根據中國傳統醫學，冬蟲夏草性味甘，功能益肺腎、補虛損，有調節免疫與防治慢性腎功能衰竭的作用。本文目的在探討冬蟲夏草前置處理，對大鼠腎臟缺血性急性腎衰竭的保護作用及其作用機轉。動物模式為大鼠腎臟缺血性急性腎衰竭，採雄性SD大鼠手術開腹、左腎摘除、右腎血管夾住缺血45分鐘、然後放開右腎血管恢復血流。冬蟲夏草組先行給予每天每公斤600毫克冬蟲夏草兩天，腎臟缺血手術後第1, 3, 6, 16, 48及120 小時抽血進行腎功能分析，並進行腎臟病理組織PSA-D染色及SDF-1α、CXCR4、Ki67免疫組織染色與西方墨點法分析。腎臟組織老化分析則透過β-galactosidase活性分析。冬蟲夏草組在缺血灌流後48 小時肌酸酐顯著較控制組為低(p=0.04)。在PAS-D染色上、冬蟲夏草組在6及16小時呈現較輕微的腎小管擴張、脫落沉澱、刷狀物邊緣喪失、腎小管壞死、外髓質細胞脫落。免疫組織染色顯示冬蟲夏草組相較於控制組，在1、3、6及16小時有較多的SDF-1α表現於腎臟遠曲小管及集尿管。控制組中的CXCR4免疫組織染色顯示CXCR4在缺血再灌流1到6小時會逐漸增加，在16小時後會降低趨近正常。在冬蟲夏草組，CXCR4的表現呈現持續穩定至第16小時。在老化相關的β-galactosidase活性研究上，控制組在1到6小時腎臟在近曲與遠曲小管位置呈現較強的活性。而在冬蟲夏草組活性在第1及3小時出現，第6小時隨即消褪。細胞修復酵素Ki67在冬蟲夏草組呈現較深的細胞核染色。冬蟲夏草前置處理可在缺血再灌流後48小時時顯著降低肌酸酐，病理形態分析上也顯示冬蟲夏草可提供缺血性急性腎衰竭的保護。冬蟲夏草可以提早引發SDF-1α的表現並使CXCR4持續穩定表現。冬蟲夏草可降低腎臟老化的β-galactosidase活性，並刺激修復酵素Ki67的表現，意即其可可減緩急性腎衰竭後細胞老化，並促進組織的修復。此結果可提供未來臨床急性腎衰竭發生時使用冬蟲夏草的瞭解與依據。

According to traditional Chinese medicine , Cordyceps sinensis (CS) can prevent subjects from renal failure. The aim of this study was to investigate the protective effect of CS preconditioning on ischemic renal acute failure in rats and to assess its mechanism. The animal model of ischemic acute renal failure was performed by left nephrectomy and clamping right renal vessel for 45 mins in S-D rats. Cordyceps group had been pretreated with two-day 600 mg/kg CS before I/R injury. Rats were sacrificed at 1, 3, 6, 16, 48 and 120 h after reperfusion for evaluation of renal function and histopathological PASD staining. The immunohistochemistry and Western blotting of SDF-1α, CXCR4 and Ki67 were also performed. Β-galactosidase activity was detected with the senescence staining. The results showed that the level of creatinine in Cordyceps group were significant lower after 48 hours I/R injury (p =0.04). PSAD staining in Cordyceps group revealed less tubular necrosis, tubular dilatation, and cast formation at 6 and 16 hour than in control group. Immunohistochemistry of SDF-1α in Cordyceps group demonstrated staining in the distal tubules and collecting ducts at 1, 3, 6, and 16 h. The CXCR4 signal of control group had gradually intensified from 1 to 6 hr after I/R . In Cordyceps group, the CXCR4 expression had been stabilized until 16 h after I/R. The β-galactosidase activity was higher in control group at 1, 3 and 6 hours. However, the senescence was presented at 1 and 3 hours in Cordyceps group. The nuclear staining of repair enzyme Ki67 in Cordyceps group showed higher density than in control group. Pathologic morphology indicated CS may protect subjects from ischemic acute renal failure. CS also induced SDF-1α expression in early stage of I/R injury, and maintained the stable CXCR4 expression. CS can not only reduce the activity of senescence-related β-galactosidase, but also regulate the expression of repair enzyme Ki67, indicating that CS may alleviate the ischemic-induced senescence and enhance renal repair.

中文摘要 ---------------------------------------------------------------------------------- 2
Abstract ------------------------------------------------------------------------------------- 3

Index of Figures --------------------------------------------------------------------------- 4

I. Abbreviation:----------------------------------------------------------------------------- 5
I. Introduction ------------------------------------------------------------------------------ 6

II. Materials and Methods ---------------------------------------------------------------- 17

III.Results ----------------------------------------------------------------------------------23

IV. Discussion ------------------------------------------------------------------------------ 39

V. Conclusion ------------------------------------------------------------------------------ 45

VI.Literature Cited -----------------------------------------------------------------------46

1. Tsan MF, Gao B. Endogenous ligands of Toll-like receptors. J Leukoc Biol.2004; 76: 514–9.
2. Zhang D, Zhang G, Hayden MS, Greenblatt MB, Bussey C, Flavell RA, Ghosh S. A toll-like receptor that prevents infection by uropathogenic bacteria. Science.2004; 303: 1522-6.
3. Cook DN, Pisetsky DS, Schwartz DA. Toll-like receptors in the pathogenesis of human disease. Nat Immunol. 2004; 5: 975–9.
4. Wu HS, Zhang JX, Wang L, Tian Y, Wang H, Rotstein O. Toll-like receptor 4 involvement in hepatic ischemia/reperfusion injury in mice. Hepatobiliary Pancreat Dis Int.2004; 3: 250–3.
5. Peng Y, Gong JP, Liu CA, Li XH, Gan L, Li SB. Expression of toll-like receptor 4 and MD-2 gene and protein in Kupffer cells after ischemia-reperfusion in rat liver graft. World J Gastroenterol. 2004; 10: 2890–2903.
6. Schumer M, Colombel MC, Sawczuk IS, Gobé G, Connor J, O'Toole KM, Olsson CA, Wise GJ, Buttyan R. Morphologic, biochemical, and molecular evidence of apoptosis during the reperfusion phase after brief periods of renal ischemia. Am J Pathol. 1992; 140: 831-8.
7. de Vries B, Walter SJ, Peutz-Kootstra CJ, Wolfs TG, van Heurn LW, Buurman WA. The mannose-binding lectin-pathway is involved in complement activation in the course of renal ischemiareperfusion injury. Am J Pathol. 2004 ; 165: 1677–88.
8. Collard CD, Väkevä A, Morrissey MA, Agah A, Rollins SA, Reenstra WR, Buras JA, Meri S, Stahl GL. Complement activation after oxidative stress: role of the lectin complement pathway. Am J Pathol. 2000; 156:1549-56.
9. Pratt JR, Basheer SA, Sacks SH. Local synthesis of complement component C3 regulates acute renal transplant rejection. NatMed 2002; 8:582–7.
10.Fearon DT, Carroll MC. Regulation of B lymphocyte responses to foreign and self-antigens by the CD19/CD21 complex. Annu Rev Immunol. 2000; 18:393–422.
11.Jankovic D, Kullberg MC, Hieny S, Caspar P, Collazo CM, Sher A. In the absence of IL-12, CD4(+) T cell responses to intracellular pathogens fail to default to a Th2 pattern and are host protective in an IL-10 (−/−) setting. Immunity. 2002; 16:429–39.
12. Coates PT, Duncan FJ, Colvin BL, Wang Z, Zahorchak AF, Shufesky WJ, Morelli AE, Thomson AW. In vivo-mobilized kidney dendritic cells are functionally immature, subvert alloreactive T-cell responses, and prolong organ allograft survival. Transplantation. 2004; 77:1080-9.
13. Hutchings A, Wu J, Asiedu C, Hubbard W, Eckhoff D, Contreras J, Thomas FT, Neville D, Thomas JM. The immune decision toward allograft tolerance in non-human primates requires early inhibition of innate immunity and induction of immune regulation. Transpl Immunol. 2003; 11:335-44.
14. He H, Stone JR, Perkins DL. Analysis of differential immune responses induced by innate and adaptive immunity following transplantation. Immunology. 2003; 109:185–96.
15. Price PM, Megyesi J, Safirstein RL. Cell cycle regulation: Repair and regeneration in acute renal failure. Kidney Int. 2004; 66: 509–14.
16. Blau HM, Brazelton TR, Weimann JM. The evolving concept of a stem cell: entity or function? Cell. 2001; 105: 829-41.
17. Lin F, Cordes K, Li L, Hood L, Couser WG, Shankland SJ, Igarashi P. Hematopoietic stem cells contribute to the regeneration of renal tubules after renal ischemia-reperfusion injury in mice. J Am Soc Nephrol. 2003; 14:1188-99.
18. Cottler-Fox MH, Lapidot T, Petit I, Kollet O, DiPersio JF, Link D, Devine S. Stem cell mobilization. Hematology Am Soc Hematol Educ Program. 2003: 419-437.
19. Lapteva N, Yang AG, Sanders DE, Strube RW, Chen SY. CXCR4 knockdown by small interfering RNA abrogates breast tumor growth in vivo. Cancer Gene Ther. 2005; 12: 84-89.
20. Balkwill F. The significance of cancer cell expression of the chemokine receptor CXCR4. Semin Cancer Biol. 2004; 14:171-179.
21. Tavor S, Petit I, Porozov S, Avigdor A, Dar A, Leider-Trejo L, Shemtov N, Deutsch V, Naparstek E, Nagler A, Lapidot T. CXCR4 regulates migration and development of human acute myelogenous leukemia stem cells in transplanted NOD/SCID mice. Cancer Res. 2004; 64:2817-2824.
22. Spiegel A, Kollet O, Peled A, Abel L, Nagler A, Bielorai B, Rechavi G, Vormoor J, Lapidot T. Unique SDF-1-induced activation of human precursor-B ALL cells as a result of altered CXCR4 expression and signaling. Blood. 2004; 103:2900-2907.
23. Kollet O, Shivtiel S, Chen YQ, Suriawinata J, Thung SN, Dabeva MD, Kahn J, Spiegel A, Dar A, Samira S, Goichberg P, Kalinkovich A, Arenzana-Seisdedos F, Nagler A, Hardan I, Revel M, Shafritz DA, Lapidot T. HGF, SDF-1, and MMP-9 are involved in stress-induced human CD34+ stem cell recruitment to the liver. J Clin Invest. 2003; 112:160-169.
24. Abbott JD, Huang Y, Liu D, Hickey R, Krause DS, Giordano FJ. Stromal cell-derived factor-1alpha plays a critical role in stem cell recruitment to the heart after myocardial infarction but is not sufficient to induce homing in the absence of injury. Circulation. 2004; 110:3300-3305.
25. Shyu W C, Lee YJ, Liu DD, Lin SZ, Li H. Homing genes, cell therapy and stroke. Front Biosci.2006; 11:899-907.
26. Tögel F, Isaac J, Hu Z, Weiss K, Westenfelder C. Renal SDF-1 signals mobilization and homing of CXCR4-positive cells to the kidney after ischemic injury. Kidney Int. 2005 May; 67:1772-1784.
27. Helbig G, Christopherson KW 2nd, Bhat-Nakshatri P, Kumar S, Kishimoto H, Miller KD, Broxmeyer HE, Nakshatri H. NF-kappaB promotes breast cancer cell migration and metastasis by inducing the expression of the chemokine receptor CXCR4. J Biol Chem. 2003; 278:21631-21638.
28. Schioppa T, Uranchimeg B, Saccani A, Biswas SK, Doni A, Rapisarda A, Bernasconi S, Saccani S, Nebuloni M, Vago L, Mantovani A, Melillo G, Sica A. Regulation of the chemokine receptor CXCR4 by hypoxia. J Exp Med. 2003; 198:1391-1402.
29. Ceradini DJ, Kulkarni AR, Callaghan MJ, Tepper OM, Bastidas N, Kleinman ME, Capla JM, Galiano RD, Levine JP, Gurtner GC. Progenitor cell trafficking is regulated by hypoxic gradients through HIF-1 induction of SDF-1. Nat Med 2004; 10:858–864.
30. Lotan D, Sheinberg N, Kopolovic J, Dekel B. Expression of SDF-1/CXCR4 in injured human kidneys. Pediatr Nephrol.2008; 23:71-77.
31. Piovan E, Tosello V, Indraccolo S, Masiero M, Persano L, Esposito G, Zamarchi R, Ponzoni M, Chieco-Bianchi L, Dalla-Favera R, Amadori A. Differential regulation of hypoxia-induced CXCR4 triggering during B-cell development and lymphomagenesis. Cancer Res. 2007; 67:8605-8614.
32. Lagaaij EL, Cramer-Knijnenburg GF, van Kemenade FJ, van Es LA, Bruijn JA, van Krieken JH. Endothelial cell chimerism after renal transplantation and vascular rejection. Lancet 2001; 357:33–37.
33. Penn MS, Zhang M, Deglurkar I, Topol EJ. Role of stem cell homing in myocardial regeneration. Nat Med 2003; 9:1370–1376
34. Aicher A, Heeschen C, Mildner-Rihm C, Urbich C, Ihling C, Technau-Ihling K, Zeiher AM, Dimmeler S. Essential role of endothelial nitric oxide synthase for mobilization of stem and progenitor cells. Nat Med. 2003; 9:1370-1376.
35. van Es A, Hermans J, van Bockel JH, Persijn GG, van Hooff JP, de Graeff J. Effect of warm ischemia time and HLA (A and B) matching on renal cadaveric graft survival and rejection episodes. Transplantation. 1983; 36:255-258.
36. Sanfilippo F, Vaughn WK, Spees EK, Lucas BA. The detrimental effects of delayed graft function in cadaver donor renal transplantation. Transplantation 1984; 38:643-648.
37. Cole E, Naimark D, Aprile M, Wade J, Cattran D, Pei Y, Fenton S, Robinette M, Zaltsman J, Bear R. An analysis of predictors of long-term cadaveric renal allograft survival. Clin Transplant. 1995; 9:282-288.
38. Troppmann C, Gillingham KJ, Benedetti E, Almond PS, Gruessner RW, Najarian JS, Matas AJ. Delayed graft function, acute rejection, and outcome after cadaver renal transplantation. The multivariate analysis. Transplantation. 1995; 59:962-968.
39. Goes N, Urmson J, Ramassar V, Halloran PF. Ischemic acute tubular necrosis induces an extensive local cytokine response: evidence for induction of interferon-r, transforming growth factor, interleukin-2, and interleukin-10. Transplantation. 1995; 59:565-572.
40. Hardy KJ, McClure DN, Subwongcharoen S.Ischaemic preconditioning of the liver: a preliminary study. Aust N Z J Surg. 1996; 66:707-710.
41. Soncul H, Oz E, Kalaycioglu S. Role of ischemic preconditioning on ischemia-reperfusion injury of the lung. Chest. 1999; 115:1672-1677.
42. Przyklenk K, Bauer B, Ovize M, Kloner RA, Whittaker P. Regional ischemic 'preconditioning' protects remote virgin myocardium from subsequent sustained coronary occlusion. Circulation. 1993; 87:893-899.
43. Verdouw PD, Gho BC, Koning MM, Schoemaker RG, Duncker DJ. Cardioprotection by ischemic and nonischemic myocardial stress and ischemia in remote organs. Implications for the concept of ischemic preconditioning. Ann N Y Acad Sci. 1996; 30; 793:27-42.
44. Takaoka A, Nakae I, Mitsunami K, Yabe T, Morikawa S, Inubushi T, Kinoshita M. Renal ischemia/reperfusion remotely improves myocardial energy metabolism during myocardial ischemia via adenosine receptors in rabbits: effects of "remote preconditioning". J Am Coll Cardiol. 1999; 33:556-564.
45. Günaydin B, Cakici I, Soncul H, Kalaycioglu S, Cevik C, Sancak B, Kanzik I, Karadenizli Y. Does remote organ ischaemia trigger cardiac preconditioning during coronary artery surgery? Pharmacol Res. 2000; 41:493-496.
46. Schoemaker RG, van Heijningen CL. Bradykinin mediates cardiac preconditioning at a distance. Am J Physiol Heart Circ Physiol. 2000; 278:1571-1576.
47. Heusch G, Schulz R. Remote preconditioning. J Mol Cell Cardiol. 2002; 34:1279-1281.
48. Cumming DV, Heads RJ, Coffin RS, Yellon DM, Latchman DS. Pharmacological preconditioning of primary rat cardiac myocytes by FK506. Basic Res Cardiol. 1996 ; 91:367-373.
49. Maulik N, Engelman RM, Wei Z, Liu X, Rousou JA, Flack JE, Deaton DW, Das DK. Drug-induced heat-shock preconditioning improves postischemic ventricular recovery after cardiopulmonary bypass. Circulation. 1995; 92:381-388.
50. Sakr MF, Hassanein TI, Zetti GM, Van Thiel DH. FK 506 ameliorates the hepatic injury associated with ischemia. Life Sci. 1990; 47:687-691.
51. Sakr M, Zetti G, McClain C, Gavaler J, Nalesnik M, Todo S, Starzl T, Van Thiel D. The protective effect of FK506 pretreatment against renal ischemia/reperfusion injury in rats. Transplantation. 1992; 53:987-991.
52. Edelstein CL, Ling H, Schrier RW. The nature of renal cell injury. Kidney Int. 1997; 51:1341-1351.
53. Schmidt JA, Abdulla E. Down-regulation of IL-1 beta biosynthesis by inducers of the heat-shock response. J Immunol. 1988; 141:2027-2034.
54. Fuller TF, Serkova N, Niemann CU, Freise CE. Influence of donor pretreatment with N-acetylcysteine on ischemia/reperfusion injury in rat kidney grafts. J Urol. 2004; 171:1296-300.
55. Foglieni C, Fulgenzi A, Ticozzi P, Pellegatta F, Sciorati C, Belloni D, Ferrero E, Ferrero ME. Protective effect of EDTA preadministration on renal ischemia. BMC Nephrol. 2006; 7: 5.
56. Kelly KJ, Williams WW Jr, Colvin RB, Bonventre JV. Antibody to intercellular adhesion molecule 1 protects the kidney against ischemic injury. Proc Natl Acad Sci USA. 1994; 91:812-816.
57. Shi N, Wu MP: Apolipoprotein A-I: Apolipoprotein A-I attenuates renal ischemia/reperfusion injury in rats. J Biomed Sci. 2008; 15:577–583.
58. Liu JC, Chan P, Hsu FL, Chen YJ, Hsieh MH, Lo MY, Lin JY. The in vitro inhibitory effects of crude extracts of traditional Chinese herbs on 3-hydroxy- 3-methylglutaryl- coenzyme A reductase on Vero cells. Am J Chin Med. 2002; 30:629-636.
59.Zhu JS, Halpern GM, Jones K. The scientific rediscovery of an ancient Chinese herbal medicine: Cordyceps sinensis: part I. J.Alternat. Complement. Med.1998; 4: 289–303.
60. Liu YK, Shen W. Inhibitive effect of cordyceps sinensis on experimental hepatic fibrosis and its possible mechanism. World J. Gastroenterol.2003; 9: 529–533.
61.Zhu JS, Halpern GM, Jones K. The scientific rediscovery of an ancient Chinese herbal medicine: Cordyceps sinensis: part I. J.Alternat. Complement. Med. 1998; 4:289–303.
62.Koh JH, Yu KW, Suh HJ, Choi YM, Ahn TS. Activation of macrophages and the intestinal immune system by an orally administered decoction from cultured mycelia of Cordyceps sinensis. Biosci Biotechnol Biochem. 2002; 66: 407–411
63. Li SP, Zhao KJ, Ji ZN, Song ZH, Dong TT, Lo CK, Cheung JK, Zhu SQ, Tsim KW. A polysaccharide isolated from Cordyceps sinensis, a traditional Chinese medicine, protects PC12 cells against hydrogen peroxide-induced injury. Life Sci. 2003; 73:2503-2513.
64. Shahed AR, Kim SI, Shoskes DA. Down-regulation of apoptotic and inflammatory genes by Cordyceps sinensis extract in rat kidney following ischemia/reperfusion. Transplant. Proc. 2001; 33:2986–2987.
65. Zhou X, Gong Z, Su Y, Lin J, Tang K. Cordyceps fungi: natural products, pharmacological functions and developmental products. J Pharm Pharmacol. 2009; 61:279-291
66. Chen YP, Liu WZ, Shen LM, Xu SN. Comparisons of fermented Cordyceps mycelia and natural Cordyceps sinesis in treating 30 patients with renal failure. Chin. Tradit. Herbal Drugs 1986; 17:256–258.
67. Zurovsky Y, Haber C. Antioxidants attenuate endotoxin-gentamicin induced acute renal failure in rats. Scan. J. Urol. Nephrol.1995; 29:147–154.
68. Zheng F, Li LS, Chu XM. Effects of fermented cordyceps on acute renal damage induced by gentamycin in rats. China J. Chin. Material Med. 1994; 19:494–497.
69. Morigi M, Imberti B, Zoja C, Corna D, Tomasoni S, Abbate M, Rottoli D, Angioletti S, Benigni A, Perico N, Alison M, Remuzzi G. Mesenchymal stem cells are renotropic, helping to repair the kidney and improve function in acute renal failure. J Am Soc Nephrol. 2004; 15:1794-1804.
70. Lin F. Stem cells in kidney regeneration following acute renal injury. Pediatr Res. 2006; 59:74R-8R.
71. Tögel F, Isaac J, Hu Z, Weiss K, Westenfelder C. Renal SDF-1 signals mobilization and homing of CXCR4-positive cells to the kidney after ischemic injury. Kidney Int. 2005; 67:1772-1784.
72. Lotan D, Sheinberg N, Kopolovic J, Dekel B. Expression of SDF-1/CXCR4 in injured human kidneys. Pediatr Nephrol. 2008; 23:71-77.
73. Mazzinghi B, Ronconi E, Lazzeri E, Sagrinati C, Ballerini L, Angelotti ML, Parente E, Mancina R, Netti GS, Becherucci F, Gacci M, Carini M, Gesualdo L, Rotondi M, Maggi E, Lasagni L, Serio M, Romagnani S, Romagnani P. Essential but differential role for CXCR4 and CXCR7 in the therapeutic homing of human renal progenitor cells. J Exp Med.2008; 205:479-490.
74. Scholzen T, Gerdes J. The Ki-67 protein: from the known and the unknown. J Cell Physiol. 2000; 182:311-322.
75. Liu W, Schöb O, Pugmire JE, Jackson D, Zucker KA, Fry DE, Glew RH. Glycohydrolases as markers of hepatic ischemia-reperfusion injury and recovery. Hepatology. 1996; 24:157-162.
76. Matsunaga H, Handa JT, Aotaki-Keen A, Sherwood SW, West MD, Hjelmeland LM. Beta-galactosidase histochemistry and telomere loss in senescent retinal pigment epithelial cells. Invest Ophthalmol Vis Sci. 1999; 40:197-202.
77. Sagrinati C, Ronconi E, Lazzeri E, Lasagni L, Romagnani P. Stem-cell approaches for kidney repair: choosing the right cells. Trends Mol Med. 2008; 14:277-285.