探討紅麴二次代謝物monascin和lovastatin對過度糖化終產物誘導的第二型糖尿病小鼠之影響

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୯ҥᆵ᡼εᏢғڮࣽᏢଣғϯࣽמᏢس ᅺγፕЎ

Department of Biochemical Science and Technology College of Life Science

National Taiwan University Master Thesis

௖૸आ᜿Βԛжᖴނ monascin ک lovastatin ჹၸࡋᑗϯ ಖౢނᇨᏤޑಃΒࠠᑗֿੰλႵϐቹៜ

The effects of monascin and lovastatin on type 2 diabetes in advanced glycation end-product (AGEs) induced mice

ֈࡘ၃ Si-Shi Lu

ࡰᏤ௲௤Ǻዐηܴ റγ (Tzu-Ming Pan, Ph.D.) ύ๮҇୯ 102 ԃ 7 Д

July 2013

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I

ᇞᖴ!

᠐࿤ڔਜǴՉ࿤ٚၡǶ᠐ਜکՉၡ೿ࢂӕኬёаวචᙦ൤ԾρϣЈޑբҔǴ ѝԖԾρޑϣЈᙦ൤ΑǴωૈѐགڙ೭ঁШࣚޑऍӳǶךϐ܌аٰᆵ᡼᠐ਜΨࢂ

рܭ೭ٿঁҞޑǴອԃᇸޑਔংᔈ၀ӭрѐوو࣮࣮Ǵӭ࣮࣮όӕӦБޑԃᇸΓ ޑғࢲǴௗڙόӕࡘགྷޑ࿘ናǴ೭ኬғڮωό཮Ⴝ΋α࢏ϔΑคғ፪Ƕ!

ٿԃӧѠ᡼ޑࣴزғғఱ΋ᙯ౳൩ा่״Ǵ२Ӄ૱ЈགᖴךޑࡰᏤ௲௤ዐη

ܴԴৣǴᜫཀௗڙך຾Εдޑჴᡍ࠻ǶགᖴԴৣჹךޑх৒ک௲ᇧǴᡣᐱӧ౦ໂ ޑך७གྕធǶԴৣჹܭࣴزࣽᏢᙣ཈ޑᄊࡋکख़ຎౢᏢ่ࣴӝޑᢀᗺᡣךڙ੻

ঘభǶᖴᖴԴৣٿԃٰޑႴᓰکਭ୻Ǵӧ҂ٰޑВηۓ྽аԴৣࣁᄦኬᝩុᏢಞ کуݨǶགᖴύᑫεᏢᛌᙴੰ౛ғނࣴز܌ᄃߪܮ܌ߏکֆࡿችᏢۆӧϪТࢉՅ Бय़๏ϒךޑࡰᏤکᔅշǶགᖴ๏ϒࡰᏤޑα၂ہ঩Ǵ୯ҥѠ᡼εᏢғϯࣽמᏢ س᝵ᇻד௲௤ǴύԀࣽמεᏢමቼᛁਠߏǵ୯ҥύᑫεᏢБᝩ௲௤аϷ୯ҥѠ᡼

εᏢ१ࠔࣽמࣴز܌ᖴలृ௲௤ǴགᖴӚՏԴৣԭԆύኘϧୖуα၂Ǵ๏ϒࡌ᝼

ᆶࡰ҅Ǵ٬ளፕЎϣ৒׳уֹ๓ǴӧԜ߄Ң૱ЈޑགᖴǶ!

གᖴჴᡍ࠻ޑεৎ೭ٿԃٰޑх఼کᔅշǶགᖴߥֻᏢߏکλ޸ᏢۆޑಒЈ

ࡰᏤᆶᔅշǴஒך೭ঁࣽࣴޑߐѦᅇሦ຾ߐǶགᖴ◖൛Ꮲۆӧჴᡍය໔کα၂ਔ ޑᔅԆᗋԖӧך௃ᆣեပޑਔংޑЈᡫᇶᏤǶགᖴד㚌Ꮲ׌ၸԃය໔όᜏٌधᔅ ךྣ៝ԴႵǶགᖴࡏቺǵ܍ᑣǵӀᝬᏢ׌کॕዝᏢۂӧ୏ނ឴࣊ਔ๏ϒךޑᔅշǶ གᖴӕᏆד࠹ǵ೾ᅴکࠧ֡ѳதϕ࣬уݨѺ਻Ǵ๏ϒךᆒઓ΢ޑವεႴᆸǶགᖴ ࡌճᏢߏکΚοᏢߏӧ meeting ਔ๏ϒޑࡰᗺǴᡣךӧࣽࣴ΢ِೲԋߏǶ!

നࡕགᖴךᒃངޑݿݿ༰༰Ǵӵ݀ؒԖգॺޑεΚЍ࡭ךΨؒԖᐒ཮ٰѠ᡼

ۺਜǴᡏᡍό΋ኬޑΓғǶགᖴ༰༰፥ϒךޑ҅ૈໆǴਔਔ໒ှध඀ޑךǴᡣך

ૈ୼୲࡭ΠѐǶ!

ᖴᖴεৎჹךޑЍ࡭کᔅշǴ׆ఈӧ҂ٰёаԋࣁ౛གྷύޑԾρǴ߿ඪޑѐ य़ჹаࡕΓғၡ΢ޑ਋שԂଓ೴ფགྷǴᡏ౜ԾيޑሽॶǶ!

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Ҟᒵ!

ᕭቪ߄………... ...9,,

ᄔा... ,; Abstract………..; Ҟᒵ……….... ... Ċ კҞᒵ………... ,9

߄Ҟᒵ………...9,

ಃ΋ക ߻قق……….………..1

ಃΒക Ў᝘ӣ៝………...3

΋ǵᑗֿੰ………...3

Βǵၸࡋᑗϯനಖౢނ………...4

Οǵી৞નૻ৲໺ሀၡ৩………...11

Ѥǵી৞નߔל………..………..………...15

ϖǵਡӢη-आՈౚϐ 2 ࣬ᜢӢη-2 (nuclear factor-erythroid 2 related factor-2; Nrf2) ᆶᑗֿੰϐᜢس...16

Ϥǵל਼ϯٛᑇس಍………...17

Ύǵआ᜿………...21

(΋) आ᜿ϐ๵ᅿ੝܄ ………...21

(Β) आ᜿ғౢϐΒભжᖴౢނ ………...22

(Ο) आ᜿ჹᑗֿੰϐᔈҔ ………...26

(Ѥ) आ᜿ׯ๓ી৞નߔלϐёૈᐒڋ ………...28

ΖǵࣴزεᆜᆶҞޑ………...………...28

ಃΟക ׷਑ᆶБݤ………...31

΋ǵჴᡍ׷਑………...31

(΋) ᛰࠔ၂Ꮚ ………...31

!!(Β)!ሺᏔ೛ഢ ………..……….…31

(Ο) ځд ………..………….32

(Ѥ) ྋనᇙഢ ………...32

ΒǵჴᡍБݤ……….……….….32

(΋) ၸࡋᑗϯനಖౢނᇙഢ ………..…... ……….33

(Β) ୏ނჴᡍ ………. ……….33

(Ο) ғނ಍ीϩ݋Бݤ!………..… ………....40

ಃѤക่݀ᆶ૸ፕ………...42

ಃ΋࿯ Monascin Ϸ lovastatin ჹ AGE ӧط᠌ύಕᑈϐቹៜ………...42

ಃΒ࿯ Monascin ک lovastatin ჹ AGE ᇨᏤϐλႵી৞નל܄ϐቹៜ……...42

(΋) ࿣१Ոᑗ ………...42!

(Β) αܺဟ๻ᑗऐໆჴᡍ (oral glucose tolerance test, OGTT) Ϸી৞નऐڙჴᡍ

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III

(insulin tolerance test, ITT)……….45

!!(Ο) Ոమી৞ન ………...48

(Ѥ) HOMA-IR ࡰኧ ………...50

ಃΟ࿯ Monascin Ϸ lovastatin ჹી৞નೈқ፦߄౜ϐቹៜ………53

(΋) ી৞નೈқ፦ϐ߄౜ ………...53

(Β) PDX-1 (pancreatic duodenal homeobox-1, PDX-1) mRNA ߄౜ ………...ġ53 (Ο) CCAAT enhancer binding protein (C/EBP) ϐ߄౜ ………...ġįį56 (Ѥ) ಃΒࠠဟ๻ᑗᙯၮೈқ (glucose transporter 2; GLUT2) mRNA ϐ߄౜…...60

ಃѤ࿯ Monascin Ϸ lovastatin ჹ AGE ᇨᏤᑗֿੰλႵિ፦ಕᑈቹៜ……...62

(΋) Ոિ፦……….………...….62

(Β) ط᠌િ፦………...64

ಃϖ࿯ Monascin Ϸ lovastatin ჹ AGE ᇨᏤᑗֿੰλႵߦวݹಒझᐟનϐቹៜ…...65

(΋)ી᠌ߦวݹಒझᐟન………...65

(Β)ط᠌ߦวݹಒझᐟન………..………….68

(Ο)๝᠌ߦวݹಒझᐟન………...72

ಃϤ࿯ Monascin ک lovastatin ჹ AGE ೷ԋ਼ϯᓸΚϐቹៜ…...74

(΋) ط᠌ p47phox (NADPH oxidase subunit) ϐ߄౜ ………...74

!!(Β) ל਼ϯሇનϐ߄౜ ………...78

(Ο) ط᠌Ϸ๝᠌ੰ౛ϩ݋ ………...82

(Ѥ) λ่ ………...90

ಃϖക ᆕӝ૸ፕ………...93

ಃϤക ୖԵЎ᝘………...98

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კҞᒵ!

კ 2-1 AGEs ޑ׎ԋၸำ………..6

კ 2-2 १ނύ AGEs ޑжᖴၸำ………...………...7

კ 2-3 ж߄܄ AGEs ϐϯӝނ่ᄬ………...………8

კ 2-4 ၸࡋᑗϯനಖౢނᇨᏤวݹϐᐒڋკ………...……....10

კ 2-5 ી৞નૻ৲໺ሀၡ৩………...……12

䡾 2-6 ਡӢη-आՈౚϐ 2 ࣬ᜢӢη-2 ࢲϯϐᐒڋკ………...18

კ 2-7 ಒझϣϐל਼ϯٛᑇس಍………...19

კ 2-8 आ᜿วሇౢނ monascin ک lovastatin ϐ่ᄬ………...24

კ 2-9 ҁፕЎϐࣴزࢎᄬ………...30

კ 4-1 Monascin ک lovastatin ჹ AGE ᇨᏤ 22 ڬᑗֿੰλႵط᠌ AGE ಕᑈϐቹ ៜ………... 43

კ 4-2 Monascin ک lovastatin ჹ AGE ᇨᏤ 22 ڬᑗֿੰλႵط᠌ AGE ಕᑈϐቹ ៜ………...………...44

კ 4-3 Monascin ک lovastatin ჹ AGE ᇨᏤ 22 ڬᑗֿੰλႵ࿣१Ոᑗϐቹ ៜ………... 46

კ 4-4 Monascin ک lovastatin ჹ AGE ᇨᏤ 22 ڬᑗֿੰλႵ OGTT ϐቹ ៜ………... 47

კ 4-5 Monascin ک lovastatin ჹ AGE ᇨᏤ 22 ڬᑗֿੰλႵ ITT ϐቹៜ...49

კ 4-6 Monascin ک lovastatin ჹ AGE ᇨᏤ 22 ڬᑗֿੰλႵՈమી৞નቹ ៜ………...51

კ 4-7 Monascin ک lovastatin ჹ AGE ᇨᏤ 22 ڬᑗֿੰλႵ HOMA-IR index ϐ ቹៜ………...52

კ 4-8 Monascin ک lovastatin ჹ AGE ᇨᏤ 22 ڬᑗֿੰλႵી৞નೈқ፦߄౜ϐ ቹៜ……….……….54

კ 4-9 Monascin ک lovastatin ჹ AGE ᇨᏤ 22 ڬᑗֿੰλႵી৞નೈқ፦߄౜ϐ ቹៜ………..55

კ 4-10 Monascin ک lovastatin ჹ AGE ᇨᏤ 22 ڬᑗֿੰλႵ PDX-1 mRNA ߄ ౜ໆቹៜ………...57

კ 4-11 Monascin ک lovastatin ჹ AGE ᇨᏤ 22 ڬᑗֿੰλႵط᠌ C/EBP ߄౜ ቹៜ………..58

კ 4-12 Monascin ک lovastatin ჹ AGE ᇨᏤ 22 ڬᑗֿੰλႵط᠌ C/EBP ໚܄ ࢉՅय़ᑈК౗ቹៜ………..59

კ 4-13 Monascin ک lovastatin ჹ AGE ᇨᏤ 22 ڬᑗֿੰλႵ GLUT2 mRNA ߄ ౜ໆቹៜ………..61

კ 4-14 Monascin ک lovastatin ჹ AGE ᇨᏤ 22 ڬᑗֿੰλႵՈమΟለҒݨ✊Ϸᕴ ᖌڰᎇϐቹៜ………...63

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V

კ 4-15 Monascin ک lovastatin ჹ AGE ᇨᏤ 22 ڬᑗֿੰλႵط᠌ΟለҒݨ✊Ϸᕴ ᖌڰᎇᐚࡋቹៜ………...64 კ 4-16 Monascin ک lovastatin ჹ AGE ᇨᏤ 22 ڬᑗֿੰλႵી᠌ߦวݹಒझᐟ

નቹៜ………..66 კ 4-17 Monascin ک lovastatin ჹ AGE ᇨᏤ 22 ڬᑗֿੰλႵط᠌ߦวݹಒझᐟ

નቹៜ………..69 კ 4-18 Monascin ک lovastatin ჹ AGE ᇨᏤ 22 ڬᑗֿੰλႵ๝᠌ߦวݹಒझᐟન

ቹៜ...71 კ 4-19 Monascin ک lovastatin ჹ AGE ᇨᏤ 22 ڬᑗֿੰλႵط᠌ p47phox ߄౜ޑ

ቹៜ...73 კ 4-20 Monascin ک lovastatin ჹ AGE ᇨᏤ 22 ڬᑗֿੰλႵط᠌ p47phox ೈқ፦

߄౜ቹៜ………...……...75 კ 4-21 Monascin ک lovastatin ჹ AGE ᇨᏤ 22 ڬᑗֿੰλႵી᠌ל਼ϯሇનࢲ܄

ቹៜ………...…...76 კ 4-22 Monascin ک lovastatin ჹ AGE ᇨᏤ 22 ڬᑗֿੰλႵط᠌ל਼ϯሇનࢲ

܄ቹៜ………...80 კ 4-23 Monascin ک lovastatin ჹ AGE ᇨᏤ 22 ڬᑗֿੰλႵ๝᠌ל਼ϯሇનࢲ

܄ቹៜ………...…...83 კ 4-24 Monascin ک lovastatin ჹ AGE ᇨᏤ 22 ڬᑗֿੰλႵط᠌ಔᙃቹៜ…….85 კ 4-25 Monascin ک lovastatin ჹ AGE ᇨᏤ 22 ڬᑗֿੰλႵ๝᠌ಔᙃቹៜ….….86 კ 4-26 Monascin ک lovastatin 㛱ჹ AGE ᇨᏤ 22 ڬᑗֿੰλႵط๝ڙཞࡰ኱ϐቹ

ៜ………..88 კ 5-1 Monascin ک lovastatin ׯ๓ી᠌фૈϐёૈᐒڋკ………...94 კ 5-2 ่ፕკ………...…95

!

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߄Ҟᒵ!

߄ 2-1Ǻဟ๻ᑗ࣬ᜢᙯၮೈқ……….…14 ߄ 3-1Ǻ!ջਔۓໆᆫӝ䁙᜘ϸᔈ (real-time PCR) ܌٬Ҕϐ primers……….…41 ߄ 4-1Ǻ AGE ᇨᏤᑗֿੰλႵϐط๝ڙཞғϯࡰ኱………..….87 ߄ 4-2Ǻmonascin ک lovastatin ჹ AGE ᇨᏤᑗֿੰλႵߦวݹಒझӢηቹៜϐλ

่………..91 ߄ 4-3;!monascin ک lovastatin ჹ AGE ᇨᏤᑗֿੰλႵל਼ϯሇનࢲ܄ቹៜϐλ

่………..92

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VII

ᕭቪ߄

मЎᕭቪ मЎӄӜ! ύЎӄӜ!

ADA American diabetes association ऍ୯ᑗֿੰڐ཮!

ALT alanine aminotransferase Чữለᙯữ䁙!

AGEs advanced glycation end-products ၸࡋᑗϯനಖౢނ ARE antioxidant response element ל਼ϯᏊϸᔈ୔

AST aspartate aminotransferase Ϻߐоữለᙯữ䁙!

BUN blood urea nitrogen ֿનේ!

BSA bovine serum albumin ФՈమೈқ!

CAT catalase ᝻䁙!

C/EBP CCAAT/enhancer-binding protein CCAATቚ㳾η䶌ӝೈқ倽 DMSO dimethyl sulfoxide ΒҘ୷٥㿲!

ELISA enzyme-linked immunosorbent assay ሇનխࣝϩ݋!

GABA -aminobutyric acid .ữ୷΍ለ!

GLUT glucose transporter ဟ๻ᑗᙯၮೈқ

GSH glutathione ᎽાҒ两

GSIS glucose-stimulated insulin secretion ဟ๻ᑗڈᐟી৞નϩݜ!

GPx glutathione peroxidase ᎽાҒ两ၸ਼ϯނ䁙!

H&E hematoxylin-eosine ᝵Е๋.Ҳआ!

H2O2 hydrogen peroxide ၸ਼ϯణ!

HOMA homeostatic model assessment ી৞નܢלࡰኧ!

i.p. interaperitoneal ဎ๚

IDDM insulin-dependent diabetes mellitus ી৞ન٩ᒘࠠᑗֿੰ!

IFG impaired fasting glucose ޜဎՈᑗ౦த!

IGF-1 insulin-like growth factor 1 ൘ࠠી৞નኬғߏӢη!

IGT impaired glucose tolerance ဟ๻ᑗऐڙ౦த!

IHC stain immunohistochemistry stain խࣝಔᙃϯᏢࢉՅ

IL-1 interleukin-1 ϟқન-1

IL-6 interleukin-6 ϟқન-6

IR insulin receptor ી৞નڙᏔ!

IRS insulin receptor substrate ી৞નڙᏔ୷፦!

ITT insulin tolerance test ી৞નऐڙ܄၂ᡍ!

LDL-C low density lipoprotein cholesterol եஏࡋિೈқᖌڰᎇ!

MG methylglyoxal Ҙ୷ΌΒ⾺

NAC N-acetylcysteines Όㄽъાữለ

NADPH nicotinamide adenine dinucleotide phosphate

๨ᡵㄽữဏ❥ᩫΒਡ㧿ለᕗለ

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NAFLD non-alcoholic fatty liver disease ߚଚᆒ܄િެط!

NF-B nuclear factor-B ਡᙯᒵӢη.B!

NIDDM non-insulin-dependent diabetes mellitus

ߚી৞ન٩ᒘࠠᑗֿੰ!

Nrf2 nuclear factor-erythroid 2 related factor-2

ਡӢη-आՈౚϐ 2 ࣬ᜢӢη-2

OGTT oral glucose tolerance test αܺဟ๻ᑗऐڙ၂ᡍ PBS phosphate-buffered saline ᕗለ጗ፂྋన

PI3K phosphatidylinositol-3 kinase ᕗለԼᎇ.3!ᐟ䁙!

PKB/Akt protein kinase B/serine protein kinase ೈқᐟ䁙!B/ํữለೈқᐟ䁙!

PKC protein kinase C ೈқᐟ䁙 C

PPAR peroxisome proliferator-activated receptor

ၸ਼ϯނλᡏቚ෗ނ௴୏ڙᡏ

RAGE receptors for AGEs ၸࡋᑗϯനಖౢނڙᏔ!

ROS reactive oxygen species ࢲ܄਼!

SEM standard error of the mean ኱ྗᇤৡ!

SHR spontaneously hypertensive rat চว܄ଯՈᓸԴႵ!

SOD superoxide dismutase ຬ਼ϯނݔϯ䁙!

TC total cholesterol ᕴᖌڰᎇ!

TG triglyceride ΟለҒݨ✊!

TNF- tumor necrosis factor- ဍዦᚯԝӢη VCAM vascular cell adhesion molecule ՈᆅಒझᗹߕӢη!

! !

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IX

ᄔा!

ҁࣴزаၸࡋᑗϯಖౢނ (advanced glycation end-products, AGEs) ࡌҥᑗֿ

ੰλႵϐ୏ނኳԄǴ٠ӕਔаဎ๚ݙ৔ monascin (MS) ک lovastatin (LOV)Ǵຑ՗

MSک LOV ૈցׯ๓ી৞નߔלکՈᑗፓ࿯ᐒڋǶ!

ࣴز่݀ᡉҢǴ AGE ᇨᏤૈ୼फ़եλႵϐી৞ન௵ག܄کဟ๻ᑗऐڙ܄Ƕ MS ک LOV ૈᙖҗගϲી৞ύી৞નޑӝԋکל਼ϯሇનޑ߄౜аफ़ե AGE ᇨ ᏤౢғϐଯՈᑗکી৞નߔלϐੱރǶ่݀ᡉҢ AGE ёૈ཮ᏤठλႵી᠌ ಒझ фૈምᛖک਼ϯᓸΚவԶౢғી৞નל܄Ƕ

ҁࣴزว౜ᆶ௓ڋಔ࣬К AGE ಔԖી৞નߔלϐ౜ຝǴхࡴՈᑗ΢ϲǴՈమ

ી৞ન֖ໆቚуǴԶՈమ TGǴTC Ψϩձ΢ϲǶלวݹБय़ǴAGE ಔߦวݹಒझ ᐟન TNF-ǵIL-6 ک IL-1 ߄౜ໆᡉ๱ගϲǴԶ MS ک LOV ೀ౛ࡕ཮Ԗ׭ڋߦว ݹಒझᐟન߄౜ϐਏ݀Ƕל਼ϯБय़ǴAGE ಔط᠌ p47phox (NADPH oxidase subunit)

ೈқ፦߄౜ᡉ๱ቚу (p < 0.05)ǴЪຬ਼ϯނݔϯ䁙 (superoxide dismutase, SOD)ǵ

᝻䁙 (catalase, CAT) کᎽાҒ两 (glutathione, GSH) ࣣᡉ๱ၨ௓ڋಔեǶӕਔҔ MSک LOV ೀ౛ࡕǴط᠌ p47phox ೈқ፦߄౜ໆڙډ׭ڋǴל਼ϯሇન SODǵCAT ᆶ GSH ҭԖ΢ϲޑᖿ༈Ƕ

ᜢᗖຒǺ monascinˣၸࡋᑗϯಖౢނǵી৞નߔלǵל਼ϯሇન

(12)

Abstract

The aim of this study is to investigate the improvement of moanscin (MS) and lovastatin (LOV) on insulin resistance and the mechanism of MS and LOV for blood glucose regulation.

In this study we also provide evidence that advanced glycation end-products (AGEs) are able to affect insulin sensitivity and glucose tolerance in vivo. AGE also reduced expression of islet insulin. Together these results suggest that the AGEs pathway may play an important role in the loss of functional -cell mass in diabetes, at least in part through the induction of oxidative stress and the alternation of -cell differentiation and survival.

We found that AGE induction significantly resulted in hyperglycemia in BALB/c mice, but MS and LOV effectively inhibited inflammation and hyperglycemia caused by AGE. MS and LOV inhibited TNF-, IL-6, IL-1 expression in pancreases, liver and kidney. We also found that MS and LOV inhibited p47phox (NADPH subunit) expression in liver. Results demonstrated that MS and LOV elevated antioxidant enzyme: superoxide dismutase (SOD), catalase (CAT) and glutathione (GSH) in pancreases, liver and kidney.

Key words: monascin, advanced glycation end-products, insulin resistance, antioxidant enzymes

(13)

1

ಃ΋ക ߻ق

Ҟ߻җܭ໯१ಞᄍᆶғࢲ׎ᄊޑׯᡂǴᑗֿੰςԋࣁӄౚ໒วύکς໒ว୯ ৎޑ୯଼҇நୢᚒǶᑗֿੰ཮೷ԋ΋سӈٳวੱǴځวੰᐒڋ΋ޔࢂࣴز዗ᗺǴ

਼ϯᓸΚ໾্ࢂᑗֿੰٳวੱޑख़ाวੰᐒڋϐ΋Ƕᑗֿੰٳวੱޑੰ౛ၸำന Ь ा ᆶ ၸ ࡋ ᑗ ϯ ಖ ౢ ނ (advanced glycation end-products, AGEs) Ϸ ځ ڙ ᡏ (receptors for AGEs, RAGE) ࣬ᜢǶ

ӧܿБ୯ৎǴआ᜿ࢂ໺಍ޑϺฅ१ҔՅનǵٛᆭᏊǶआ᜿๵ (Monascus)ࢂ

΋ᅿλࠠํރᆭғ੿๵Ǵឦܭ੿๵ࣚǴη៶๵ߐ (Plectomycetes)Ǵη៶๵ᆜ (Ascomycetes)Ǵό᏾η៶๵Ҟ (Plectascales)Ǵआ᜿๵ࣽ (Monascaceae)Ǵआ᜿๵

ឦ (Monascus) (݅Ǵ1983)Ƕ߈ԃٰჹआ᜿ϐࣴزаځΒԛжᖴނࣁࣴز዗ᗺǶ Ԗӭ໨ࣴزᡉҢआ᜿ޑΒԛжᖴނڀԖӭᅿғ౛ࢲ܄٠ЪڀԖߥ଼фਏǶځύ lovastatin (monacolin K) ࣁआ᜿ғౢϐᖌڰᎇӝԋ׭ڋᏊǴ࿶୏ނϷΓᡏ၂ᡍ᛾

ჴڀफ़եՈિϷ෧ϿЈՈᆅ੯ੰ॥ᓀϐਏ݀ (Alberts, 1988; Juzlova et al., 1996)Ƕ ٠ЪԖࣴزൔᏤࡰрǴstatins ᜪϯӝނڀԖፓ࿯Ոᑗϐфਏǹआ᜿Βԛжᖴނύ ӭᅿ໳Յનς೏ϩᚆрٰǴځύ ankaflavin ᆶ monascin ନΑڀלวݹǵ׭ڋᕎ ಒझғߏ฻ (Su et al., 2005; Lee et al., 2006b) ӭᅿߥ଼фਏѦǴΨ೏᛾ჴڀԖׯ ๓ᑗֿੰੱރϐфਏ (Shi and Pan, 2010a; Shi and Pan, 2010b; Shi et al., 2012; Lee et al., 2013a)ǴڀԖ໒วࣁߥ଼१ࠔϐወΚǶ

ਡӢኧ-आՈౚϐ 2 ࣬ᜢӢη-2 (nuclear factor-erythroid 2 related factor-2, Nrf2) ࣁל਼ϯၡ৩ޑख़ाፓ௓ӢηǶ྽ᡏϣڙډ਼ϯᓸΚ໾্ਔǴԜӢηёᙯ

౽Կਡϣᆶל਼ϯᏊϸᔈ୔ (antioxidant response element, ARE) ่ӝǴௗ๱ᇨᏤ ځፓ௓ޑΠෞ୷ӢǴхࡴ೚ӭᆶל਼ϯ࣬ᜢޑሇનᆶೈқ፦ϐ߄౜Ǵ٬ځวචբ

(14)

ҔவԶफ़ե਼ϯᓸΚ஥ٰޑ໾্ǶӢԜҁࣴزа໢܄ BALB/c λႵբࣁ୏ނჴ ᡍຑ՗ኳԄǴаᕕှ monascin ک lovastatin ჹ AGEs ᇨᏤᑗֿੰ୏ނી৞નߔ לک਼ϯᓸΚᆶวݹϐፓ௓ૈΚǴ׆Ꮗ monascin ک lovastatin ё໒วԋࣁ෧Ͽી

৞ ಒझઇᚯǴׯ๓ી৞નל܄ϐཥᑉݯᕍಃΒࠠᑗֿੰϐᛰނǶ

(15)

3

ಃΒക Ў᝘ӣ៝

΋ǵᑗֿੰ (diabetes)

ᑗֿੰࢂ΋ᅿᆶᒪ໺ӢનکӭᅿᕉნӢન࣬ᜢޑᄌ܄ӄيжᖴ܄੯ੰǶ߈ԃ

ٰǴҗܭғࢲНྗޑගଯǵ໯१่ᄬޑׯᡂǵВᖿᆙ஭ޑғࢲ࿯ࠩаϷό଼நޑ ғࢲБԄ฻ፏӭӢનǴᏤठᑗֿੰԋࣁӄౚ܄ޑᝄख़࠶ુΓᜪ଼நϐ੯ੰǶՋϡ 2000ԃӄౚᑗֿੰΓαࣁ 1.71 ሹǴႣෳԿՋϡ 2030 ԃᑗֿੰΓαஒԋߏԿ 3.66 ሹ (Wild et al., 2004)ǶᑗֿੰନΑ཮Ꮴठ஻ޣၸଯޑֿᑗѦǴ׳཮Їଆځдٳว

ੱǴхࡴຎᆛጢ໾্ǵ๝᠌ੰᡂϷတཞ໾ǴӢԜ௓ڋᑗֿੰচ߾ϐ΋ࣁፓ࿯Ոᑗ аᗉխٳวੱวғǶ!

ਥᏵऍ୯ᑗֿੰڐ཮ (American Diabetes Association: ADA) Ϧթϐᑗֿੰ

ղۓ኱ྗǴޜဎ 8 λਔࡕՈᑗεܭ܈฻ܭ 126 mg/dL ܈αܺ 75 g ဟ๻ᑗऐڙ၂ ᡍǴ 2 λਔՈᑗϝεܭ܈฻ܭ 200 mg/dLǴջёບᘐࣁᑗֿੰǶWeyer ฻Γ೯ ၸᑗֿੰޑࢬՉੰᏢࣴزගрᑗֿੰวੰޑ“ٿ໘ࢤኳԄ”Ꮲᇥ (Weyer et al., 1999)Ƕಃ΋໘ࢤǺ२Ӄᐒᡏҗܭી৞નܢלᏤठᑗऐໆ҅தᄽᡂࣁᑗऐໆ౦த (impaired glucose tolerance, IGT)ǹಃΒ໘ࢤǺી৞નܢלک ಒझϩݜфૈڙ ཞǴ٬ IGT ᄽᡂࣁಃ 2 ࠠᑗֿੰǶ IGT ϐղۓ኱ྗࣁ໭ࡕ 2 λਔޑՈᑗॶຬၸ 140 mg/dLǴՠϝ҂ၲډ 200 mg/dLǶ

Andrew฻Γࡰрᑗֿੰϩࣁϖεᜪ (Andrew et al., 1984)Ǵхࡴ (1) ಃ 1 ࠠ ᑗֿੰ (type 1 diabetes)ǴΞᆀࣁી৞ન٩ᒘࠠᑗֿੰ (insulin-dependent diabetes mellitus, IDDM)ǴεӭኧӢࣁ ಒझڙઇᚯ೷ԋી᠌ϩݜી৞નόى܌ЇଆǴ஻

ޣሡݙ৔ી৞નωૈׯ๓ՈᑗǶҞ߻ჹಃ 1 ࠠᑗֿੰϐੰӢکวੰᐒڋόֹӄ ᕕှǶ(2) ಃ 2 ࠠᑗֿੰ (type 2 diabetes)ǴΞᆀࣁߚી৞ન٩ᒘࠠᑗֿੰ

(16)

(non-insulin-dependent diabetes mellitus, NIDDM)Ǵ9095%ᑗֿੰឦܭԜᜪǴЬा

ੰӢࣁી৞નߔלᏤठଯՈᑗ (hyperglycemia)ǶԜᜪ஻ޣᗨฅી৞ ಒझૈ୼

҅தϩݜી৞નǴՠӢڬᜐಔᙃ (طǵԼԺᆶિެ฻) ჹી৞ન௵ག܄फ़եǴ຾

Զቹៜဟ๻ᑗឪڗբҔԶ೷ԋଯՈᑗǴԶ࡭ុ܄ޑଯՈᑗڈᐟી৞ ಒझόᘐӦ ϩݜી৞નǴӢԶౢғଯી৞નՈੱ (hyperinsulinemia)ǶBouche ฻Γࡰрߏය ଯՈᑗᆶଯՈિ཮೷ԋી৞ ಒझཞ໾Ǵ٠೴ᅌ٬ಃ 2 ࠠᑗֿੰ஻ޣી৞ ಒ झѨѐϩݜી৞નфૈǴ٬ϐനಖሡा٩ᒘݙ৔ી৞નᆢ࡭҅தғ౛ᐒૈ

(Bouche et al., 2004)Ƕ(3) ֳৄᑗֿੰ (gestational diabetes mellitus) ࣁ஁ζᚶѫਔ և౜ޑᑗֿੰǴᇸޣሡा໯१௓ڋǴᝄख़ޣሡा຾Չી৞નݯᕍǶ(4) ੝ਸᜪࠠ

ޑᑗֿੰ: ӵ୷Ӣલഐ೷ԋ ಒझ౦தǵી৞નϩݜ౦தǵѦӢ܄ી᠌фૈ౦ தǵᛰނ܈ϯᏢ໾্೷ԋ౦தǶ(5) ဟ๻ᑗऐڙ౦த (impaired glucose tolerance, IGT)ǵޜဎՈᑗ౦த (impaired fasting glucose, IFG)Ǻёۓကࣁᑗֿੰ߻යǴ೭೽

ҽΓဂύԖ࣬྽ଯКٯ཮ว৖ԋᑗֿੰǶ!

ᑗֿੰ཮ౢғ೚ӭᄌ܄ٳวੱǴ཮ቹៜόሡाી৞નޑᏔ۔ǴႽࢂЈ᠌Ոᆅǵ

౳࿊ǵ๝᠌ک೷ԋઓ࿶Бय़ޑੰᡂǶҗܭᑗֿੰ஻ޣߏයኪ៛ܭଯՈᑗΠǴόՠ Ոᑗᐚࡋၸଯ཮٬ளၸࡋᑗϯಖౢނ (AGEs) ε൯ቚуǴԶЪ཮ගϲ୏ેฯϯǵ ЈՈᆅٳวੱǵֿࢥ᝛Ϸ๝᠌ੰᡂ฻ӭᅿ੯ੰϐวғ൳౗ǶՠҞ߻೭٤ٳวੱठ

ੰᐒᙯۘ҂ֹӄᕕှǶ

Βǵၸࡋᑗϯಖౢނ (advanced glycation end-products, AGEs)

AGEs ޑғԋ೼৩Ǻ྽ಒझೀܭ΋ۓำࡋޑଯՈᑗᕉნΠ܈ࢂ१ࠔуπၸำ ύǴೈқ፦ޑ amino group کᗋচᑗޑ carbonyl group วғߚሇન܄ϸᔈ (nonenzymatic reaction)Ǵ ׎ԋόᛙۓЪଯࡋё଍ޑ Schiff baseǴӧ೭ঁၸำύ

(17)

5

཮ғԋල໵Յޑεϩηނ፦ᜪ໵ᆒ܈ᆀᔕ໵નǶନౢғᜪ໵ᆒѦǴϸᔈၸำύᗋ

཮ౢғԋԭ΢ίঁԖόӕ਻ښޑύ໔ᡏϩηǴхࡴᗋচ✉ǵ⾺کᚇᕉϯӝނǴ೭ ٤ނ፦ࣁ१ࠔගٮΑەΓёαޑ॥ښکᇨΓޑՅᐛǶϐࡕ཮࿶җ amadori rearrangement ౦ᄬϯ׎ԋ amadori product炻ځၨᛙۓՠϝё଍Ǵനಖ࿶җϩη໔ ख़ಔ (molecular rearrangement)ǵಥН (dehydration) Ϸᐚᕭ (condensation) ฻ϸ ᔈ຾Զ׎ԋόё଍ޑϯӝނǴѬૈᆶ࣬ᎃೈқ΢ϐၯᚆữ୷Ӆሽ่ӝǴᆶೈқ፦

Ҭΰ࣬ϕೱௗ(cross-linking) ࡕǴև౜ፃՅ٠วрᑻӀ (Amore et al., 1997)Ƕ!

ဟ๻ᑗޑԾي਼ϯౢނΌΒ⾺ (glyoxal) ϷҘ୷ΌΒ⾺ (methylglyoxal) ฻ жᖴނࢂ΋ᜪΒ♐୷ϯӝނ (dicarbonyl compounds) ǴԖࣴز᛾ჴࢂᙖҗϩηϣ

܈ϩη໔Ҭᖄᗖ่܌ౢғǴԜҬᖄϸᔈЬाҗΌΒ⾺܈Ҙ୷ΌΒ⾺ᆶᚆữለ (lysine) ܈ᆒữለ (arginine) ϸᔈ܌ठǴӆᆶೈқ፦ϸᔈ׎ԋ AGEsǶAGEs ғ ӝԋၡ৩ӵკ 2-1 ܌ҢǶӧᑗֿੰ஻ޣޑಔᙃύததӸӧଯᐚࡋޑ AGEsǴԶѬ ൩ ࢂ Ꮴ ठ ε ӭ ኧ ᑗ ֿ ੰ ٳ ว ੱ ޑ ന Ь ा চ Ӣ Ǵ ٯ ӵ Ǻ ୏ ે Ո ᆅ ๆ ރ ฯ ϯ (atherosclerosis) ൩ࢂ΋ᅿᑗֿੰٳวੱǶBrownlee ᇡࣁଯᐚࡋޑ AGEs ᆶᑗֿ

ੰϷځٳวੱวғԖ҅࣬ᜢ܄ (Brownlee, 1991)Ƕ

Γᡏϣ AGEs ޑٰྍԖٿᅿБԄǺ΋ᅿࢂᡏϣౢғǴќ΋ᅿ൩ࢂѦྍ܄ᑧ १ឪΕǴᆀࣁѦྍ܄ AGEsǴٯӵྟ૛܈ޣё኷ύ೿֖Ԗଯᐚࡋޑ AGEsǴ੘ᓥ

࿶ၸଯྕొፓϐࡕ AGEs ޑ֖ໆ཮΢ϲ 100 ७ (Koschinsky et al., 1997)Ƕ१ނύ ޑ AGEs жᖴ೼৩ӵკ 2-2 ܌ҢǶAGEs ࢂ೚ӭόӕᅿޑ่ᄬނޑᕴᆀǴж߄܄

AGEsϐϯӝނ่ᄬӵკ 2-3 ܌ҢǶ౜ςޕၰхࡴ N-caroxymethyllysine (CML)ǵ pentosidineǵpyrralineǵcrosslineǵglyoxal-lysine dimer (GOLD)ǵmethylglyoxal-lysine

(18)

კ 2-1 AGE Fig. 2-1 Fo

Esޑ׎ԋၸ ormation of

ၸำ AGEs

(Reddy andd Beyaz, 20006)

(19)

კ 2-2 १ Fig. 2-2

१ނύ AGE Schematic

Esޑжᖴၸ representati

7

ၸำ!

ion of the faate of diet-dderived AGE (Koschins

Es

sky et al., 19997)

(20)

კ 2-3!ж߄ Fig. 2-3 Str

߄܄!AGEs ructures of s

s ϐϯӝނ selected adv

ނ่ᄬ!

vanced glyc

(Redd

cation end-p

dy and Beya

products (AG

az, 2006)

GEs)

(21)

9

dimer (MOLD) ฻ǴᗋԖ΋٤ނ፦౜ӧϝόޕၰځڀᡏ่ᄬǶځύ CML ᆶ pentosidine ࢂڂࠠޑ਼ϯޑ AGEsǴ܌аΨћ଺ᑗϯ਼ϯౢނǶԶ pyrraline ࢂ ߚ਼ϯޑ AGEsǶCML ೏ᇡࣁࢂᑗϯ኱ޑނǴԶ parralineǵcrossline ک pentosidine

೏ᇡࣁࢂೈқ࣬ϕҬᖄޑ኱ޑނǶAminoguanidine ࢂ΋ᅿёа׭ڋ AGEs ׎ԋ ϐᛰނǴځբҔᐒڋࣁஒఘયౢނύϐᅹ਼ᚈᗖ (C=O) ׯࣁᅹේᚈᗖ (C=N)Ǵ

຾Զ׭ڋ AGEs ғԋǴ෧጗ᑗֿੰٳวੱϐੱރǴхࡴຎᆛጢੰᡂǵ๝᠌ੰᡂ ϷՈᆅ౦த (Brownlee et al., 1986; Amore et al., 1997)Ƕ

Ԗࣴزࡰр AGEs ୖᆶᑗֿੰٳวੱǵ๝фૈόӄੰΓޑಔᙃཞ໾ǵՈિ

౦தϷԴϯၸำǴҞ߻ჹ AGEs ޑࣴزЬा໣ύӧᆶᑗֿੰٳวੱ࣬ᜢϐᐒڋ

௖૸ǴӢࣁᑗֿੰ஻ޣՈᑗၨଯǴಔᙃೈқܰ೏ᑗϯǶAGEs ׎ԋЇଆޑੰ౛ׯ ᡂЬा೯ၸΟঁၸำ (Brownlee, 2001)Ǻಃ΋ǴAGEs ׯᡂΑಒझѦ୷፦ೈқޑ

܄፦ϷಒझѦ୷፦ᇨวޑߞဦᙯᏤ೼৩ǶಃΒǴ೯ၸ AGEs ᆶځ੝౦ޑಒझڙ ᡏ࣬ϕբҔǴׯᡂΑёྋ܄ߞဦϩηӵಒझӢηǵᐟનکԾҗ୷ޑНྗǶಃΟǴ җဟ๻ᑗǵ݀ᑗ฻ӧಒझϣ׎ԋޑ AGEs کϸᔈࢲ܄ଯޑύ໔ౢނૈޔௗׯᡂ ႢಔᙃೈқфૈǶ

RAGE (receptor for AGE) ࢂ AGE ޑЬाڙᏔǴឦܭխࣝౚೈқຬભৎ௼Ǵ җ 400 ӭঁữ୷ለ܌ᄬԋǴόڀԖႀữለᐟ䁙ࢲ܄Ƕӧ೚ӭख़ाಒझύ೿ว౜Ԗ RAGE ޑӸӧǴхࡴϣҜಒझ (endothelial cells)ǵൂਡտᏘಒझ (mononuclear phagocyte)ǵՈᆅѳྖԼಒझ (smooth muscle cells) Ϸط᠌ࢃރಒझ (hepatic stallete cells)ǶRAGE ޑϩηໆаϷک AGE ่ӝޑૈΚ཮ӢಒझᅿᜪԶԖৡ౦Ƕ ӢԜёૈӧόӕಒझ΢ޑ RAGE ࢂឦܭځৎ௼ύόӕޑ subtypeǶ྽ AGEs ᆶ

(22)

კ 2 Fig.

2-4ၸࡋᑗϯ . 2-4 Mecha

ϯനಖౢނ anism for in

ނᇨᏤวݹϐ nflammation

ϐᐒڋკ n induction

(Fatehi-H

by AGEs

Hassanabadd et al., 20100)

(23)

11

RAGE ่ӝࡕ཮೷ԋΠෞૻ৲໺ሀǴ٬ p47phox (NADPH oxidase subunit) ࢲϯǴ ගϲಒझ਼ϯᓸΚ຾Զࢲϯ nuclear factor-kappa B (NF-B)Ǵ NF-B ࢲϯࡕ཮ᙯ

౽ډಒझਡϣߦ٬Πෞ୷ӢϐᙯᒵǴ຾ԶౢғวݹಒझᐟનǴхࡴ TNF- Ϸ IL-1 (Guha et al., 2000) کՈᆅಒझᗹߕϩη-1 (vascular cell adhesion molecule-1, VCAM-1) аϷߦ٬ RAGE ҁيޑεໆ߄౜ (Wantier et al., 2001) Ǵ຾Զ٬ளӭ ᅿಒझوӛ঒ΫǴཞ໾ಔᙃᏔ۔фૈ(Huttuen et al.,1999)ǶAGEs ᇨᏤวݹ೼৩ ӵკ 2-4 ܌ҢǶ

!

Οǵી৞નૻ৲໺ሀၡ৩

ી৞ન (insulin) җી᠌ ಒझϩݜౢғǴࢂᆢ࡭Ոᑗॶ㚌ۓᆶಔᙃғߏว ػޑख़ा಻ᅟᆾϐ΋Ǵી৞નૻ৲໺ሀၡ৩ӵკ 2-5 ܌ҢǶી৞નϐЬाբҔࣁ ߦ຾ಒझճҔՈనύဟ๻ᑗԶፓ࿯ՈᑗǴቚуڬᜐಔᙃٯӵԼԺᆶિެಔᙃჹဟ

๻ᑗޑឪڗᆶճҔǴ٠ᙖҗિ፦ғӝԋ (lipognesis) Ϸطᑗӝԋ (glycogenesis) բҔǴ٬ط᠌ϷԼԺᆶિެಔᙃᓯӸيᡏύၸӭϐૈໆǴ٠ჹિှ (lipolysis) ᆶ طᑗϩှ (glycogenolysis)Ԗ׭ڋբҔǶ

ી৞નૻဦ໺ሀ໒ۈܭી৞નᆶಒझ߄य़ڙᏔ่࣬ӝǴௗ๱຾ՉΠෞૻ৲໺

ሀǶી৞નڙᏔ (insulin receptor, IR) ӧথ٢୏ނޑಔᙃಒझύቶݱӸӧǴЀځ ӧԼԺǵિެϷط᠌฻ી৞નբҔಔᙃύεໆ߄౜ǶIR ࢂ΋ᅿऀጢޑᗐೈқǴ Ьाҗٿঁ -٥୷ (subunit) Ϸٿঁ -٥୷ಔԋǴ-٥୷ڀԖႀữለೈқ፦ᐟ䁙 (tyrosine protein kinase) ࢲ܄Ǵᙖҗᚈ౷ᗖᄬԋѤᆫᡏǶ྽ಒझጢѦޑ -٥୷ᆶ

ી৞ન่ӝࡕǴ཮Їวಒझጢ΢ -٥୷วғԾᕗለϯ (auto-phophorylation)ǴԶ

׎ԋڀԖࢲ܄ޑ p-IRǴځᒿջஒ (insulin receptor substrate, IRS) IRS-1 Ϸ IRS-2

(24)

კ Fi

კ 2-5 ી৞ન g. 2-5 Insul

નૻ৲໺ሀ lin signaling

ሀၡ৩

g mechanismm

(Saaltiel and KKahn, 2001)

(25)

13

ޑႀữለՏ࿼຾Չᕗለϯ (phosphorylation) வԶ٬ IRS-1 Ϸ IRS-2 ࢲϯǴа໺

ଌΠෞૻဦ٠ගϲી৞ન௵ག܄ǶIRS-1 ࢂ΋ঁϩηໆऊࣁ 180 kDa ϐεϩηೈ

қǶѬڀԖ PTB domainǴ٠аԜ่ӝډᕗለϯޑ IRǴ೷ԋ IRS-1 ޑႀữለᕗ ለϯǶᕗለϯޑ IRS-1 ёᆶ೚ӭڀԖ src homology 2 domain (SH2 domain) ޑϩ η่ӝǶ!IRS-1 ޑ߄౜फ़եΨࢂᏤठી৞નߔלޑख़ाӢનϐ΋ (Saltiel and Kahn, 2001)ǶIRS-2 ڙډ insulin-like growth factor 1 (IGF-1) کځдߦಒझวݹᐟ ન܌ፓ௓ǴIRS ୷Ӣޑᕗለϯ໪ᙖҗ๱ insulin receptor ௗԏډ insulinǴԶ٬ள receptor΢ޑ tyrosine kinase ᕗለϯٰ௴୏Ƕ

IRS-1ೈқᕗለϯ཮Їଆ phosphatidylinositol 3-kinase (PI3K) ޑࢲϯǴ ߦ٬

PIP2ӛ PIP3 ᙯᡂǴࢲϯޑ PI3K ໺ሀӭख़ૻ৲Їว PtdIns ᙯԋ PtdIns-(3, 4 and 5)-P3Ǵ٬ځѐࢲϯԋڀԖ pleckstrin homology domains (PH) ޑڙᡏǴ٠ᝩុΠ

ෞϐૻ৲໺ሀբҔ(Satiel and Kahn, 2001)Ƕ

PIP3཮٬ PKB/Akt ᕗለϯǴ ځᕗለϯޑՏ࿼Ьाӧ threonine 308 ک serine 473ǶPKB ೏ᕗለϯϐࡕ཮٬চҁӧಒझϣޑ glucose transporter 4 (GLUT4) ᙯՏ (translocation) ډಒझጢ߄य़΢߄౜ǄGLUT4 ࣁી৞ન௵ག܄ဟ๻ᑗᙯၮೈқ, ЬाӸӧܭମᓝԼکિެಒझǶനಖ٬ဟ๻ᑗ৒ܰ຾Εډಒझϣ೏ճҔکжᖴǴ ᆢ࡭Ոᑗ㚌ۓ (Wang et al., 1999)Ƕ

ဟ๻ᑗࢂথ٢୏ނख़ाޑૈໆౢғޑٰྍϷғӝԋբҔϐ୷፦ǴЬा࿶җᐘ ણ ᜪ ނ ፦ Н ှ ࡕ ౢ ғ ٠ ࿶ λ ဉ ֎ ԏ ᓯ Ӹ ճ Ҕ Ǵ ܈ җ ط ᑗ ϩ ှ ౢ ғ (glycogenolysis)Ƕҗܭဟ๻ᑗࢂᒃН܄ޑނ፦Ǵคݤ೯ၸિ፦ᚈቫޑಒझጢǴӢ ԜՈనύဟ๻ᑗࣣሡाဟ๻ᑗᙯၮೈқ (glucose transporter, GLUTs) ೸ၸߦ຾

ᘉණ (facilitate diffusion) ޑБԄᙯၮԿಒझϣճҔǶGLUTs ᙯၮೈқৎ௼Ҟ߻ !

(26)

߄ 2-1!ဟ๻

Table 2-1 T

๻ᑗ࣬ᜢၮ The glucose

ၮᒡೈқ!

transporter

r (GLUT) fa

amily of fac

(Wo

cilitative sug

ood and Tra

gar transpor

ayhurn, 200 rters

3)

(27)

15

Ԗ GLUT 1-12 ฻όӕ౦ᄬނǴܭಔᙃύϩթӵ߄ 2-1 ܌ҢǶᇶշ܄ဟ๻ᑗᙯၮ

ೈ қ х ֖ GLUT1 Կ GLUT2 Ǵ ࿶ җ ữ ୷ ለ ׇ ӈ К ჹ ள ޕ ۶ Ԝ ໔ Ӹ ӧ ๱ 39%-65% ޑ࣬՟܄ǴਥᏵಔᙃϩթՏ࿼ৡ౦کځ೏಻ᅟᆾ܌ፓ࿯όӕԶϩࣁΟ ᜪǴջ Class 1 (GLUT1-4)ǴClass 2 (GLUT5, 7, 9, 11)!ࢂ݀ᑗᙯၮೈқϷ Class 3 (GLUT6, 8, 10, 12)Ҟ߻фૈۘόమཱǶGLUT1 ࣁന୷ᘵϐဟ๻ᑗၮᒡೈқǴЬ

ाϩթܭჹૈໆሡ؃നܮ౰ޑआՈౚϷတಒझύǶGLUT5 Ьाॄೢၮᒡ݀ᑗǴ Ӹӧܭλဉಒझ (Davidson et al., 1992)ǶGLUT2 ࢂ΋ᅿکဟ๻ᑗ่ӝΚࡐեޑ ဟ๻ᑗᙯၮೈқǴЬाϩթܭطǵ๝ǵλဉکી৞ ಒझǶӧી৞ ಒझύǴ

GLUT2 բࣁဟ๻ᑗགڙᏔǴᙯၮဟ๻ᑗ຾ΕಒझύǴ࿶΋سӈߞဦ໺ሀǴനࡕ

ߦ٬ી৞નញܫǶGLUT4 ࢂҞ߻ςޕڙી৞નፓ࿯നЬाޑဟ๻ᑗᙯၮೈқǴ ЬाϩթܭિެಔᙃǵЈ᠌ᆶԼԺύǴࡕٰ೏ว౜ΨӸӧܭတಔᙃύ (Rayner et al., 1994)ǶGLUT4 ӧી৞નל܄ౢғ܌೷ԋޑᑗֿੰύתᄽख़ाفՅ (Garvey et al., 1987)Ƕڙډી৞નޑڈᐟբҔࡕǴ཮٬চҁӸӧಒझ፦ϣ֖Ԗ GLUT4 ޑ vesicleᙖҗझӗբҔ!(exocytosis) ஒ GLUT4 ᙯ౽Կಒझጢ߄य़ (Holman et al., 1990)ǴԶගϲಒझჹဟ๻ᑗտᏘբҔǴ྽ી৞નڈᐟ੃ѨࡕǴಒझጢ΢ޑ GLUT4

཮ӆ࿶җझ໯բҔ (endocytosis) ख़ཥӣډಒझ፦ϣ฻ࡑΠ΋ԛી৞નڈᐟǶ!

!

Ѥǵી৞નߔל (insulin resistance)

ಃ 2 ࠠᑗֿੰ஻ޣวੰ߻தԖી৞નߔל౜ຝǶી৞નߔלࢂࡰՈనൻᕉύ ϐી৞નፓ࿯ᡏϣૈໆжᖴϐૈΚफ़ե (Zick et al., 2001)ǶΨ൩ࢂӧ҅தᐚࡋޑ

ી৞નғ౛ᐚࡋΠǴيᡏჹી৞નޑ௵ག܄फ़եࣗԿჹϐడคϸᔈǶી৞નߔל ޑϩηᐒڋӸӧӭᅿᏢᇥǴхࡴ୷Ӣલഐǵᛰނǵޥऔǵ໯१ǵғࢲ׎ᄊ฻ቹៜǶ

(28)

ၸӭޑၯᚆિެለΨࢂ೷ԋી৞નߔלޑख़ाӢનǶӧޥऔੰΓᡏϣǴيᡏϣၸ ӭޑિެಔᙃ཮ቚу adipokine ᆶၯᚆિެለϐញܫ٠࿶Ոనଌډط᠌܈ԼԺ฻

ಔᙃǶLeclercq ฻Γࡰр adipokine ύޑဍዦᚯԝӢη- (tumor necrosis factor-, TNF-)ǵϟқન-6 (interleukin-6, IL-6)ġ ᆶ resistin ཮೷ԋط᠌ౢғી৞નߔל (Leclercq et al., 2007)Ƕ ી৞નߔל೷ԋޑଯી৞નՈੱΨ཮ቚமᡏϣ໊ᚆηӆ

֎ԏǴ຾ԶቚуଯՈᓸ (hypertension) ᆶ୏ેฯϯ (atherosclerosis) ϷځдЈՈ ᆅ੯ੰ฻॥ᓀ (Hanley et al., 2002)Ƕ

ط᠌ࣁፓ௓ՈᑗޑᏔ۔ϐ΋ǴՈనύޑဟ๻ᑗЬा೯ၸဟ๻ᑗᙯၮೈқ 2 (GLUT2) ຾Εط᠌Ƕ྽ՈᑗᐚࡋϲଯǴી৞નᙖҗࢲϯطᑗᆶિެӝԋ࣬ᜢޑ ሇનவԶߦ຾ط᠌ؕڗՈనύޑဟ๻ᑗ٠ஒځᙯϯࣁطᑗ܈િެǴી৞નΨёа

׭ڋط᠌ύᑗ፦ཥғ࣬ᜢ୷Ӣ߄౜Ƕط᠌Ψࢂ௓ڋՈనύી৞નᐚࡋޑख़ाᏔ۔Ǵ Meshkani฻Γว౜ಃ 2 ࠠᑗֿੰ஻ޣᡏϣط᠌ી৞નమନૈΚ (insulin clearance) फ़ե (Meshkani et al., 2009)Ƕ

྽ط᠌ύી৞નߔלϐ௃׎วғࡕǴط᠌ፓ௓Ոనύဟ๻ᑗᆶી৞નૈΚό ىǴ೷ԋଯՈᑗᆶଯી৞નՈੱǴߏය࡭ុΠѐё೷ԋಃ 2 ࠠᑗֿੰǵ୏ેฯϯ

܈ߚଚᆒ܄િެط (non-alcoholic fatty liver, NAFLD)ǶGastaldelli ฻Γࡰрᑗֿ

ੰ஻ޣޜဎਔط᠌ᑗ፦ཥғբҔၨ଼நޣቃਗ਼Ǵ೷ԋځޜဎଯՈᑗ౜ຝǴЪόܰ

ڙી৞ન܌׭ڋ (Gastaldelli et al., 2001)Ƕ

!

ϖǵਡӢη.आՈౚϐ!2 ࣬ᜢӢη.2ġ(nuclear factor-erythroid 2 related factor-2,

Nrf2) ᆶᑗֿੰޑᜢس!

ଯՈᑗ཮Ꮴठ AGEs ౢғǴ٠ᇨᏤխࣝಒझᇙ೷ϷϩݜวݹӢηǶଯՈᑗ

(29)

17

཮ࢲϯ NADPH oxidaseǴ٬ϐౢғࢲ܄਼ (reactive oxygen species, ROS)ǴԶ ROS ቚу཮फ़եી᠌ી৞ל਼ϯሇનޑ mRNAǵೈқ፦Ϸࢲ܄߄౜Ƕ྽ી৞ન ߔלౢғਔǴࣁΑжᓭԜ౜ຝǴ཮ᙖҗቚуဟ๻ᑗޑжᖴٰቚம ಒझញܫી

৞નǴΨӢԜቚуΑಈጕᡏౢғ ROSǶ ಒझߏයኪ៛ܭ ROS ᕉნΠǴஒ཮

೷ԋфૈምᛖǶી৞નߔלวғਔǴମᓝԼύޑ਼ϯᓸΚΨ཮ቚуǴٯӵǺຬ਼

഍ᚆηǵణ਼Ծҗ୷฻ (Henriksen, 2006)Ƕᆕӝ΢ॊǴ਼ϯᓸΚࢂ೷ԋᑗֿੰϷ ځٳวੱวғޑ΋໨ख़ाӢηǶ

Nrf2 ᙯᒵӢηǴ྽ځ่ӝԿ antioxidant response element (ARE) ߾཮ୖᆶ࣬

ᜢ୷Ӣޑፓ௓ǴЬाӧှࢥᏔ۔ӵطǵ๝ϷᆶѦࣚᕉნௗ᝻ޑᏔ۔ӵҜጥǵޤک ဉၰύεໆ߄౜Ƕ҅தރᄊΠǴNrf2 Տܭझ፦ύǴЪᆶ Kelch-like ECH-associated protein-1 (Keap-1) ࣬ϕբҔࡕफ़ှǵѨࢲ (Kaspar et al., 2009)Ƕ྽ڙډѦࣚڈᐟ ࡕǴNrf2 ᆶ Keap-1 ϩ໒٠຾Ε䵿झਡǴߦ຾࣬ᜢޑל਼ϯሇન୷Ӣ߄౜ (Itoh et al., 1999)Ƕ

Ԗࣴزࡰр protein kinase C (PKC)ǵphosphoinositide 3-kinase (PI3K)Ǵ extracellular signal-regulated kinase (ERK) ฻཮ஒ Nrf2 ΢ޑ serine Ϸ threonine

ූ୷຾ՉᕗለϯǴவԶߦ຾ Nrf2 ᆶ Keap-1 ޑှᚆǴ຾Զᙯ౽Կಒझਡϣ (Numazawa et al., 2003; Kang et al., 2002; Zipper and Mulcahy, 2002)ǶNrf2 ϐࢲϯ ᐒڋӵკ 2-6 ܌ҢǶ

!

Ϥǵל਼ϯٛᑇس಍!

ࢲ܄਼ (reactive oxygen species炻ROS) ϷԾҗ୷ (free radical) ཮೷ԋғނ ᡏϩηޑཞ໾ǴᏤठ੯ੰޑౢғǶՠғނᡏϣڀԖל਼ϯٛᑇس಍Ǵа෧጗ࢲ܄!

(30)

!

კ 2-6 ਡӢ Fig. 2-6 Me

!

Ӣη.आՈౚ echanism of

ౚϐ 2 ࣬ᜢ f Nrf2 activ

Ӣη.2 ࢲϯ vation

ϯϐᐒڋკ

(Negi et a

კ!

al., 2011)

!

(31)

!

Βǵ Ȑ΋

კ 2- Fig. 2

ǵआ᜿

΋ȑआ᜿๵

7ಒझϣϐ 2-7 The anti

੝܄

ϐל਼ϯٛᑇ ioxidative d

ᑇس಍!

defense syst

19

tem in the c

(Jas

ell

ssem and HeHeaton, 20044)

(32)

਼ϷԾҗ୷ޑౢғǴځύхࡴל਼ϯނϷל਼ϯሇન (Chen and Yen, 1998)Ƕಒ झϣϐל਼ϯس಍ӵკ 2-7 ܌ҢǶל਼ϯሇનٛᑇس಍Ьाхࡴ superoxide dismutase (SOD)ǵglutathione peroxidase (GPx)ǵcatalase (CAT) ฻Ƕ

1. ຬ਼ϯނݔϯ䁙 (superoxide dismutase, SOD)

SODࢂ΋ᅿߎឦሇન (metalloenzymes)ǴӸӧܭಒझ፦ᆶಈጕᡏύǴϩթܭ ӳ਼܄ғނᡏϣǶᎋል SOD (Zn/Cu-SOD) ЬाӸӧܭಒझ፦ύǴᒰ SOD (Mn-SOD)!ЬाӸӧܭಈጕᡏǶځբҔࢂమନຬ਼ϯނ഍ᚆηԾҗ୷Ǵஒϐᙯᡂ ԋ H2O2Ϸ O2ǴԶ H2O2ё೸ၸ GPx ܈ CAT ஒϐжᖴԋόڀࢥ܄ޑ H2OǶSOD

଺ࣁаΠϸᔈޑሇનǺ

O₂^ˤ+ O₂^ˤ+ 2 H+ ɦ!H2O₂+ O₂

2. ᎽાҒ两ၸ਼ϯނ䁙 (glutathione peroxidase, GPx)

GPx ࢂΓᡏϣߚதख़ाޑל਼ϯሇન-ࡪځϩթՏ࿼ёϩࣁಒझ፦ᆶಈጕᡏ

ύ ޑ cellular glutathione peroxidase (cGPx) ǵ Ո ዀ Ϸ Ո మ ύ ޑ extracellular glutathione peroxidase (eGPx) Ϸ Տ ܭ ಒ झ ጢ ΢ ޑ phospholipid hydroperoxide glutathione peroxidase (phGPx)ǶGPx ཮ஒિ፦ၸ਼ϯނᗋচԋࣁคࢥ্ౢނǶGPx ӧғނᡏϣॄೢమନ H2O2Ǵ ځஒ H2O2 ᗋচԋ H2OǴ٠ஒ਼ϯᄊ GSH ᗋচ ԋᗋচᄊ GSSGǴаफ़եғނᡏϣ਼ϯᓸΚޑౢғǶGSH ᙖҗ GPx ᙯᡂࣁ GSSGǴ ஒಒझϣѦόӕ೽Տౢғޑ H2O₂Ϸિ፦ၸ਼ϯނᗋচԋН܈ࢂ lipid alcoholsǴ ӆ࿶ᎽાҒ两ᗋচሇન (glutathione reductaseǹGR) ᗋচԋࣁ GSSGǶځమନ H2O2 ᐒڋӵΠǺ

2 GSH + H₂O₂ʈ GSSG + 2 H2O 3

(33)

21

3. ᝻䁙 (Catalase, CAT)

CATЬाӸӧܭၸ਼ϯᡏ (peroxisome)Ǵځё੝౦܄Ӧஒ H2O₂ᙯࣁ H2OǶ ၸ਼ϯᡏϣޑሇનӵ amino acid oxidase ک a- hydroxy-acid oxidase ౢғεໆޑ H2O2Ƕ܌а catalase ࣁၸ਼ϯᡏύжᖴၸ਼ϯణޑख़ाሇનǶCAT ଺ࣁаΠϸᔈ ޑሇનǺ

2 H₂O₂ʈ 2 H2O + O₂

4. ᎽાҒ两 (Glutathione, GSH)

ᎽાҒ两ࣁख़ाНྋ܄ל਼ϯނǶᎽાҒ两ӧ౦ғނ፦ཥഋжᖴёૈޔௗӦ ύک ROS ٯӵݨિၸ਼ϯނǴ٠ЪתᄽЬفǶғނᡏύόӕ೽Տޑ GSH ֖ໆ Ԗৡ౦ǶGSH ёޔௗᆶԾҗ୷բҔ܈ک GPx Ӆӕమନᡏϣޑ H2O2Ϸځдၸ਼

ϯނ፦Ǵа෧ᇸ ROS ჹғނᡏ೷ԋ਼ϯ܄໾্Ƕ

߈ԃٰǴ೚ӭࣴز჋၂җϺฅٰྍа൨؃ӼӄЪڀԖமל਼ϯਏΚϐל਼ϯ ᏊǴځύаӭ⇌Ϸᜪ໳✉ࣁ೏ࣴزനӭϐל਼ϯނ፦Ǵځёբࣁל਼ϯᏊǴૈ୼

ԖਏಖЗԾҗ୷᜘ᙹϸᔈǶӢԜ൨؃ڀԖமל਼ϯ܄ޑϺฅל਼ϯނ፦຾Զၲډ ғނᡏߥ଼ϐҞޑǴςԋࣁ१ࠔᔼᎦठΚࣴزޑख़ाፐᚒǶ!

!

Ύǵआ᜿!

Ȑ΋ȑआ᜿ϐ๵ᅿ੝܄!

आ᜿๵ឦ (Monascus) ࢂ΋ᅿλࠠํރᆭғ੿๵Ǵҗݤ୯Ꮲޣ Van Tieghem ܭ 1884 ԃϩᚆǵ!ࡌҥǶ!ӧϩᜪس಍ឦܭ੿๵ࣚ (Fungi)ǵη៶๵ߐ (Ascomycota)ǵ η៶๵ᆜ (Ascomycetes)ǵණ៶๵Ҟ(Eurotiales)ǵआ᜿๵ࣽ (Monascaceae) (Van

(34)

Tieghem, 1884)Ƕआ᜿๵ឦޑ੝ቻࣁ๵ํևคՅǴፃՅ܈आՅǴڀԖᐉ႖ (septa)Ǵ ᙖ҃ᆄౢғ΋ঁεࠠޑԖ܄ࠆᏛη៶ (ascocarp) ܈аൂ΋ಒझϐϩғ᪐η຾Չ ᕷ෗Ƕ

आ᜿๵ӧ٥ࢪӦ୔ᔈҔςԖ΢ίԃᐕўǴЬाܭ໯१ǵᙴᛰǵπ཰฻Бय़ᔈ ҔǶआ䍍๵ӧኻࢪ΋ࡋ೏ຎࣁ΋ᅿᆭ௳๵Ǵՠӧܿ٥Ӧ୔Ǵआ䍍๵ࠅࢂ໺಍१ࠔ уπޑઓڻ༾ғނǶԾчֺ߃යߡԖ࣬ᜢЎ᝘૶ၩǴҁ૛ᆜҞύΨ૶ၩआ᜿Ьݯ

੃१ࢲՈǴ଼๠ᔿगǶआ᜿දၹҔܭ१ࠔ๱ՅᏊϷВத໯१চ਑Ƕаԯ଺ࣁ୷፦

຾Չڰᄊวሇ܌ғౢϐआ᜿ᆀࣁआ᜿ԯǴӧ٥ࢪ୯ৎࣁதҔϐ१ࠔϷ໺಍ύ૛ᛰǶ ߈ԃࣴزว౜Ǵआ᜿ԯ֖Ԗ೚ӭڀғނࢲ܄ϐжᖴౢނǴځύࢌ٤ϯӝނς೏પ ϯϷ᠙ۓǶ!

!

ȐΒȑआ᜿ғౢϐΒભжᖴౢނ

Ԑයᜢܭआ᜿ޑࣴزЬा໣ύӧ䁙ᜪکՅનБय़Ƕ1979 ԃǴВҁᇻᛯ௲௤

ϩᚆр monacolin K ϐࡕǴЇଆӚ୯ࣽᏢৎޑቶݱᜢݙǴआ᜿ύޑ΋٤ғ౛ࢲ܄

ނ፦ഌុ೏ว౜Ǵ຾΋؁ቚуआ᜿ޑᎶΚǶ!

1. ᖌڰᎇӝԋ׭ڋᏊ (monacolins)

Lovastatin ջࣁ monacolin KǴࣁआ᜿๵܌ғౢϐफ़ᖌڰᎇϯӝނǶϩηԄ ࣁ C24H36O5Ǵϩηໆ 404.55ǴࣁқՅ่඲Ǵёྋܭ methanolǵethanol Ϸ acetonitrile

฻ԖᐒྋᏊύՠόྋܭНǶനε֎ԏݢߏࣁ 231ǵ238ǵ247 nm (Budavari et al., 1989)Ƕ

1979ԃǴВҁܿ٧ၭπεᏢᇻᛯ௲௤Ծआ᜿๵ M. ruber ޑ୻Ꭶనύϩᚆр Ϻฅޑᖌڰᎇӝԋ׭ڋᏊ monacolin K (Endo, 1979)Ǵ൚ۓΑВࡕआ᜿ޑࣴز୷ᘵǶ

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23

monacolin Kޑ่ᄬᆶ HMG-CoA (3-hydroxy-3-methylgutaryl coenzyme A) ᜪ՟Ǵ

཮ᆶ HMG-CoA reductase ᝡݾǴ຾Զ׭ڋ၀ሇનǴ٬ mevalonic acid คݤғԋǴ Զ໔ௗ׭ڋᖌڰᎇӝԋǴၲډफ़եಒझϣᖌڰᎇ֖ໆϐਏ݀Ƕ྽ಒझϣᖌڰᎇޑ

֖ ໆ Π फ़ ਔ ཮ ቚ у LDL receptor ޑ ߄ ౜ ຾ Զ ග ϲ low-density lipoprotein cholesterol (LDL-C) ϐжᖴǴΨၲډफ़ե LDL-C ֖ໆϐਏ݀ǶԜѦǴᖏ׉ࣴز

ࡰрǴmonacolin K ڀԖफ़եᡏϣวݹϸᔈޑբҔ (Lefer, 2002)ǴҭԖלᕎϐф ਏ (Ho and Pan, 2009)Ǵ٠ЪڀԖफ़եߚી৞ન٩ᒘ܄ᑗֿੰ஻ޣϐՈᑗфਏ (Goldberg et al.,1990; Ko et al., 1998ȑǶ

ҁࣴز࠻ว౜ǴӧೈᚊޑႩ਑ύబу֖ monacolin K ϐआ᜿Ǵڀफ़եᚊೈ

ೈ໳ύᖌڰᎇ֖ໆ 14% ϐфਏ (Wang and Pan, 2003)Ƕҭૈफ़եଯિ໯१ᇨᏤঊ ႵՈమύᖌڰᎇ(Lee et al., 2006a)ǶԖൔᏤࡰрǴstatin ᜪᛰނڀԖלวݹϐфਏǴ फ़եЈՈᆅ੯ੰǵύ॥ǵᑗֿੰǵମ፦౧᚞ੱϷߓ૟ੇᓨੱ฻ੰϐวੰᐒ౗ (ഋ炻 1999)Ƕ

2. आ᜿Յન!

आ᜿Յનࢂआ᜿๵ӧғߏၸำύౢғޑ΋سӈՅનޑᕴᆀǴѬࢂШࣚ΢Ҟ߻

໻ԖޑճҔ༾ғނғౢޑ१ҔϺฅՅનǴߏයаٰբࣁ१ࠔ๱ՅᏊک३਑٬ҔǶ ЪӭኧύѦᏢޣޑࣴزൔ֋Ψࡰрआ᜿ՅનޑӼӄ܄ཱུଯǴӢԜआ᜿Յનቶݱ೏

բࣁϺฅޑ१ࠔ๱ՅᏊ٬ҔǶ!

आ ᜿ Յ ન ς ೏ ዴ ۓ р ϯ Ꮲ ่ ᄬ Ъ ቶ ࣁ Γ ޕ ޑ Ь ा Ԗ Ο ᜪ Ǵ ջ आ Յ ન (monascorubramineϷ rubropunctamine)ǵᐊՅન(monascorubrin Ϸ rubropunctatin) Ϸ໳Յન (monascinǵankaflavinǵyellowɛϷ xanthomonascin A) ΟεᜪǶᐊՅ નޑғԋࢂ࿶җሇનϸᔈғԋǴmonascorubrin Ϸ rubropunctatin ڀࢲዃޑ CO ୷Ǵ

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ġ ġ ġ კ Fig.

mon

!

ġ ġ ġ ġ ġ ġ ġ ġ ġ 2-8 आ᜿ว . 2-8 Structu nacolin K (l

ġ ġ ġ

วሇౢނ mo

ure of Mona lovastatin)!

onascinک ascus-femen

lovastatin nted produc

ϐ่ᄬ!

cts, includin

(Patak

ng monascin

kova. 2012)

n and

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25

ჹữ୷ለԖଯࡋᒃکΚǴ྽ځکữ୷ለϸᔈࡕǴ߾཮ғԋНྋ܄ޑआՅનǴԶ໳

Յન׎ԋᐒڋҞ߻ۘ҂ֹӄᕕှ (Juzlova et al., 1996; Blanc, 1994)ǶMonascin ᆶ lovastatinϐ่ᄬҢӵკ 2-8Ƕ໳Յન monascin Ϸ ankaflavin ς೏᛾ჴڀԖӭᅿ ғ౛ࢲ܄Ǵmonascin ڀԖלวݹਏ݀ (Lee et al., 2006b)ǴЪૈ׭ڋҜጥᕎǹ ankaflavin ᙖҗᇨวᕎಒझوӛಒझ঒Ϋၲԋࢥఠಒझϐਏ݀ǶԜѦǴmonascin ᆶ ankaflavin ჹܭԴႵ๠᠌ T ಒझڀԖխࣝ׭ڋբҔ (Martinkova et al., 1999)Ƕ

Yasukawa ฻Γว౜ᐊՅન monascorubrin ё׭ڋ 12-o-tetradecanpylphorbol 13-acetate (TPA) ܌ᇨวޑҜጥᕎϯ (Yasukawa et al., 1994)ǶMartinkova ฻Γว

౜ᐊՅન rubropunctatin ک monascorubrin ჹ Bacillus subtilis Ϸ Candida pseudotropicolis ޑ ғ ߏ ڀ Ԗ ׭ ڋ բ Ҕ Ǵ ᡉ Ң ၀ ᜪ Յ ન ڀ Ԗ ؼ ӳ ׭ ๵ ਏ ݀ (Martinkova et al., 1995)Ƕ

ҁࣴز࠻ճҔ Monascus purpureus NTU 568 ๵ਲ਼Ǵଞჹόӕ୷፦຾Չวሇ చҹᆶΒԛжᖴౢނғԋޑ௖૸Ǵࣴز่݀ᡉҢǴаξᛰڗж໺಍ԯᜪวሇǴڀ Ԗၨଯޑ໳Յનғԋໆ (Lee et al., 2006)Ƕ

3. ઓ࿶໺Ꮴᆶफ़Ոᓸނ፦ (-aminobutyric acid, GABA)

-ữ୷΍ለ (-aminobutyric acid, GABA) ࢂ΋ᅿύኰઓ࿶׭ڋ܄໺Ꮴނ፦Ǵ ࣁ L-glutamate ࿶ glutamate decarboxylase ຾Չಥ♐բҔ (decarboxylation) ࡕ܌

ளޑౢނǶGABA ନڀफ़ՈᓸϐфਏѦǴᗋԖלยᠱϷלኁᢠϐфਏǴӕਔӢځ ऐ዗܄٫Ǵࡺӧ१ࠔуπၸำύόܰ೏ઇᚯǶӧғނᡏύǴGABA ཮ᆶ GABAA

Ϸ GABAB่ӝǴቚуँ᝻ࡕጢෛᚆηޑ໺ᏤԶ׭ڋँ᝻ࡕႝՏǴफ़ե҃ఋઓ࿶

ޑ௵གࡋǴڈᐟҬགઓ࿶ࢲ୏٠Ъڈᐟ๝΢ဏનϩݜٽૡ⇌ữ (catecholamine)

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வԶౢғߏਏޑफ़ՈᓸբҔǶӢԜǴ྽ՈᆅၸࡋԏᕭЇଆଯՈᓸਔǴGABA ૈ׭

ڋઓ࿶ፂ୏ǴԖਏ௓ڋՈᓸޑ΢ϲ (Sawynok, 1987)Ƕ

Kohama ฻Γࡰрआ᜿ԯ܌֖ϐ GABA ࿶୏ނჴᡍ᛾ჴڀफ़եՈᓸϐфਏ (Kohama et al., 1987)ǶGABA ନΑૈԖਏफ़եՈᓸѦǴҭԖ೚ӭൔ֋ࡰр GABA ڀԖלขቾᆶኁᢠ (Kalueff and Nutt, 2007) аϷלᡜ⻯ޑਏ݀ (Meldrum, 1975)Ƕ

ϐ߻ҁࣴز࠻ճҔ Monascus purpureus NTU 568 ๵ਲ਼วሇ܌ள֖ଯ GABA

ౢໆϐआ᜿ξᛰᗯ१ӃϺ܄ଯՈᓸႵ (spontaneously hypertensive rat; SHR) ᛾ჴ ڀԖफ़Ոᓸϐਏ݀!(Wu et al., 2009)ǶWu ฻ࡰрआ᜿ξᛰКआ᜿ԯਏ݀׳٫ё

ૈࢂҗܭआ᜿ξᛰύ֖Ԗၨଯޑ GABAǵmonascin ᆶ ankaflavinǶ!

4. ל਼ϯނ፦ (dimerumic acid)

1999 ԃǴВҁᏢޣ Aniya ගрआ᜿ڀל਼ϯૈΚǴ٠ว౜ל਼ϯૈΚԋϩ ࣁ dimerumic acidǶдॺว౜ dimerumic acid ڀమନ , -diphenyl--picrylhydrazyl (DPPH) Ծҗ୷Ϸלݨિၸ਼ϯ܄፦ϐૈΚǴ٠җ acetaminophen (AAP) ܌ᇨว ط ᚯ ԝ (hepatic necrosis) ޑ ୏ ނ ኳ Ԅ ᛾ ჴ Ǵ M. anka ว ሇ ๧ р ނ ૈ ቚ у glutathione-s-transferase (GST) ޑࢲ܄Ǵ෧ᇸϯᏢᛰᏊჹܭط᠌ޑ໾্ǴႣٛط

᠌ޑཞ໾ (Aniya et al., 2000)Ƕ

Dimerumic acid ޑל਼ϯᐒڋࣁගٮ΋ঁႝη๏਼ϯނவԶᗋচ਼ϯނǴԶ ҁي਼ϯԋࣁ nitroxide radicalǴௗ๱ nitroxide radical ཮ӆ೏మନǴ࿶΋ೱՍޑ

਼ϯᗋচൻᕉ٬ளຬ਼഍ᚆηǵణ਼Ծҗ୷ǵalkyl radicalǵalkoxyl radical ک peroxyl radicalளаమନၲډל਼ϯϐਏ݀!(Yasukawa et al., 1994)ˤ

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27

ȐΟȑआ᜿ჹᑗֿੰϐᔈҔ

ၸѐςԖ೚ӭࣴزᡉҢआ᜿ޑԋҽёҔٰႣٛ೚ӭ੯ੰǴхࡴלଯՈᓸǵל གࢉǵफ़եՈᖌڰᎇǵל਼ϯǵ෧ϿЈՈᆅ੯ੰ॥ᓀǵלဍዦཥғϷᑗֿੰǵफ़ ՈિǵቚமխࣝբҔǴ౜ӧаआ᜿ჹܭᑗֿੰБय़ٰуа௖૸Ƕ!

ВҁᏢޣᇻᛯ௲௤฻ӧ຾Չआ᜿๵୻Ꭶफ़եՈమᖌڰᎇ၂ᡍޑӕਔǴว౜܌

Ԗ၂ᡍټηܺҔࡕ 0.5 h ϣՈᑗफ़ե 23%~29%ǴԶӧ 1 h ࡕՈᑗϝᆶჹྣಔΠ फ़ 19%~29%ǴՠځԖਏԋҽҞ߻҂ܴǶआ᜿วሇౢނޑफ़Ոિکफ़ՈᓸբҔӧ ᑗֿੰႣٛکᇶշݯᕍ΢ᕍਏޭۓǴԖ΋ۓޑᛙۓՈᑗբҔǴёаፓ࿯ӄيфૈǴ ԶЪ࣬КҞ߻໒วޑफ़ՈᑗᛰނǴڀԖคࢥୋբҔǴᕍਏᡉ๱฻ᓬᗺǶ

ϐ߻ҁࣴز࠻ճҔ M. purpureus NTU 568 аԯǵξᛰϷ⽋Ϙ଺ࣁวሇ୷፦Ǵ ஒวሇౢނೱុΖڬᗯ१ streptozotocin (STZ) ᇨวᑗֿੰεႵ (200 mg/kg)Ǵ่

݀ᡉҢᗯ१आ᜿ёफ़եεႵՈᑗǴҭૈफ़եᐘણ䁙ǵΟለҒݨ✊ϷᖌڰᎇǴआ᜿

ξᛰڀԖؼӳל਼ϯૈΚǴૈ୼ᡉ๱फ़եᑗֿੰεႵી᠌ಔᙃϐ ROSǴ٠ૈග ଯ glutathione disulfide reductaseǵglutathione reductase Ϸ catalase (CAT) ฻ל਼

ϯሇનࢲ܄ӢԶૈ෧ᇸ STZ ೷ԋϐી᠌ಔᙃ਼ϯ໾্Ǵ຾Զׯ๓ᑗֿੰεႵੱ

ރǴԶЪआ᜿วሇξᛰჹՈᑗϐׯ๓ਏ݀ၨआ᜿วሇԯϷआ᜿วሇ⽋Ϙࣁ٫!

)Shi and Pan, 2010a; Shi and Pan, 2010b)Ƕ

ࣴزᡉҢԼԺಔᙃޑᄌ܄วݹ཮Ꮴठી৞નל܄ک 2 ࠠᑗֿੰǶၸ਼ϯނ λᡏቚ෗ނ௴୏ڙᡏ (peroxisome proliferator-activated receptor, PPAR) ޑଛᡏ཮

ࢲϯᕗિㄽԼᎇ 3 ᐟ䁙 (phosphatidylinositol 3-kinase, PI3K)/Akt ೼৩Ƕ

ҁࣴز࠻аဍዦᚯԝӢη- (TNF-; 20 ng/mL) ᇨᏤ C2C12 ԼԺಒझౢғ

ી৞નל܄ϐಒझኳԄǴ௖૸ monascin ෧ᇸી৞નל܄ޑᐒڋǶࣴزว౜आ᜿

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ޑжᖴౢނ monascin (MS) ڀלวݹࢲ܄Ƕ

่݀ᡉҢǴMS ׭ڋ p-JNK ޑࢲ܄٠ЪߔЗ PPAR- ᕗለϯǴӢԜᏤठ PPAR- ࢲ ϯ ک PI3K/Akt ೼ ৩ ೏ ࢲ ϯ Ǵ ന ࡕ C2C12 Լ Ժ ಒ झ ឪ ڗ 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino]-2-deoxy-D-glucose (2- NBDG) ቚ уǶԜѦǴӧ PPAR- ࡮לᏊ (GW9662) ޑჴᡍǴࣴز่݀ᡉҢǴMS ёૈբ ࣁ PPAR- ߦਏᏊӢԶගଯી৞નޑ௵ག܄ǶMS ޑբҔᜪ՟㞓਱ӈ✉Ǵᛙۓ PPAR- ޑ่ᄬک෧Ͽ PPAR- ᕗለϯǴӢԶׯ๓ી৞નל܄ (Lee et al., 2011)Ƕ

ȐѤȑआ᜿ׯ๓ી৞નߔלϐёૈᐒڋ!

आ᜿ڀԖל਼ϯբҔǶԖࣴزࡰрǴӧ STZ ᇨวᑗֿੰεႵύǴځᡏϣՈ ዀǵआՈౚǵط᠌ǵ๝᠌ޑિ፦ၸ਼ϯ཮ቚуǴЪញрԾҗ୷Ǵቚуᡏϣ਼ϯᓸ ΚǴᡏϣ਼ϯᓸΚගଯǴી৞નߔלΨᒿ๱ቚуǶӢԜ೸ၸל਼ϯሇનس಍ٯӵ glutathioneǵsuperoxide dismutaseǵcatalase फ़ե ROSǴஒԖշܭી৞નߔלޑ ׯ๓Ƕ

ΖǵࣴزεᆜᆶҞޑ!

ᑗֿੰࢂᝄख़࠶ુΓᜪيᡏ଼நޑ΋ᅿதـ੯ੰǴวੰ౗ВᖿቚଯǶᑗֿੰ

୷ҁੰ౛੝ᗺࣁી৞નϩݜ๊ჹ܈࣬ჹόىǴ܈Ѧڬಔᙃჹી৞ન௵ག܄Πफ़Ǵ ЇଆᑗǴિެǴೈқ፦жᖴધ໶ޑ΋ᅿӄي܄੯ੰǶᒿ๱ੰำว৖ǴёаᏤठӭ ಔᙃӭᏔ۔ޑᄌ܄຾Չ܄ੰᡂǴวғӭᅿ࡚ǵᄌ܄ٳวੱǶ!

आ᜿ࣁ໺಍ϐวሇ๵ᅿǴޔԿϞВǴआ᜿ӧᙴᏢሦୱᆶߥ଼фਏ΢ϐ࣬ᜢࣴ

زςӭόയኧǶआ᜿วሇނύޑՅનϩης೏᛾ჴڀԖؼӳޑғނࢲ܄Ǵځύ໳

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29

Յન monascin ᆶ ankaflavin ନΑڀלวݹǵ׭ڋᕎಒझғߏ฻ӭᅿߥ଼фਏѦǴ ΨڀԖל୏ેๆރฯϯǴόܰ׎ԋᡏિެ฻ӭᅿߥ଼фਏǶआ᜿Βԛжᖴނ lovastatin ΨڀԖלวݹϐфਏǴफ़եЈՈᆅ੯ੰǵύ॥ǵᑗֿੰǵମ፦౧᚞ੱ

Ϸߓ૟ੇᓨੱ฻ੰϐวੰᐒ౗ˤҁჴᡍ࠻ς᛾ჴआ᜿วሇౢނڀԖ෧጗ᑗֿੰε ႵੱރϐфਏǴЪගϲᡏϣל਼ϯሇનϐࢲ܄Ƕ

ӢԜǴҁࣴزЬाճҔᡏϣ୏ނჴᡍǴຑ՗आ᜿໳Յન monascin ک!lovastatin ჹၸࡋᑗϯಖౢނ!(advanced glycation end-products, AGEs) ᇨᏤϐ୏ނી৞ન ߔלϐቹៜǶ߈ԃٰǴၸࡋᑗϯಖౢނᆶځڙᡏ!(receptor for AGEs, RAGE)ġ ޑ

࣬ϕբҔ܌Їวޑੰ౛ғ౛ਏᔈຫٰຫڙډΓॺޑख़ຎǶҁࣴزа໢܄!BALB/c!

λႵբࣁჴᡍ୏ނϩ݋!monascin ک lovastatin ჹ AGEs ᇨᏤϐ୏ނી᠌ǵط᠌

Ϸ๝᠌ύל਼ϯሇનϷߦวݹಒझᐟન߄౜ϐፓ௓Ϸ AGEs ჹી৞નૻဦ໺ሀ ϐቹៜǴаᕕှ monascin ک lovastatin ჹ AGEs ᇨᏤી৞નߔלᆶી᠌ ಒझф

ૈϐ໔ᜢ߯Ǵ׆Ꮗ monascin ک lovastatin ё໒วԋࣁ෧Ͽી৞ ಒझઇᚯǴቚу

ી৞નϩݜϐཥᑉݯᕍಃΒࠠᑗֿੰϐᛰނǶࣴزࢎᄬӵკ!2-9 ܌ҢǺ!

(42)

Monascin & Lovastatin

ᡏϣ୏ނჴᡍ!

фਏ܄ຑ՗! Ոమғϯॶຑ՗!

ط᠌ಔᙃϩ݋! ી᠌ಔᙃϩ݋!

࿣१Ոᑗ!

Ոమી৞ન TCǵTG ֖ໆ!

ߦวݹϸᔈӢη!

TCǵTG ֖ໆ

კ 2-9 ҁፕЎϐࣴزࢎᄬ!

Fig. 2-9 The framework of the study

๝᠌ಔᙃϩ݋!

ל਼ϯሇનϩ݋

ߦวݹϸᔈӢη

ל਼ϯሇનϩ݋ ל਼ϯሇનϩ݋!

ી৞નૻဦ໺ሀ ፓ௓!

ߦวݹϸᔈӢη!

ၸࡋᑗϯಖౢނᇨᏤ!

AGEૻဦ໺ሀ!

ፓ௓!

αܺဟ๻ᑗ ऐڙ܄ෳ၂!

ી৞ન!

ऐڙ܄ෳ၂!

(43)

31

ಃΟക! ׷਑ᆶБݤ!

΋ǵჴᡍ׷਑

Ȑ΋ȑᛰࠔ၂Ꮚ

1. D-glucoseǵbovine serum albumin (BSA)ǵTris-HClǵdimethyl sulfoxide (DMSO)ǵ N-acetylcysteine (NAC)ǵfructose Ϸ Triton-X 100 ᖼԾ Sigma Ϧљ (Sigma Co., St. Louis, MO, USA)

2. NaOHϷ HCl ᖼԾ Merck Ϧљ (Merck Co., Darmstadat, Germany) 3. MethanolᖼԾ Mallinckrodt Baker (Phillipsburg, NJ, USA)

4. Anti-p47phox antibody Ϸ anti-C/EBP antibody ᖼ Ծ Santa Cruz Biotechnology Inc., (Burlingame, CA, USA)

5. Anti-insulin antibody ᖼԾ!Cell Signaling Technology (Beverly, MA, USA) 6. Anti-AGE antibody ᖼԾ Abcam (Cambridge, MA, USA)

7. Anti-rabbit antibody (secondary antibody) ᖼԾ (GeneTex, Inc., San Antonio, TX, USA)

8. Lovastatin ᖼԾғၲϯᏢڋᛰިҽԖज़Ϧљ! (ᆵࠄǴᆵ᡼)

9. Bio-Rad ೈқ፦ۓໆࢉᏊᖼԾ Bio-Rad Laboratories (Hercules, CA, USA) 10. Monascin җఃཧғמϦљගٮ

ȐΒȑሺᏔ೛ഢ

1. ϩӀӀࡋी (Model U-2001, Hitachi Co., Tokyo, Japan) 2. ᚆЈᐒ (Himac CR-21, Hitachi Co., Tokyo, Japan) 3. પНᇙ೷ᐒ (Milli-Q, Millipore Co., Molsheim, France) 4. Н੎㚌ྕኲ (Model DB, Deng Yng Co., Taipei, Taiwan)

(44)

5. ॹҥԄᡉ༾᜔ (ECLIPSE TS100, Nikon Co., Tokyo, Japan) 6. ELISA reader (Thermo Labsystem, Beverly, MA, USA) 7. pH meter (Model SS-320, Tomy Co., Tokyo, Japan) 8. ྐ๵ଠ (Model SS-320, Tomy Co., Tokyo, Japan) 9. ኧՏ࣬ᐒ (COOLPIX5200, Nikon Co., Tokyo, Japan)

10.ຬॣݢ᎜ᑲᏔ (Model 690D, ETL testing laboratories Inc., Cortland, NY, USA) 11.֡፦ᐒ (Model PRO 200, PRO Scientific Inc., Oxford, CT, USA)

12.ႝηϺѳ (Model AG204, Mettler-Toledo Co., Greifense, Switzerland)

)Ο*!ځд!

MonascinҗఃཧғמϦљගٮǶჴᡍ܌Ҕϐኬࠔྋှܭ DMSO ύǴ٠௓ڋ ځᐚࡋեܭ 0.1% (v/v)Ƕ!!

!

)Ѥȑྋనᇙഢ

Phosphate buffered saline (PBS)

ᛰࠔ ख़ໆ (g)

NaCl 80.1 KCl 2.0

KH2PO4 2.0

Na2HPO4 11.5

΢ॊᛰࠔуΕ 800 mL ଍ᅖ೸НύǴፓ᏾ pH Կ 7.2ǴуНۓໆԿ 1000 mL ࣁ 10X stockǶ

ΒǵჴᡍБݤ

(45)

33

Ȑ΋ȑၸࡋᑗϯനಖౢނ炷AGEs炸ᇙഢ

ࣴزύ܌٬Ҕޑ AGE s ࢂҗ݀ᑗ (fructose) ᆶФՈమೈқ (BSA) ܌ӝԋ ϐ AGEsǴځᇙഢБݤࢂҔค๵ޑ BSA (60 mg/mL) ک݀ᑗ (1.5 M) ྋܭ 0.2 M potassium phosphate buffer ( pH 7.4, ֖Ԗ 0.06% sodium azide)Ƕڗ 100 L BSA ྋనǵ100 L ݀ᑗྋనک 100 L ค๵Н࿼ܭ 1.5 mL ᚆЈᆅǴ٠ܭ 50±C Πϸ ᔈ 24 λਔ (Wang et al., 2011)Ƕ

AGEϐᐚࡋෳۓБݤࢂஒ BSA ک AGE ϐኬࠔճҔ ELISA reader ෳໆ 435 nmݢߏϐ֎ӀॶӆਥᏵ BSA ϐᐚࡋඤᆉр AGE ᐚࡋˤ

ȐΒȑ୏ނჴᡍ 1. ୏ނኳԄࡌҥ

(1) ჴᡍ୏ނႩᎦᆶྣ਑

ࣴزύ܌ҔλႵࣁѳ֡ᡏख़ 20 g Ѥڬε BALB/c ࠔسλႵǴᖼԾᆵεᙴଣ ჴᡍ୏ނύЈǴ4-5 ڬសǶ௦ڗԾҗ຾१БԄǴߥ࡭໯ҔНϷႩ਑кىǴ؂ຼෳ

ۓႩ਑ឪڗໆᆶᡏख़炻؂ٿϺඤ΋ԛ໯Нᆶპ਑Ǵ٠ᒿਔᆢៈ୏ނ܊ϐమዅ٠ߥ

࡭ӼᓉǴЪᒥൻՉࡹଣၭ཰ہ঩཮୏ނႩᎦಒ߾ᆶೕۓ (Չࡹଣၭہ཮ύ๮҇୯ 99 ԃ 1 Д 27 Вঅ҅ϐ୏ނߥៈݤ (Animal Protection Law))ǶႩᎦਔ௓ڋ࣬ჹ ᔸࡋ 60%Ǵྕࡋ௓ڋܭ 25^°CǴӀྣਔ໔ࣁ 8:00-20:00 ϐ 12 λਔӀྣൻᕉǴჴᡍ ࣁය 22 ڬǶ୏ނ (n=5~6) ϩԋ:

(a) bovine serum albumin (BSA; 100 mg/kg bw; ؂Ϻ຾Չ 1 ԛဎ๚ݙ৔)

(b) advanced glycation end-products (AGEs; 100 mg/kg bw; ؂Ϻ຾Չ 1 ԛဎ๚ݙ

৔)

(c) AGE + monascin (MS; 10 mg/kg bw; ؂ٿϺ຾Չ΋ԛဎ๚ݙ৔)

(46)

(d) AGE + lovastatin (LOVA; 10 mg/kg bw; ؂ٿϺ຾Չ΋ԛဎ๚ݙ৔) (e) AGE + NAC (NAC; 100 mg/kg bw; ؂ٿϺ຾Չ΋ԛဎ๚ݙ৔)

Ꮚໆᒧ᏷΢Ǵ monascin ک lovastatin ᏊໆୖԵҁࣴز࠻ Lee ฻Γϐ߻ࣴز (Lee et al., 2013)ǶBSA ಔբࣁ control ಔǴӢࣁ AGE ᇙբၸำύ٬Ҕډ BSAǴ

܌аᒧ᏷ BSA բࣁ control ௨ନ BSA ҁيёૈ೷ԋޑቹៜǶNAC ࢂ΋ᅿதҔޑ ל਼ϯᏊǴբࣁҁჴᡍޑ໚܄ჹྣಔǶ NAC ᏊໆୖԵ Rana ฻Γޑࣴز (Rana et al., 2006)ˤNAC ޑל਼ϯբҔࢂᙯϯࣁ GSHǴԶ GSH ߾ࢂӝԋᡏϣനԖਏޑ ל਼ϯሇન܌όё܈લޑϡનǴԖᖏ׉ࣴز᛾ჴǴံк NAC ૈ୼ׯ๓ϷႣٛᆶ Ծҗ୷࣬ᜢ੯ੰǴӵ୏ેฯϯǵલ਼܄ЈԼੰᡂǵᑗֿੰǵᕎੱǵԴԃᛔքੱ฻

ޑวғϷ෧጗ൾϯ܈ගଯᘰࡕૈΚǴӕਔΨૈፓ᏾խࣝᐒૈǴׯ๓ၸ௵܈ܢלΚ ό٫ޑᡏ፦Ƕ

(2) αܺဟ๻ᑗऐڙ၂ᡍ (oral glucose tolerance test, OGTT ) Ϸી৞નऐڙ၂ᡍ!

(insulin tolerance test, ITT)!

αܺဟ๻ᑗऐڙ၂ᡍΏஒԴႵ࿣१ 8 λਔࡕǴෳځޜဎՈᑗॶǴӆஒԴႵа αܺБԄឲ१ဟ๻ᑗН (2 g/kg of bw) ϷኬࠔࡕǴෳۓ 30ǵ60ǵ90 ᆶ 120 ϩដ ՈᑗᡂϯǶી৞નऐڙ၂ᡍΏஒԴႵ࿣१ 4 λਔࡕǴෳۓޜဎՈᑗǴӆаဎ๚ݙ

৔БԄ๏ϒ insulin (0.5 U/kg bw)Ǵฅࡕෳۓ 30ǵ60ǵ90 ᆶ 120 ϩដՈᑗᡂϯǴ а௖૸୏ނჹી৞નϐ௵ག܄Ƕ

(3) ୏ނ឴࣊ᆶኬࠔ௦໣

࿣१ 12 λਔࡕǴаᖍᓌ௦Ոݤ឴࣊ԴႵ٠ڗӄՈးΕ 10 mL ֖ EDTA ל ᏉՈᆅύǴ຾ՉᚆЈ (800 g, 15 min)Ƕԏ໣΢ቫՈమǴᓯӸܭ -20^°CǴٮВࡕෳ

ۓՈિˣՈమી৞નᆶวݹӢኧǶ୊Πી᠌ǵط᠌ǵЈ᠌ᆶ๝᠌Ƕط᠌ˣી᠌ᆶ

๝᠌ڗΠࡕӃϪр΋λ༧੆ݰܭ 10% formaldehyde ڰۓǴࡑࡕុ຾ՉಔᙃϪТ

(47)

35

ϩ݋ǶഭᎩϐط᠌ˣી᠌Ϸ๝᠌ಔᙃးΕ֨᜘೓Ǵِೲ࿼ܭనᄊේύǴܭ -80^°C ߥӸǴٮВࡕෳۓߦಒझวݹᐟનϷל਼ϯሇન֖ໆǶ

2. фਏ܄ຑ՗

(1) ಔᙃ֡፦నᇙഢ

ϩձڗ 0.5 g ી᠌ǵط᠌Ϸ๝᠌уΕ 9 ७ (w/v) ϐᕗለ጗ፂྋనаಔᙃ֡

፦ᐒᑃ࿗Ǵܭ 4^°CΠа 12,000 xg ᚆЈ 5 ϩដǴڗ΢మనࣁಔᙃ֡፦నǴᓯӸ ܭ -80^°CΠаٮВࡕϩ݋ל਼ϯሇનǵߦวݹಒझᐟનǶ

(2)!ط᠌િ፦ෳۓ!

ڗ୏ނط᠌ಔᙃ֡፦నኬҁаΟለҒݨ✊!(triglyceride, TG)ǵᕴᖌڰᎇ!

(total cholesterol, TC) ϐѱ୧঺ಔ!(Randox, Antrim, UK) ຾ՉෳۓǶ!

1. TGෳۓБݤǺ

ϩձڗ!5 L ׇӈีញϐ኱ྗࠔϷ!5 L ኬࠔǴуΕ 200 L ሇન၂Ꮚ٠షӝ

֡ϬǴܭ࠻ྕϸᔈ 10 ϩដǶӧ 60 ϩដϣճҔ!ELISA reader ෳۓ኱ྗࠔϷኬࠔ ܭݢߏ!500 nm ϐ֎ӀॶǴᛤᇙ኱ྗԔጕа௢ᆉрኬࠔᐚࡋǶ!

2. TCෳۓБݤǺ

ϩձڗ!5 L ׇӈีញϐ኱ྗࠔϷ 5 L ኬࠔǴуΕ 200 L ሇન၂Ꮚ٠షӝ

֡ϬǴܭ࠻ྕϸᔈ 10 ϩដǶӧ 60 ϩដϣճҔ ELISA reader ෳۓ኱ྗࠔϷኬࠔ ܭݢߏ!500 nm ϐ֎ӀॶǴᛤᇙ኱ྗԔጕа௢ᆉрኬࠔᐚࡋǶ!

!

(3) Ոమғϯॶຑ՗

1. Ոమિ፦ෳۓ

(48)

ڗ୏ނՈమኬҁаΟለҒݨ✊ (triglyceride, TG)ǵᕴᖌڰᎇ (total cholesterol, TC) ϐѱ୧঺ಔ (Randox, Antrim, UK) ຾ՉෳۓǶ

2. Ոమી৞નෳۓ

ճҔ Mouse Insulin ELISA Kit (Mercodia, Uppsala, Sweden) ϩ݋Ոమી৞નǴ аޜဎી৞ન/ՈᑗКॶբࣁી৞ન௵གࡋࡰ኱Ƕ

ճҔሇનխࣝϩ݋ݤ (enzyme-linked immunosorbent assay, ELISA) ෳۓՈమ

ી৞નᐚࡋǴ96 Ͼዬ΢ςӃڰۓ΋ቫ insulin לᡏǴёᆶኬࠔύޑ insulin բҔǶ ஒ insulin ่ӝԿϾዬࡕǴуΕᆶၸ਼ϯ䁙 (peroxidase) ᗖ่ϐ insulin לᡏǴന ࡕуΕ peroxidase, ڙ፦ϸᔈևՅǶ

Ӄஒ 50 mL conjugate solution stock ᆶ 500 mL buffer షӝǴᇙԋ conjugate solutionǴ٠ஒ 40 mL washing concentrate ᆶ 800 mL ଍ᅖ೸НషӝǴᇙԋ wash bufferǶڗ 25 L ՈమኬࠔуԿ ELISA 96 ϾዬύǴௗ๱уΕ 50 L conjugate solution, ܭ࠻ྕΠϸᔈ 2 λਔǶа wash buffer ࢱѐߚ஑΋܄่ӝނ፦ǴуΕ 200

L 3,3’5,5’-tetramethy-benzidine (TMB) ଺ࣁ peroxidase ڙ፦Ǵܭ࠻ྕΠϸᔈ 15 minǶനࡕуΕ 50 L stop solution (֖ 1 M H2SO₄) ಖЗϸᔈǴషӝ֡ϬࡕǴճҔ ELISA readerෳໆ 450 nm ݢߏϐ֎ӀॶǴ٠ᆶ኱ྗԔጕКၨǴ຾ՉۓໆǶ

(4) ಔᙃੰ౛ϪТࢉՅ (Hematoxylin-Eosine ࢉՅ)

᝵Е๋ (hematoxylin) ࣁ༐ᡵ܄ࢉ਑Ǵૈஒಒझਡࢉԋ๋ՅǹҲआ (eosine) ࣁ

༐ለ܄ࢉ਑Ǵஒಒझ፦ࢉԋ఩आՅǴа຾Չੰ౛ϪТϐղ᠐Ƕ

ஒϪڗϐλႵط᠌ಔᙃа 10%ύ܄ᅽଭ݅੆ݰڰۓǴ຾ՉҡៃхশᆶϪТ (3-5 m/Т)ǶஒಔᙃϪТ࿼ܭ superfrost coating slide ΢Ǵа 45^°C੗Т႖ڹǶӆ ஒϪТ࿼ܭ xylene ύಥៃ 3 ԛǴ؂ԛ 5 ϩដǶᒿࡕ٩ׇ࿼Ε 100%ǵ95%ǵ90%ǵ

(49)

37

80%ǵ60%ϐΌᎇྋనύѐନ xyleneǶᅀу hematoxylin solution ᇂၸϪТ߄य़ऊ 1ϩដǴаᄌೲНࢬࢱǶᅀу eosine solutionǴӆ౽ԿНύНࢱǴаֹԋࢉՅǶϪ Тӆ٩ׇ੆ܭ 50%ǵ75%ǵ95%ǵคНϐΌᎇྋనϷ xylene Ӛऊ 1 λਔаֹԋ ಥНǶϪТᅀу Canada Balsam-Xylene solutionǴуᇂ࣒Тᓉ࿼ၸڹǴֹԋ࠾ТǶ

ੰ౛ϪТаӀᏢᡉ༾᜔ᢀჸϷྣ࣬Ƕ

(5) ೈқ፦ᐚࡋෳۓ

ճҔ Bio-Rad Dc protein assay kit (Bio-Rad Laboratories, Inc., Hercules, CA, USA) ຾Չೈқ፦ᐚࡋϐෳۓǶஒ 5 L ೈқ፦ኬࠔᆶ 25 L reagent A Ϸ 200 L reagent B షӝ֡ϬࡕǴ࿼ܭ࠻ྕΠϸᔈ 10 ϩដǶճҔ ELISA readerǴܭݢߏ 750 nmΠǴෳۓྋనϐ֎ӀॶǶа BSA բࣁ኱ྗࠔᇙբೈқ፦ᐚࡋ኱ྗԔጕǶᇙբ

኱ྗԔጕǴஒኬࠔ֎ӀॶжΕǴीᆉೈқ፦ᐚࡋǶ

(6) วݹᐟનෳۓ

ڗ፾ໆط᠌کી᠌ಔᙃ຾Չ֡፦Ϸೈқ፦ۓໆࡕᓯӸܭ -80^°C аٮ၂ᡍǶа ELISA kits (eBioscience, San Diego, CA, USA) ୀෳวݹ࣬ᜢಒझᐟન tumor necrosis factor- (TNF-)ǵinterleukin-1 (IL-1)ǵinterleukin-6 (IL-6) ֖ໆǶБݤ ӵΠǺஒ capture antibody а 1:250 ีញ७౗ϩձ coating ܭ 96 ϾዬύǴ4^°Cܫ࿼

ၸڹǶ႖Вॹ༸ ELISA plateǴа 260 L wash buffer ࢱϖԛǴஒ buffer ॹ༸ǴӚ ϾࢰуΕ 200 L block bufferǴ࠻ྕᓉ࿼΋λਔǴஒ buffer ॹ༸Ǵа wash buffer ࢱϖԛǶௗ๱ӚϾࢰуΕ 200 L ኬࠔܭӚᐚࡋϐ኱ྗࠔǴ࠻ྕᓉ࿼ 2 λਔǴௗ

๱а wash buffer ࢱϖԛǴӚϾࢰύуΕа 1:250 ีញ७౗ϐ detection antibodyǴ

࠻ྕΠϸᔈ 1 λਔࡕа wash buffer ࢱϖԛǴуΕ avidin-HRPǴܭ࠻ྕϸᔈ 30

(50)

ϩដࡕа wash buffer ࢱΎԛǴӆуΕϸᔈ୷፦ TMB ࡑځբҔǴ15 ϩដࡕа stop solutionಖЗϸᔈǴճҔ ELISA readerǴܭ 450 nm ک 570 nm!(ਠ҅) Πෳۓځ֎

ӀॶǶᇙբ኱ྗԔጕǴஒኬࠔ֎ӀॶжΕǴीᆉځ֖ໆǶ

(7) Ոమطǵ๝фૈࡰ኱ϩ݋

ALTǴԜᅿᙯữ䁙ѳਔ໻ϿໆӸӧՈమύǴ྽ಔᙃಒझڙډཞ໾Ǵ߾ሇન ࢲ܄ҥջϲଯǶځϲଯޑำࡋᆄຎط᠌ಔᙃ໾্ޑጄൎᆶமࡋǴத೏Ҕٰຑ՗ط фૈǶԼለὢ (creatinine) ࢂ֖ේϯނǴࣁԼለޑжᖴౢނǴε೿җ๝᠌௨рǴ ᆶ blood urea nitrogen (BUN) Ϸ albumin ӕࣁ๝фૈϐࡰ኱ǶႵᡏ឴࣊௦ՈࡕǴ

຾ՉᚆЈǴڗՈమ೽ϩǴ٬ҔғϯԾ୏ϩ݋ሺ຾Չ ALTǵCREǵBUNǵalbumin

֖ໆϩ݋Ƕ

(8) ל਼ϯሇનࢲ܄ϩ݋

1. ᎽાҒ两 (glutathione, GSH) ֖ໆϩ݋

ڗ፾ໆી᠌ǵط᠌ک๝᠌ಔᙃన຾Չ၂ᡍǴа glutathione assay kit!(Cayman Chemical Company, Ann Arbor, MI, USA) ୀෳ GSH!֖ໆǶБݤӵΠ;!ܭӚϾࢰ ύуΕ 50 L ኬࠔᆶӚᐚࡋϐ኱ྗࠔǴଛ࿼ϸᔈనǺܭ 50 mL ᚆЈᆅύуΕ 11.25 mL MES buffer,!0.45 mL reconstituted cofactor mixtureǴ2.1 mL reconstituted enzyme mixture-!2.3 mL ଍ᅖ೸НǴ0.45 mL reconstituted 5, 5'-dithiobis (2-nitrobenzoic acid) (DTNB)ˤଛӳషϬࡕǴ؂ϾࢰуΕ 150 L ϸᔈనǶճҔ ELISA readerǴܭ 405 nm!

Π؂႖ 5 ϩដෳۓځ֎ӀॶǴෳۓ 30 ϩដϣޑ֎ӀॶᡂϯǶڗ 25 ϩដਔޑኧᏵ ᇙբ኱ྗԔጕǴஒኬࠔ֎ӀॶжΕǴीᆉځ֖ໆǶ!

2. ᝻䁙 (catalase, CAT) ࢲ܄ෳۓ

(51)

39

ڗ፾ໆી᠌ǵط᠌ک๝᠌ಔᙃన຾Չ၂ᡍǴа catalase assay kit (Cayman Chemical Company) ୀෳ CAT ࢲ܄ǶБݤӵΠǺܭӚϾࢰύуΕ 100 L assay bufferǴ30 L!methanol!ک 20!L ኬࠔᆶӚᐚࡋϐ኱ྗࠔǶௗ๱ܭӚϾࢰύуΕ 20 L hydrogen peroxideܭ࠻ྕΠਁᕏϸᔈ 20 ϩដǶௗ๱ܭӚϾࢰύуΕ 30 L potassium hydroxide!ಖЗϸᔈ٠ЪуΕ 30 L catalase purpald (chromagen) ӚϾ ࢰύуΕ 10 L!catalase potassium periodate ܭ࠻ྕΠਁᕏϸᔈ 5 ϩដǶճҔ ELISA readerǴܭ 540 nm Πෳۓځ֎ӀॶǴᇙբ኱ྗԔጕǴஒኬࠔ֎ӀॶжΕǴीᆉځ ࢲ܄Ƕ!

3. ຬ਼ϯނݔϯ䁙 (superoxide dismutase, SOD) ࢲ܄ෳۓ

ڗ፾ໆી᠌ǵط᠌ک๝᠌ಔᙃన຾Չ၂ᡍ炻а SOD kit (Cayman Chemical Company) ຾Չࢲ܄ෳۓǶБݤӵΠǺܭӚϾࢰύуΕ 200 L radical detector Ϸ Ӛᐚࡋϐ኱ྗࠔǶௗ๱ܭӚϾࢰύуΕ 20 L!xanthione oxidase தྕΠϸᔈ 20 ϩដǶճҔ ELISA readerǴܭ 450!nm Πෳۓځ֎ӀॶǴᇙբ኱ྗԔጕǴஒኬࠔ

֎Ӏॶ஥ΕǴीᆉځࢲ܄Ƕ!

!

(9) խࣝಔᙃϯᏢࢉՅ

ӃஒϪТ࿶ xylene Ϸ ethanol ಥៃೀ౛Ǵௗ๱а 3% H2O2ઇᚯၸ਼ϯ䁙ࢲ

܄Ǵӆ࿶ 5%!ಥિФѪ຾Չ༤ံ (blocking) ࡕǴа΋ભלᡏ (primary antibody)

ೀ౛ 12 λਔ٠࿶җ PBS మࢱࡕǴӆаΒભלᡏ (secondary antibody) ೀ౛ 1 λ ਔ Ǵ ന ࡕ ӆ а 3’-3-diaminobenzen (DAB) chromogen ϸ ᔈ Կ և Յ Ǵ ӆ а haematoxylin ຾ՉൺࢉǴϪТ࿶ಥНଳᔿࡕ຾Չ࠾ТǶ

௦Ҕ Image-Pro Plus კႽϩ݋೬ᡏǴ ϪТӧӀ᜔Πࡪ಍΋ܫε७ኧ Ȑ10 X

(52)

40 ७ȑ຾Չϩ݋Ƕஒ؂஭ϪТࡪ฻ຯܜኬচ߾ᒿᐒឪڗ 5 ঁຎഁǴᒡΕीᆉᐒ բࣁෳۓკႽǶीᆉ໚܄य़ᑈэᕴϪТय़ᑈϐКٯǶ

(10)!ջਔۓໆᆫӝ䁙᜘ϸᔈ!(real-time PCR)

٬ҔሺᏔࣁ StepOne TM Real-time PCR system (Applied Biosystems)Ƕڗ!5 L ޑ complimentary DNA բࣁ҆ިǴ٩ׇуΕ 10!L Power SYBR^® Green PCR Master Mix (Applied Biosystems)ǵ0.5 L 20 mM primer (forward and reverse) Ϸ 4

L DEPC-dH₂O, ᕴᡏᑈࣁ 20 LǶϸᔈచҹ೛ۓࣁ: 95^°C 15ϩដǴௗ๱а 95^°C 15

ࣾǴ60^°C 1 ϩដޑచҹǴբҔ 40 ঁ cycleǶҁࣴز܌ୀෳޑ୷Ӣࣣа GAPDH ྽

଺ internal controlǴ٠а Ct БݤीᆉӚঁ୷Ӣ mRNA ߄౜຾Չ࣬ჹۓໆǶ٬

Ҕϐ primers (Yessoufou et al., 2010; Zhang et al., 2011; Wu et al., 2006) ӵ Table 3-1 ܌ҢǶ

!

Ȑϖȑғނ಍ीϩ݋Бݤ!

܌Ԗჴᡍࣣ຾ՉΟख़ൺǴჴᡍ่݀аѳ֡ॶ (mean) ²!኱ྗୃৡ (standard deviation, SEM) ߄ҢǴа SPSS ຾Չ಍ीϩ݋Ǵ٠а Student’s t-test բᡉ๱܄ৡ ౦ϐКၨǴа p < 0.05 բࣁڀԖᡉ๱܄ৡ౦Ƕ

(53)

41

߄!3-1Ǻջਔۓໆᆫӝ䁙᜘ϸᔈ!!(real-time PCR) ܌٬Ҕϐ primers

Table 3-1: Primers used in real-time PCR

Primers Sense Antisense

PDX-1 5’-CTACTGCCTTCGGGCCTTAG-3’ 5’-TTGGAACGCTCAAGTTTGTACC-3’

GLUT2 5’-CTGGGTCTGCAATTTTGTCA-3’ 5’-TGTAAACAGGGTGAAGACCA-3’

GAPDH 5’-ACGACCCCTTCATTGACC-3’ 5’-AGACACCAGTAGACTCCACG-3’

(54)

ಃѤക ่݀ᆶ૸ፕ

ಃ΋࿯ Monascin Ϸ lovastatin ჹ AGE ӧط᠌ύಕᑈϐቹៜ

ҁࣴزаխࣝಔᙃϯᏢ (IHC) ࢉՅຑ՗ AGE ӧط᠌ύಕᑈำࡋ (ፃՅౢ

ނࣁ҅ϸᔈ)Ǵ่݀ӵკ 4-1 ܌ҢǴ AGE ಔλႵط᠌ಔᙃύ AGE ֖ໆ֡ቚଯǴ ᡉҢဎ๚ݙ৔ AGE ཮ቚуطಔᙃύޑ AGE ֖ໆ (IHC ໚܄ࢉՅ่݀ևලፃ Յ)Ƕᆶ AGE ಔ࣬КၨǴ MS 炻LOV ک NAC ֡ё෧Ͽط᠌ AGE ֖ໆǴᡉҢ MSǴ LOV ک NAC ჹط᠌ύޑ AGE ಕᑈԖమନਏ݀ǶӚಔط᠌ύ AGE ಕ ᑈำࡋໆϯ่݀ӵკ 4-2 ܌ҢǴ22 ڬޑ AGE ᇨᏤ٬ط᠌ύ AGE ᡉ๱ଯܭ௓

ڋಔ (BSA ಔ) (p < 0.05)Ǵӕਔဎ๚ݙ৔ MSǴ LOV ᆶ NAC ёϩձ෧Ͽط᠌ύ AGE ᐚࡋǴफ़ե౗ϩձࣁ 50%Ǵ!57%ᆶ 37%Ǵځύ MS ᆶ AGE ࣬КԖᡉ๱ৡ ౦ (p < 0.05)ǶNAC ಔձၟ AGE ಔ࣬КΨԖफ़եط᠌ύ AGE ಕᑈϐфਏǶϩ݋

а΢่݀ǴMSǴ LOV ᆶ NAC ڀԖफ़ե AGE ӧط᠌ύಕᑈϐਏ݀ǴЪёૈ

ჹ AGE ܌ᏤठଯՈᑗཥғԋϣྍ܄ AGE Ԗ׭ڋբҔǶ

ಃΒ࿯ Monascin ک lovastatin ჹ AGE ᇨᏤλႵϐી৞નל܄ቹៜ

΋ǵ࿣१Ոᑗ

Ҟ߻ٛݯಃ 2 ࠠᑗֿੰϐπբख़ᗺࣁӧဟ๻ᑗऐڙ܄ό٫ (impaired glucose tolerance, IGT)೭΋ё଍܄жᖴ౦த໘ࢤ຾ՉႣٛǶӢࣁᒿ๱࿶ᔮޑว৖Ǵғࢲ БԄޑׯᡂǴᑗֿੰޑ஻ੰ౗όᘐᚹϲǴ٠ЪӄౚԖ IGT ஻ޣஒӧ 10 ԃϣว৖

ࣁಃ 2 ࠠᑗֿੰǴ೭ߦวΑಃ 2 ࠠᑗֿੰٛݯ฼ౣޑᙯᡂǶԖࣴزᡉҢǴӧ

IGT ໘ࢤ຾Չᛰނ܈ғࢲБԄ༸ႣǴёफ़ե܈ۯ጗ᑗֿੰޑวғǶી৞નߔל

ک ಒझϩݜલഐࢂځวੰޑΒঁЬाᕉ࿯Ǵӧ IGT ໘ࢤǴ ಒ

(55)

კ 4 Ёε Fig.

mic BSA MS grou NA

4-1 Monasc ελࣁ 200 . 4-1 The ef ce with 22 w A: control g ǺAGE ind up and 10 m

C/kg bw gr

cinک lovas

m

ffect of mon weeks of i.p.

group; AGE duced group

mg LOV/kg roup

BSA

AGE + M

tatinჹ AG

nascin and l . injection o E: advanced p and 10 mg g bw group;

43

A

MS

GEᇨᏤλႵ

lovastatin o of advanced d glycation g MS/kg bw

; AGE + NA

AGE + NA Ⴕط᠌ AGE

on hepatic A d glycation e n end-produ

w group; A AC: AGE i

A

AC

Eಕᑈϐቹ

AGE level o end-product uct induced AGE + LOV induced gro

AGE

AGE + LOV

ቹៜǶკύК

of male BAL ts

group; AG V: AGE indu

oup and 100 V

Кٯ

LB/c

GE + uced 0 mg

(56)

კ 4-2 Monascin ک lovastatin ჹ AGE ᇨᏤλႵط᠌ AGE ಕᑈϐቹៜ Fig. 4-2 The effect of monascin and lovastatin on hepatic AGE of male BALB/c mice with 22 weeks of i.p. injection of advanced glycation end-products

BSA: control group; AGE: advanced glycation end-product induced group; AGE + MSǺAGE induced group and 10 mg MS/kg bw group; AGE + LOV: AGE induced group and 10 mg LOV/kg bw group; AGE + NAC: AGE induced group and 100 mg NAC/kg bw group

#p < 0.05, significantly different from control (BSA) group; *p < 0.05, significantly different from AGE group

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