臺灣博碩士論文加值系統

Propofol、thiopental、citosol 與midazolam是在重症加護病房中常用於
鎮靜安眠的靜脈麻醉劑。這些病人常面臨各式各樣的氧化壓力與自由基損
傷，尤其需要抗氧化劑以幫助抵抗自由基的傷害。因此，這些常用的靜脈
麻醉劑的抗氧化能力更顯現其重要性。本實驗即設計評估這些臨床上常被
使用的藥物的抗氧化特性。經由透過menadione、hydrogen peroxide 及
cumene hydroperoxide 在人類單核白血球THP-1 細胞誘導產生氧化壓力傷
害，我們利用流式細胞儀去評估propofol、thiopental、citosol 及 midazolam
對THP-1 細胞面對氧化壓力狀態的保護作用上，做一番釐清與探討。在上
述四種麻醉劑中，Propofol有明顯抑制menadione處理後THP-1 細胞內超氧
自由基的濃度；也可以有效地抑制細胞內GSH的減少及細胞內粒線體膜電
位(ΔΨm)的下降。Menadione處理後的THP-1 細胞會抑制二種抗氧化酵素
superoxide dismutase (SOD)與gamma-glutamylcysteine (γ-GCS)的表現，而在
細胞前處理propofol後，可以維持menadione處裡的THP-1 細胞內SOD與
γ-GCS 表現。另一個抗氧化酵素catalase (CAT) 的表現並沒有受到
menadione及propofol的影響。實驗結果顯示，在四種被測試的靜脈鎮靜劑
中，Propofol是較其他藥物更具有抗氧化能力與清除活性氧物質活性的靜脈
麻醉劑。

Propofol, thiopental, citosol, and midazolam are widely used as sedatives for
the critically ill in the intensive care unit. The patients usually suffer from critical
conditions caused by oxidative stress due to the lack of a sufficient defense
system against reactive oxygen species (ROS). Therefore, the antioxidant
activities of intravenous anesthtics may be clinically important. We attempted to
evaluate the antioxidant and ROS scavenging activities of these clinically relevant
intravenous anesthtics. By using menadione, hydrogen peroxide and cumene
hydroperoxide to create oxidative stress in human acute monocytic leukemia cell
line (THP-1), we evaluated the antioxidative activity of the four intravenous
anesthetics by flow cytometry to investigate the intracellular ROS scavenging
ability in THP-1 cells. Propofol induced an excellent scavenging activity
compared with the other intravenous anesthetics, especially in menadione-induced
THP-1 cells. Propofol also significantly inhibited the intracellular GSH depletion
and the decrease of mitochondrial transmembrane potential (ΔΨm) induced by
menadione-treated THP-1 cells. Although menadione treatment could inhibit the
expression of two antioxidant enzymes, superoxide dismutase (SOD) and
gamma-glutamylcysteine synthetase (γ-GCS), pretreatment with propofol
maintained the expression of SOD and γ-GCS in menadione-treated THP-1 cells.
The expression of another antioxidant enzyme, catalase (CAT), was not obviously
affected by menadione and propofol. Among the four anesthetics, propofol
showed the most antioxidant and ROS scavenging activities.

目錄.......................................................I
縮寫表.....................................................V
中文摘要..................................................VII
英文摘要...................................................IX
第一章 緒論.................................................1
第二章 文獻探討.............................................2
第一節 疾病與氧化壓力........................................2
第二節 靜脈麻醉藥物的抗氧化能力...............................3
一、巴比妥鹽類..............................................4
二、苯二酚類................................................5
三、異丙酚..................................................6
四、K它命...................................................6
第三節 自由基與氧化壓力.....................................12
一、超氧陰離子基(superoxide anion radical；O2‧–)..........13
二、過氧化氫(hydrogen peroxide；H2O2 ).....................14
三、氫氧自由基(hydroxyl radical；OH‧)......................14
四、一氧化氮(nitric oxide; NO·)............................15
第四節 麩胱甘肽(Glutathione；GSH)與γ-glutamycsteine synthetase
(γ-GCS)...................................................18
第五節 氧化壓力與粒線體膜電位的改變(mitochondrial
transmembrane potential)..................................23
第六節 抗氧化酵素...........................................23
一、Superoxide Dismutase (SOD)............................24
二、Glutathione peroxidease (GSHPx).......................24
三、Catalase(CAT).........................................25
第三章 研究目的及動機.......................................26
第四章 材料與實驗方法.......................................27
第一節 材料................................................27
一、細胞株及細胞培養........................................27
二、靜脈麻醉劑.............................................27
三、抗氧化及自由基清除能力...................................28
四、抗體...................................................28
五、實驗儀器...............................................29
第二節 實驗方法............................................29
一、懸浮細胞培養............................................30
二、利用流式細胞儀(flow cytometry)分析細胞內抗氧化及自由基清除實驗........................................................30
1. 分析人類單核白血球THP-1細胞株細胞內H2O2自由基的含量........................................................30
2. 分析人類單核白血球THP-1 細胞株細胞內peroxide 自由
基的含量...................................................31
3. 分析人類單核白血球THP-1 細胞株細胞內superoxide 自
由基的含量.................................................32
4. 分析propofol 對於人類單核白血球THP-1 細胞株細胞內
glutathione 的含量........................................33
5. 分析propofol對於人類單核白血球THP-1細胞株細胞內粒線體膜電位的變化........................................................33
6. 流式細胞儀..............................................34
三、利用西方墨點法分析propofol 對於人類單核白血球THP-1 細
胞株細胞內抗氧化酵素(superoxide dismutase、catalase)及麩胱甘
肽合成速率決定步驟酵素(γ-GCS)的表現.........................36
1. 抽取細胞內總蛋白.........................................36
2. 蛋白質濃度定量試驗.......................................36
3. 蛋白質電泳(sodium dodecyl sulfate-polyacrylamide gel
electrophoresis；SDS-PAGE) ...............................37
4. 西方墨點法..............................................37
III
四、統計分析...............................................39
第五章 結果................................................40
第一節 靜脈麻醉劑對細胞內抗氧化能力的探討......................40
第二節 Propofol可以抑制menadione處理後THP-1細胞內麩胱甘肽
損耗(GSH depletion)的程度..................................42
第三節 Propofol可以維持menadione處理後THP-1細胞麩胱甘肽合
成速率決定步驟酵素(γ-GCS)的表現量............................43
第四節Propofol可以抑制menadione處理後THP-1細胞內粒線體膜電
位變化的程度...............................................43
第五節 Propofol 對於menadione 處理後THP-1 細胞內抗氧化酵素
(superoxide dismutase、catalase)表現的探討.................44
第六章 討論................................................46
第七章 結論................................................52
第八章 參考文獻............................................53
第九章 圖表................................................63
第十章 附錄................................................76

1. Galley H.F., Davies M.J., Webster N.R.: Xanthine oxidase activity and
free radical generation in patients with sepsis syndrome. Crit Care Med.
1996; 24: 1649-1653.
2. Goodyear-Bruch C., Pierce J.D.: Oxidative stress in critically ill
patients. Am J Crit Care. 2002; 11: 543-551.
3. Berger M.M., Chioléro R.L.: Antioxidant supplementation in sepsis
and systemic inflammatory response syndrome. Crit Care Med. 2007; 35:
584-590.
4. Tanelian D.L., Kosek P., Mody I., MacIver M.B.: The role of the
GABAA receptor/chloride channel complex in anesthesia. Anesthesiology.
1993; 78: 757-776.
5. Dogan Z., Yuzbasioglu M.F., Kurutas E.B., Yildiz H., Coskuner I.,
Senoglu N., Oksuz H., Bülbüloglu E.: Thiopental improves renal
ischemia-reperfusion injury. Ren Fail. 2010; 32: 391-395.
6. Nishiyama K., Hirakawa M.: Effect of thiamylal on superoxide
generation in the cells and on lipid peroxidation of biological membranes.
Masu. 1989; 38: 202-210.
53
7. Almaas R., Saugstad O.D., Pleasure D., Rootwelt T.: Effect of
barbiturates on hydroxyl radicals, lipid peroxidation and hypoxic cell
death in human NT2-N neurons. Anesthesiology. 2000; 92: 764-774.
8. Bateson A.N.: The benzodiazepine site of the GABAA receptor: an old
target with new potential ? Sleep Med. 2004; 5: 9-15.
9. Nishina K., Akamatsu H., Mikawa K., Shiga M., Maekawa N., Obara
H., Niwa Y.: The inhibitory effects of thiopental, midazolam, and
ketamine on human neutrophil functions. Anesth Analg. 1998; 86:
159-165.
10. Duke T.: A new intravenous anesthetic agent: propofol. Can Vet J.
1995; 36: 181-183.
11. Mirenda J. & Broyles G.: Propofol as used for sedation in the ICU.
Chest 1995; 108: 539-548.
12. Hara M., Kai Y., & Ikemoto Y.: Propofol activates GABAA
receptor-chloride ionophore complex in dissociated hippocampal
pyramidal neurons of the rat. Anesthesiology. 1993; 79: 781-788.
13. Liu K.X., Rinne T., He W., Wang F., Xia Z.: Propofol attenuates
intestinal mucosa injury induced by intestinal ischemia-reperfusion in the
rat. Can J Anaesth. 2007; 54: 366-374.
54
14. Shao H., Li J., Zhou Y., Ge Z., Fan J., Shao Z., Zeng Y.:
Dose-dependent protective effect of propofol against mitochondrial
dysfunction in ischaemic/reperfused rat heart: role of cardiolipin. Br J
Pharmacol. 2008; 153: 1641-1649.
15. Yumoto M., Nishida O., Nakamura F, Katsuya H.: Propofol
attenuates oxidant-induced acute lung injury in an isolated perfused
rabbit-lung model. J Anesth. 2005; 19: 287-294.
16. Cámara C.R., Guzmán F.J., Barrera E.A., Cabello A.J., Garcia A.,
Fernández N.E., Caballero E., Ancer J.: Ketamine anesthesia reduces
intestinal ischemia/reperfusion injury in rats. World J Gastroenterol.
2008; 14: 5192-5196.
17. Choi S.J., Kim M.H., Lim S.W., Gwak M.S.: Effect of ketamine on
apoptosis by energy deprivation in astroglioma cells using flow
cytometry system. J Korean Med Sci 2005; 20: 113-120.
18. Walker S.M., Westin B.D., Deumens R., Grafe M., Yaksh T.L.:
Effects of intrathecal ketamine in the neonatal rat: evaluation of apoptosis
and long-term functional outcome. Anesthesiology. 2010; 113: 147-159.
19. Bertram G.K., Susan B.M., Anthony J.T.: Basic and Clinical
Pharmacology. Lange 2010, 11th edition: 331-337
55
20. Montiel Duarte C., Ansorena E., Lopez Zabalza M.J., Cenarruzabeitia
E., Iraburu M.J.: Role of reactive oxygen species, glutathione and
NF-kappa B in apoptosis induced by 3,4-methylenedioxymethamphetamine
("Ecstasy") on hepatic stellate cells. Biochem Pharmacol.
2004; 67: 1025-1033.
21. Pardhasaradhi B.V., Reddy M., Ali A.M., Kumari A.L., Khar A.:
Differential cytotoxic effects of Annona squamosa seed extracts on
human tumour cell lines: role of reactive oxygen species and glutathione.
J Biosci. 2005; 30: 237-244.
22. Kaur P., Aschner M., Syversen T.: Role of glutathione in determining
the differential sensitivity between the cortical and cerebellar regions
towards mercury-induced oxidative stress. Toxicology. 2007; 230:
164-177.
23. Dringen R., Gutterer J.M., Hirrlinger J.: Glutathione metabolism in
brain metabolic interaction between astrocytes and neurons in the defense
against reactive oxygen species. Eur J Biochem. 2000; 267: 4912-4916.
24. Gatti R., Belletti S., Uggeri J., Vettori M.V., Mutti A., Scandroglio R.,
Orlandini G.: Methylmercury cytotoxicity in PC12 cells is mediated by
56
primary glutathione depletion independent of excess reactive oxygen
species generation. Toxicology. 2004; 204: 175-185.
25. Higuchi Y.: Glutathione depletion-induced chromosomal DNA
fragmentation associated with apoptosis and necrosis. J Cell Mol Med.
2004; 8: 455-464.
26. Rosi A., Grande S., Luciani A.M., Palma A., Giovannini C., Guidoni
L., Sapora O., Viti V.: Role of glutathione in apoptosis induced by
radiation as determined by 1H MR spectra of cultured tumor cells. Radiat
Res. 2007; 167: 268-282.
27. Biroccio A., Benassi B., Fiorentino F., Zupi G.: Glutathione depletion
induced by c-Myc downregulation triggers apoptosis on treatment with
alkylating agents. Neoplasia. 2004; 6: 195-206.
28. Lin C.H., Huang C.C., Wang T.W., Wang Y.J., Lin P.H.: Disparity in
the induction of glutathione depletion, ROS formation, poly (ADP-ribose)
polymerase-1 activation, and apoptosis by quinonoid derivatives of
naphthalene in human cultured cells. Chem Biol Interact. 2007; 165:
200-210.
57
29. Swamy S.M., Huat B.T.: Intracellular glutathione depletion and
reactive oxygen species generation are important in alpha-hederininduced
apoptosis of P388 cells. Mol Cell Biochem. 2003; 245: 127-139.
30. Nakabeppu Y., Tsuchimoto D., Ichinoe A., Ohno M., Ide Y., Hirano
S., Yoshimura D., Tominaga Y., Furuichi M., Sakumi K.: Biological
significance of the defense mechanisms against oxidative damage in
nucleic acids caused by reactive oxygen species: from mitochondria to
nuclei. Ann N Y Acad Sci. 2004; 1011: 101-111.
31. Zang Q., Maass D.L., White J., Horton J.W.: Cardiac mitochondrial
damage and loss of ROS defense after burn injury: the beneficial effects
of antioxidant therapy. J Appl Physiol. 2007; 102: 103-112.
32. Degli Esposti M.: Mitochondria in apoptosis: past, present and future.
Biochem Soc Trans. 2004; 32:493-495.
33. Voortman J., Checinska A., Giaccone G., Rodriguez J.A., Kruyt F.:
Bortezomib, but not cisplatin, induces mitochondria-dependent apoptosis
accompanied by up-regulation of noxa in the non-small cell lung cancer
cell line NCI-H460. Mol Cancer Ther. 2007; 6:1046-1053.
34. Kuwana T., Newmeyer D.D.: Bcl-2-family proteins and the role of
mitochondria in apoptosis. Curr Opin Cell Biol. 2003; 15: 691-699.
58
35. Cichorek M., Kozlowska , Bryl E.: Mitochondrial transmembrane
potential in spontaneous and camptothecin-induced apoptosis of
melanotic and amelanotic melanoma cells. Neoplasma. 2007; 54: 29-36.
36. auf dem Keller U., Kümin A., Braun S., Werner S.: Reactive oxygen
species and their detoxification in healing skin wounds. J Investig
Dermatol Symp Proc. 2006; 11: 106-111.
37. Higashit T. and Peters T.: Studies on rat liver catalase. J Biol Chem.
1963; 238: 3952-3954.
38. Petrova V.Y., Rasheva T.V., Anna V.K.: Catalase enzyme in
mitochondria of Saccharomyces cerevisiae. Electron J Biotechnol. 2002;
5:42-54.
39. Mézes M., Erdélyi M, Shaaban G, Virág G, Balogh K, Wéber M:
Genetics of glutathione peroxidase. Acta Biol Szegediensis. 2003; 47:
135-138.
40. Oztürk E., Demirbilek S., Körođlu A., But A., Begeç Z.O., Gülec M.,
Akyol O., Ersoy M.O.: Propofol and erythropoietin antioxidant properties
in rat brain injured tissue. Prog Neuropsychopharmacol Biol Psychiatry.
2008; 32: 81-86.
59
41. Gao J., Zhao W.X., Zhou L.J., Zeng B.X., Yao S.L., Liu D., Chen
Z.Q.: Protective effects of propofol on lipopolysaccharide-activated
endothelial cell barrier dysfunction. Inflamm Res. 2006; 55: 385-392.
42. Song H.K., Jeong D.C.: The effect of propofol on cytotoxicity and
apoptosis of lipopolysaccharide-treated mononuclear cells and
lymphocytes. Anesth Analg. 2004;98:1724-1728.
43. Cavalca V., Colli S., Veglia F., Eligini S., Zingaro L., Squellerio I.,
Rondello N., Cighetti G., Tremoli E., Sisillo E.: Anesthetic propofol
enhances plasma gamma-tocopherol levels in patients undergoing cardiac
surgery. Anesthesiology. 2008; 108: 988-997.
44. Ploppa A., Kiefer R.T., Nohé B., Haeberle H.A., Dieterich H.J.,
Unertl K.E., Durieux M.E. Krueger W.A.: Monocyte phagocytosis of
viable Staphylococcus aureus is impaired by barbiturates, but not by
propofol. Infection. 2008; 36: 220-225.
45. Krumholz W., Reussner D., Hempelmann G.: The influence of
several intravenous anaesthetics on the chemotaxis of human monocytes
in vitro. Eur J Anaesthesiol. 1999; 16: 547-549.
46. Cinnella G., Vendemiale G., Dambrosio M., Serviddio G., Pugliese
P.L., Aspromonte G., Altomare E.: Effect of propofol, sevoflurane and
60
desflurane on systemic redox balance. Int J Immunopathol Pharmacol.
2007; 20: 585-593.
47. Daskalopoulos R., Korcok J., Farhangkhgoee P., Karmazyn M., Gelb
A.W., Wilson J.X.: Propofol protection of sodium-hydrogen exchange
activity sustains glutamate uptake during oxidative stress. Anesth Analg.
2001; 93: 1199-1204.
48. De La Cruz J.P., Zanca A., Carmona J.A., De La Cuesta F.S.: The
effect of propofol on oxidative stress in platelets from surgical patients.
Anesth Analg. 1999; 89: 1050-1055.
49. Da Silva-Azevedo L., Jähne S., Hoffmann C., Stalder D., Heller M.,
Pries A.R., Zakrzewicz A., Baum O.: Up-regulation of the peroxiredoxin-
6 related metabolism of reactive oxygen species in skeletal muscle of
mice lacking neuronal nitric oxide synthase. J Physiol. 2009; 587:
655-668.
50. Allaouchiche B., Debon R., Goudable J., Chassard D., Duflo F.:
Oxidative stress status during exposure to propofol, sevoflurane and
desflurane. Anesth Analg. 2001; 93: 981-985.
61
51. Xu J.J., Wang Y.L.: Propofol attenuation of hydrogen peroxidemediated
oxidative stress and apoptosis in cultured cardiomyocytes
involves haeme oxygenase-1. Eur J Anaesthesiol. 2008; 25: 395-402.
52. Turan R., Yagmurdur H., Kavutcu M., Dikmen B.: Propofol and
tourniquet induced ischaemia reperfusion injury in lower extremity
operations. Eur J Anaesthesiol. 2007; 24: 185-189.
53. Lin C.S., Liu C.Y., Sun Y.L., Chang L.C., Chiu Y.T., Huang S.Y.,
Lin J.H., Yang P.C., Chu R., Huang M.C., Mao S.J.: Alteration of
endogenous antioxidant enzymes in naturally occurring hypertrophic
cardiomyopathy. Biochem Mol Biol Int. 1997; 43: 1253-1263.
54. Pinho R.A., Andrades M.E., Oliveira M.R., Pirola A.C., Zago M.S.,
Silveira P.C., Dal-Pizzol F., Moreira J.C.: Imbalance in SOD/CAT
activities in rat skeletal muscles submitted to treadmill training exercise.
Cell Biol Int. 2006; 30: 848-853.