腹膜炎是一種腹腔發炎所導致的疾病，其發生的原因是腹腔內器官破裂、發炎或細菌感染所致。嚴重的腹膜炎會造成多重器官衰竭而導致死亡。本研究以小鼠腹膜炎為模式，探討腹膜炎引發肺部損傷時，c-Jun N-terminal kinase (JNK)所扮演的角色。首先，誘發小鼠發生腹膜炎後，分別測定血液中reactive oxygen species (ROS)的含量；測定肺部嗜中性白血球浸潤的程度及肺部血管滲漏程度；並觀察肺部外觀型態的受損情形；接著，藉由施予JNK抑制劑、採用JNK基因剔除小鼠、基因嵌合小鼠(chimeric mice)，觀察JNK訊號傳遞路徑所扮演的角色。實驗結果顯示，小鼠發生腹膜炎後會引發肺部損傷，在施打JNK抑制劑SP600125或lefunomide後，腹膜炎引發肺部損傷的情形均獲得改善。當進一步使用JNK基因剔除小鼠時，腹膜炎引發肺部損傷情形亦較為舒緩。最後利用基因嵌合小鼠證實，腹膜炎引發肺部損傷是經由造血細胞的JNK訊號傳遞路徑，進一步引發肺部組織的iNOS產生NO，最後產生過量的peroxynitrite而造成組織器官的傷害。

Abdominal sepsis is a common, life-threatening condition in the critically ill patients. The c-Jun N-terminal kinase (JNK) is known as a stress-activated protein kinase, in order to study the role of JNK on peritonitis-induced lung injury, the changes of plasma dihydrorhodamine 123 (DHR 123) oxidation level; the myeloperoxidase (MPO) and extravasations of Evans blue dye (EBD) of lung in wild-type (WT) mice with P. aeruginosa-induced peritonitis were determined first. Second, the specific JNK inhibitor, SP600125 or lefunomide, was given to WT mice immediately after P. aeruginosa injection and DHR oxidation, MPO activity, and EBD extravasations were examined. Third, JNK1-/- mice and JNK1+/- mice were subjected to peritonitis and assayed for DHR 123 oxidation, MPO activity, EBD extravasations, and reactive oxygen species (ROS). Fourth, chimeric mice (WT → WT, JNK1-/- → WT, WT→JNK1-/-) were generated and used to determine the role of hematopoietic cells in peritonitis-induced lung damage. The results show that peritonitis induced DHR 123 oxidation; MPO activity and EBD extravasations in lungs and administration of specific JNK inhibitor decreased the peritonitis-induced DHR oxidation and lung damage. Also, both JNK1-/- and JNK1+/- mice showed a decreased DHR oxidation and lung damage after peritonitis. Finally, the decrease of DHR 123 oxidation, ROS, and lung damage in JNK1-/- → WT chimeric mice suggests that that peritonitis-induced expression of iNOS and subsequent peroxynitrite production and lung damage depends on the JNK1 signaling of the hematopoietic cells.

Abstract in Chinese …………………………………………………1

Abstract in English ………………………………………………… 2

Introduction …………………………………………………………3

Materials and Methods………………………………………………9

Results……………………………………………………………....19

Discussion…………………………………………………………..25

Figures……………………………………………………………....30

References…………………………………………………………..39

1. Cohen J: The immunopathogenesis of sepsis. Nature 2002; 420:885-891.
2. Hotchkiss RS, Karl IE: The pathophysiology and treatment of sepsis. N Engl J Med 2003; 348:138-150.
3. van der Poll T: Immunotherapy of sepsis. Lancet Infect Dis 2001; 1:165-174.
4. Wiggins KJ, Craig JC, Johnson DW, Strippoli GF. Treatment for peritoneal dialysis-associated peritonitis: Cochrane Database of Systematic Reviews 2008, Issue 1. Art. No.: CD005284. DOI: 10.1002/14651858.CD005284.pub2
5. Bernard GR, Wheeler AP, Russell JA, Schein R, Summer WR, Steinberg KP, Fulkerson WJ, Wright PE, Christman BW, Dupont WD et al: The effects of ibuprofen on the physiology and survival of patients with sepsis. The Ibuprofen in Sepsis Study Group. N Engl J Med 1997; 336:912-918.
6. Lorber B, Swenson RM: The bacteriology of intra-abdominal infections. Surg Clin North Am 1975; 55:1349-1354.
7. Li X, Xiang J, Liao Z: [Statistical analysis of 4,547 burn patients and the mortality in different periods]. Chung Hua Cheng Hsing Shao Shang Wai Ko Tsa Chih 1995; 11:335-338.
8. Cuzzocrea S, Zingarelli B, Sautebin L, Rizzo A, Crisafulli C, Campo GM, Costantino G, Calapai G, Nava F, Di Rosa M et al: Multiple organ failure following zymosan-induced peritonitis is mediated by nitric oxide. Shock 1997; 8:268-275.
9. Chen LW, Hsu CM, Wang JS, Chen HL, Chen JS: Inhibition of inducible nitric oxide synthase (iNOS) prevents lung neutrophil deposition and damage in burned rats. Shock 2001; 15:151-156.
10. Chen LW, Hwang YC, Chen CJ, Wang JS, Chen JS, Hsu CM: Burn-induced lung damage in rat is mediated by a nitric oxide/cGMP system. Shock 2003; 20:369-374
11. Arbak S, Ercan F, Hurdag CG, et al: Acute lung injury following thermal insult to the skin: a light and transmission electron microscopial study. Acta Histochem 1999; 101:255-262.
12. Arbak S, Ercan F, Hurdag CG, Karabulut O, Gurbuz V, Corak A, Alican I: Acute lung injury following thermal insult to the skin: a light and transmission electron microscopial study. Acta Histochem 1999; 101:255-262.
13. Moncada E, Subirá ML, Oleaga A, Goñi F, Sánchez-Ibarrola A, Monreal M, Sevilla M, Goñi MJ, Yoldi A, Terán D, et al. Insulin requirements and residual beta-cell function 12 months after concluding immunotherapy in type I diabetic patients treated with combined azathioprine and thymostimulin administration for one year. J Autoimmun. 1990 Oct;3(5):625-38.
14. Moncada, S. and Higgs, A., 1993. The L-arginine NO pathway. New England Journal of Medicine 329:2002-2012.
15. Nathan N, Denizot Y, Feiss P, Laskar M, Arnoux B, Variations of blood PAF-acether levels during coronary artery surgery. J Cardiothorac Vasc Anesth. 1992 Dec;6(6):692-6.
16. Deitch, E.A., 1992. Multiple organ failure. Pathophysiology and potential future therapy. Annals Surgery 216:117-134.
17. Szabó PE, Mann JR. Allele-specific expression and total expression levels of imprinted genes during early mouse development: implications for imprinting mechanisms.Genes Dev. 1995 Dec 15;9(24):3097-108.
18. Southan, G. and Szabò, C., 1996. Selective pharmacological inhibition of distinct NOS isoforms. Biochemical Pharmacology 51:383-394.
19. Moncada E, Subirá ML, Oleaga A, Goñi F, Sánchez-Ibarrola A, Monreal M, Sevilla M, Goñi MJ, Yoldi A, Terán D, et al. Insulin requirements and residual beta-cell function 12 months after concluding immunotherapy in type I diabetic patients treated with combined azathioprine and thymostimulin administration for one year. J Autoimmun. 1990 Oct;3(5):625-38.
20. Crow, J.P., Beckman, J.S. and McCord, J.M., 1995. Sensitivity of the essential zinc-thiolate moiety of yeast alcohol dehydrogenase to hypochlorite and peroxynitrite. Biochemistry 34:3544-52.
21. Beckman BS, Mason-Garcia M, Martinez MI, Fermin C. Enhanced expression of the beta II subspecies of protein kinase C in differentiating erythroleukemia cells. Exp Cell Res. 1990 Dec;191(2):319-22.
22. Rao, A.V. and Balachandran, B., 2002. Role of oxidative stress and antioxidants in neurodegenerative diseases. Nutritional Neuroscience 5:291-309.
23. Iuliano, L., 2001. The oxidant stress hypothesis of atherogenesis. Lipids 36:41-44 (Suppl).
24. Ishii, M., Suzuki, Y., Takeshita, K., Miyao, N., Kudo, H., Hiraoka, R., Nishio, K., Sato, N., Naoki, K., Aoki, T., and Yamaguchi, K., 2004. Inhibition of c-Jun NH2-terminal kinase activity improves ischemia/reperfusion injury in rat lungs. J. Immunol. 172:2569-2577.
25. Cuzzocrea, S., Riley, D.P., Caputi, A.P. and Salvemini, D., 2001. Antioxidant therapy: a new pharmacological approach in shock, inflammation, and ischemia/reperfusion injury. Pharmacological Review 53:135-159.
26. Salvemini, D., Wang, Z.Q., Bourdon, D.M., Stern, M.K., Currie, M.G. and Manning, P.T., 1996. Evidence of peroxynitrite involvement in the carrageenan-induced rat paw edema. European Journal Pharmacology 303:217-220.
27. Nathan C. Inducible nitric oxide synthase: what difference does it make J Clin Invest. 1997, 100:2417-23. Review.
28. Lowenstein CJ, Dinerman JL, Snyder SH. Nitric oxide: a
physiologic messenger. Ann Intern Med. 1994, 120:227-37.
Review.
29. Schmidt HH, Walter U. NO at work. Cell. 1994, 78:919-25.
Review.
30 Robinson MJ, Cobb MH. Mitogen-activated protein kinase
pathways. Curr Opin Cell Biol. 1997, 9:180-6. Review.
31. Tibble LA, Woodgett JR. The stress-activated protein kinase
pathways. Cel lMol Life Sci. 1999, 55:1230–1254.
32. Zhang X, Bedard EL, Potter R, Zhong R, Alam J, Choi AM, Lee PJ: Mitogen-activated protein kinases regulate HO-1 gene transcription after ischemia-reperfusion lung injury. Am J Physiol Lung Cell Mol Physiol 2002; 283:L815-829.
33. Li LF, Yu L, Quinn DA: Ventilation-induced neutrophil infiltration depends on c-Jun N-terminal kinase. Am J Respir Crit Care Med 2004; 169:518-524. Epub 2003 Nov 2025.
34. Jiyan Zhang, Jing Liu, Chenfei Yu and Anning Lin. BAD Ser128 Is Not Phosphorylated by c-Jun NH2-Terminal Kinase for Promoting Apoptosis. Cancer Research 65, 8372-8378, September 15, 2005
35. Chen Lee-Wei ,Chang Wei-Jung ,Wang Jyh-Seng , Thermal injury-induced peroxynitrite production and pulmonary inducible nitric oxide synthase expression depend on JNK/AP-1 signaling. Critical care medicine. 2006, vol. 34, no1, pp. 142-150
36. Matute-Bello G, Lee JS, Frevert CW, Liles WC, Sutlief S, Ballman K, Wong V, Selk A, Martin TR: Optimal timing to repopulation of resident alveolar macrophages with donor cells following total body irradiation and bone marrow transplantation in mice. J Immunol Methods 2004; 292:25-34.
37. Bennett BL, Sasaki DT, Murray BW, O'Leary EC, Sakata ST, Xu W, Leisten JC, Motiwala A, Pierce S, Satoh Y et al: SP600125, an anthrapyrazolone inhibitor of Jun N-terminal kinase. Proc Natl Acad Sci U S A 2001; 98:13681-13686.
38. Han Z, Boyle DL, Chang L, Bennett B, Karin M, Yang L, Manning AM, Firestein GS: c-Jun N-terminal kinase is required for metalloproteinase expression and joint destruction in inflammatory arthritis. J Clin Invest 2001; 108:73-81.
39. Magnotti LJ, Upperman JS, Xu DZ, Lu Q, Deitch EA: Gut-derived mesenteric lymph but not portal blood increases endothelial cell permeability and promotes lung injury after hemorrhagic shock. Ann Surg 1998; 228:518-527.
40. Kooy NW, Royall JA, Ischiropoulos H, Beckman JS: Peroxynitrite-mediated oxidation of dihydrorhodamine 123. Free Radic Biol Med 1994; 16:149-156.
41. Patterson CE, Rhoades RA, Garcia JG: Evans blue dye as a marker of albumin clearance in cultured endothelial monolayer and isolated lung [see comments]. J Appl Physiol 1992; 72:865-873.
42. Smith JA: Neutrophils, host defense, and inflammation: a double-edged sword. J Leukoc Biol 1994; 56:672-686.
43. Balk RA: Pathogenesis and management of multiple organ dysfunction or failure in severe sepsis and septic shock. Crit Care Clin 2000; 16:337-352, vii.
44. Kooy NW, Royall JA, Ye YZ, Kelly DR, Beckman JS: Evidence for in vivo peroxynitrite production in human acute lung injury. Am J Respir Crit Care Med 1995; 151:1250-1254.
45. Szabo A, Hake P, Salzman AL, Szabo C: Beneficial effects of mercaptoethylguanidine, an inhibitor of the inducible isoform of nitric oxide synthase and a scavenger of peroxynitrite, in a porcine model of delayed hemorrhagic shock. Crit Care Med 1999; 27:1343-1350.
46. Chen LW, Wang JS, Chen HL, Chen JS, Hsu CM: Peroxynitrite is an important mediator in thermal injury-induced lung damage. Crit Care Med 2003; 31:2170-2177.
47. Nabeyrat E, Jones GE, Fenwick PS, Barnes PJ, Donnelly LE: Mitogen-activated protein kinases mediate peroxynitrite-induced cell death in human bronchial epithelial cells. Am J Physiol Lung Cell Mol Physiol 2003; 284:L1112-1120. Epub 2003 Feb 1121.
48. Shrivastava P, Pantano C, Watkin R, McElhinney B, Guala A, Poynter ML, Persinger RL, Budd R, Janssen-Heininger Y: Reactive nitrogen species-induced cell death requires Fas-dependent activation of c-Jun N-terminal kinase. Mol Cell Biol 2004; 24:6763-6772.
49. Lahti A, Jalonen U, Kankaanranta H, Moilanen E: c-Jun NH2-terminal kinase inhibitor anthra(1,9-cd)pyrazol-6(2H)-one reduces inducible nitric-oxide synthase expression by destabilizing mRNA in activated macrophages. Mol Pharmacol 2003; 64:308-315.
50. Razavi HM, Wang le F, Weicker S, Rohan M, Law C, McCormack DG, Mehta S: Pulmonary neutrophil infiltration in murine sepsis: role of inducible nitric oxide synthase. Am J Respir Crit Care Med 2004; 170:227-233. Epub 2004 Apr 2001.
51. Bultinck J, Sips P, Vakaet L, Brouckaert P, Cauwels A: Systemic NO production during (septic) shock depends on parenchymal and not on hematopoietic cells: in vivo iNOS expression pattern in (septic) shock. Faseb J 2006; 20:2363-2365.