㸪ࡸ ࡸపୗࡍࡿഴྥࡀㄆࡵࡽࢀࡓࡀ㸪᭷ព࡞ኚ໬࡛ࡣ࡞࠿ࡗࡓࠋCelastrol ࡛ฎ⌮ࡋࡓ ⣽⬊࡟࠾࠸࡚ࡣ㸪TNF-α ࡢ⏘⏕ࡣㄆࡵࡽࢀ࡞࠿ࡗࡓ (Fig. 8)ࠋ

PGE2࡟㛵ࡋ࡚㸪U0126㸪SB203580 ཬࡧ SP600125 ࡛ฎ⌮ࡋࡓ RAW264.7 ⣽⬊

࡟࠾࠸࡚ࡣ㸪LPS ㄏⓎᛶࡢ PGE2⏘⏕ࡣinhibitor ฎ⌮ࡋ࡞࠿ࡗࡓ⣽⬊ (Fig. 5) ࡜

ẚ㍑ࡋ࡚1/5 ⛬ᗘ࡟ᢚไࡉࢀࡓࠋ࡞࠾㸪ࡇࢀࡽ LPS ㄏⓎᛶࡢ PGE2⏘⏕ࡣ㸪HT ࡟

ࡼࡗ࡚⃰ᗘ౫Ꮡⓗ࡟ᢚไࡉࢀࡓࠋCelastrol ࡛ฎ⌮ࡋࡓ⣽⬊࡟࠾࠸࡚ࡣ㸪LPS ㄏⓎ

ᛶࡢPGE2⏘⏕ࡢኚ໬ࡣㄆࡵࡽࢀ࡞࠿ࡗࡓ (Fig. 9)ࠋ

29

Western blotting ἲࢆ⏝࠸࡚㸪ྛ inhibitor ࡛ฎ⌮ࡋࡓ RAW264.7 ⣽⬊࡟ࡘ࠸࡚㸪 HT Ꮡᅾࡲࡓࡣ㠀Ꮡᅾୗ࡛ࡢ pNF-κB ཬࡧ pERK1/2 ࡢࢱࣥࣃࢡ㉁Ⓨ⌧ࢆホ౯ࡋࡓࠋ Celastrol ฎ⌮⣽⬊࡛ࡣ㸪LPS (0.25 μg/mL) ࡢῧຍ࡟ࡼࡗ࡚ࡶ㸪pNF-κB ࡢⓎ⌧ࡣ඲

ࡃㄆࡵࡽࢀ࡞࠿ࡗࡓ (Fig. 10A)ࠋU0126 ฎ⌮⣽⬊࡛ࡣ㸪inhibitor ฎ⌮ࡋ࡞࠿ࡗࡓ⣽

⬊ (Fig. 6B) ࡜ẚ㍑ࡋ࡚㸪pERK1/2 ࡢⓎ⌧ࡀ୙᫂░࡛࠶ࡗࡓࠋࡲࡓ㸪pERK1/2 ࡢ

Ⓨ⌧ࡣ㸪HT ࡢᏑᅾ࡟ࡼࡗ࡚ᢚไࡉࢀ࡞࠿ࡗࡓ (Fig. 10B)ࠋp38 ཬࡧ JNK ࡟㛵ࡋ࡚

ࡣLPS ่⃭࡟ࡼࡿࣜࣥ㓟໬ࡀ☜ㄆ࡛ࡁ࡞࠿ࡗࡓࡓࡵ㸪inhibitor ࢆ⏝࠸ࡓᐇ㦂ࡣᐇ

30 ⪃ᐹ HT ࡀస⏝ࡍࡿࢩࢢࢼࣝఏ㐩⤒㊰ࢆ᫂ࡽ࠿࡟ࡍࡿࡓࡵ㸪NF-κB ཬࡧ ERK1/2㸪p38㸪 JNK ࡢ 3 ࡘ MAPK ࡟ᑐࡋ࡚㸪inhibitor ࢆ⏝࠸࡚ྛ⤒㊰ࢆ㜼ᐖࡋࡓ㝿ࡢ HT ࡢస⏝ ࡢኚ໬ࢆホ౯ࡋࡓࠋ NF-κB 㜼ᐖ⸆࡛࠶ࡿ celastrol ࡣ RAW264.7 ⣽⬊࡟࠾ࡅࡿ NO ࡢ⏘⏕ࢆ᏶඲࡟ᢚ ไࡋࡓࡀ㸪ERK1/2 㜼ᐖ⸆࡛࠶ࡿ U0126㸪p38 㜼ᐖ⸆࡛࠶ࡿ SB203580㸪JNK 㜼ᐖ ⸆࡛࠶ࡿSP600125 ࡟ࡘ࠸࡚ࡣ㸪NO ⏘⏕ࡢᢚไస⏝ࡣㄆࡵࡽࢀ࡞࠿ࡗࡓࠋLPS ㄏ ⓎᛶࡢNO ⏘⏕࡜ྛ MAPK ࡢ㛵ಀࡣࡇࢀࡲ࡛࡟」ᩘሗ࿌ࡉࢀ࡚࠸ࡿࠋChan ࡽ (32) ࡣ㸪ERK1/2 㜼ᐖ⸆࡛࠶ࡿ PD-98059 ཬࡧ p38 㜼ᐖ⸆࡛࠶ࡿ SB-203580 ࢆ RAW264.7 ⣽⬊࡟ฎ⌮ᚋ࡟ LPS ่⃭ࡋࡓ⤖ᯝ㸪PD-98059 ฎ⌮⣽⬊࡛ࡣ NO ࡢ⏘⏕ࡀᢚไࡉ ࢀࡓࡀ㸪SB203580 ฎ⌮⣽⬊࡛ࡣᢚไࡣㄆࡵࡽࢀ࡞࠿ࡗࡓ࡜ሗ࿌ࡋ࡚࠸ࡿࠋ୍᪉ ࡛㸪Bhat ࡽ (50) ࡣࢢࣜ࢔⣽⬊ࢆ⏝࠸ࡓᐇ㦂ࢆ⾜࠸㸪ERK1/2㸪p38 ࠸ࡎࢀࡢ⤒㊰ ࢆ㜼ᐖࡋࡓሙྜ࡛ࡶ㸪LPS ㄏⓎᛶࡢ NO ࡣᢚไࡉࢀࡓ࡜ሗ࿌ࡋ࡚࠸ࡿࠋࡲࡓ㸪Chen ࡽ (51) ࡣ LPS ㄏⓎᛶࡢ iNOS ࡢୖ᪼ࡣ p38 ⤒㊰࡟ࡼࡗ࡚ไᚚࡉࢀࡿࡀ㸪ERK1/2 ⤒㊰ࡣ㛵୚ࡋ࡞࠸࡜ሗ࿌ࡋ࡚࠸ࡿࠋJNK ⤒㊰࡟㛵ࡋ࡚ࡶ㸪LPS ㄏⓎᛶࡢ NO ⏘⏕ ࡟ಁ㐍ⓗ࡟ാࡃ࡜࠸࠺ሗ࿌ࡶ࠶ࡿ୍᪉࡛ (52)㸪JNK ⤒㊰ࢆ㜼ᐖࡋ࡚ࡶ NO ⏘⏕࡟ ኚ໬ࡣ࡞࠸࡜࠸࠺ሗ࿌ࡶ࠶ࡿ (32)ࠋ௒ᅇࡢᐇ㦂࡛ࡣ㸪NF-κB 㜼ᐖ⸆࡛࠶ࡿ celastrol ௨እࡣNO ⏘⏕࡟┤᥋ᙳ㡪ࢆ୚࠼࡞࠿ࡗࡓࠋᮏ⤖ᯝࡼࡾ㸪ྛ MAPK ࡣ LPS (0.25 μg/mL) ่⃭᮲௳ୗࡢ RAW264.7 ⣽⬊࡟࠾࠸࡚ࡣ༢⊂࡛ࡣ NO ࡢ⏘⏕ࢆಁ㐍ࡍࡿ ࡇ࡜ࡣ࡞ࡃ㸪NF-κB ࡢάᛶ໬ࡀ NO ࡢ⏘⏕࡟୺యⓗ࡟㛵ࢃࡗ࡚࠸ࡿྍ⬟ᛶࡀ♧၀ ࡉࢀࡓࠋᮏ⤖ᯝࡣ㸪ྛMAPK ࡀ NF-κB ࡢάᛶ໬࡟⿵㊊ⓗ࡟ാࡃ࡜࠸࠺◊✲ሗ࿌

31

(53) ࡜୍⮴ࡍࡿࡶࡢ࡛࠶ࡿࠋࡲࡓ㸪p38 ཬࡧ JNK ⤒㊰࡛ࡣ activator protein 1 (AP-1)

ࡢάᛶ໬ࢆ௓ࡋ࡚iNOS ࡢ⏘⏕ࢆቑຍࡉࡏࡿࡀ㸪AP-1 ࡢάᛶࡣ LPS ่⃭࡟ࡼࡗ࡚ ㉳ࡇࡽ࡞࠸࡜࠸࠺ሗ࿌ࡶ࠶ࡿ (54, 55)ࠋࡇࡢሗ࿌ࡣ㸪௒ᅇ Western blotting ࡟࠾࠸ ࡚LPS ่⃭࡟ࡼࡿ p38 ཬࡧ JNK ࡢࣜࣥ㓟໬ࢆᤊ࠼ࡽࢀ࡞࠿ࡗࡓ஦ᐇࢆ⿬௜ࡅࡿ ࡶࡢ࡜⪃࠼ࡽࢀࡿࠋ HT ࡣྛ MAPK ࡢ㜼ᐖࡢ᭷↓࡟㛵ࢃࡽࡎ㸪⃰ᗘ౫Ꮡⓗ࡟ NO ⏘⏕ࢆᢚไࡋࡓࡇ ࡜࠿ࡽ㸪HT ࡢస⏝Ⅼࡣ ERK1/2㸪p38㸪JNK ⤒㊰ୖ࡟ࡣ↓ࡃ㸪࠿ࡘ NF-κB ⤒㊰ࡼ ࡾࡶୗὶ࡟࠶ࡿྍ⬟ᛶࡀ㧗࠸࡜⪃࠼ࡽࢀࡓࠋ➨ 1 ❶ࡢ⤖ᯝࢆ㋃ࡲ࠼ࡿ࡜㸪HT ࡣ 12.5 μg/mL ࡢ⃰ᗘ࡛ࡣ NF-κB ࢩࢢࢼࣝఏ㐩⤒㊰ࢆᢚไࡍࡿࡇ࡜࡛㸪iNOS㸪COX-2 ཬࡧIL-1β 㑇ఏᏊࡢⓎ⌧ࢆᢚไࡋ㸪12.5 μg/mL ࡼࡾప࠸⃰ᗘ࡛ࡣ NF-κB ⤒㊰ࡼࡾ ࡶୗὶ࡟࠶ࡿࢩࢢࢼࣝ⤒㊰࡟స⏝ࡋ࡚㸪ୖグࡢ㑇ఏᏊ⩌ࢆᢚไࡋ࡚࠸ࡿ࡜⪃࠼ࡽ ࢀࡿࠋ TNF-α ࡟㛵ࡋ࡚㸪U0126 (ERK1/2 ⤒㊰㜼ᐖ)㸪SB203580 (p38 㜼ᐖ) ཬࡧ SP600125 (JNK 㜼ᐖ) ࡛ฎ⌮ࡋࡓ⣽⬊࡛ࡣ㸪LPS ㄏⓎᛶࡢ TNF-α ⏘⏕ࡣ inhibitor ฎ⌮ࡋ࡞ ࠿ࡗࡓ⣽⬊࡜ẚ㍑ࡋ࡚ 1/5ࠥ1/10 ⛬ᗘ࡟ᢚไࡉࢀࡓࠋᮏ⤖ᯝࡣ㸪ྠᵝ࡟ inhibitor ࢆ⏝࠸ࡓ㐣ཤࡢሗ࿌࡜ࡶ୍⮴ࡋ࡚࠾ࡾ㸪ERK1/2 ཬࡧ JNK ⤒㊰ࡀ TNF 㑇ఏᏊ㌿ ෗࡟㸪ࡲࡓ p38 ⤒㊰ࡀ㌿෗ᚋࡢㄪ⠇㐣⛬࡟㛵୚ࡋ࡚࠸ࡿ࡜⪃࠼ࡽࢀࡓ (56)ࠋ Celastrol ࡛ฎ⌮ࡋࡓ⣽⬊࡟ࡘ࠸࡚ࡣ㸪TNF-α ࡢ⏘⏕ࡣ඲ࡃㄆࡵࡽࢀ࡞࠿ࡗࡓࡇ࡜ ࠿ࡽ㸪NO ࡜ྠᵝ࡟ NF-κB ࡢάᛶ໬ࡀ TNF-α ࡢ⏘⏕࡟୺యⓗ࡟㛵ࢃࡗ࡚࠸ࡿࡇ࡜ ࡀ♧၀ࡉࢀࡓࠋ࡞࠾㸪ࡇࢀࡽࡢ TNF-α ⏘⏕࡟ࡘ࠸࡚㸪SB203580 ཬࡧ SP600125 ࡛ฎ⌮ࡋࡓ⣽⬊࡟ࡘ࠸࡚ࡣHT ฎ⌮࡟ࡼࡗ࡚పୗഴྥࢆ♧ࡋ㸪U0126 ࡛ฎ⌮ࡋࡓ ⣽⬊࡟ࡘ࠸࡚ࡣ㸪HT ࡟ࡼࡗ࡚⃰ᗘ౫Ꮡⓗ࡟᭷ព࡟ᢚไࡉࢀࡓࠋ➨ 1 ❶࡟࠾࠸࡚㸪

32

HT ࡣ TNF-α ⏘⏕࡟ᑐࡋᢚไⓗ࡞స⏝ࢆ♧ࡋࡓࡇ࡜࠿ࡽ㸪௒ᅇࡢ⤖ᯝࡣ๓ᅇࡢ⤖ ᯝ࡜୍⮴ࡍࡿࡶࡢ࡛࠶ࡗࡓࠋTNF-α ⏘⏕ᢚไ࡟㛵ࡋ࡚㸪HT ࡣ U0126㸪SB203580

ཬࡧSP600125 ࡜┦஌ຠᯝࢆ♧ࡋࡓ࡜ᤊ࠼ࡿ࡜㸪HT ࡢస⏝Ⅼࡣ ERK1/2㸪p38㸪JNK

⤒㊰ୖ௨እ࡟࠶ࡿ࡜⪃࠼ࡽࢀࡓࠋ࡞࠾㸪Western blotting ࡟࠾࠸࡚㸪U0126 ฎ⌮᮲

௳ୗ࡛ HT ฎ⌮࡟ࡼࡿ pERK1/2 ࡢᢚไࡣㄆࡵࡽࢀ࡚࠾ࡽࡎ㸪HT ࡢస⏝Ⅼࡀ

ERK1/2 ⤒㊰ୖ࡟↓࠸࡜࠸࠺௬ㄝࢆ⿬௜ࡅࡿ⤖ᯝ࡛࠶ࡗࡓࠋ

PGE2࡟㛵ࡋ࡚㸪U0126 (ERK1/2 ⤒㊰㜼ᐖ)㸪SB203580 (p38 㜼ᐖ) ཬࡧ SP600125

(JNK 㜼ᐖ) ࡛ฎ⌮ࡋࡓ⣽⬊࡛ࡣ㸪LPS ㄏⓎᛶࡢ PGE2⏘⏕ࡣinhibitor ฎ⌮ࡋ࡞࠿ ࡗࡓ⣽⬊࡜ẚ㍑ࡋ࡚1/5 ⛬ᗘ࡟ᢚไࡉࢀࡓࠋTNF-α ྠᵝ࡟ PGE2ࡀMAPK ࡟ࡼࡾ ไᚚࡉࢀ࡚࠸ࡿࡇ࡜ࡣከᩘሗ࿌ࡉࢀ࡚࠸ࡿ (57–59)ࠋࡲࡓ㸪NO ࡜ TNF-α ྠᵝ࡟ celastrol ࡛ฎ⌮ࡋࡓ⣽⬊࡟ࡘ࠸࡚ࡣ㸪LPS ㄏⓎᛶࡢ PGE2⏘⏕ቑຍࡣㄆࡵࡽࢀ࡞࠿ ࡗࡓࠋࡇࢀࡽࡢ⤖ᯝ࠿ࡽ㸪LPS ㄏⓎᛶࡢ⅖⑕࡟ࡣ NF-κB ⤒㊰ࡀ୺య࡛㛵ࢃࡗ࡚࠸ ࡿࡇ࡜ࡀ☜ㄆࡉࢀࡓࠋ࡞࠾㸪TNF-α ࡜␗࡞ࡾ㸪ࡇࢀࡽࡢ PGE2⏘⏕ࡣ㸪U0126㸪 SB203580 ཬࡧ SP600125 ฎ⌮⣽⬊ࡢ࠸ࡎࢀ࡟࠾࠸࡚ࡶ㸪HT ࡟ࡼࡗ࡚⃰ᗘ౫Ꮡⓗ ࡟ᢚไࡉࢀࡓࠋ ➨2 ❶࡟࠾ࡅࡿ⤖ᯝ࡟ࡼࡾ㸪HT ࡢస⏝Ⅼࡣ ERK1/2㸪p38㸪JNK ⤒㊰ୖ࡟ࡣ↓ ࡃ㸪࠿ࡘ NF-κB ⤒㊰ࡼࡾࡶୗὶ࡟࠶ࡿྍ⬟ᛶࡀ㧗࠸ࡇ࡜ࡀ᫂ࡽ࠿࡜࡞ࡗࡓ (Fig. 11)ࠋࡲࡓ㸪HT ࡣ㸪NO㸪TNF-α ཬࡧ PGE2⏘⏕࡟ᑐࡋ࡚ᢚไⓗ࡟ാࡃࡇ࡜ࡀ☜࠿ ࡵࡽࢀࡓࠋ➨1 ❶ཬࡧ➨ 2 ❶ࡢ⤖ᯝ࠿ࡽ㸪HT ࡣ NSAIDs ࡸ COX-2 㜼ᐖ๣➼ࡢ᪤ Ꮡࡢᢠ⅖⑕⸆࡜ࡣ␗࡞ࡿ࣓࢝ࢽࢬ࣒࡛ᢠ⅖⑕స⏝ࢆ♧ࡍࡇ࡜ࡀ᫂ࡽ࠿࡜࡞ࡗࡓࠋ ḟ❶࡛ࡣ㸪ື≀ࣔࢹࣝࢆ⏝࠸࡚HT ࡢᢠ⅖⑕స⏝ࢆホ౯ࡋࡓࠋ

33 ᑠᣓ ➨1 ❶࡟࠾࠸࡚㸪HT ࡀ㸪࣐࢘ࢫ࣐ࢡࣟࣇ࢓࣮ࢪᵝ⣽⬊ᰴ RAW264.7 ࡟࠾࠸࡚ LPS ่⃭࡛ㄏᑟࡉࢀࡿ NO ཬࡧ PGE2⏘⏕ࢆᢚไࡍࡿࡇ࡜ࢆ᫂ࡽ࠿࡟ࡋࡓࠋ HT ࡀస⏝ࡍࡿࢩࢢࢼࣝఏ㐩⤒㊰ࢆ᫂ࡽ࠿࡟ࡍࡿࡓࡵ㸪NF-κB ཬࡧ ERK1/2㸪p38㸪 JNK ࡢ 3 ࡘ MAPK ࡟ᑐࡋ࡚㸪inhibitor ࢆ⏝࠸࡚ྛ⤒㊰ࢆ㜼ᐖࡋࡓ㝿ࡢ HT ࡢస⏝ ࡢኚ໬ࢆホ౯ࡋࡓࠋ ⤖ᯝ㸪MAPK ࡢ㜼ᐖ࡟ࡼࡾ TNF-α ཬࡧ PGE2ࡢ⏘⏕ࡣపୗࡋ㸪HT ࡢฎ⌮࡟ࡼ ࡾ⏘⏕㔞ࡣࡉࡽ࡟పୗࡋࡓࠋࡲࡓ㸪NO ࡢ⏘⏕㔞ࡣ MAPK ࡢ㜼ᐖ࡟ࡼࡾኚ໬ࡋ࡞ ࠿ࡗࡓࡀ㸪HT ࡢฎ⌮࡟ࡼࡾపୗࡋࡓࠋᮏ⤖ᯝࡼࡾ㸪HT ࡢస⏝Ⅼࡣ ERK1/2㸪p38㸪 JNK ⤒㊰ୖ࡟ࡣ↓ࡃ㸪࠿ࡘ NF-κB ⤒㊰ࡼࡾࡶୗὶ࡟࠶ࡿྍ⬟ᛶࡀ㧗࠸ࡇ࡜ࡀ᫂ࡽ ࠿࡜࡞ࡗࡓࠋ➨1 ❶ࡢ⤖ᯝࢆ㋃ࡲ࠼ࡿ࡜㸪HT ࡣ 12.5 μg/mL ࡢ⃰ᗘ࡛ࡣ NF-κB ࢩ ࢢࢼࣝఏ㐩⤒㊰ࢆᢚไࡍࡿࡇ࡜࡛㸪iNOS㸪COX-2 ཬࡧ IL-1β 㑇ఏᏊࡢⓎ⌧ࢆᢚไ ࡋ㸪12.5 μg/mL ࡼࡾప࠸⃰ᗘ࡛ࡣ NF-κB ⤒㊰ࡼࡾࡶୗὶ࡟࠶ࡿࢩࢢࢼࣝ⤒㊰࡟స ⏝ࡋ࡚࠸ࡿ࡜⪃࠼ࡽࢀࡓࠋ ࡲࡓ㸪HT ࡣ㸪NO ཬࡧ PGE2⏘⏕࡟ᑐࡋ࡚ࡣᢚไⓗ࡟ാࡃࡀ㸪TNF-α ࡟ᑐࡋ࡚ ࡣస⏝ࢆ♧ࡉ࡞࠸Ⅼࡀ☜࠿ࡵࡽࢀࡓࠋࡇࢀࡣ᪤Ꮡࡢᢠ⅖⑕⸆࡛࠶ࡿ NSAIDs ࡸ COX-2 㜼ᐖ๣࡟ࡣぢࡽࢀ࡞࠸స⏝࡛࠶ࡗࡓࠋ ➨1 ❶ཬࡧ➨ 2 ❶ࡢ⤖ᯝ࠿ࡽ㸪HT ࡣ᪤Ꮡࡢᢠ⅖⑕⸆࡜ࡣ␗࡞ࡿ࣓࢝ࢽࢬ࣒࡛ ᢠ⅖⑕స⏝ࢆ♧ࡍࡇ࡜ࡀ᫂ࡽ࠿࡜࡞ࡗࡓࠋḟ࡟㸪ື≀ࣔࢹࣝࢆ⏝࠸࡚HT ࡢᢠ⅖ ⑕స⏝ࢆホ౯ࡋࡓࠋ

34

ᅗ⾲

Fig. 7. NO production by RAW264.7 cells cultured with LPS (0.25 μg/mL) and HT (0–12.5 μg/mL) for 24 h following the treatment with cellular signaling pathway inhibitors.

Cells were treated with (A) 10 μM SB203580, (B) 10 μM SP600125, (C) 10 μM U0126, or (D) 10 μM celastrol for 2 h before the LPS stimulation. NO concentration in the culture supernatant of RAW264.7 cells was measured by Griess method. Mean ± S.E. *P < 0.05, **P < 0.01, ***P < 0.001 (Williams’ multiple comparison test) (n = 3).

SB203580 U0126

35

Fig. 8. TNF-α production by RAW264.7 cells cultured with LPS (0.25 μg/mL) and HT (0–12.5 μg/mL) for 24 h following the treatment with cellular signaling pathway inhibitors.

Cells were treated with (A) 10 μM SB203580, (B) 10 μM SP600125, (C) 10 μM U0126, or (D) 10 μM celastrol for 2 h before the LPS stimulation. TNF-α concentration in the culture supernatant of RAW264.7 cells was measured by ELISA. Mean ± S.E. *P < 0.05, ***P < 0.001 (Williams’ multiple comparison test) (n = 3).

SB203580

SP600125

U0126

36

Fig. 9. PGE2 production by RAW264.7 cells cultured with LPS (0.25 μg/mL) and

HT (0–12.5 μg/mL) for 24 h following the treatment with cellular signaling pathway inhibitors.

Cells were treated with (A) 10 μM SB203580, (B) 10 μM SP600125, (C) 10 μM U0126,

or (D) 10 μM celastrol for 2 h before the LPS stimulation. PGE2 concentration in the

culture supernatant of RAW264.7 cells was measured by ELISA. Mean ± S.E. *P < 0.05, **P < 0.01, ***P < 0.001 (Williams’ multiple comparison test) (n = 3).

SB203580

SP600125

U0126

37

Fig. 10. Expression of pNF-κB (A), pERK1/2 (B) in RAW264.7 cells stimulated with LPS (0.25 μg/mL) and HT (0–12.5 μg/mL) following the treatment with cellular signaling pathway inhibitors.

Western blotting analysis was used to determine the levels of these proteins in the cells treated for 15 min. Representative results of duplicate experiments are shown. Cells were treated with (A) 10 μM celastrol or (B) 10 μM U0126 for 2 h before the LPS stimulation. The graph shows the relative protein expression levels measured by Quantity One Ver 4.5.

Celastrol

38

39

➨3 ❶

40

⥴ゝ

⌧ᅾ㸪㛵⠇࣐ࣜ࢘ࢳཬࡧኚᙧᛶ㛵⠇⑕࡞࡝៏ᛶ⅖⑕ᛶ⑊③࡟ᑐࡋ࡚᪤Ꮡࡢ NSAIDs ཬࡧ COX-2 㑅ᢥⓗ㜼ᐖ๣࡜ྠ➼௨ୖࡢᢠ⅖⑕࣭㙠③ຠᯝࢆ♧ࡋ㸪ᾘ໬⟶ 㞀ᐖ➼ࡢ๪స⏝ࣜࢫࢡࡀ㍍ῶࡉࢀࡓ᪂つᾘ⅖㙠③⸆ࡢ㛤Ⓨࡀᮃࡲࢀ࡚࠸ࡿ (60)ࠋ

ᡃࠎࡣin vitro ◊✲࡟࠾࠸࡚㸪HT ࡀ㸪LPS ㄏⓎᛶࡢ iNOS㸪COX-2 ཬࡧ IL-1β 㑇

ఏᏊࡢⓎ⌧ࢆᢚไࡋ㸪⤖ᯝ࡜ࡋ࡚ NO ཬࡧ PGE2ࡢ⏘⏕㔞ࢆῶᑡࡉࡏࡿࡇ࡜ࢆぢ ฟࡋࡓࠋᮏ◊✲࡛ࡣ㸪HT ࡢ᪂つᾘ⅖㙠③⸆࡜ࡋ࡚ࡢྍ⬟ᛶࢆぢᤣ࠼㸪in vivo ࡟ ࠾ࡅࡿᢠ⅖⑕άᛶࢆホ౯ࡍࡿࡇ࡜ࢆ┠ⓗ࡜ࡋࡓࠋ ࢝ࣛࢤࢽࣥㄏⓎࣛࢵࢺ⫥ᾋ⭘ࣔࢹࣝࡣ㸪ᢠ⅖⑕⸆ࡢຠᯝࢆホ౯ࡍࡿࡢ࡟㐺ࡋ࡚ ࠸ࡿ࡜⪃࠼ࡽࢀ࡚࠸ࡿ (61, 62)ࠋ࢝ࣛࢤࢽࣥㄏⓎࣛࢵࢺ⫥ᾋ⭘ࣔࢹࣝ࡟࠾ࡅࡿ⭘ ⬽ࡣᛴᛶⁱฟ࡟ࡼࡿ⭘⬽࡛࠶ࡿ (63)ࠋ࢝ࣛࢤࢽࣥࡣ PG㸪ࣟ࢖ࢥࢺ࢚ࣜࣥ㸪ࣄࢫ ࢱ࣑ࣥ㸪ࢭࣟࢺࢽࣥ㸪ࣈࣛࢪ࢟ࢽࣥཬࡧTNF-α ࢆྵࡴ⅖⑕ㄏⓎᛶ࣓ࢹ࢕࢚࣮ࢱ࣮ ࡢᨺฟࢆ่⃭ࡍࡿࠋࡇࢀࡽࡢ⅖⑕ㄏⓎᛶ࣓ࢹ࢕࢚࣮ࢱ࣮࡟ࡼࡾ㸪ᒁᡤ࡛ᚤᑠᚠ⎔ ⣔ࡢᣑᙇ࡜඘⾑㸪⾑⟶㏱㐣ᛶࡢஹ㐍ࡀ㉳ࡇࡾ㸪⥆࠸࡚ⓑ⾑⌫ࡢ⾑⟶እ࡛ࡢ㐟㉮ࡀ ㉳ࡇࡿࠋ⾑⟶㏱㐣ᛶஹ㐍ࡣ⾑₢⺮ⓑࢆకࡗ࡚Ỉࡀ⾑⟶ࡢእ࡬₃ࢀฟࡍࡇ࡜࡟ࡼࡗ ࡚㉳ࡇࡿࠋࡇࢀ࡟ࡼࡾᢞ୚ᚋ5 ᫬㛫௨ෆ࡟㐍⾜ᛶࡢᾋ⭘ࢆㄏⓎࡍࡿ (61)ࠋ ᮏ◊✲࡛ࡣ㸪࢝ࣛࢤࢽࣥㄏⓎࣛࢵࢺ⫥ᾋ⭘ࣔࢹࣝࢆ⏝࠸࡚HT ࡢᢠ⅖⑕άᛶࢆ ホ౯ࡋࡓࠋຍ࠼࡚㸪௦⾲ⓗ࡞NSAIDs ࡢ࢖ࣥࢻ࣓ࢱࢩࣥ (64) ཬࡧ㑅ᢥⓗ COX-2 㜼ᐖ๣࡛࠶ࡿࢭࣞࢥ࢟ࢩࣈ (65) ࡞࡝ࡢ᪤Ꮡ἞⒪⸆࡜᭷ຠᛶࢆẚ㍑ࡋࡓࠋ

41 ᮦᩱཬࡧ᪉ἲ ヨ㦂≀㉁ࡢ⏤᮶ HT ࡢ⏤᮶ࡣ➨ 1 ❶࡜ྠᵝ࡛࠶ࡿࠋ࢖ࣥࢻ࣓ࢱࢩࣥ㸪࢝ࣛࢤࢽࣥཬࡧ፹య࡜ࡋ ࡚⏝࠸ࡓ0.5 w/v% ࣓ࢳࣝࢭ࣮ࣝࣟࢫ⁐ᾮ (0.5 % MC) ࡣ㸪࿴ග⣧⸆ᕤᴗᰴᘧ఍♫ ࠿ࡽ㸪ࢭࣞࢥ࢟ࢩࣈࡣᮾி໬ᡂᕤᴗᰴᘧ఍♫࠿ࡽࡑࢀࡒࢀ㉎ධࡋࡓࠋ ヨ㦂≀㉁ࡢㄪ〇 (1) HT ࡢㄪ〇 100 mg/mL ࡢ⁐ᾮࢆ⵨␃Ỉ࡛ 2 ಸᕼ㔘ࡋ࡚㸪50 mg/mL ࡟ㄪ〇ࡋࡓࠋ (2) ࢖ࣥࢻ࣓ࢱࢩࣥᠱ⃮ᾮࡢㄪ〇 ࢖ࣥࢻ࣓ࢱࢩࣥࢆ㐺㔞⛗㔞ࡋ㸪࣓ࣀ࢘ங㖊ࢆ⏝࠸࡚඘ศ࡟⢊○ࡋ㸪0.5% MC ࢆ ᚎࠎ࡟ຍ࠼࡚3 mg/mL ࡢᠱ⃮ᾮ࡟࡞ࡿࡼ࠺࡟⏝᫬ㄪ〇ࡋࡓࠋ (3) ࢭࣞࢥ࢟ࢩࣈᠱ⃮ᾮࡢㄪ〇 ࢭࣞࢥ࢟ࢩࣈࢆ㐺㔞⛗㔞ࡋ㸪࣓ࣀ࢘ங㖊ࢆ⏝࠸࡚඘ศ࡟⢊○ࡋ㸪0.5% MC ࢆ ᚎࠎ࡟ຍ࠼࡚10 mg/mL ⁐ᾮࡢᠱ⃮ᾮ࡟࡞ࡿࡼ࠺࡟⏝᫬ㄪ〇ࡋࡓࠋ (4) ࢝ࣛࢤࢽࣥ⁐ᾮࡢㄪ〇 ࢝ࣛࢤࢽࣥࢆ㐺ᙜ㔞⛗㔞ࡋ㸪1.0%࡟࡞ࡿࡼ࠺࡟⵨␃Ỉࢆຍ࠼㸪ࢫࢱ࣮࣮ࣛࢆ⏝ ࠸୍࡚ᬌ᧠ᢾࡋ࡚ㄪ〇ࡋࡓࠋ

42 ౪ヨື≀ ᙜヨ㦂ࡣ㸪⛉◊〇⸆ᰴᘧ఍♫ (⸨ᯞ㸪㟼ᒸ┴㸪᪥ᮏ㸪HS ㈈ᅋㄆᐃ␒ྕ㸸15-047) ࡢື≀ᐇ㦂ࡢᐇ᪋࡟㛵ࡍࡿつᐃཬࡧࡑࡢ௚ࡢ㛵ಀἲ௧ࢆ㑂Ᏺࡋ㸪ᢎㄆࡉࢀࡓື≀ ᐇ㦂ィ⏬᭩ (チྍ␒ྕ㸸S16-012) ࡟ᚑࡗ࡚ᐇ᪋ࡋࡓࠋ5 㐌㱋ࡢ Sprague–Dawley (Crl:CD (SD) ) ⣔⤫ࡢ㞝ᛶࣛࢵࢺࢆ᪥ᮏࢳ࣮ࣕࣝࢫ࣭ࣜࣂ࣮ᰴᘧ఍♫ (ཌᮌ⦾Ṫࢭ ࣥࢱ࣮㸪⚄ዉᕝ┴㸪᪥ᮏ) ࠿ࡽ㉎ධࡋ㸪7 ᪥㛫ᐇ㦂⎔ቃ࡟㥆໬ࡉࡏࡓࠋ㣫⫱ᐊࡣ  ᗘ23 ± 3 ºC㸪‵ᗘ 50 ± 20%࡟タᐃࡋ㸪㣫⫱ᐊෆࡢ᥮Ẽ㢖ᗘࡣ 1 ᫬㛫࠶ࡓࡾ 17 ᅇ௨ୖ࡜ࡋࡓࠋ᫂ᬯࢧ࢖ࢡࣝࢆ12 ᫬㛫࡜ࡋ㸪7:00–19:00 ࢆ᫂ᮇ࡜ࡋࡓࠋ1 ࢣ࣮ࢪ (W 450×D 500×H 350mm㸪㔠⥙ࢣ࣮ࢪ) ᙜࡓࡾ 1 ༉ࡢࣛࢵࢺࢆ཰ᐜࡋࡓࠋ㥆໬ᮇ 㛫୰㸪ᇶ♏㣫ᩱ (࢚࢜ࣜࣥࢱࣝ㓝ẕᕤᴗᰴᘧ఍♫㸪ᮾி㸪᪥ᮏ) ཬࡧୖỈ (⸨ᯞ ᕷ㸪㟼ᒸ┴㸪᪥ᮏ) ࢆ⮬⏤ᦤྲྀࡉࡏࡓࠋ㥆໬ᚋ㸪ࣛࢵࢺࢆᒙู↓సⅭ໬ἲ࡟࡚㸪 ᭱᪂ࡢయ㔜࡟ᇶ࡙࠸࡚ྛ⩌࡟᣺ࡾศࡅヨ㦂࡟౑⏝ࡋࡓࠋ ᐇ㦂᪉ἲ ⩌ᵓᡂࢆ௨ୗ࡟♧ࡍࠋ ⩌␒ྕ ⩌ ㊊㋲⓶ୗᢞ୚ ヨ㦂≀㉁ ᢞ୚ᐜ㔞 ౛ᩘ 1 ↓ฎ⨨ᑐ↷⩌ ⏕⌮㣗ሷᾮ 0.5% MC 10 mL/kg 5 2 㐺ྜᑐ↷⩌ 1.0% ࢝ࣛࢤࢽࣥ⁐ ᾮ 0.5% MC 3 HT 500 mg/kg ⩌ HT 50 mg/mL 4 ࢭࣞࢥ࢟ࢩࣈ 100 mg/kg ⩌ ࢭࣞࢥ࢟ࢩࣈ 10 mg/mL 5 ࢖ࣥࢻ࣓ࢱࢩࣥ 30 mg/kg ⩌ ࢖ࣥࢻ࣓ࢱࢩࣥ 3 mg/mL

43 ↓ฎ⨨ᑐ↷⩌ࡣ⾑ᾮᏛ᳨ᰝ㸪⾑ᾮ⏕໬Ꮫ᳨ᰝ࡟࠾ࡅࡿẚ㍑ᑐ↷࡜ࡋ࡚タᐃࡋࡓࠋ ⏝㔞タᐃ࡟㛵ࡋ࡚㸪HT ࡣ 2 g/kg ࡢ⏝㔞࡛ࣛࢵࢺ࡟ẘᛶࢆ♧ࡉ࡞࠸࡜ሗ࿌ࡉࢀ࡚ ࠸ࡿࡇ࡜࠿ࡽ (36), ẘᛶᙳ㡪ࡀ↓ࡃ⸆ຠࡀᮇᚅ࡛ࡁࡿ⏝㔞࡜ࡋ࡚ 500 mg/kg ࢆタ ᐃࡋࡓࠋ➨ 4 ❶࡟࠾࠸࡚࢔ࢫࣆࣜࣥ࡜ࡢే⏝࡟ࡼࡿ๪స⏝ᙳ㡪ࢆ᳨ウࡍࡿࡓࡵ㸪 ࢭࣞࢥ࢟ࢩࣈ࡟㛵ࡋ࡚ࡣ㸪ẚ㍑ᑐ↷࡜ࡋ࡚㸪⸆ຠࢆ♧ࡋ࠿ࡘ༢⊂ࡢᢞ୚࡛ࡣᾘ໬ ⟶㞀ᐖࢆ♧ࡉ࡞࠸᪤▱⏝㔞 (66) ࢆタᐃࡋࡓࠋ࢖ࣥࢻ࣓ࢱࢩࣥ࡟㛵ࡋ࡚ࡣ㸪㝧ᛶ ᑐ↷࡜ࡋ࡚⸆ຠࢆ♧ࡋ࠿ࡘ༢⊂ࡢᢞ୚࡛ᾘ໬⟶㞀ᐖࢆ♧ࡍ᪤▱⏝㔞 (67) ࢆタᐃ ࡋࡓࠋ 1. ⫥ᐜ✚ ᐃཬࡧᾋ⭘⋡࡜ᾋ⭘ᢚไ⋡ࡢ⟬ฟ ヨ㦂≀㉁ࢆ10 mL/kg ࡢᐜ㔞࡛ࣛࢵࢺ࡟ᙉไ⤒ཱྀᢞ୚ࡋࡓࠋ᪤ሗ (68) ࡟ᇶ࡙ࡁ ᢞ୚1 ᫬㛫ᚋ㸪1.0%࢝ࣛࢤࢽࣥ⁐ᾮ 0.1mL ࢆࣛࢵࢺྑᚋ㊊ࡢ㊊㋲࡟⓶ୗᢞ୚ࡋࡓࠋ

⫥ᐜ✚ࡣ㸪ࣉࣞࢳࢫ࣓࣮ࣔࢱ࣮ (Model 7141, Ugo Basile, Milan, Italy) ࢆ⏝࠸࡚㸪

࢝ࣛࢤࢽࣥᢞ୚๓ཬࡧᢞ୚ᚋ1㸪2㸪3㸪4 ཬࡧ 5 ᫬㛫࡟ ᐃࡋࡓࠋ᪤ሗ (61) ࡟ᇶ

࡙ࡁ௨ୗࡢᘧࢆ౑⏝ࡋ࡚㸪ᾋ⭘⋡ (% swelling) ཬࡧᾋ⭘ᢚไ⋡ (% inhibition) ࢆ ィ⟬ࡋࡓࠋ

% swelling = (VX − V0) /V0 × 100 (x = 1–5)

% Inhibition = (Control – Test) /Control × 100

V0ཬࡧVXࡣࡑࢀࡒࢀ࢝ࣛࢤࢽࣥᢞ୚๓ (0 ᫬㛫) ཬࡧ 1㸪2㸪3㸪4 ཬࡧ 5 ᫬㛫

44 ⩌࡟࠾ࡅࡿᖹᆒᐜ✚࡛࠶ࡿࠋ 2. ⫥⤌⧊ࡢ⑓⌮⤌⧊Ꮫⓗ᳨ᰝ ࡍ࡭࡚ࡢື≀ࢆ㸪⫥ᐜ✚ࡢ᭱⤊ ᐃᚋ㸪࢖ࢯࣇࣝࣛࣥ྾ධ㯞㓉ୗ࡛⭡㒊኱㟼⬦ ཬࡧ኱ື⬦ࢆษ᩿ࡋ࡚ᨺ⾑࡟ࡼࡾᏳᴦẅࡋࡓࠋᏳᴦẅᚋ㸪⫥⤌⧊ࢆ᥇ྲྀࡋ10 vol% ୰ᛶ⦆⾪࣐࣍ࣝࣜࣥᾮ࡛ᅛᐃᚋ㸪⬺⬡ཬࡧࢠ㓟㺃ࢡ࢚ࣥ㓟ࢼࢺ࣒ࣜ࢘ἲ࡟ࡼࡿ⬺ ⅊ࢆ⾜ࡗࡓࠋᅛᐃᚋ㸪⬺Ỉ㸪⬺⬡㸪ࣃࣛࣇ࢕ࣥᾐ㏱ࡋ㸪ࣃࣛࣇ࢕ࣥໟᇙࡋࡓࠋࣃ ࣛࣇ࢕ࣥࣈࣟࢵࢡࡣⷧษࡋ㸪࣐࣊ࢺ࢟ࢩ࣭࢚ࣜࣥ࢜ࢪࣥ (H.E.) ᰁⰍࢆ᪋ࡋࡓࠋ ᰁⰍᶆᮏࢆ᳨㙾ࡋ㸪௨ୗࡢᇶ‽࡟ᚑ࠸㸪ΰྜ⣽⬊ᾐ₶ཬࡧᾋ⭘ࡢ⛬ᗘ࡟ࡘ࠸࡚⑓

⌮Ꮫⓗ࡞ࢢ࣮ࣞࢻ௜ࡅࢆ⾜ࡗࡓ㸸0, negative; 1, minimal; 2, slight; 3, moderate.

3. ⾑ᾮᏛⓗ᳨ᰝ

Ᏻᴦẅ᫬࡟ EDTA-2K ࢆ⏝࠸࡚᥇ྲྀࡋࡓ⾑ᾮࢆ⏝࠸࡚㸪⥲ྜ⾑ᾮ᳨ᰝ⿦⨨

(ADVIA120, Bayer AG, Leverkusen, Germany) ࡟ࡼࡾ௨ୗࡢ᳨ᰝࢆ⾜ࡗࡓࠋ ࣭⥲ྜ⾑ᾮ᳨ᰝ⿦⨨ (ADVIA120) ࡟ࡼࡿ᳨ᰝ㡯┠㸪༢఩ཬࡧ ᐃ᪉ἲ ㉥⾑⌫ᩘ _{RBC ×10}6_/μL 2 ゅᗘ䝺䞊䝄䞊䝣䝻䞊䝃䜲䝖䝯䝖䝸 䞊ἲ ࣊ࣔࢢࣟࣅࣥ⃰ᗘ Hemoglobin g/dL 䝅䜰䞁䝯䝖䝦䝰䜾䝻䝡䞁ኚἲ ࣐࣊ࢺࢡࣜࢵࢺ್ Hematocrit % ィ⟬ᘧ䠖 (ᖹᆒ㉥⾑⌫ᐜ✚×㉥⾑ ⌫) 䠋10 ᖹᆒ㉥⾑⌫ᐜ✚ MCV fL 2 ゅᗘ䝺䞊䝄䞊䝣䝻䞊䝃䜲䝖䝯䝖䝸 䞊ἲ ᖹᆒ㉥⾑⌫࣊ࣔࢢࣟࣅࣥ㔞 _{MCH pg} ィ⟬ᘧ䠖 (䝦䝰䜾䝻䝡䞁㔞×㉥⾑ ⌫ᩘ) 䠋10 ᖹᆒ㉥⾑⌫࣊ࣔࢢࣟࣅࣥ⃰ ᗘ MCHC g/dL ィ⟬ᘧ䠖䝦䝰䜾䝻䝡䞁㔞䠋 (㉥⾑ ⌫ᩘ×ᖹᆒ㉥⾑⌫ᐜ✚) ×1000 ⾑ᑠᯈᩘ _{Platelet ×10}3_{/μL 2 ゅᗘ䝺䞊䝄䞊䝣䝻䞊䝃䜲䝖䝯䝖䝸}

45 䞊ἲ ⓑ⾑⌫ᩘ WBC ×103/μL 2 ゅᗘ䝺䞊䝄䞊䝣䝻䞊䝃䜲䝖䝯䝖䝸 䞊ἲ ⥙≧㉥⾑⌫ᩘཬࡧⓒศ⋡ Reticulocyte ×103/μL RNA ᰁⰍἲ䛻䜘䜛䝺䞊䝄䞊䝣䝻 䞊䝃䜲䝖䝯䝖䝸䞊ἲ ࣜࣥࣃ⌫ᩘཬࡧⓒศ⋡ Lymphocyte ×103/μL 䝨䝹䜸䜻䝅䝎䞊䝊ᰁⰍ䛻䜘䜛䝣䝻 䞊䝃䜲䝖䝯䝖䝸䞊ἲ ዲ୰⌫ᩘཬࡧⓒศ⋡ _{Neutrophil㻌 ×10}3_/μL ዲ㓟⌫ᩘཬࡧⓒศ⋡ Eosinophil ×103/μL ዲሷᇶ⌫ᩘཬࡧⓒศ⋡ _{Basophil ×10}3_/μL ༢⌫ᩘཬࡧⓒศ⋡ Monocyte ×103/μL 4. ⾑ᾮ⏕໬Ꮫⓗ᳨ᰝ Ᏻᴦẅ᫬࡟࣊ࣃࣜࣥࣜࢳ࣒࢘ࢆ⏝࠸࡚᥇ྲྀࡋࡓ⾑ᾮࢆ㸪┤ࡕ࡟෭༷㐲ᚰศ㞳ᶵ (05PR-22㸪᪥❧ᕤᶵᰴᘧ఍♫㸪ᮾி㸪᪥ᮏ) ࢆ⏝࠸ࡓ㐲ᚰศ㞳 (4 Υ㸪ẖศ 3,000 ᅇ㌿㸪15 ศ㛫) ࢆ⾜࠸㸪⾑₢ࢆศ㞳ࡋࡓࠋᚓࡽࢀࡓ⾑₢ࢆ⏝࠸࡚㸪⏕໬Ꮫ⮬ືศ ᯒ⿦⨨ (LABOSPECT 006) ࡟ࡼࡾ௨ୗࡢ᳨ᰝࢆ⾜ࡗࡓࠋ ࣭⮬ືศᯒ⿦⨨ (LABOSPECT 006) ࡟ࡼࡿ᳨ᰝ㡯┠㸪༢఩ཬࡧ ᐃ᪉ἲ ⥲⺮ⓑ್ _{T.Protein g/dL} ࣅ࢘ࣞࢵࢺἲ ࢔ࣝࣈ್࣑ࣥ Albumin g/dL BCG ἲ ࢔ࣝࣈ࣑࣭ࣥࢢࣟࣈࣜࣥẚ A/G ratio − ⥲⺮ⓑཬࡧ࢔ࣝࣈ್࣑ࣥ࠿ࡽ⟬ฟ ࢔ ࢫ ࣃ ࣛ ࢠ ࣥ 㓟 ࢔ ࣑ࣀ ࢺ ࣛࣥࢫࣇ࢙࣮ࣛࢮάᛶ AST U/L JSCC ᶆ‽໬ᑐᛂἲ ࢔ ࣛ ࢽ ࣥ ࢔ ࣑ ࣀ ࢺ ࣛࣥ ࢫ ࣇ࢙࣮ࣛࢮάᛶ ALT U/L JSCC ᶆ‽໬ᑐᛂἲ ࢔ ࣝ ࢝ ࣜ ࣍ ࢫ ࣇ ࢓ ࢱ࣮ ࢮ άᛶ ALP U/L JSCC ᶆ‽໬ᑐᛂἲ ⾑⢾್ Glucose mg/dL ࣊࢟ࢯ࢟ࢼ࣮ࢮ࣭G-6-PDH ἲ

46

⤫ィゎᯒ

⤖ᯝࡣ㸪ᖹᆒ ± ᶆ‽ㄗᕪ (S.E.) ࡜ࡋ࡚♧ࡋࡓࠋᐃ㔞ⓗࢹ࣮ࢱ (ᾋ⭘⋡㸪⾑ᾮ

Ꮫཬࡧ⾑ᾮ⏕໬Ꮫ᳨ᰝ㡯┠) ࡟ࡘ࠸࡚ࡣ㸪Bartlett ࡢ᳨ᐃ࡟ࡼࡾ➼ศᩓᛶࢆ᳨ウࡋ㸪

➼ศᩓࡢሙྜࡣStudent’s t-test ἲࢆ⏝࠸᳨࡚ᐃࢆ⾜ࡗࡓࠋ➼ศᩓ࡜ㄆࡵࡽࢀ࡞࠿

ࡗࡓሙྜ࡟ࡘ࠸࡚ࡣWilcoxon rank sum test ࡢ᳨ᐃࢆ⾜ࡗࡓࠋᐃᛶⓗࢹ࣮ࢱ (⑓⌮

ホ౯ࢢ࣮ࣞࢻ) ࡟ࡘ࠸࡚ࡣ⣼✚ χ2 ᳨ᐃࢆ⾜ࡗࡓࠋ᭷ពᕪ᳨ᐃࡢ᭱ప᭷ពỈ‽ࡣ୧ ഃ5%࡜ࡋࡓࠋᾋ⭘⋡ཬࡧ⑓⌮ホ౯ࢢ࣮ࣞࢻ࡟㛵ࡋ࡚ࡣ㸪㐺ྜᑐ↷⩌ (2 ⩌) ࡜ヨ 㦂≀㉁ᢞ୚⩌ (3–5 ⩌) ࡜ࡢ㛫࡛⤫ィࢆᐇ᪋ࡋࡓࠋ⾑ᾮᏛཬࡧ⾑ᾮ⏕໬Ꮫ᳨ᰝ࡟ 㛵ࡋ࡚ࡣ㸪↓ฎ⨨ᑐ↷⩌ (1 ⩌) ࡜㐺ྜᑐ↷⩌ (2 ⩌) ཬࡧ㐺ྜᑐ↷⩌ (2 ⩌) ࡜ヨ 㦂≀㉁ᢞ୚⩌ (3–5 ⩌) ࡜ࡢ㛫࡛⤫ィࢆᐇ᪋ࡋࡓࠋ⤫ィゎᯒ࡟ࡣ SAS ཬࡧ EXSUS ࢆ౑⏝ࡋࡓࠋ ᒀ⣲❅⣲್ Ureanitrogen mg/dL ࢘ࣞ࢔࣮ࢮ࣭GlDH ἲ㸪࢔ࣥࣔࢽ࢔ ᾘཤἲ ࢡࣞ࢔ࢳࢽ್ࣥ Creatinine mg/dL ࢡࣞ࢔ࢳࢽࢼ࣮ࢮ࣭HMMPS ἲ ⥲ࢥࣞࢫࢸ್࣮ࣟࣝ Cholesterol mg/dL ࢥ ࣞ ࢫ ࢸ ࣟ ࣮ ࣝ ࢜ ࢟ ࢩ ࢲ ࣮ ࢮ ࣭ HMMPS ἲ ࢺࣜࢢࣜࢭࣜࢻ್ Triglyceride mg/dL GPO࣭HMMPS ἲ㸪ࢢࣜࢭࣜࣥᾘཤ ἲ ࣜࣥ⬡㉁್ Phospholipid mg/dL ࢥࣜࣥ࢜࢟ࢩࢲ࣮ࢮ࣭DAOS ἲ ↓ᶵ್ࣜࣥ Inor-P mg/dL PNP࣭XDH ἲ ࢼࢺ್࣒ࣜ࢘ Sodium mEq/L ࢖࢜ࣥ㑅ᢥ㟁ᴟἲ ್࣒࢝ࣜ࢘ _{Potassium mEq/L ࢖࢜ࣥ㑅ᢥ㟁ᴟἲ} ࢡ್࣮ࣟࣝ Chloride mEq/L ࢖࢜ࣥ㑅ᢥ㟁ᴟἲ

47 ⤖ᯝ 1. ࣛࢵࢺ࢝ࣛࢤࢽࣥ⫥ᾋ⭘ࣔࢹࣝ࡟࠾ࡅࡿ HT ࡢᢠᾋ⭘స⏝ ࢝ࣛࢤࢽࣥㄏⓎࣛࢵࢺ⫥ᾋ⭘ࣔࢹࣝ࡟࠾ࡅࡿHT ࡢᢠ⅖⑕స⏝ࢆホ౯ࡋࡓࠋ⫥ ᐜ✚ࡣ㸪ࡍ࡭࡚ࡢ⩌࡟࠾࠸࡚࢝ࣛࢤࢽࣥᢞ୚ᚋ࡟ቑຍࡋࡓ (Fig. 12)ࠋHT ᢞ୚⩌ ࡛ࡣ㸪࢝ࣛࢤࢽࣥᢞ୚ 2㸪3 ཬࡧ 5 ᫬㛫ᚋ࡟࠾࠸࡚㸪㐺ྜᑐ↷⩌ࡼࡾࡶ᭷ព࡟ᾋ ⭘⋡ࡀపୗࡋࡓ (p <0.05)ࠋࢭࣞࢥ࢟ࢩࣈᢞ୚⩌࡛ࡣ㸪࢝ࣛࢤࢽࣥᢞ୚ 1㸪2 ཬࡧ 3 ᫬㛫ᚋ࡛㐺ྜᑐ↷⩌ࡼࡾࡶ᭷ព࡟ᾋ⭘⋡ࡀపୗࡋࡓ (p <0.05)ࠋ࢖ࣥࢻ࣓ࢱࢩࣥ ᢞ୚⩌࡛ࡣ㸪࢝ࣛࢤࢽࣥᢞ୚2㸪3㸪4 ཬࡧ 5 ᫬㛫ᚋ࡛㐺ྜᑐ↷⩌ࡼࡾࡶ᭷ព࡟ᾋ ⭘⋡ࡀపୗࡋࡓ (p <0.01) (Fig. 13)ࠋ ࢝ࣛࢤࢽࣥᢞ୚1㸪2㸪3㸪4 ཬࡧ 5 ᫬㛫ᚋࡢᾋ⭘ᢚไ⋡ࡣ㸪HT ᢞ୚⩌࡛ࡣࡑࢀ ࡒࢀ 0.34%㸪13.3%㸪19.2%㸪20.9%ཬࡧ 22.9%࡛࠶ࡗࡓࠋࢭࣞࢥ࢟ࢩࣈᢞ୚⩌࡛ ࡣ49.6%㸪33.4%㸪24.0%㸪17.6%ཬࡧ 14.2%㸪࢖ࣥࢻ࣓ࢱࢩࣥᢞ୚⩌࡛ࡣ 52.7%㸪 61.5%㸪52.1%㸪51.6%ཬࡧ 47.9%࡛࠶ࡗࡓ (Fig. 14)ࠋ ᡭᢏⓗ࣑ࢫ࡟ࡼࡾ㸪㐺ྜᑐ↷⩌ࡢ1 ༉࡟ࡣ࢝ࣛࢤࢽࣥࢆ㐺ษ࡟ᢞ୚࡛ࡁ࡞࠿ࡗ ࡓࠋᚑࡗ࡚㸪ࡇࡢື≀ࡣホ౯࠿ࡽ㝖እࡋࡓࠋᮏᥐ⨨࡟ࡼࡾ1 ౛Ḟᦆ್࡜࡞ࡗࡓࡀ㸪 ⤫ィゎᯒ࡟ᚲせ࡞ࢹ࣮ࢱᩘࡣᚓࡽࢀ࡚࠾ࡾ㸪ヨ㦂ࡢಙ㢗ᛶ࡟ᙳ㡪ࢆཬࡰࡉ࡞࠸࡜ ุ᩿ࡋࡓࠋ 2. ⫥⤌⧊࡟࠾ࡅࡿ⑓⌮⤌⧊Ꮫⓗ᳨ᰝ ࢝ࣛࢤࢽࣥᢞ୚5 ᫬㛫ᚋ࡟࠾ࡅࡿ⑓⌮⤌⧊Ꮫⓗホ౯࡟࠾࠸࡚㸪㊊⏥㒊࡟㍍ᗘࡢ ΰྜ⣽⬊ᾐ₶ཬࡧ୰⛬ᗘࡢᾋ⭘ࡀࡍ࡭࡚ࡢື≀࡛ㄆࡵࡽࢀࡓࠋࡋ࠿ࡋ㸪⑓⌮Ꮫⓗ

48 ࢢ࣮ࣞࢻ௜ࡅ࡟ᇶ࡙ࡃΰྜ⣽⬊ᾐ₶ࡢ⛬ᗘࡣ㸪HT ⩌ (p <0.05)㸪ࢭࣞࢥ࢟ࢩࣈ⩌ (p <0.01) ཬࡧ࢖ࣥࢻ࣓ࢱࢩࣥ⩌ (p <0.05) ࡟࠾࠸࡚㐺ྜᑐ↷⩌ࡼࡾ᭷ព࡟ప࠿ ࡗࡓࠋࡉࡽ࡟㸪ᾋ⭘ࡢ⛬ᗘࡣࢭࣞࢥ࢟ࢩࣈ⩌ (p <0.01) ཬࡧ࢖ࣥࢻ࣓ࢱࢩࣥ⩌ (p <0.01) ࡟࠾࠸࡚㐺ྜᑐ↷⩌ࡼࡾ᭷ព࡟ప࠿ࡗࡓ (Fig. 15, Fig. 16)ࠋ 3. ⾑ᾮᏛ᳨ᰝཬࡧ⾑ᾮ⏕໬Ꮫ᳨ᰝ ⾑ᾮᏛ᳨ᰝ࡟࠾࠸࡚㸪↓ฎ⨨ᑐ↷⩌࡜ẚ㍑ࡋ࡚㸪㐺ྜᑐ↷⩌࡛ࡣዲ୰⌫ᩘࡢ᭷ ព࡞ቑຍཬࡧࣜࣥࣃ⌫ࡢ᭷ព࡞ቑຍࡀㄆࡵࡽࢀࡓ (p <0.05)ࠋ㐺ྜᑐ↷⩌࡜ヨ㦂≀ ㉁ᢞ୚⩌࡜ࡢẚ㍑࡛ࡣ㸪᭷ព࡞ᕪࡣㄆࡵࡽࢀ࡞࠿ࡗࡓ (Table 2)ࠋ ⾑ᾮ⏕໬Ꮫ᳨ᰝ࡟࠾࠸࡚㸪↓ฎ⨨ᑐ↷⩌࡜ẚ㍑ࡋ࡚㸪㐺ྜᑐ↷⩌࡛ࡣ࣒࢝ࣜ࢘ ࡢ᭷ព࡞పୗࡀㄆࡵࡽࢀࡓࠋ㐺ྜᑐ↷⩌࡜ヨ㦂≀㉁ᢞ୚⩌࡜ࡢẚ㍑࡛ࡣ㸪HT ⩌ ࡛↓ᶵࣜࣥࡢ᭷ព࡞ቑຍ㸪ࢭࣞࢥ࢟ࢩࣈ⩌࡛࢔ࣝࣈ࣑ࣥࡢ᭷ព࡞పୗ㸪࢔ࢫࣃࣛ ࢠࣥ㓟࢔࣑ࣀࢺࣛࣥࢫࣇ࢙࣮ࣛࢮάᛶ㸪ᒀ⣲❅⣲್㸪↓ᶵࣜࣥ㸪࣒࢝ࣜ࢘ࡢ᭷ព ࡞ቑຍࡀㄆࡵࡽࢀࡓࠋ࢖ࣥࢻ࣓ࢱࢩࣥ⩌࡛ࡣ㸪⥲⺮ⓑཬࡧ࢔ࣝࣈ࣑ࣥࡢ᭷ព࡞ప ୗ㸪↓ᶵࣜࣥ㸪࣒࢝ࣜ࢘ࡢ᭷ព࡞ቑຍࡀㄆࡵࡽࢀࡓ (Table 3)ࠋ

49 ⪃ᐹ ᮏヨ㦂࡛ࡣ㸪ࣛࢵࢺ࢝ࣛࢤࢽࣥ⫥ᾋ⭘ࣔࢹࣝࢆ⏝࠸࡚ HT㸪ࢭࣞࢥ࢟ࢩࣈཬࡧ ࢖ࣥࢻ࣓ࢱࢩࣥࡢᢠ⅖⑕ຠᯝࢆ᳨ウࡋࡓࠋ㐺ྜᑐ↷⩌࡟ㄆࡵࡽࢀࡓ⤒᫬ⓗ࡞⫥ᐜ ✚ࡢቑຍ࡟ᑐࡋ࡚㸪HT㸪ࢭࣞࢥ࢟ࢩࣈཬࡧ࢖ࣥࢻ࣓ࢱࢩࣥࡣ⫥ᐜ✚ࡢቑຍࢆ᭷ព ࡟ᢚไࡋࡓࠋHT ࡢ᭷ព࡞ᢚไຠᯝࡣ࢝ࣛࢤࢽࣥᢞ୚ᚋ 2 ᫬㛫࠿ࡽㄆࡵࡽࢀ㸪ᢞ ୚ᚋ5 ᫬㛫࡛᭱኱ࡢᢚไస⏝ࢆ♧ࡋࡓ (% inhibition㸸22.9%)ࠋ ᑐ↷ⓗ࡟㸪ࢭࣞࢥ࢟ࢩࣈཬࡧ࢖ࣥࢻ࣓ࢱࢩࣥࡣ㸪ࡑࢀࡒࢀ࢝ࣛࢤࢽࣥᢞ୚ᚋ 1 ᫬㛫 (% inhibition㸸49.6%) ཬࡧ 2 ᫬㛫 (% inhibition㸸61.5%) ࠿ࡽ᭷ព࡟⫥ᾋ⭘ ࢆᢚไࡋࡓࠋHT ࡣࢭࣞࢥ࢟ࢩࣈཬࡧ࢖ࣥࢻ࣓ࢱࢩࣥࡼࡾᢚไస⏝ࡣᙅ࠿ࡗࡓࡀ㸪 ࡑࡢຠᯝࡣ᫬㛫࡜࡜ࡶ࡟ቑຍࡍࡿഴྥ࡟࠶ࡗࡓࠋ୍᪉࡛㸪ࢭࣞࢥ࢟ࢩࣈཬࡧ࢖ࣥ ࢻ࣓ࢱࢩࣥࡢຠᯝࡣ᫬㛫࡜࡜ࡶ࡟ῶᑡࡋࡓࠋᚑࡗ࡚㸪HT ࡣ㐜⾜ᛶࡢᢠ⅖⑕స⏝ ࢆᣢࡘྍ⬟ᛶࡀ࠶ࡾ㸪HT ࡜ࢭࣞࢥ࢟ࢩࣈࡲࡓࡣ࢖ࣥࢻ࣓ࢱࢩࣥ࡜ࡢ⤌ྜࡏ࡟ࡼ ࡾ㸪ᢞ୚ึᮇ࠿ࡽᚋᮇࡲ࡛ᣢ⥆ⓗ࡞ᢠ⅖⑕ຠᯝࡀᚓࡽࢀࡿྍ⬟ᛶࡀ♧၀ࡉࢀࡓࠋ ⑓⌮⤌⧊Ꮫⓗ᳨ᰝ࡟࠾࠸࡚㸪ΰྜ⣽⬊ᾐ₶ࡢ⛬ᗘࡣ㸪HT㸪ࢭࣞࢥ࢟ࢩࣈཬࡧ࢖ ࣥࢻ࣓ࢱࢩࣥᢞ୚⩌࡛㐺ྜᑐ↷⩌ࡼࡾ᭷ព࡟ప࠿ࡗࡓࠋᾐ₶⣽⬊ࡣ㸪ࣜࣥࣃ⌫㸪 ዲ୰⌫㸪ዲ㓟⌫ཬࡧ༢᰾⣽⬊࠿ࡽᵓᡂࡉࢀ࡚࠸ࡓࠋ⅖⑕ᛶ⣽⬊ᾐ₶ཬࡧయᾮࡢ₃ ฟࡣ㸪ࣄࢫࢱ࣑ࣥ㸪ࢭࣟࢺࢽࣥཬࡧPG ࡢᨺฟ࡟ࡼࡗ࡚ᘬࡁ㉳ࡇࡉࢀ㸪⅖⑕㒊఩ ࡛ࡢᾋ⭘ࢆࡶࡓࡽࡍ (69)ࠋᚑࡗ࡚㸪ࢭࣞࢥ࢟ࢩࣈཬࡧ࢖ࣥࢻ࣓ࢱࢩࣥ࡟ࡘ࠸࡚▱ ࡽࢀࡿࡼ࠺࡟㸪HT ࡣ㸪PGE2ࡢ⏘⏕ࢆῶᑡࡉࡏࡿࡇ࡜࡟ࡼࡗ࡚㸪ࣄࢫࢱ࣑ࣥ㸪ࢭ ࣟࢺࢽࣥཬࡧPG ࡞࡝ࡢ⅖⑕ᛶ࣓ࢹ࢕࢚࣮ࢱ࣮ࡢ⏘⏕ࢆᢚไࡋ㸪ᢠ⅖⑕ຠᯝࢆ♧ ࡍࡶࡢ࡜⪃࠼ࡽࢀࡓ (70, 71)ࠋ࡞࠾㸪HT ࡣΰྜ⣽⬊ᾐ₶ࢆᢚไࡋࡓ୍᪉㸪ᾋ⭘ࢆ

50

ᢚไࡋ࡞࠿ࡗࡓ⌮⏤࡜ࡋ࡚㸪ᾋ⭘ࡣ⅖⑕ࡢึᮇ࡟ẟ⣽⾑⟶ࡢᣑᙇ࡟ࡼࡾ⏕ࡌࡿ཯ ᛂ࡛࠶ࡿࡢ࡟ᑐࡋ㸪ΰྜ⣽⬊ᾐ₶ࡣ⅖⑕ࡢึᮇ௨㝆࡟⅖⑕ᛶ⣽⬊ࡀ㞀ᐖ㒊఩࡟㐟 ㉮ࡋ࡚࠾ࡇࡿ཯ᛂ࡛࠶ࡿࡓࡵ (72)㸪㐜ຠᛶࡢ HT ࡣ⅖⑕ึᮇ཯ᛂ࡛࠶ࡿᾋ⭘ࢆᢚ ไ࡛ࡁ࡞࠿ࡗࡓྍ⬟ᛶࡀ⪃࠼ࡽࢀࡓࠋ

࢝ࣛࢤࢽࣥࡣ COX-2 ࢆάᛶ໬ࡋ㸪COX-2 ࡣ࢔ࣛ࢟ࢻࣥ㓟ࢆᇶ㉁࡜ࡋ࡚ PGE2

࡞࡝ࢆ⏕ᡂࡍࡿࡇ࡜࡛⅖⑕཯ᛂࢆಁ㐍ࡍࡿࠋࡲࡓNO ࡣ COX-2㸪࣍ࢫ࣍ࣜࣃ࣮ࢮ A2 (PLA2) ࢆ S-ࢽࢺࣟࢩࣝ໬ࡍࡿࡇ࡜࡛άᛶ໬ࡋ㸪PGE2ࡢ⏘⏕ࢆቑᙉࡉࡏ⅖⑕ ཯ᛂࢆஹ㐍ࡉࡏࡿ (63)ࠋ➨ 1 ❶࡟࠾࠸࡚㸪ᡃࠎࡣ HT ࡀ NO ཬࡧ PGE2ࡢ⏘⏕ࢆ ᢚไࡍࡿࡇ࡜ࢆ᫂ࡽ࠿࡟ࡋ࡚࠾ࡾ㸪ࡇࢀࡽࡢస⏝ࡀᮏᐇ㦂࡟࠾࠸࡚ᾋ⭘ᙧᡂᢚไ స⏝࡟⧅ࡀࡗࡓ࡜⪃࠼ࡽࢀࡓࠋ➨ 1 ❶ཬࡧ 3 ❶࡟࠾࠸࡚㸪HT ࡢ NO ཬࡧ PGE2 ⏘⏕ᢚไస⏝㸪࢝ࣛࢤࢽࣥᾋ⭘ᙧᡂᢚไస⏝ࢆࡑࢀࡒࢀ☜ㄆࡋ㸪HT ࡢᢠ⅖⑕స ⏝ࡢ᭷ຠᛶࢆ☜ㄆࡍࡿࡇ࡜ࡀ࡛ࡁࡓࠋࡑࡇ࡛㸪ḟ࡟⌧ᅾࡢ἞⒪⸆࡛ㄆࡵࡽࢀࡿ๪ స⏝ࡢ୍ࡘ࡛࠶ࡿᾘ໬⟶㞀ᐖࡢㄏⓎࡢ᭷↓࡟ࡘ࠸࡚HT ࢆホ౯ࡋࡓࠋ

51

ᑠᣓ

ᡃࠎࡣin vitro ◊✲࡟࠾࠸࡚㸪HT ࡀ㸪LPS ㄏⓎᛶࡢ iNOS㸪COX-2 ཬࡧ IL-1β

㑇ఏᏊࡢⓎ⌧ࢆᢚไࡋ㸪⤖ᯝ࡜ࡋ࡚ NO ཬࡧ PGE2ࡢ⏘⏕㔞ࢆῶᑡࡉࡏࡿࡇ࡜ࢆ ぢฟࡋࡓࠋᮏ◊✲࡛ࡣ㸪HT ࡢ᪂つᾘ⅖㙠③⸆࡜ࡋ࡚ࡢྍ⬟ᛶࢆぢᤣ࠼㸪in vivo ࡟࠾ࡅࡿᢠ⅖⑕άᛶࢆホ౯ࡍࡿࡇ࡜ࢆ┠ⓗ࡜ࡋࡓࠋ ヨ㦂≀㉁࡜ࡋ࡚㸪0.5% MC (㐺ྜᑐ↷⩌)㸪HT (500 mg/kg)㸪ࢭࣞࢥ࢟ࢩࣈ (100 mg/kg)㸪࢖ࣥࢻ࣓ࢱࢩࣥ (30 mg/kg) ࢆ㸪࢝ࣛࢤࢽࣥᢞ୚ 1 ᫬㛫๓࡟⤒ཱྀᢞ୚ࡋࡓࠋ ࢝ࣛࢤࢽࣥᢞ୚ᚋ࡟⫥ᐜ✚ࢆ ᐃࡋ㸪⫥ᐜ✚ࡢኚ໬㔞ࢆᣦᶆ࡟ᢠ⅖⑕ຠᯝࢆホ౯ ࡋࡓࠋHT ࡣࢭࣞࢥ࢟ࢩࣈཬࡧ࢖ࣥࢻ࣓ࢱࢩࣥࡼࡾ⫥ᾋ⭘ᢚไస⏝ࡣᙅ࠿ࡗࡓࡀ㸪 ࡑࡢຠᯝࡣ᫬㛫࡜࡜ࡶ࡟ቑຍࡋࡓࠋ୍᪉࡛㸪ࢭࣞࢥ࢟ࢩࣈཬࡧ࢖ࣥࢻ࣓ࢱࢩࣥࡢ ຠᯝࡣ᫬㛫࡜࡜ࡶ࡟ῶᑡࡋࡓࠋᚑࡗ࡚㸪HT ࡣ㐜⾜ᛶࡢᢠ⅖⑕స⏝ࢆᣢࡘྍ⬟ᛶ ࡀ࠶ࡾ㸪HT ࡜ࢭࣞࢥ࢟ࢩࣈࡲࡓࡣ࢖ࣥࢻ࣓ࢱࢩࣥ࡜ࡢ⤌ྜࡏ࡟ࡼࡾ㸪ᢞ୚ึᮇ ࠿ࡽᚋᮇࡲ࡛ᣢ⥆ⓗ࡞ᢠ⅖⑕ຠᯝࡀᚓࡽࢀࡿྍ⬟ᛶࡀ♧၀ࡉࢀࡓࠋ ➨1 ❶ཬࡧ➨ 3 ❶࡟࠾࠸࡚㸪NO ཬࡧ PGE2⏘⏕ᢚไస⏝㸪࢝ࣛࢤࢽࣥᾋ⭘ᙧᡂ ᢚไస⏝ࢆࡑࢀࡒࢀ☜ㄆࡋ㸪HT ࡢᢠ⅖⑕స⏝ࢆ☜ㄆࡍࡿࡇ࡜ࡀ࡛ࡁࡓࠋHT ࡟ᢠ ⅖⑕స⏝ࡀㄆࡵࡽࢀࡓࡇ࡜࠿ࡽ㸪ḟ࡟⌧ᅾࡢ἞⒪⸆࡟ㄆࡵࡽࢀࡿ๪స⏝ࡢ୰࡛᭱ ࡶၥ㢟࡜࡞ࡿᾘ໬⟶㞀ᐖㄏⓎࡢ᭷↓࡟ࡘ࠸࡚HT ࢆホ౯ࡋࡓࠋ

52

ᅗ⾲

Table 2.

Hem

atology in the rat paw

edem a model Gro u p No. 1 2 3 4 5 Gro u p 0.5% M C + S al in e 0.5% M C + C ar ra ge ena n H T + C ar ra ge en an Ce lec ox ib + Ca rr ag ee na n Indo m et haci n+ C arrag een an N 4 5 5 5 5 RBC 5. 96 8 ± 0. 19 5 6. 26 8 ± 0. 40 8 6. 08 4 ± 0. 20 8 5. 85 8 ± 0. 23 9 5. 97 8 ± 0. 22 0 He m og lo bin 13. 60 ± 0.3 1 14. 00 ± 0.7 8 13. 72 ± 0.3 2 13. 18 ± 0.3 4 13. 64 ± 0.4 2 He m ato crit 40. 14 ± 0.8 5 41. 58 ± 2.0 3 41. 60 ± 1.3 1 39. 38 ± 0.9 8 40. 10 ± 1.1 0 MCV 67. 26 ± 1.7 5 66. 43 ± 2.6 4 68. 42 ± 2.0 3 67. 26 ± 2.3 9 67. 12 ± 1.6 2 MCH 22. 76 ± 0.4 2 22. 33 ± 1.0 3 22. 58 ± 0.8 1 22. 50 ± 0.8 5 22. 80 ± 0.5 2 MCHC 33. 88 ± 0.5 9 33. 63 ± 0.3 3 33. 00 ± 0.4 7 33. 46 ± 0.2 1 33. 98 ± 0.4 3 platelet 109 7. 2 ± 214 .1 136 4. 5 ± 124 .1 134 1. 2 ± 92. 0 132 8. 4 ± 160 .8 127 3. 8 ± 122 .3 WB C 9. 49 0 ± 1. 77 5 9. 01 5 ± 1. 76 6 6. 98 2 ± 0. 80 2 8. 48 6 ± 1. 46 9 8. 52 4 ± 1. 11 0 Reticu lo cy te 537 .4 8 ± 44. 43 524 .5 3 ± 46. 99 497 .6 6 ± 45. 43 467 .7 0 ± 33. 36 460 .3 4 ± 38. 79 Ne ut ro ph il 1. 86 8 ± 0. 66 4 4. 74 3 ± 0.7 41 a 3. 87 4 ± 0. 71 1 4. 12 8 ± 0. 85 4 4. 88 0 ± 0. 85 1 L ym phocy te 7. 06 0 ± 0. 93 8 3. 85 3 ± 1.2 67 a 2. 75 2 ± 0. 43 2 4. 00 4 ± 0. 78 4 3. 29 2 ± 0. 40 2 Mon oc yte 0. 38 2 ± 0. 16 8 0. 28 0 ± 0. 07 4 0. 24 8 ± 0. 07 9 0. 23 0 ± 0. 06 0 0. 22 4 ± 0. 03 4 E os ino ph il 0. 13 4 ± 0. 12 1 0. 10 0 ± 0. 00 8 0. 07 2 ± 0. 02 6 0. 08 0 ± 0. 04 9 0. 11 0 ± 0. 03 7 B as ophi l 0. 02 6 ± 0. 00 9 0. 01 3 ± 0.0 05 a 0. 01 0 ± 0. 00 0 0. 01 6 ± 0. 00 9 0. 01 0 ± 0. 00 0 a : p < 0.05 com pare d w ith c ontro l ( Stud ent’s t -t est ). Ea ch v al ue rep rese nts th e m ean ± s tan dard error of the m ean of d ata from fiv e anim als, excep t f or the c ont rol g roup , in w hi ch da ta from only f our an im al s was u sed.

53 Table 3. Blood chem ist ry in th e rat p aw ed em a m od el Gro u p No. 1 2 3 4 5 Gro u p 0.5% M C + S al in e 0.5% M C + C ar ra ge ena n H T + C ar ra ge en an Ce lec ox ib + Ca rr ag ee na n Indo m et haci n+ C arrag een an N 4 5 5 5 5 T .P ro tei n 4.9 8 ± 0.0 8 5.1 5 ± 0.1 7 5.2 0 ± 0.1 0 4.9 6 ± 0.1 1 4.3 8 ± 0.2 8 㼎 Al bu mi n 3.9 6 ± 0.1 1 4.0 0 ± 0.0 8 4.0 8 ± 0.0 4 3.8 4 ± 0.0 5 c 3.4 2 ± 0.2 6 c A/ G r at io 3. 90 8 ± 0. 40 8 3. 51 0 ± 0. 37 3 3. 66 2 ± 0. 28 0 3. 45 4 ± 0. 32 5 3. 58 4 ± 0. 42 1 AS T 92. 2 ± 6.8 99. 3 ± 4.3 99. 0 ± 3.5 104 .4 ± 4.3 b 83. 8 ± 7.6 ALT 43. 2 ± 3.6 40. 8 ± 3.5 42. 8 ± 5.1 44. 6 ± 4.2 38. 2 ± 5.3 AL P 133 0. 6 ± 358 .2 144 8. 5 ± 487 .9 133 3. 8 ± 240 .2 112 9. 8 ± 173 .2 965 .4 ± 190 .0 Gl uc os e 183 .0 ± 10. 9 163 .5 ± 20. 0 159 .8 ± 12. 1 161 .2 ± 20. 8 149 .0 ± 12. 6 U re ani tr og en 9.0 6 ± 1.4 5 7.4 5 ± 1.5 9 8.7 4 ± 1.2 7 12. 22 ± 1.4 6 c 16. 26 ± 4.7 8 c Creatin in e 0. 18 2 ± 0. 01 8 0. 18 0 ± 0. 01 8 0. 19 6 ± 0. 01 1 0. 17 8 ± 0. 01 3 0. 19 2 ± 0. 01 3 Cho lesterol 77. 4 ± 11. 6 79. 3 ± 12. 1 81. 0 ± 10. 0 80. 0 ± 7.4 71. 4 ± 10. 5 T rigl ycerid e 50. 2 ± 23. 7 30. 5 ± 9.1 21. 8 ± 8.0 38. 0 ± 21. 5 44. 2 ± 27. 0 Phos ph ol ip id 130 .0 ± 16. 6 134 .5 ± 14. 8 124 .4 ± 11. 1 140 .2 ± 11. 8 126 .2 ± 15. 0 Inor-P 9.4 4 ± 0.4 6 8.8 8 ± 0.5 0 9.5 4 ± 0.3 0 b 9.8 4 ± 0.5 2 b 9.3 2 ± 0.6 5 Sodi um 144 .6 ± 0.9 144 .8 ± 2.1 144 .4 ± 1.3 145 .0 ± 1.6 142 .8 ± 1.5 Po tassium 4.3 2 ± 0.2 9 3.6 0 ± 0.1 2 a 3.8 4 ± 0.3 4 4.5 6 ± 0.6 1 b 4.5 0 ± 0.3 7 b Chlo ri de 10 2. 8 ± 1.9 103 .0 ± 0.8 102 .2 ± 0.8 102 .6 ± 1.5 103 .0 ± 0.7 a : p < 0.05 com pare d w ith g roup 1 (Stu den t’s t-t es t) . b : p < 0.05 com pared wi th g ro up 2 ( St uden t’ s t -t es t) . c : p < 0.05 c om pared wi th g rou p 2 (Wilco xon rank sum test). Each v al ue rep res ents the m ean ± st anda rd e rro r of th e m ean of da ta from fiv e an im al s, exc ept fo r th e g roup 2, in w hich d at a fr om only four ani m al s was used.