臺灣博碩士論文加值系統

蟬花(Cordyceps cicadae)為蟲草屬之真菌，為傳統之中藥材，應用於免疫調節與腎功能之改善。研究指出蟬花菌絲體具有預防慢性腎衰竭進展之功能，然而蟬花菌絲體是否具有預防藥物引起之腎功能損傷與細胞之凋亡，目前仍不清楚。本研究以環孢靈(cyclosporine A, CsA)藥物誘導損傷之模式，探討蟬花菌絲體是否對於環孢靈所產生之腎毒性，具有保護腎臟之功能。研究以八週齡Sprague-Dawley大鼠透過管灌餵食菌絲體及環孢靈，並共分成六組(一)對照組;(二)環孢靈 (CsA；10 mg/kg)組;(三)低劑量蟬花 (LCC；40 mg/kg)組(四)高劑量蟬花(HCC；400 mg/kg)組; (五)低劑量蟬花+環孢靈(LCC+CsA)組；(六)高劑量蟬花+環孢靈(HCC+CsA)組。實驗為期七天，環孢靈與蟬花菌絲體皆溶於0.9%生理食鹽水中進行管灌餵食，實驗在第七天分別給予環孢靈及蟬花菌絲體後，置於代謝籠內24小時收集尿液，之後犧牲、收集血液及腎臟組織進行分析。結果發現給予環孢靈之組別尿素氮(urea nitrogen)與肌酸酐(creatinine)的廓清率明顯下降，預先給予蟬花菌絲體，依劑量之增加，有明顯舒緩環孢靈損傷之現象；其尿素氮及肌酸酐之廓清率，亦皆有顯著之改善。利用腎臟組織透過蘇木素-伊紅染色(hematoxylin and eosin stain、H&E stain)分析，明顯觀察到給予環孢靈，其腎臟組織中的腎小管會有裂解及腎絲球產生萎縮的現象，給予蟬花菌絲體預先處裡，可明顯改善此種現象。環孢靈會造成腎小管末端的遠端小管上皮細胞的鎂離子通道蛋白質TRPM6與TRPM7表現下調現象，導致鎂離子的重吸收產生障礙，在免疫組織化學染色法(immunohistochemistry; IHC)染色切片上可以清楚顯示環孢靈會造成TRPM6及TRPM7鎂離子通道蛋白表現的降低，而蟬花菌絲體之預先處裡，顯示具有上調此鎂離子通道蛋白的表現；此外，腎臟組織蛋白質電泳的部分也發現到，在給予蟬花菌絲體後，能夠降低環孢靈(CsA)所造成的的損傷，並且能夠降低內質網中氧化壓力指標Grp78蛋白質的表現，此結果推測蟬花具有保護腎臟之功能，部分原因係來自於減緩內質網壓力，以避免細胞的凋亡；又鎂離子通道之改善，亦可減緩腎臟細胞的凋亡，使腎臟在鎂離子的重吸收的功能達到保護之作用。透過液相層析串聯質譜分析，鑑定蟬花菌絲體中含高含量之N6-(2-hydroxyethyl)-adenosine (HEA)活性成分，經由腎臟細胞株HK-2與HEA培養方式，顯示在HEA濃度達45 M時仍有90%以上存活率，且在低濃度下(0.1-0.9 M) 即具有預防環孢靈誘導細胞損傷之效果，詳細之抑制環孢靈誘導之腎毒性機制正進一步探討中。

The traditional Chinese medicinal mushroom, Cordyceps cicadae (CC), is one of the species described in Cordyceps. It has been applied in pharmacological effects for the protections of immune modulatory and renal functions. However, it is still unclear whether CC mycelium has protective effects against drug-induced renal function damage and cell apoptosis. This study aimed to investigate the protective effects of CC mycelia against the cyclosporine A (CsA)-induced renal toxicity in vivo. Eight-week-old Sprague-Dawley (SD) rats were divided into six groups, including control group (0.9 % normal saline), CsA group (10 mg/kg BW), LCC group (low dose of CC at 40mg/kg for 7 days), HCC (high dose of CC at 400mg/kg for 7 days), LCC + CsA (low dose of CC at 40mg/kg for 7 days before CsA administration), HCC + CsA (high dose of CC at 400 mg/kg for 7 days before CsA administration). Both of CC mycelia and CsA were dissolved in 0.9% normal saline and were received with oral administrations. Before sacrifice, the animals were allocated into metabolic cages for 24 h for urine collection. After blood and kidney tissues sampling, biochemical analysis and tissues staining analysis were investigated. Results reveal that CC mycelia treatment significantly inhibited CsA-induced the increased levels of urea nitrogen (BUN) and creatinine (CRE) in a dose responsive manner. The hematoxylin and eosin staining of kidney tissues indicated that epithelial vacuolization and tubular dilatation in kidney tissues were observed in the CsA group, while the treatment of CC mycelia resulted in a remarkable amelioration. In addition, the expression of glucose regulated protein 78 (GRP78), one of the endoplasmic reticulum stress biomarkers, was downregulated significantly after the treatment of CC mycelia by Western blotting analysis. CC mycelia also ameliorated the expression of the magnesium ion channel proteins transient receptor potential melastin 6 (TRPM6) and transient receptor potential melastin 7 (TRPM7). It was speculated that the protective effects of CC mycelia on CsA-induced kidney injury were from the improvements of magnesium transporter, TRPM6 and TRPM7, to reduce the endoplasmic reticulum stress induced cell apoptosis. By using the HPLC tandem mass spectrometer analysis, a high content of N6-(2-hydroxyethyl)-adenosine (HEA) with a low toxicity on kidney cell lines HK-2 (0.1-45 M, 90% cell survival) was found from CC mycelia. Under the low concentrations of 0.1-0.9 M of HEA, the preventive effects of HEA on CsA-induced cell damages indicated that CC mycelia might be a promising complementary natural product for protecting kidney tissues from the CsA-induced nephrotoxicity. Details regarding the mode of action of HEA for inhibition of CsA-induced nephrotoxicity are under investigation.

目錄
中文摘要.............................................................................................................I
英文摘要.............................................................................................................II
圖目錄.................................................................................................................V
表目錄...............................................................................................................VII
第一章、緒論……………………………………………………………………1
第二章、文獻回顧..............................................................................................3
第一節、環孢靈(Cyclosporine A).......................................................3
第二節、蟬花(Cordyceps cicadae) ..................................................5
第三節、蟬花的目前研究及現況....................................................6
第四節、腎臟組織介紹....................................................................11
第五節、血液及尿液生化值............................................................15
第六節、環孢靈腎毒性對於鎂離子通道蛋白TRPM 6及TRPM 7
之影響..............................................................................19
第七節、內質網壓力指標蛋Glucose-regulated protein 78(GRP
78)………………….............................................................20
第八節、未摺疊蛋白質反應……………………………………....21
第三章、研究目的…………………………………………………………….22
第四章、材料與方法………………………………………………………….23
第一節、實驗材料與試劑………………….…………………….24
第二章、蟬花萃取物一般成分分析……….……………….........26
第三節、高效液相層析串聯質譜分析蟬花萃取物中核苷類之化合物…………………….……………………………...27
第四節、細胞實驗設計………………………….……………….28
第五節、動物實驗設計…………………………….…………….31
第六節、腎臟組織及生化值樣品之備製…………………….….32
第七節、組織切片………………………………………………..33
第八節、西方墨點法分…………………………………..………34
第九節、統計分法……………………………………………….36
第五章、實驗結果……….................................................................................38
第一節、蟬花萃取物一般成分分析……………………………..37
第二節、大鼠腎功能生化值分析………………………………..37
第三節、大鼠腎臟組織HE染色切片……………………………49
第四節、大鼠腎臟組織IHC染色切片………………………..... 52
第五節、蟬花菌絲體萃取物高效液相層析與串聯質譜分析…..56
第六節、大鼠腎臟組織Glucose-regulated protein 78(GRP 78)蛋白質表現…………………………………………………..57
第七節、Cyclosporine A與N6-(2-Hydroxyethyl)-adenosine(HEA)
對腎臟細胞(HK-2)之影響.............................................58
第六章、討論…………………………………………………………………..61第七章、結論…………………………………………………………………..63
第八章、參考文獻……………………………………………………………..65


圖目錄

圖一、Cyclosporine A化學結構圖…………………………………………..3
圖二、蟬花(Cordyceps cicadae) ………………………………………….….6
圖三、蟬花萃取物新的化合物………………………………………...………7
圖四、蟲草素化學結構式……………………………………………………...9
圖五、腎臟位置圖…………………………………………………………...12
圖六、未摺疊蛋白質反應圖……………………………………………….…21
圖七、細胞添加環孢靈實驗設計圖……………...…………………………..28
圖八、細胞添加活性成分HEA實驗設計圖………..…………………….…29
圖九、動物實驗流程圖……………………………………………………….31
圖十、蟬花菌絲體與環孢靈處理之大鼠中鈣離子排泄分率(FE-Ca)...........39
圖十一、蟬花菌絲體與環孢靈處理之大鼠中鎂離子排泄分率(FE-Mg)…..40
圖十二、蟬花菌絲體與環孢靈處理之大鼠中鈉離子排泄分率(FE-Na).......42
圖十三、蟬花菌絲體與環孢靈處理之大鼠中鉀離子排泄分率(FE-K)….....44
圖十四、蟬花菌絲體與環孢靈處理之大鼠中磷離子排泄分率(FE-P)…......45
圖十五、大鼠腎臟尿素氮及肌酸酐廓清率………………………………….48
圖十六、蟬花處理之大鼠HE切片染色腎臟型態圖(放大倍率×200) …....50
圖十七、蟬花處理之大鼠HE切片染色腎臟型態圖(放大倍率×400) …....51
圖十八、大鼠經過大蟬花及環孢靈處理之免疫病理切片TRPM6蛋白質含量…....................................................................................................53
圖十九、大鼠經過大蟬花及環孢靈處理之免疫病理切片TRPM7蛋白質含量…....................................................................................................54

圖二十、蟬花菌絲體與環孢靈處理大鼠中的免疫切片TRPM 6與TRPM7蛋白質量化圖....................................................................................55
圖二十一、蟬花菌絲體高效液相層析串聯質譜分析儀之分析圖.................56
圖二十二、不同處理之大鼠中GRP 78蛋白質表現量..................................57
圖二十三、環孢靈對於HK-2細胞生長之影響.............................................58
圖二十四、蟬花活性成分HEA對於 HK-2細胞生長之影響………………59
圖二十五、HEA保護環孢靈對於HK-2細胞生長之影響…........................60
表目錄

表一、臺灣地區十大死亡原因………………………………………………..2
表二、蟬花菌絲體一般成份分析……............................................................38
表三、大鼠在不同組別中鈣離子排泄分率…………………………………..39
表四、大鼠在不同組別中鎂離子排泄分率………………………………….41
表五、大鼠在不同組別中鈉離子排泄分率………………………………….43
表六、大鼠在不同組別中鉀離子排泄分率………………………………….44
表七、大鼠在不同組別中磷離子排泄分率………………………………….46
表八、大鼠在不同組別中尿素氮及肌酸酐廓清率……………………….....48

台灣地區十大死亡原因 (2013)。衛生福利部統計資料。
陳玉明 (2012) 腎臟病人不可不知高血磷的飲食策略。國泰醫訊
張基隆，胡祐甄，黃姿菁，鄭筱翎，謝保萱(2013)。生物化學。華杏出版有限
公司。
吳永義 (2007)。慢性腎臟病病人的磷結合劑使用。台北榮民總醫院腎臟科。 腎
臟與透析第20卷第3期
洪敏元，楊育麟，劉良慈，林育娟，何明聰，賴明華 (2008)。當代生理學。
華杏出版社有限公司。第四版第五刷。
鄭士豪 (2010)。蟬花發酵產物對 Cisplatin 誘導 SD 大鼠腎損傷的保護作用。
弘光科技大學生物科技研究所碩士論文。
陳俊章(2012)。Cylosporine 誘導腎損傷引起鎂離子流失與蟬花多醣體之保護
作用。弘光科技大學生物科技研究所碩士論文。
陳勁初，鄭劍廷 (2010)。蟬花保護腎臟之研究。菇類產業發展研究研討會專
刊。
徐瑞霞，陳勁初，陳炎鍊，葉淑幸 (2015)。蟬花之保護功效。食品工業研究
所專題報導。第47卷第05期。
賴明德，曹瓊方，李英中，王錠釧，吳俐慧，沈賈堯，林秋霞，戴瑄，阮勝
威，張宏洲，王明仁，張林松，林淑玟 (2012)。最新生理學。6月初版
郭錦輯，周鈺翔，李柏葒，陳昶旭，王介立，蔡壁如，吳允升，林水龍 陳永
銘，吳寬墩，蔡敦仁，柯文哲，吳明修 (2009)。急性腎損傷與重症透析之
最新進展。內科學誌 (國立台灣大學附設醫院內科部) ；20：320-334。



Cheung, Y. I., Liu. X. X., Wang, W. Q., Wu, J. Y. 2014. Ultrasonic disruption of fungal mycelia for efficient recovery of polysaccharide–protein complexes from viscous fermentation broth of a medicinal fungus. Ultrasonics Sonochemistry 22, 243-248.
FellstrÖm, B.2004. Cyclosporine Nephrotoxicity. Transplantation Proceedings, 36 (Suppl 2S), 220S-223S.
Finnerud, C. W. 2002. Calcineurin Inhibitors. Dermatologic Therapy 15, 325-332.
Luo S, Mao C, Lee B, Lee AS. 2006. GRP78/BiP is required for cell proliferation and protecting the inner cell mass from apoptosis during early mouse embryonic development. Molecular and Cellular Biology, 26, 5688-5697.
Matsuda, S., Koyasu, S. 2000. Mechanisms of action of cyclosporine. Immunopharmacology 47, 119–125.
Meng, Z., Kang, J., Wen, T., Lei, B., Hyde, K. D. 2015. Cordycepin and N6-(2-hydroxyethyl)-adenosine from Cordyceps pruinosa and their interaction with human serum albumin. PLoS One, 10(3): e0121669.
Nakamura, K., Konoha, K., Yoshikawa, N., Yamaguchi, Y., Kagota, S., Shinozuka, K., Kunitomo, M. 2005. Effect of Cordycepin (3’-Deoxyadenosine) on hematogenic lung metastatic model mice. In Vivo 19, 137–141.
Overgaard-Hansen, H. 1964. The inhibition of 5-phosphoribosyl-1-
pyrophosphate formation by cordycepin triphosphate in extracts of Ehrlich ascites tumor cells. Biochim Biophys Acta 80:504–507.
Pfaffenbach, K. T., Lee A. S. 2010. The critical role GRP 78 in physiologic and pathologic stress. Current Opinion Cell Biology 23, 150-156.
Tuli, H. .S, Sandhu, S. S., Sharma, A. K. 2014. Pharmacological and therapeutic potential of Cordyceps with special reference to cordycepin. 3 Biotech 4(1): 1–12.
Ukai, .S, Kiho, T., Hara, C., Morita, M., Goto, A., Imaizumi, N., Hasegawa, Y. 1983. Polysaccharides in fungi. XIII. Antitumor activity of various polysaccharides isolated from Dictyophora indusiata, Ganoderma japonicum, Cordyceps cicadae, Auricularia auricula-judae, and Auricularia species. Chem Pharm Bull (Tokyo) 31, 741-744.
Wang, Q., Liu, Z.Y. 2004. Advances in studies on medicinal fungi Cordyceps cicadae. Chinese Traditional and Herbal Drugs 34, 469–471.
Wang, H., Zhang, J., Sit, W. H., Jetty, C. H., Wan, M. F. 2014. Cordyceps cicadae induces G2/M cell arrest in MHCC97H human hepatocellular carcinoma cell: a proteomic study. Chinese Medicine 9:15.
Weng, S. C.,, Chou, C. J., Lin, L. C., Tsai, W. J., Kuo, Y. C. 2002. Immunomodulatory functions of extracts from the Chinese medicinal fungus Cordyceps cicadae. Journal of Ethnopharmacology, 83, 79-85.
Yakugaku, Z. 1990. Polysaccharides in fungi. XXV. Biological activities of two galactomannans from the insect-body portion of Chán hua (fungus: Cordyceps cicadae). 110, 286-288.
Zeng, W. B., Yu, H., Ge, F., Yang, J. Y., Chen, H. Z., Wang, Y. B., Dai, Y. D. 2014. A Distribution of nucleosides in populations Cordyceps cicadae. Molecules 19, 6123-6141.
Zhang, S. W., Xuan, L. J. 2007. Five aromatics bearing a 4-O-Methylglucose Unit from Cordyceps cicadae. Helvetica Chimi Acta 90, 404-410.
Zhang, S. W., Xuan, L. J. 2008. Cyclopentnone and Furan Derivative from the Mycelia of Cordyceps cicadae. The Journal of Antibiotics 61(1):43-45.
Zhu, R., Chen, Y. P., Deng, Y. Y., Zheng, R., Zhong, Y. F., Wang, L., Du, L. P. 2011. Cordyceps cicadae extracts ameliorate renal malfunction in a remnant kidney model. Biomed & Biotechonl 12(12): 1024-1033.