中文摘要
鏈球菌致熱性外毒素B (SPE B) 是一種cysteine protease，所有A群鏈球菌 (GAS) 都攜帶致熱性外毒素B之基因，在A群鏈球菌侵入性感染的病程中，致熱性外毒素B扮演一個重要的致病角色。根據實驗室先前的研究結果顯示，致熱性外毒素B能夠引發U937細胞的凋亡結果，同時降低U937細胞的吞噬能力。冬蟲夏草是中國傳統的中草藥，被認為具有調節免疫能力的功效。本論文係利用人類單核細胞株U937細胞及A群鏈球菌感染模式來探討冬蟲夏草之免疫調節機轉，我們證實冬蟲夏草水提液確實可以活化U937細胞之吞噬能力，若是冬蟲夏草水提液經加熱或trypsin酵素處理後，並不會對冬蟲夏草的功能造成影響。由於致熱性外毒素B能夠降低U937細胞的吞噬能力，我們進一步利用A群鏈球菌感染小鼠的模式以及外給U937細胞SPE B的條件下，探討冬蟲夏草是否能夠活化吞噬能力。實驗結果顯示，冬蟲夏草能夠回復致熱性外毒素B對U937細胞所引起之吞噬能力下降。另一方面，冬蟲夏草與U937細胞作用十個小時能觀察到其吞噬能力上升，但作用六小時則不具活化效果。根據這樣的結果，我們取冬蟲夏草與U937細胞共同作用之培養液進一步與U937細胞共同培養六小時後測試其吞噬能力，實驗結果發現U937細胞受冬蟲夏草刺激會釋放某些因子，而這些因子具有增強吞噬能力、可以緩解致熱性外毒素B的抑制效果。當細胞培養液經加熱處理，其活化吞噬能力便明顯降低，因此推測U937細胞釋放增強吞噬能力的因子，可能是如細胞激素之類的蛋白質。我們發現冬蟲夏草確實能夠活化U937細胞在TNF-, IL-12 p35, IL-12 p40以及IFN-這些細胞激素mRNA的表現，在抗體中和試驗中，培養液活化吞噬能力的效果可被IFN-、IL-12及TNF-抗體所抑制。利用小鼠動物模式進一步探討胃管餵食冬蟲夏草之小鼠，在背部氣囊感染A群鏈球菌後，其存活率有回復增加的結果，根據菌血量以及氣囊回抽液菌數，餵食冬蟲夏草的小鼠較控制組之菌數為低，而且在控制組的小鼠能夠觀察到A群鏈球菌菌血症及細菌散佈到脾臟、肝藏及腎臟，而餵食冬蟲夏草的小鼠則沒有這些的情形。在血清中AST和ALT的表現，控制組小鼠感染A群鏈球菌後較蟲草組為高。這些實驗結果顯示冬蟲夏草對A群鏈球菌的感染具有修復的效果，冬蟲夏草的成份中，具有能夠調節免疫反應的特殊因子，特別是可以活化U937細胞的吞噬能力及刺激細胞激素的產生、提高感染A群鏈球菌小鼠的存活率，對於冬蟲夏草的免疫調節能力值得更深入研究。

Abstract:

Streptococcal pyrogenic exotoxin B (SPE B) is a cysteine protease and carried by every strain of Streptococcus pyogenes. SPE B has been shown to play an important role in invasive group A streptococcal (GAS) infection. We previously showed that SPE B could induce apoptosis and reduce the phagocytic activity in U937 cells. Cordyceps sinensis (CS) is a Chinese herbal medicine, which is known to possess immunomodulatory activity. In this study, we use human monocytic U937 cells and GAS infection mouse model to study the mechanisms of immunomodulatory effects of CS. We had demonstrated the elevation of phagocytic activity of U937 cells by CS extract. Heat or trypsin treatment did not affect the enhancing effect of CS. Whether the phagocytic enhancing properties of CS may exert modulatory effect on the pathogenicity of GAS, especially the role played by SPE B, was further investigated. Results showed that SPE B-mediated suppression of U937 phagocytic activity could be abrogated by CS extract. The enhancing effect was detectable at 10 h after coincubation with CS, but not at 6 h. The conditioned medium containing factors released from CS extract-treated U937 cells augmented the phagocytic activity and also abrogated SPE B-mediated suppression of phagocytosis. Moreover, heat treatment eliminated the enhancing effect of conditioned medium, suggesting that the protein products, such as cytokines, released by U937 cells caused augmentation of phagocytosis. CS can induce TNF-, IL-12 p35, IL-12 p40 and IFN- mRNA expression in U937 cells. In antibody neutralization test, the enhancing effect of conditioned medium on phagocytosis was suppressed by anti-IFN-, anti-IL-12 and anti-TNF- antibodies. Further studies in the mouse model showed that force-feeding with CS was able to increase the survival rate after air pouch inoculation with GAS. Bacterial numbers in the air pouch exudates from CS-treated mice were lower than those from control mice. Bacteremia and bacterial dissemination to spleen, liver and kidney could be observed in mice infected with GAS, but not in those pretreated with CS. The levels of AST and ALT in control mice sera after GAS infection were higher than CS-pretreated mice. These results suggest that CS confers protective effect in the animal model of GAS infection. The CS contains factors that can modulate the immune responses, especially phagocytic activity of U937 cells and cytokine expression. The CS extract can increase survival rate and decrease bacteremia in GAS infection. The immunomodulatory activity of CS is worthy for further investigation.

參考文獻

1. Cunningham, M. W. Pathogenesis of group A streptococcal infections. Clin Mocrobiol Rev 13: 470-511, 2000.
2. Efstratiou, A. Group A streptococci in the 1990s. J Antimicrob Chemother 45, Topic T1: 3-12, 2000.
3. Reid, S. D., Hoe, N. P., Smoot, L. M., and Musser, J. M. Group A Streptococcus: allelic variation, population genetics, and host-pathogen interactions. J Clin Invest 107: 393-9, 2001.
4. Lukomski, S., Burns, E. H. Jr., Wyde, P. R., Podbielski, A., Rurangirwa, J., Moore-Poveda, D. K., and Musser, J. M. Genetic inactivation of an extracellular cysteine protease (Spe B) expressed by Streptococcus pyogenes decreases resistance to phagocytosis and dissemination to organs. Infect Immun 66: 771-6, 1998.
5. Kuo, C. F., Wu, J. J., Lin, K. Y., Tsai, P. J., Lee, S. C., Jin, Y. T., Lei, H. Y., and Lin, Y. S. Role of streptococcal pyrogenic exotoxin B in the mouse model of group A streptococcal infection. Infect Immun 66: 3931-5, 1998.
6. Tsai, P. J., Kuo, C. F., Lin, K. Y., Lin, Y. S., Lei, H. Y., Chen, F. F., Wang, J. R., and Wu, J. J. Effect of group A streptococcal cysteine protease on invasion of epithelial cells. Infect Immun 664: 1460-6, 1998.
7. Tsai, P. J., Lin, Y. S., Kuo, C. F., Lei, H. Y., and Wu, J. J. Group A streptococcus induces apoptosis in human epithelial cells. Infect Immun 67: 4334-9, 1999.
8. Kuo, C. F., Wu, J. J., Tsai, P. J., Kao, F. J., Lei, H. Y., Lin, M. T., and Lin, Y. S. Streptococcal pyrogenic exotoxin B induces apoptosis and reduces phagocytic activity in U937 cells. Infect Immun 67: 126-30, 1999.
9. Chen, Y. J., Shiao, M. S., Lee, S. S., and Wang, S. Y. Effect of Cordyceps sinensis on the proliferation and differentiation of human leukemic U937 cells. Life Sci 60: 2349-59, 1997.
10. Raeder, R. H., B. M. L., Lester, T., and Boyle, M. D., and Metzger, D. W. A pivotal role for interferon-gamma in protection against group A streptococcal skin infection. J Infect Dis 181: 639-45, 2000.
11. Metzger D. W., Raeder, R., Van Cleave, V. H., and Boyle, M. D. Protection of mice from group A streptococcal skin infection by interleukin-12. J Infect Dis 171: 1643-5, 1995.
12. 郭志峰 熱原性外毒素B於A群鏈球菌感染之致病機轉中扮演角色之探討 國立成功大學基礎醫學研究所博士論文 1999.
13. 蔡宛樺 熱原性外毒素B引發A549細胞凋亡之探討 國立成功大學生物化學研究所碩士論文 2000.
14. 羅月霞 熱原性外毒素B在A群鏈球菌感染小鼠模式中與組織病變、NO及cytokine產生的相關性 國立成功大學微生物暨免疫學研究所碩士論文 2001.
15. 黃懷玉 補充冬蟲夏草配合重量訓練對於身體組成、血液生化值及最大總肌力之影響 國立體育學院運動科學研究所碩士論文 2000.
16. 李建良 液態培養生產冬蟲夏草菌絲體與冬蟲夏草多醣之研究 國立交通大學生物科技研究所碩士論文 2000.
17. 程麗菁 冬蟲夏草發酵菌絲體之免疫調節作用 國立成功大學微生物暨免疫學研究所碩士論文 1999.
18. Manabe, N., Sugimoto, M., Azuma, Y., Taketomo, N., Yamashita, A., Tsuboi, H., Tsunoo, A., Kinjo, N., Nian-Lai, H., and Miyamoto, H. Effects of the mycelial extract of cultured Cordyceps sinensis on in vivo hepatic energy metabolism in the mouse. Jpn J Pharmacol 70: 85-8, 1996.
19. Liu P., Zhu, J., Huang, Y., and Liu, C. Influence of Cordyceps sinensis (Berk.) Sacc. and rat serum containing same medicine on IL-1, IFN and TNF produced by rat Kupffer cells. Zhongguo Zhong Yao Za Zhi 21: 367-9, 1996.
20. Nakamura, K., Yamaguchi, Y., Kagota, S., Shinozuka, K., and Kunitomo, M. Activation of in vivo Kupffer cell function by oral administration of Cordyceps sinensis in rats. Jpn J Pharmacol 79: 505-8, 1999.
21. Zhou, L., Yang, W., Xu, Y., Zhu, Q., Ma, Z., Zhu, T., Ge, X., and Gao, J. Short-term curative effect of cultured Cordyceps sinensis (Berk.) Sacc. Mycelia in chronic hepatitis B. Zhongguo Zhong Yao Za Zhi 15: 53-5, 1990.
22. Li, L. S., Zheng, F., and Liu, Z. H. Experimental study on effect of Cordyceps sinensis in ameliorating aminoglycoside induced nephrotoxicity. Zhongguo Zhong Xi Yi Jie He Za Zhi 16: 733-7, 1996.
23. Zhen, F., Tian, J., and Li, L. S. Mechanisms and therapeutic effect of Cordyceps sinensis (CS) on aminoglycoside induced acute renal failure (ARF) in rats. Zhongguo Zhong Xi Yi Jie He Za Zhi 12: 288-91, 1992.
24. 李立仁 冬蟲夏草菌萃取液對大白鼠腎上腺皮質細胞皮質酮分泌功能及脂肪滴形態的影響 國立台灣大學解剖學研究所碩士論文 1996.
25. Wang, S. M., Lee, L. J., Lin, W. W., and Chang, C. M. Effects of a water-soluble extract of Cordyceps sinensis on steroidogenesis and capsular morphology of lipid droplets in cultured rat adrenocortical cells. J Cell Biochem 69: 483-9, 1998.
26. Xu, F., Huang, J. B., Jiang, L., Xu, J., and Mi, J. Amelioration of cyclosporin nephrotoxicity by Cordyceps sinensis in kidney-transplanted recipients. Nephrol Dial Transplant 10: 142-3, 1995.
27. Cheng, Q. Effect of Cordyceps sinensis on cellular immunity in rats with chronic renal insufficiency. Chung-Hua I Hsueh Tsa Chin (Chinese Medical Journal) 72: 27-9, 1992.
28. Ikumoto, T., Sasaki, S., Namba, H., Toyama, R., Horitoki, H., and Mouri, T. Physiologically active compounds in the extracts from tochukaso and cultured mycelia of Cordyceps and Isaria. Yakugaku Zasshi 111: 504-9, 1991.
29. Zhao, Y. Inhibitory effects of alcoholic extract of Cordyceps sinensis on abdominal aortic thrombus formation in rabbits. Chung-Hua I Hsueh Tsa Chin (Chinese Medical Journal) 71: 612-5, 1991.
30. 張佩靖 人工培養冬蟲夏草菌絲對大白鼠血管作用之探討 國立陽明大學藥理學研究所碩士論文 1996.
31. Chiou W. F., Chang P. C., Chou C. J., and Chen, C. F. Protein constituent contributes to the hypotensive and vasorelaxant activities of Cordyceps sinensis. Life Sci 66: 1369-76, 2000.
32. Mei, Q. B., Tao, J. Y., Gao, S. B., Xu, G. C., Chen, L. M., and Su, J. K. Antiarrhythmic effects of Cordyceps sinensis (Berk.) Sacc. Zhongguo Zhong Yao Za Zhi 14: 616-8, 1989.
33. Kuo, Y. C., Lin, C. Y., Tsai, W. J., Wu, C. L., Chen, C. F., and Shiao, M. S. Growth inhibitors against tumor cells in Cordyceps sinensis other than cordycepin and polysaccharides. Cancer Invest 12: 611-5, 1994.
34. Bok, J. W., Lermer, L., Chilton, J., Klingeman, H. G., and Towers, G. H. Antitumor sterols from the mycelia of Cordyceps sinensis. Phytochemistry 51: 891-8, 1999.
35. Yoshida, J., Takamura, S., Yamaguchi, N., Ren, L. J., Chen, H., Koshimura, S., and Suzuki, S. Antitumor activity of an extract of Cordyceps sinensis (Berk.) Sacc. against murine tumor cell lines. Jpn J Exp Med 59: 157-61, 1989.
36. Xu, R. H., Peng, X. E., Chen, G. Z., and Chen, G. L. Effects of Cordyceps sinensis on natural killer activity and colony formation of B16 melanoma. Chin Med J 105: 97-101, 1992.
37. Nakamura, K., Yamaguchi, Y., Kagota, S., Shinozuka, K., and Kunitomo, M. Activation of in vivo Kupffer cell function by oral administration of Cordyceps sinensis in rats. Jpn J Pharmacol 79: 505-8, 1999.
38. Chiu, J. H., Ju, C. H., Wu, L. H., Lui, W. Y., Wu, C. W., Shiao, M. S., and Hong, C. Y. Cordyceps sinensis increases the expression of major histocompatibility complex class II antigens on human hepatoma cell line HA22T/VGH cells. Am J Chin Med 26: 159-70, 1998.
39. Chen, G. Z., Chen, G. L., Sun, T., Hsieh, G. C., and Henshall, J. M. Effects of Cordyceps sinensis on murine T lymphocyte subsets. Chin Med J 104: 4-8, 1991.
40. Zhu, X. Y., and Yu, H. Y. Immunosuppressive effect of cultured Cordyceps sinensis on cellular immune response. Zhong Xi Yi Jie He Za Zhi 10: 485-7, 1990.
41. Liu, C., Lu, S., Ji. and M. R. Effects of Cordyceps sinensis on in vitro natural killer cells. Chung-Kuo Chung Hsi I Chieh Ho Tsa Chih. 12:267-9, 1992.
42. Guan, Y. J., Hu, Z., and Hou, M. Effect of Cordyceps sinesis on T-lymphocyte subsets in chronic renal failure. Zhongguo Zhong Xi Yi Jie He Za Zhi 12: 338-9, 1992.
43. Chen, J. R., Yen, J. H., Lin, C. C., Tasi, W. J., Liu, W. J., Tsai, J. J., Lin, S. F., and Lin, H. W. The effects of Chinese herbs on improving survival and inhibiting anti-ds DNA antibody production in lupus mice. Am J Chin Med 21: 257-62 , 1993.
44. 楊令瑀 研究臺灣兒童狼瘡性腎炎及以冬蟲夏草單純天然物H1-A治療之可行性及機制 國立陽明大學臨床醫學研究所碩士論文 1999.
45. Kuo, Y. C., Tsai, W. J., Wang, J. Y., Chang, S. C., Lin, C. Y., and Shiao, M. S. Regulation of bronchoalveolar lavage fluids cell function by the immunomodulatory agents from Cordyceps sinensis. Life Sci 68: 1067-82, 2001.
46. 許毓芬 篩選可抑制血小板活化因子以治療氣喘之冬蟲夏草天然物 國立陽明大學傳統醫藥學研究所碩士論文 1994.
47. 錢震莊 以棕色挪威鼠為模式探討冬蟲夏草天然物對卵白蛋白誘發氣喘後期氣管收縮與脂多醣體誘發急性肺傷害之療效 輔仁大學生物學系碩士論文 1995.
48. Shin, K. H., Lim, S. S., Lee, S. H., Lee, Y. S., and Cho, S. Y. Antioxidant and immunostimulating activities of the fruiting bodies of Paecilomyces japonica, a new type of Cordyceps sp. Ann NY Acad Sci 928: 261-73, 2001.
49. Kiho, T., Yamane, A., Hui, J., Usui, S., and Ukai, S. Polysaccharides in fungi. XXXVI. Hypoglycemic activity of a polysaccharide (CS-F30) from the cultural mycelium of Cordyceps sinensis and its effect on glucose metabolism in mouse liver. Biol Pharm Bull 19: 294-6, 1996
50. Kneifel, H., Konig, W. A., Loeffler, W., and Muller, R. Ophiocordin, an antifungal antibiotic of Cordyceps ophioglossoides. Arch Microbiol 113: 121-30, 1977.
51. Huang, B. M., Hsu, C. C., Tsai, S. J., Sheu, C. C., and Leu, S. F. Effects of Cordyceps sinensis on testosterone production in normal mouse Leydig cells. Life Sci 69: 2593-602, 2001.
52. Wang, S. Y., and Shiao, M. S. Pharmacological functions of Chinese medicinal fungus Cordyceps sinensis and related species. 藥品食品分析 8: 248-57, 2000.
53. Kuo, Y. C., Tsai, W. J., Shiao, M. S., Chen, C. F., and Lin, C. Y. Cordyceps sinensis as an immunomodulatory agent. Am J Chin Med 24: 111-25, 1996.
54. Smiley, S. T., King, J. A., and Hancock, W. W. Fibrinogen stimulates macrophage chemokine secretion through toll-like receptor 4. J Immunol 167: 2887-94, 2001.