Sirtuin抑制劑 (sirtinol, splitomicin)為一含貝它萘酚(Naphthol)成份並具細胞穿膜功能之內酯化合物，它們是Sir2 (沉默信息調節2)蛋白酶之強效選擇性抑制劑。過去的研究顯示含萘酚化合物具有抑制及血小板及對抗白血球發炎反應之效果，而這些萘酚成份之化學結構，很可能具有抑制血小板凝集及降低出血性休克傷害的效果。故我們研究目的即在檢驗Sirtuin抑制劑在血小板凝集及出血性休克上之可能作用機轉，藉以在心血管、腦血管疾病上發展新的抗血小板治療方法及在出血性休克傷害上發展新的治療方式。
對於Sirtuin抑制劑在抑制血小板凝集研究中，我們使用人類洗滌血小板，並監測血小板被凝血酶 thrombin (0.1 U/ml)、膠原蛋白 collagen (2 μg/ml)、花生四烯酸 arachidonic acid (AA) (0.5 mM)、血栓素類似物 U46619 (2 μM)和二磷酸腺苷 ADP (10 μM)這些凝集誘導劑刺激後所產生之血小板凝集及三磷酸腺苷ATP釋放情形，此外利用螢光監測器測量Sirtuin抑制劑對血小板內鈣離子湧入 (Ca2+ influx)之影響，以及利用流式細胞儀偵測血小板表面選擇素-P (P-selection) 表現狀況，並使用酵素免疫連結分析對血栓素A2 (Thromboxane A2)及環磷腺甘磷酸雙酯酶 (cAMP phosphodiesterase)做進一步測量分析。
對於Sirtuin抑制劑在出血性休克研究中，我們使用雄性Sprague-Dawley大鼠產生創傷出血性休克，在引發肝臟損傷後，監測包括肝臟中過氧化酶 (MPO)的活性、嗜中性白血球趨化吸引分子-1 (CINC-1)、嗜中性白血球趨化吸引分子-3 (CINC-3)、細胞間黏著分子-1 (ICAM-1)和介白質-6 (IL-6)含量及血漿中丙氨酸轉胺酵素 (ALT)之濃度。在引發肺臟損傷後，則監測包括肺中過氧化酶的活性、腫瘤壞死因子-α (TNF-α)、介白質-6、和介白質-10 (IL-10)及第一型血紅素氧化酶 (HO-1)含量及肺泡沖洗液中蛋白質之濃度。
在血小板凝集研究中，顯示在不同濃度範圍之sirtinol和splitomicin均可抑制由thrombin、 collagen、 arachidonic acid、 ADP和U46619刺激所產生之血小板凝集作用。此外，sirtinol和splitomicin對U46619、 collagen和arachidonic acid刺激所引起之細胞內鈣離子流動和血栓素 (Thromboxane A2)合成也有抑制作用。而洗滌血小板在sirtinol或splitomicin合併前列腺素El (PGE1)之下，會增加cAMP表現量。但它們與IBMX合併作用時卻不會增加cAMP表現。這個結果顯示sirtinol和splitomicin會使cAMP增加乃是藉由抑制磷雙酯鍵酶活性而來。
在出血性休克研究中，給予sirtinol可降低創傷出血後引發之肝臟損傷，其原因與sirtinol可減少促發炎介質 (CINC-1, CINC-3, ICAM-1和IL-6 )含量、過氧化酶活性及血漿中丙氨酸轉胺酵素之濃度有關。而sirtinol對大鼠創傷出血後肺臟損傷之治療在於其能增加肺中第一型血紅素氧化酶 (HO-1)表現，並在給予sirtinol後可降低肺泡沖洗液中蛋白質之總量和降低肺中腫瘤壞死因子-α、介白質-6與過氧化酶的活性增加的程度。
總結，Sirtuin抑制劑 (sirtinol和splitomicin)在人類洗滌血小板凝集實驗中，其對血小板抑制機轉，包括主要是對環磷腺甘磷酸雙酯酶活性具抑制作用而使得cAMP含量增加，進而對細胞內鈣離子流動、血栓素B2形成和ATP之釋放產生抑制使用。而在雄性大鼠創傷出血研究中，sirtinol可藉由減少促發炎細胞激素之產生來達到保護並減低肝臟損傷的功能。另外，sirtinol也具有上調並增加第一型血紅素氧化酶表現作用，且藉此達到降低創傷出血後肺臟損傷之功能。

ABSTRACT
Sirtuin inhibitors (sirtinol, splitomicin) are cell-permeable lactone derived from β-naphthol, Which are the potent selectively inhibitor of Sir2 (silent information regulator 2) enzyme. Previous studies have demonstrated that some naphthoic compounds possess an inhibitory effect on platelets and anti-inflammatory effect on neutrophils. Therefore, their naphthoic moiety might be responsible for their inhibitory effects on platelet aggregation and attenuation injury effect on hemorrhagic shock. The major aims of our studies were to examine possible mechanisms of action of sirtuin inhibitors on platelet aggregation and hemorrhagic shock in order to promote development of a new anti-platelet therapy in cardiovascular and cerebrovascular diseases and find a novel treatment method for hemorrhagic shock injury.
To study the inhibitory effects of sirtuin inhibitors on platelet aggregation, we used washed human platelets, and monitored platelet aggregation and ATP release induced by thrombin (0.1 U/ml), collagen (2μg/ml), arachidonic acid (AA) (0.5 mM), U46619 (2 μM) or ADP (10 μM). Cytosolic Ca++ influx concentration was detected by fluorescence spectrophotometer. P-selection expression was analyzed by flowcytometry. The concentration of TXB2 and cAMP were measured by enzyme immunoassay kits.
To study the inhibitory effects of sirtuin inhibitors on hemorrhagic shock, we used male Sprague-Dawley rats following trauma-hemorrhage shock, and monitor include hepatic myeloperoxidase activity, CINC-1, CINC-3, ICAM-1, and IL-6 levels and plasma ALT concentrations on hepatic injury and monitor include myeloperoxidase activity, TNF-α, IL-6, IL-10 and hemeoxygenase-1 levels and protein concentrations of bronchoalveolar lavage fluid on lung injury.
In platelets aggregation studies, sirtinol and splitomicin inhibited platelet aggregation induced by thrombin, collagen, AA and U46619 in a concentration dependent manner. Moreover, sirtinol and splitomicin attenuated intracellular Ca++ release and thromboxane B2 formation stimulated by thrombin, collagen, AA and U46619 in human washed platelets. Increasing cAMP was noted when sirtinol and splitomicin were treated with Prostaglandin E1 in washed platelets. They did not further increase cAMP when combined with IBMX. This data indicated that sirtinol and splitomicin increase cAMP by inhibiting activity of phosphodiestease.
In hemorrhage shock studies, sirtinol administration on attenuation of hepatic injury following trauma-hemorrhage are related to reduction of pro-inflammatory mediators (CINC-1, CINC-3, ICAM-1, and IL-6 levels ), hepatic MPO activity, and plasma ALT concentrations. Sirtinol treatment following trauma-hemorrhage leads to increase in HO-1 expression of lung injury in male Sprague-Dawley rats. Sirtinol administration also attenuated the increase in bronchoalveolar lavage fluid total protein content and attenuated the increase in lung TNF-α, IL-6, MPO activity.
In conclusion, the inhibitory mechanism of sirtuin inhibitors (sirtinol and splitomicin) on human washed platelet aggregation may include an increase cyclic AMP levels via inhibition of cyclic AMP phosphodiesterase activity and subsequent inhibition of intracellular Ca++ mobilization, TXB2 formation and ATP release. Sirtinol administration following trauma-hemorrhage may decrease pro-inflammatory cytokine production and protect against hepatic injury in male Sprague-Dawley rats. Sirtinol also attenuates of lung injury following trauma-hemorrhage are mediated via upregulation of hemeoxygenase-1 expression.

Table of Contents
授權書 III
誌謝 V
中文摘要 VII
英文摘要 X
ABBREVIATION 1
CHAPTER I INTRODUCTION 4
1-1 BACKGROUND 5
1-2 SPECIFIC AIMS OF THIS PROJECT 11
1-3 FIGURES AND FIGURE LEGENDS 12
CHAPTER II MATERIALS AND METHODS 16
IN VITRO PLATELETS AGGREGATION STUDIES
2-1 BLOOD SAMPLING 18
2-2 CHEMICALS 18
2-3 PREPARATION OF PLATELETS 19
2-4 PFA-100 ANALYZER STUDIES 20
2-5 PLATELET AGGREGATION AND ATP RELEASE
REACTION 20
2-6 MEASUREMENT OF CA2+ INFLUX 21
2-7 PLATELET CAMP AND CGMP LEVEL ASSAY 22
2-8 THROMBOXANE B2 ASSAY 23
2-9 P-SELECTIN EXPRESSION 23
2-10 WESTERN BLOTTING ANALYSIS 24
2-11 STATISTIC ANALYSIS 24
IN VIVO HEMORRHAGIC SHOCK STUDIES
2-12 HEMORRHAGIC SHOCK PROCEDURE 26
2-13 MEASUREMENT OF HEPATIC INJURY 27
2-14 MEASUREMENT OF MYELOPEROXIDASE (MPO)
ACTIVITY OF LIVER 28
2-15 DETERMINATION OF CINC-1, CINC-3, ICAM-1, AND IL-
6 LEVELS OF LIVER 28
2-16 PREPARATION OF LUNG TISSUE AND COLLECTION OF
BRONCHOALVEOLAR LAVAGE FLUID 29
2-17 PROTEIN ASSAY IN LUNG LAVAGE 29
2-18 MEASUREMENT OF MYELOPEROXIDASE (MPO) ACTIVITY
OF LUNG 30
2-19 DETERMINATION OF LUNG TNF-Α, IL-6, AND IL-10
LEVELS OF LUNG 30
2-20 WESTERN BLOT ASSAY (HO-1) OF LUNG 31
2-21 HISTOLOGICAL ANALYSIS OF LUNG 32
2-22 STATISTICAL ANALYSIS 32
2-23 FIGURES AND FIGURE LEGENDS 32
CHAPTER III ANTI-PLATELET EFFECTS OF SIRTINOL IN PLATELETS AGGREGATION VIA CYCLIC AMP PHOSPHODIESTERASE 35
3-1 SIRTINOL RESTRAINED CLOT FORMATION WITH
PROLONGED CEPI-CT AND CADP-CT 36
3-2 SIRTINOL INHIBITED THE AGGREGATION AND ATP
RELEASE IN WASHED PLATELET WITH DOSE-DEPENDENT
MANNER 36
3-3 SIRTINOL INHIBITED THE INTRACELLULAR CALCIUM
MOBILIZATION 37
3-4 SIRTINOL AND PGE1 INCREASED INTRACELLULAR CAMP
LEVEL SYNERGISTICALLY, BUT NOT CGMP 37
3-5 SIRTINOL ATTENUATED THROMBOXANE B2 IN PLATELETS
INDUCED BY COLLAGEN, AA, THROMBIN AND U46619 BUT
NOT ADP 38
3-6 SIRTINOL INHIBITED THE WASHED PLATELET P-
SELECTIN EXPRESSION BY THROMBIN AND U46619 IN
WITH DOSE-DEPENDENT MANNER 39
3-7 SIRTINOL INCREASED THE WASHED PLATELET VASP-
SER157 PHOSPHORYLATION 39
3-8 TABLES AND FIGURES 40
CHAPTER IV SPLITOMICIN SUPPRESSES HUMAN PLATELET AGGREGATION VIA INHIBITION OF CYCLIC AMP PHOSPHODIESTERASE AND INTRACELLULAR CA++ RELEASE 57
4-1 SPLITOMICIN RESTRAINED THE BLOOD-CLOTTING WITH
PROLONGED CEPI-CT AND CADP-CT 58
4-2 SPLITOMICIN DOSE-DEPENDENTLY INHIBITED PLATELET
AGGREGATION AND ATP RELEASE IN WASHED
PLATELETS 58
4-3 SPLITOMICIN INHIBITED INTRACELLULAR CALCIUM
MOBILIZATION 59
4-4 SPLITOMICIN AND PROSTAGLANDIN E1 (PGE1)
INCREASED INTRACELLULAR CAMP LEVELS
SYNERGISTICALLY, BUT NOT CGMP LEVELS 59
4-5 SPLITOMICIN ATTENUATED THROMBOXANE B2 LEVELS IN
PLATELETS INDUCED BY COLLAGEN, AA, THROMBIN AND
U46619 BUT NOT BY ADP 60
4-6 SPLITOMICIN INHIBITED THE EXPRESSION OF PLATELET
P-SELECTIN BY THROMBIN IN A DOSE-DEPENDENT
MANNER IN WASHED PLATELETS 60
4-7 TABLES AND FIGURES 61
CHAPTER V SIRTINOL ATTENUATES HEPATIC INJURY AND PRO-INFLAMMATORY CYTOKINE PRODUCTION FOLLOWING TRAUMA-HEMORRHAGE IN MALE SPRAGUE–DAWLEY RATS 72
5-1 ALTERATION IN PLASMA ALANINE AMINOTRANSFERASE
(ALT) LEVEL 73
5-2 ALTERATION IN HEPATIC MPO ACTIVITY 73
5-3 ALTERATION IN HEPATIC IL-6 LEVELS 73
5-4 ALTERATION IN HEPATIC CINC-1, CINC-3, ANDICAM-1
EXPRESSIONS 73
5-5 FIGURES AND FIGURE LEGENDS 74
CHAPTER VI HEMEOXYGENASE-1 UPREGULATION IS CRITICAL
FOR SIRTINOL-MEDIATED ATTENUATION OF LUNG INJURY AFTER TRAUMA-HEMORRHAGE IN A RODENT MODEL 79
6-1 HO-1 PROTEIN EXPRESSION IN LUNG 80
6-2 HO-1 AND TOTAL PROTEIN CONTENT IN
BRONCHOALVEOLAR LAVAGE FLUID 80
6-3 HO-1 AND LUNG MPO ACTIVITY 80
6-4 HO-1 AND LUNG TNF-Α, IL-6, AND IL-10 LEVELS 81
6-5 HISTOLOGICAL ANALYSIS OF LUNG 82
6-6 FIGURES AND FIGURE LEGENDS 82
CHAPTER VII DISCUSSION 88
7-1 ANTI-PLATELET EFFECTS OF SIRTINOL IN PLATELETS
AGGREGATION 89
7-2 ANTI-PLATELET EFFECTS OF SPLITOMICIN IN
PLATELETS AGGREGATION 94
7-3 EFFECTS OF SIRTINOL ON HEPATIC INJURY AFTER
HEMORRHAGIC SHOCK 98
7-4 EFFECTS OF SIRTINOL ON LUNG INJURY AFTER
HEMORRHAGIC SHOCK 100
7-5 FIGURES AND FIGURE LEGENDS 104
CHAPTER VIII CONCLUSION 107
8-1 A NEW INSIGHT OF ANTI-PLATELET EFFECTS OF
SIRTINOL IN PLATELETS AGGREGATION VIA CYCLIC AMP
PHOSPHODIESTERASE 108
8-2 SPLITOMICIN SUPPRESSES HUMAN PLATELET
AGGREGATION VIA INHIBITION OF CYCLIC AMP
PHOSPHODIESTERASE AND INTRACELLULAR CA++
RELEASE 108
8-3 SIRTINOL ATTENUATES HEPATIC INJURY AND PRO-
INFLAMMATORY CYTOKINE PRODUCTION FOLLOWING
TRAUMA-HEMORRHAGE IN MALE SPRAGUE-DAWLEY
RATS 109
8-4 HEMEOXYGENASE-1 UPREGULATION IS CRITICAL FOR
SIRTINOL-MEDIATED ATTENUATION OF LUNG INJURY
AFTER TRAUMA-HEMORRHAGE IN A RODENT MODEL 109
8-5 FIGURES AND FIGURE LEGENDS 110
CHAPTER IX FUTURE STUDIES 114
9-1 FUTHER DIRECTIONS 115
9-2 FIGURES AND FIGURE LEGENDS 116
REFERENCES 120
APPENDIX 134

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