2008年2月28日 星期四
2008年2月23日 星期六
樟芝效用的研究 ：膀胱出口阻塞之缺血傷害病因論
膀胱出口阻塞之缺血傷害病因論 ：樟芝的效用
Robert M. Levin PhD and 林鼎淯 醫師
序言：
膀胱是個平滑肌器官，它的功能是在平時儲存尿液，和經過高度協調的持續收縮來排出尿液；膀胱的功能表現取決於數個因子，包括神經支配、組織、收縮反應、鈣離子的儲存及釋放和新陳代謝的能量。
臨床上，五十歲以上的男人，百分之八十有良性前列腺肥大引起的膀胱排放出口阻塞，最近的證據說明，局部缺血/再灌注是良性前列腺肥大引起的膀胱排放出口阻塞的主要致病因子。我們這個計畫特定目的是探討給予口服樟芝，能保護膀胱經誘導產生局部缺血/再灌注之兩種嚴重傷害兔子的膀胱。
方法：將24隻公紐西蘭大白兔分成4組每組6隻，組別1、2每天餵食含100mg樟芝的懸浮液，組別3、4為標準組，在三星期中所有的兔子均依其組別的每日標準飲食，三星期後，第1及3組的兔子麻醉後手術使其膀胱都受到平均對稱的缺血及一個星期的再灌注，第2及4組的兔子則是對照手術之後給予一星期恢復。在恢復的時間持續按照先前分組之個別標準飲食，在結束後立即進行麻醉和膀胱內壓測量法。膀胱內壓測量法後緊接著立即割去膀胱並秤重。
個別肌肉束藥理學﹕將三個分離取得的膀胱分別置於分離槽，並以氨甲醯醇及氯化鉀來刺激其生理收縮反應。組織學﹕將兩個全皮層束的膀胱體置於10%福馬林溶液中8小時後,作為免疫組織化學研究。另外，我們將神經絲蛋白染色，做為對神經支配效應的標記；以細胞凋零作為細胞損壞的標記。
生物化學方面：將每個膀胱均衡的將膀胱肌肉和膀胱黏膜間隔分離。將其冷凍儲存於-80oC作為生物化學研究。我們測量檸檬酸合成酶活性做為是線粒體功能標記，以肌漿內質網Ca2+ -ATP酶活性作為是肌漿內質網功能標記，和膽鹼乙醯酶活性像是膽鹼能的神經功能標記。
結果：
(1)在控制組，樟芝能增強其膀胱的生理收縮功能，是值得注目的有趣現象。此外，在遭到膀胱缺血/再灌注傷害組，樟芝亦能保護其生理收縮功能。
(2) 在膀胱儲容性上，不論在控制組或缺血/再灌注傷害組，樟芝都具有保護作用。
(3)在膀胱平滑肌，樟芝能在膀胱缺血/再灌注傷害之後，提升肌漿內質網Ca2+ -ATP酶活性（代表粒腺體功能）。
(4) 膀胱缺血/再灌注傷害有顯著減少膀胱平肌的神經密度的破壞現象,而樟芝能在膀胱缺血/再灌注傷害來臨之後，顯著增加神經密度，使之回復到接近正常。
(5) 膀胱缺血/再灌注傷害會顯著引起細胞凋零，而樟芝能顯著減少細胞凋零。
結論：餵食樟芝的兔子有當程度的提升正常膀胱功能的功能表現﹔且在缺血/再灌注的傷害產生時，樟芝能有保護效用。
Ischemic Etiology of Obstructive Bladder Dysfunction:
Effect of Antrodia Camphorata
Robert M. Levin PhD and Alpha DY Lin, MD
Introduction: The urinary bladder is a smooth muscle (SM) organ whose function is to collect and store urine at low intravesical pressure and then to periodically expel the urine via a highly coordinated, sustained contraction. Bladder function depends upon several factors including innervation, structure, contractile response, calcium storage and release, and metabolism of energy. More than 80% of males over 50 years of age have varying degrees of bladder outlet obstruction secondary to benign prostatic hyperplasia (BPH). Recent evidence has demonstrated that ischemia / reperfusion are major etiological factors in bladder dysfunctions secondary to obstructive bladder disease. The specific aim of our project was to determine if orally administered Antrodia camphorata could protect rabbit bladders from the progressive dysfunctions induced by experimental bilateral ischemia / reperfusion.
Methods: 24 male NZW rabbits were separated into 4 groups of 6 animals each. Rabbits in groups 1 - 2 were fed 100 mg Antrodia camphorata suspensions daily; those in groups 3-4 received vehicle. All rabbits received their respective treatments daily for 3 weeks. After 3 weeks, all rabbits were anesthetized and each rabbit in groups 1 and 3 were subjected to bilateral ischemia and one week reperfusion. The rabbits in groups 2 and 4 were sham operated and allowed to recover for 1 week. Feeding was continued during the recovery period. Upon completion each rabbit was anesthetized and cystometry performed. Immediately after cystometry the bladder was excised rapidly and weighed. Isolated muscle strip pharmacology: Three isolated strips from each bladder were mounted in separate baths and the contractile responses to field stimulation (FS), carbachol, and KCl were determined. Histology: Two full thickness strips of bladder body were fixed for 8 hours in 10% buffered formalin and used for immunohistochemical studies. We stained for neurofilament as a marker for innervation and for apoptosis as a marker for cellular damage.
Biochemistry: The balance of each bladder was separated into its muscle and mucosal compartments, frozen and stored at -80oC for biochemical studies. We measured citrate synthase (CS) activity as a marker for mitochondrial function, Sarcoplasmic reticulum Ca2+ ATPase Activiey (SERCA) as a marker for Sarcoplasmic reticulum function and choline acetyltransferase (ChAT) activity as a marker for cholinergic nerve function.
Results: 1) Antrodia resulted in significantly increased contractile responses to all forms of stimulation in both control rabbits and those subjected to I/R. 2) Antrodia resulted in improved bladder compliance in both control and after I/R. 3) In the smooth muscle compartment Antrodia stimulated a marked increase in SERCA activity following I/R. 4) I/R resulted in a significant decrease in the nerve density of the bladder smooth muscle; whereas Antrodia significantly increased the nerve density back toward normal. 5) I/R significantly increased the level of apoptosis; whereas Antrodia reduced apoptosis back to control levels. Conclusion: Feeding rabbits Antrodia camphorata significantly improved normal bladder function; and protected the bladder against significant dysfunctions induced by ischemia / reperfusion.
Robert M. Levin PhD and 林鼎淯 醫師
序言：
膀胱是個平滑肌器官，它的功能是在平時儲存尿液，和經過高度協調的持續收縮來排出尿液；膀胱的功能表現取決於數個因子，包括神經支配、組織、收縮反應、鈣離子的儲存及釋放和新陳代謝的能量。
臨床上，五十歲以上的男人，百分之八十有良性前列腺肥大引起的膀胱排放出口阻塞，最近的證據說明，局部缺血/再灌注是良性前列腺肥大引起的膀胱排放出口阻塞的主要致病因子。我們這個計畫特定目的是探討給予口服樟芝，能保護膀胱經誘導產生局部缺血/再灌注之兩種嚴重傷害兔子的膀胱。
方法：將24隻公紐西蘭大白兔分成4組每組6隻，組別1、2每天餵食含100mg樟芝的懸浮液，組別3、4為標準組，在三星期中所有的兔子均依其組別的每日標準飲食，三星期後，第1及3組的兔子麻醉後手術使其膀胱都受到平均對稱的缺血及一個星期的再灌注，第2及4組的兔子則是對照手術之後給予一星期恢復。在恢復的時間持續按照先前分組之個別標準飲食，在結束後立即進行麻醉和膀胱內壓測量法。膀胱內壓測量法後緊接著立即割去膀胱並秤重。
個別肌肉束藥理學﹕將三個分離取得的膀胱分別置於分離槽，並以氨甲醯醇及氯化鉀來刺激其生理收縮反應。組織學﹕將兩個全皮層束的膀胱體置於10%福馬林溶液中8小時後,作為免疫組織化學研究。另外，我們將神經絲蛋白染色，做為對神經支配效應的標記；以細胞凋零作為細胞損壞的標記。
生物化學方面：將每個膀胱均衡的將膀胱肌肉和膀胱黏膜間隔分離。將其冷凍儲存於-80oC作為生物化學研究。我們測量檸檬酸合成酶活性做為是線粒體功能標記，以肌漿內質網Ca2+ -ATP酶活性作為是肌漿內質網功能標記，和膽鹼乙醯酶活性像是膽鹼能的神經功能標記。
結果：
(1)在控制組，樟芝能增強其膀胱的生理收縮功能，是值得注目的有趣現象。此外，在遭到膀胱缺血/再灌注傷害組，樟芝亦能保護其生理收縮功能。
(2) 在膀胱儲容性上，不論在控制組或缺血/再灌注傷害組，樟芝都具有保護作用。
(3)在膀胱平滑肌，樟芝能在膀胱缺血/再灌注傷害之後，提升肌漿內質網Ca2+ -ATP酶活性（代表粒腺體功能）。
(4) 膀胱缺血/再灌注傷害有顯著減少膀胱平肌的神經密度的破壞現象,而樟芝能在膀胱缺血/再灌注傷害來臨之後，顯著增加神經密度，使之回復到接近正常。
(5) 膀胱缺血/再灌注傷害會顯著引起細胞凋零，而樟芝能顯著減少細胞凋零。
結論：餵食樟芝的兔子有當程度的提升正常膀胱功能的功能表現﹔且在缺血/再灌注的傷害產生時，樟芝能有保護效用。
Ischemic Etiology of Obstructive Bladder Dysfunction:
Effect of Antrodia Camphorata
Robert M. Levin PhD and Alpha DY Lin, MD
Introduction: The urinary bladder is a smooth muscle (SM) organ whose function is to collect and store urine at low intravesical pressure and then to periodically expel the urine via a highly coordinated, sustained contraction. Bladder function depends upon several factors including innervation, structure, contractile response, calcium storage and release, and metabolism of energy. More than 80% of males over 50 years of age have varying degrees of bladder outlet obstruction secondary to benign prostatic hyperplasia (BPH). Recent evidence has demonstrated that ischemia / reperfusion are major etiological factors in bladder dysfunctions secondary to obstructive bladder disease. The specific aim of our project was to determine if orally administered Antrodia camphorata could protect rabbit bladders from the progressive dysfunctions induced by experimental bilateral ischemia / reperfusion.
Methods: 24 male NZW rabbits were separated into 4 groups of 6 animals each. Rabbits in groups 1 - 2 were fed 100 mg Antrodia camphorata suspensions daily; those in groups 3-4 received vehicle. All rabbits received their respective treatments daily for 3 weeks. After 3 weeks, all rabbits were anesthetized and each rabbit in groups 1 and 3 were subjected to bilateral ischemia and one week reperfusion. The rabbits in groups 2 and 4 were sham operated and allowed to recover for 1 week. Feeding was continued during the recovery period. Upon completion each rabbit was anesthetized and cystometry performed. Immediately after cystometry the bladder was excised rapidly and weighed. Isolated muscle strip pharmacology: Three isolated strips from each bladder were mounted in separate baths and the contractile responses to field stimulation (FS), carbachol, and KCl were determined. Histology: Two full thickness strips of bladder body were fixed for 8 hours in 10% buffered formalin and used for immunohistochemical studies. We stained for neurofilament as a marker for innervation and for apoptosis as a marker for cellular damage.
Biochemistry: The balance of each bladder was separated into its muscle and mucosal compartments, frozen and stored at -80oC for biochemical studies. We measured citrate synthase (CS) activity as a marker for mitochondrial function, Sarcoplasmic reticulum Ca2+ ATPase Activiey (SERCA) as a marker for Sarcoplasmic reticulum function and choline acetyltransferase (ChAT) activity as a marker for cholinergic nerve function.
Results: 1) Antrodia resulted in significantly increased contractile responses to all forms of stimulation in both control rabbits and those subjected to I/R. 2) Antrodia resulted in improved bladder compliance in both control and after I/R. 3) In the smooth muscle compartment Antrodia stimulated a marked increase in SERCA activity following I/R. 4) I/R resulted in a significant decrease in the nerve density of the bladder smooth muscle; whereas Antrodia significantly increased the nerve density back toward normal. 5) I/R significantly increased the level of apoptosis; whereas Antrodia reduced apoptosis back to control levels. Conclusion: Feeding rabbits Antrodia camphorata significantly improved normal bladder function; and protected the bladder against significant dysfunctions induced by ischemia / reperfusion.
2008年2月13日 星期三
新發現-----台灣特有紅樟芝的研究新進展
Levin 教授的背景介紹
Levin 教授目前擔任美國紐約有一百二十五年歷史的Albany College of Pharmacy 的終生教授職，除擔任該校的研究主管外，其所主持的實驗室活躍於國際醫學界，每年有二、三十篇的高質量研究論文發表，。在台灣，有許多的泌尿科醫學專家都曾經在他的實驗室受教於他，例如台北榮總林登龍教授、台大余宏政教授、成大唐一清教授、……等。
Levin 教授的動物模型之重要性與珍貴性
在實驗室的藥品研究上，一般都是採用實驗動物模式觀察施用前後的效果，但是藥品施用後的生理效果，一般實驗室的動物模型很難能夠直接表現出來，例如心臟功能以心博量作指標；肺功能以肺活量作指標；腦功能以思慮是否敏捷、記憶力作指標……等，但這些都不易在動物實驗上表現出效果。
難得的是， Levin教授在 1985年於賓州大學所設計發展出來的『大白兔膀胱收縮模型』就可以達到這個要求：藉由膀胱收縮能力的改變，作為生理機能的指標，是藥物功效直接的證據顯現，不僅於此，該模型對於細胞內部的微環境變化、與組織細胞活性相關的蛋白質、與實際的組織形態變化等，都可以提供一系列佐證。 1985年以來， Levin教授已經以此成熟的模型出版了超過四百篇以上的論文，作了許多藥物的功效研究，此模型也成為醫藥發展的重要依循，在醫學發展上，貢獻卓著。
當吳沙遇見 Levin教授
西元 1788年，吳沙率領漢人開墾蘭陽平原，他觀察到當地的原住民以樟芝做為藥物與增強酒力的保健品，於是吳沙也開始試用於一些疾病的治療上，發現了『樟芝』作藥的許多療效。自此『樟芝』成了台灣民間特有的民俗用藥。但天然『樟芝』因量少而價高，……兩百年多年後，應用現代科技，「台灣偉翔生技公司」以二十年的苦功研究，發展出人工栽培的方法，其中的有效成份樟芝子實體三帖類（ Tripternoids）極高。自此，『樟芝』入藥的推廣進入新里碑。
台灣特有的紅樟芝子實體之前在台灣各大醫學研究機構進行了許多的研究，證明了有抗『自由基傷害』、『抗癌』、『抗發炎』等能力，也有許多腫瘤病患當成『補充療法』來減緩癌症治療副作用使用。唯一具備人工培養並量產紅樟芝子實體的「台灣偉翔」將其產品交到 Levin教授的實驗室作驗證……
2007 年10月 17日 午夜，Levin教授的一通越洋電話讓人驚喜：「 Surprise Findings !」台灣特有的紅樟芝不僅成功地表現出強大的抗『自由基傷害』能力；實驗顯示，在器官組織遭受到『自由基傷害』時，能夠保護組織，降低『自由基傷害』所帶來的細胞損害，維護器官功能。更令 Levin教授驚奇的是……
強化作用──驚奇的意外發現！
食用紅樟芝的群組，竟然會有較一般膀胱更強的生理功能！紅樟芝不僅是在自由基傷害時，保護細胞，減少傷害；更令人意外的顯現出，食用紅樟芝的群組，竟然會有較一般膀胱更強的生理功能的膀胱，這顯示出，紅樟芝可以讓身體組織的功能在沒有任何副作用的情況下，往上提升至更健康、更強生理功能的狀態。這也表示服用紅樟芝子實體可以調節身體的組織到更佳狀態，表現出更好的生理機能，當然，在身體遭受疾病傷害時，可以有更強的抵抗力。這個發現，讓
Levin教授聯想到傳統醫學的『食補強身』──這在現在西方醫學上的藥物功能上，未曾出現！
紅樟芝可能的運用
一、抗『自由基傷害』：『自由基』是身體細胞代謝過程的自然產物，身體會有自然的『抗自由基』機制去平衡，但若是身體處於疾病或不平衡狀態，則自然的『抗自由基』機制會失衡，而使組織遭受更多傷害，就如同地球的臭氧層有了破洞。反之，若是『抗自由基』機制失衡，則身體容易處於疾病或不平衡狀態，
醫學研究顯示，幾乎所有的疾病都與『自由基傷害』有關；『自由基傷害』是諸多疾病或身體不平衡狀態如老化、疲勞、不當飲食、疾病、癌症治療（例如手術、化療、電療、標靶治療等）的重要病理機轉，若是能減少『自由基傷害』，亦較能延緩老化、消除疲勞、預防疾病發生、減緩疾病症狀，及減緩癌症治療副作用所帶來的痛苦等。
現代人的文明病－『三高：高血壓、高血脂、高血糖』，及心血管疾病、慢性阻塞性肺病、阿茲海默症（俗稱老年癡呆症）、腎臟病、皮膚老化、……等，其病理機轉皆與體內的『自由基傷害』有密切關係，紅樟芝或可作為預防或改善疾病症狀的輔助保健品。
二、日常保健－『食補強身』：預防醫學的重要觀念，即是『預防勝於治療』；平時即作好身體調養，增強對於疾病的抵抗力，延緩身體退化與老化，根據 Levin教授對於紅樟芝的研究發現，可以樂觀地預測，紅樟芝應是日常保健與『食補強身』的優先選擇。
紅樟芝──癌症治療副作用的輔助選擇
癌症病患在經歷化學藥物治療、放射線治療、或標靶治療時，身體會遭受遠超出平時的『自由基傷害』，這也是癌症治療副作用例如恶心、嘔吐、疲倦感、掉髮、口腔炎、皮膚炎、……等的病理機轉之重要部份，根據Levin 教授的研究推論，紅樟芝可以是癌症治療後的輔助選擇，應有助於癌症病患在治療後的舒適度及生活品質，服用紅樟芝也應有助於治療後迅速恢復生理機能，讓癌症病患儘速展開新生活。
Levin 教授目前擔任美國紐約有一百二十五年歷史的Albany College of Pharmacy 的終生教授職，除擔任該校的研究主管外，其所主持的實驗室活躍於國際醫學界，每年有二、三十篇的高質量研究論文發表，。在台灣，有許多的泌尿科醫學專家都曾經在他的實驗室受教於他，例如台北榮總林登龍教授、台大余宏政教授、成大唐一清教授、……等。
Levin 教授的動物模型之重要性與珍貴性
在實驗室的藥品研究上，一般都是採用實驗動物模式觀察施用前後的效果，但是藥品施用後的生理效果，一般實驗室的動物模型很難能夠直接表現出來，例如心臟功能以心博量作指標；肺功能以肺活量作指標；腦功能以思慮是否敏捷、記憶力作指標……等，但這些都不易在動物實驗上表現出效果。
難得的是， Levin教授在 1985年於賓州大學所設計發展出來的『大白兔膀胱收縮模型』就可以達到這個要求：藉由膀胱收縮能力的改變，作為生理機能的指標，是藥物功效直接的證據顯現，不僅於此，該模型對於細胞內部的微環境變化、與組織細胞活性相關的蛋白質、與實際的組織形態變化等，都可以提供一系列佐證。 1985年以來， Levin教授已經以此成熟的模型出版了超過四百篇以上的論文，作了許多藥物的功效研究，此模型也成為醫藥發展的重要依循，在醫學發展上，貢獻卓著。
當吳沙遇見 Levin教授
西元 1788年，吳沙率領漢人開墾蘭陽平原，他觀察到當地的原住民以樟芝做為藥物與增強酒力的保健品，於是吳沙也開始試用於一些疾病的治療上，發現了『樟芝』作藥的許多療效。自此『樟芝』成了台灣民間特有的民俗用藥。但天然『樟芝』因量少而價高，……兩百年多年後，應用現代科技，「台灣偉翔生技公司」以二十年的苦功研究，發展出人工栽培的方法，其中的有效成份樟芝子實體三帖類（ Tripternoids）極高。自此，『樟芝』入藥的推廣進入新里碑。
台灣特有的紅樟芝子實體之前在台灣各大醫學研究機構進行了許多的研究，證明了有抗『自由基傷害』、『抗癌』、『抗發炎』等能力，也有許多腫瘤病患當成『補充療法』來減緩癌症治療副作用使用。唯一具備人工培養並量產紅樟芝子實體的「台灣偉翔」將其產品交到 Levin教授的實驗室作驗證……
2007 年10月 17日 午夜，Levin教授的一通越洋電話讓人驚喜：「 Surprise Findings !」台灣特有的紅樟芝不僅成功地表現出強大的抗『自由基傷害』能力；實驗顯示，在器官組織遭受到『自由基傷害』時，能夠保護組織，降低『自由基傷害』所帶來的細胞損害，維護器官功能。更令 Levin教授驚奇的是……
強化作用──驚奇的意外發現！
食用紅樟芝的群組，竟然會有較一般膀胱更強的生理功能！紅樟芝不僅是在自由基傷害時，保護細胞，減少傷害；更令人意外的顯現出，食用紅樟芝的群組，竟然會有較一般膀胱更強的生理功能的膀胱，這顯示出，紅樟芝可以讓身體組織的功能在沒有任何副作用的情況下，往上提升至更健康、更強生理功能的狀態。這也表示服用紅樟芝子實體可以調節身體的組織到更佳狀態，表現出更好的生理機能，當然，在身體遭受疾病傷害時，可以有更強的抵抗力。這個發現，讓
Levin教授聯想到傳統醫學的『食補強身』──這在現在西方醫學上的藥物功能上，未曾出現！
紅樟芝可能的運用
一、抗『自由基傷害』：『自由基』是身體細胞代謝過程的自然產物，身體會有自然的『抗自由基』機制去平衡，但若是身體處於疾病或不平衡狀態，則自然的『抗自由基』機制會失衡，而使組織遭受更多傷害，就如同地球的臭氧層有了破洞。反之，若是『抗自由基』機制失衡，則身體容易處於疾病或不平衡狀態，
醫學研究顯示，幾乎所有的疾病都與『自由基傷害』有關；『自由基傷害』是諸多疾病或身體不平衡狀態如老化、疲勞、不當飲食、疾病、癌症治療（例如手術、化療、電療、標靶治療等）的重要病理機轉，若是能減少『自由基傷害』，亦較能延緩老化、消除疲勞、預防疾病發生、減緩疾病症狀，及減緩癌症治療副作用所帶來的痛苦等。
現代人的文明病－『三高：高血壓、高血脂、高血糖』，及心血管疾病、慢性阻塞性肺病、阿茲海默症（俗稱老年癡呆症）、腎臟病、皮膚老化、……等，其病理機轉皆與體內的『自由基傷害』有密切關係，紅樟芝或可作為預防或改善疾病症狀的輔助保健品。
二、日常保健－『食補強身』：預防醫學的重要觀念，即是『預防勝於治療』；平時即作好身體調養，增強對於疾病的抵抗力，延緩身體退化與老化，根據 Levin教授對於紅樟芝的研究發現，可以樂觀地預測，紅樟芝應是日常保健與『食補強身』的優先選擇。
紅樟芝──癌症治療副作用的輔助選擇
癌症病患在經歷化學藥物治療、放射線治療、或標靶治療時，身體會遭受遠超出平時的『自由基傷害』，這也是癌症治療副作用例如恶心、嘔吐、疲倦感、掉髮、口腔炎、皮膚炎、……等的病理機轉之重要部份，根據Levin 教授的研究推論，紅樟芝可以是癌症治療後的輔助選擇，應有助於癌症病患在治療後的舒適度及生活品質，服用紅樟芝也應有助於治療後迅速恢復生理機能，讓癌症病患儘速展開新生活。
2007 Research Report From Dr.Levin and Dr.Lin, Albany, New York
Protection against ischemia and ischemia followed by reperfusion by Antrodia Camphorata
Final Report
Robert M. Levin Ph.D. and Alpha D. Lin M.D.
Albany College of Pharmacy and Stratton VA Medical Center
Urology Division, National Yang-Ming University, and Taichung Poah-Ai hospital Taiwan
Taiwan Well Shine Biotech Company, Taiwan
Introduction
There is growing evidence that ischemia (reduced blood flow to an organ system) and reperfusion following ischemia (the generation of free radicals and subsequent oxidative damage to tissues and organs) are directly involved in several degenerative diseases associated with ageing including heart disease, kidney dysfunction, liver dysfunction, CNS degeneration, and urinary bladder dysfunction (1-8). In general antioxidant therapies have proven to be effective in both reducing the progression of ischemia / reperfusion injury, and in the treatment of ischemic / reperfusion damage (9-16). The major targets for ischemic / reperfusion injury are mitochondria, sarcoplasmic reticulum, and nerves (1,7,8,11,14-16).
We have found the urinary bladder to be an excellent model organ system to study ischemia / reperfusion injury (1,14-18). Urinary bladder dysfunction is a major affliction of aging men (19-21); more than 80% of men over the age of 50 require medical attention for bladder dysfunction. In men, it is a progressive disease that results from a slowly increasing prostate mass compressing the urethra, eventually causing the characteristic bladder symptoms of urgency, frequency and poor urine flow (19-21). As the prostate gradually enlarges, bladder function can remain relatively "normal" for many years as the organ compensates for the progressive increase in urethral resistance through hypertrophy (an increase in bladder wall thickness and net increase in bladder mass) (22,23). It is not until the bladder shifts to decompensated function that moderate to severe alterations occur and the patient seeks medical attention.
The progression of obstructed bladder dysfunction is related directly to the the increased wall thickness resulting in ischemia / reperfusion injury characterized by damage to nerves, synapses and smooth muscle cells within the bladder wall, and due, in part, to generation of reactive oxygen and nitrogen species that functionally damage plasma and subcellular membranes (24,25).
It is clear from published studies that Antrodia camphorate has significant cell-protective properties (26-29), which would make it an excellent candidate for the prevention and / or treatment of ischemic / reperfusion damage
In conclusion, we believe that if we reduce the level of ischemia / reperfusion, we would significantly reduce the progression of the pathogenesis of ischemic bladder dysfunction described above. From the studies in the literature, Antrodia camphorate extracts may be very effective in the treatment of ischemic bladder dysfunction. On a broader scale, the demonstration that Antrodia camphorate protects the bladder against ischemia / reperfusion injury would indicate that this agent would probably be very effective in the protection and treatment of other organs against ischemia / reperfusion damage; ie congestive heart failure, alzheimers, kidney disease, etc.
Specific Aims
The specific aim of our proposal is to show that orally administered Antrodia camphorata can protect rabbit bladders from the progressive dysfunctions induced by experimental bilateral ischemia / reperfusion. The preparation will be administered daily by gastric lavage to insure that uniform amounts of extracts are ingested.
Experimental Design
Antrodia camphoata (AC) preparation:
Each pellet of AC manufactured by Taiwan Well Shine Biotech Company contains 250 mg powder. For man, the daily dose is 6 capsules / day = 1500 mg/day. For a 4 kg rabbit, the dose would be 100 mg / day suspended in 10 ml room temperature water and given to the rabbit daily by oral gavage.
Specifically, we have removed the powder from the commercial capsules, weighed out the proper amount and stirred the powder in room temperature water slowly so we did not destroy the cell wall of AC.
Animal Procedures: Thirty six male New Zealand White rabbits (3-4 kg) were separated into 6 groups of 6 animals each. Rabbits in groups 1 - 3 were fed 100 mg Antrodia camphoata suspensions daily as described above; those in groups 4 - 6 received vehicle. All rabbits received their respective treatments daily for 3 weeks.
After 3 weeks, all rabbits were anesthetized and cystometries performed. After cystometry each rabbit in groups 1,2,4,and 5 were subjected to bilateral ischemia, created by placing mini vascular clamps on both vesical arteries for a period of 2 hours. At the end of the two hours, the clamps were removed and the rabbits in groups 1 and 4 allowed to recover for 2 hours in order to recover the cellular levels of ATP; the rabbits in groups 2 and 5 were allowed to recover for one week (reperfusion period). The rabbits in groups 3 and 6 were sham operated and allowed to recover for 1 week. Feeding was continue during the recovery period.
Upon completion of the experiment, each rabbit was anesthetized and cystometry repeated. Immediately after cystometry the bladder was excised rapidly and weighed.
Isolated muscle strip pharmacology: Then, three isolated strips from each bladder were mounted in separate baths and the contractile responses to FS, carbachol, ATP and KCl were determined by standard methods.
Biochemistry: The balance of each bladder was separated into its muscle and mucosal compartments, frozen in liquid nitrogen and stored at -70oC for biochemical analyses. We measured citrate synthase (CS) activity as a marker for mitochondrial function, SR Ca+2-ATPase (SERCA) as a marker for SR function and choline acetyltransferase (ChAT) activity as a marker for cholinergic nerve function.
These studies will answer the following questions:
1. Does Antrodia camphorate protect the urinary bladder from ischemia and ischemia followed by reperfusion?
2. Does Antrodia camphorate protect bladder nerves from ischemia and ischemia followed by reperfusion?
3. Does Antrodia camphorate protect detrusor muscle mitochondrial and SR membranes from ischemia and ischemia followed by reperfusion?
Positive results from these studies would strongly support the usefulness of Antrodia camphorate for the prevention of progressive lower urinary tract dysfunction in the aging male population.
Progress Report:
Figure 1 At this time, all physiological studies have been completed. The ischemia group followed by 2 hours recovery showed no positive (or negative) effects of Antrodia, and have been eliminated from the report and subsequent manuscript. Ischemia / Reperfusion in the following figures represent ischemia followed by 1 week reperfusion.
Figure 1 shows that ischemia reperfusion (I/R) results in a mild but significant increase in bladder weight. No significant increase in bladder weight was noted for the I/R Antrodia group.
B
A
Figure 2 Figure 2 shows the responses to field stimulation. Panel A displays the maximal contractile response while panel B displays the maximal rate of tension generation. For both parameters, Antrodia mediated a significant increase in the response in the control bladders to all frequencies of stimulation. I/R resulted in significant decreases in the responses to all frequencies of stimulation. In the presence of Antrodia, the responses to all frequencies of stimulation were significantly greater than the responses in the absence of Antrodia.
Figure 3Figure 3 displays the response to ATP, carbachol, and KCl. Antrodia resulted in a significant increase in the responses to all forms of stimulation in the control rabbits. I/R resulted in significant decreases in the responses to all forms stimulation; although the magnitude of the decrease was less than the magnitude of the decreased responses to FS. Antrodia resulted in completely inhibiting the reduced contractile responses to ATP and KCl, and a significantly reduced decrease in the magnitude of the decreased response to carbachol.
Figure 4 AFigure 4 shows the cystometric curves. For both control and I/R, the cystometric curves for the Antrodia treated rabbits were significantly below those of the no treatment group. This indicates that compliance of the control and the I/R bladders were significantly reduced under both conditions by Antrodia.
Figure 4 B
Figure 5Figure 5 shows the compliance as intravesical pressure change over the first 20% capacity. I/R resulted in a significant decrease in compliance indicating a stiffer bladder. Antrodia treatment resulted in a significantly more compliant bladder under both control and I/R conditions. In regard to the IR bladder, there was no change in compliance for the Antrodia treated bladder.
Figure 6Figure 6 shows the micturition pressure and the volume at micturition derived from the cystometry studies. Antrodia treatment had no effect on either parameter for the control bladders. I/R resulted in no change in micturition pressure and a significant decrease in the volume at micturition. Antrodia treatment of the I/R bladders showed a significantly decreased volume at micturition compared to the no treatment groups.
Figure 7 shows the effect pf Amtrodia on citrate synthase activity. I/R mediated a significant decrease in citrate synthase activity of the mucosa. No change was noted in the activity of the smooth muscle. The rabbits treated with Antrodia showed a significantly higher citrate synthase activity of the mucosa after I/R when compared to the activity of the rabbits receiving vehicle.
Figure 8 shows that I/R resulted in a significant decrease in the choline acetyltransferase (Chat) activity of both treatment groups. Antrodia had no significant effect on the Chat activity of control or I/R groups.
Figure 9 shows the calcium ATPase activity. I/R had no significant effect on the total clcium ATPase activity; nor did treatment with Antrodia.
Figure 10 shows the SERCA activity (thapsigargin-sensitive calcium ATPase activity). I/R had no effect on SERCA activity of muscle, however, I?R resulted in an increased activity of the mucosa. Antrodia treatment resulted in a significant increase in the SERCA activity of the muscle but no effect on the activity of the mucosa.
Discussion and Conclusions:
One of the most striking results of this study is the demonstration that orally feeding the rabbits Antrodia Camphorata improves urinary bladder function in the control animals. The bladders have increased contractile responses to all forms of stimulation tested, and improved bladder compliance. At this point we do not have a mechanism of action for the increased contractile responses nor for the increased compliance. The biochemical studies have demonstrated that Antrodia had no effect on citrate synthase activity, choline acetyltransferase activity, total calcium ATPase activity, nor SERCA activity. Future studies will look at the smooth muscle collagen ratio. It is possible that Antrodia may result in an improved smooth muscle to collagen ratio in the bladder body which may be involved in the beneficial effects observed in the control condition.
I/R mediated the following changes in the no treatment group: increased bladder weight, decreased contractile responses to all forms of stimulation, decreased compliance (stiffer bladder), and decreased volume at micturition. In addition, I/R mediated a decreased activity for citrate synthase and Chat; but no change in total calcium ATPase activity. Antrodia treatment of the I/R bladders resulted in the following: decreased bladder weight back to control values, increased contractile responses to all forms of stimulation; the responses to ATP and KCl returning to control values while the responses to FS and carbachol were still significantly below control levels; increased (improved) compliance and decreased volume at micturition. In addition, Antrodia treatment resulted in an increased citrate synthase activity, although the activity did not get back to control levels. Antrodia had no effect on choline acetyltransferase activity or on total calcium ATPase activity of the I/R bladders; however, Antrodia resulted in a significant increase in the SERCA activity of the smooth muscle above control activity. There was no effect of Antrodia on the SERCA activity of the mucosa. The increased SERCA activity of the smooth muscle is consistent with the increased contractile responses; since the mucosa does not have a contractile function, it was not supriosing there was no effect on mucosal SERCA activity.
These all are beneficial effects which demonstrate that Antrodia treatment both improved normal bladder function, and protected the bladder against the detrimental changes mediated by ischemian / reperfusion.
This completes the studies; however, we are continuing with additional studies to try to determine the mechanisms by which Antrodia has it’s beneficial action. These additional studies include histological studies. No additional funds are requested.
Literature References
1. Mannikarottu, AS, Kogan, B, and Levin, RM Ischemic etiology of obstructive bladder dysfunction: A review. Recent Res. Devel. Mol. Cell Biochem, 2: 15-34, 2005.
2. Gasche Y, Soccal PM, Kanemitsu M, Copin JC. Matrix metalloproteinases and diseases of the central nervous system with a special emphasis on ischemic brain. Front Biosci. 11: 1289-1301, 2006.
3. Minami M, Katayama T, Satoh M. Brain cytokines and chemokines: roles in ischemic injury and pain. J Pharmacol Sci. 100: 461-470, 2006
4. Thurman JM. Triggers of inflammation after renal ischemia/reperfusion.Clin Immunol. 123: 7-13, 2007
5. Fandella A, Pernetti R, Marchiori D, Bertaccini A. The effects of renal ischemia on kidney function in renal cancer conservative surgery. Arch Ital Urol Androl. 78: 117-122., 2006.
6. Perco P, Pleban C, Kainz A, Lukas A, Mayer B, Oberbauer R. Gene expression and biomarkers in renal transplant ischemia reperfusion injury. Transpl Int. 20: 2-11, 2007
7. Dzurik R, Krivosikova Z, Stefikova K, Spustova V. Mitochondria and mitochondrial nitric oxide synthase alterations participate in energetical dysbalance, aging and age-related diseases. Bratisl Lek Listy. 107: 405-411, 2006.
8. Dhalla NS, Saini HK, Tappia PS, Sethi R, Mengi SA, Gupta SK. Potential role and mechanisms of subcellular remodeling in cardiac dysfunction due to ischemic heart disease. J Cardiovasc Med. 8: 238-250, 2007.
9. Carreira RS, Monteiro P, Gon Alves LM, Providencia LA. Carvedilol: just another Beta-blocker or a powerful cardioprotector? Cardiovasc Hematol Disord Drug Targets. 6: 257-266, 2006.
10. Tousoulis D, Boger RH, Antoniades C, Siasos G, Stefanadi E, Stefanadis C. Mechanisms of disease: L-arginine in coronary atherosclerosis--a clinical perspective. Nat Clin Pract Cardiovasc Med. 4: 274-283, 2007.
11. Schouten JW. Neuroprotection in traumatic brain injury: a complex struggle against the biology of nature. Curr Opin Crit Care. 13: 134-142, 2007
12. Liu Q, Xie F, Rolston R, Moreira PI, Nunomura A, Zhu X, Smith MA, Perry G. Prevention and treatment of Alzheimer disease and aging: antioxidants. Mini Rev Med Chem. 7: 171-180, 2007
13. Filipcik P, Cente M, Ferencik M, Hulin I, Novak M. The role of oxidative stress in the pathogenesis of Alzheimer's disease. Bratisl Lek Listy. 107: 384-394, 2006
14. Levin, R.M., Whitbeck, C., Horan, P., and Bellamy, F. Low-dose Tadenan protects the rabbit bladder from bilateral ischemia / reperfusion-induced contractile dysfunction, Phytomedicine 12: 17-24, 2005.
15. Lin, A D-Y, Mannikarottu, A.S, Chaudhry, A, Whitbeck, C, Kogan, B.A. Chichester, P, and Levin, R,M, Protective Effects of Grape Suspension on in-vivo ischemia / reperfusion of the rabbit bladder BJU 96: 1397-1402, 2005.
16. Levin, R.M., Leggett, R.E., Whitbeck, C., Matsumoto, S., Ohto, N., Ikeda, T., and Mizutani, K. Kohki tea protects the rabbit bladder from ischemia / reperfusion induced contractile dysfunction. Urologia Internationalis (in press).
17. Erdem, E., Leggett, R.E., Dicks, B., Kogan, B., and Levin, R.M. Effect of bilateral ischemia followed by reperfusion on superoxide dismutase activity and contraction. BJU 96: 169-174, 2005
18. Erdem, E., Whitbeck, C., Kogan, B.A., and Levin, R.M. Effect of maturation and ageing on response of the rabbit bladder to bilateral in vivo ischemia / reperfusion, UROLOGY 67: 220-224, 2006
19. Barry, M.J., and Meigs, J.B. The Natural History of Benign Prostatic Hyperplasia, pp 106-115. In Prostatic Diseases (Lepor, H. Ed.). W.B. Saunders Co., Phila., 2000.
20. Girman, C.J., and Guess, H.A. Epidemiology of Benigh Prostatic Hyperplasia, pp 116-126. In Prostatic Diseases (Lepor, H. Ed.). W.B. Saunders Co., Phila., 2000.
21. Grayhack, J. T. and Kozlowski, J. M.: Benign Prostatic Hyperplasia. In Adult and Pediatric Urology (Gillenwater, J. Y., Grayhack, J. T., Howards, S. S. and Duckett, J. W., eds.), Year Book Med. Pub., Inc. (Chicago), pp 1062‑1126, 1987.
22. Levin, R. M., Yu, H.J., Kim, K.B., Longhurst, P. A. and Damaser, M. S.: Etiology of bladder dysfunction secondary to partial outlet obstruction. Scand. J. Urol. Neph. Suppl. 184: 43-50, 1997.
23. Levin, R.M., Levin, S.S., Zhao, Y., and Buttyan, R. Cellular and molecular aspects of bladder hypertrophy. Eur. Urol. 32: (supp) 15-21, 1997.
24. Levin, R.M., Haugaard, N., O=Connor, L., Buttyan, R., Das, A.K., Dixon, J.S., and Gosling, J.A., Obstructive response of human bladder to BPH vs., rabbit bladder response to partial outlet obstruction: A direct comparison. Neurourol. Urodyn. 19: 609-629, 2000.
25. Mannikarottu, A., Lin, A.D., Whitebeck, C., Leggett, R., Kogan, B., Levin, R. Effect of partial bladder outlet obstruction on nitrotyrosine levels and their correlation with contractile function. Neurourol Urodyn. 25: 397-401, 2006.
26. Yang HL, Hseu YC, Chen JY, Yech YJ, Lu FJ, Wang HH, Lin PS, Wang BC. Antrodia camphorata in submerged culture protects low density lipoproteins against oxidative modification. Am J Chin Med. 34: 217-231, 2006
27. Yang HL, Chen CS, Chang WH, Lu FJ, Lai YC, Chen CC, Hseu TH, Kuo CT, Hseu YC. Growth inhibition and induction of apoptosis in MCF-7 breast cancer cells by Antrodia camphorata. Cancer Lett. 231: 215-227, 2006
28. Peng CC, Chen KC, Peng RY, Su CH, Hsieh-Li HM. Human urinary bladder cancer T24 cells are susceptible to the Antrodia camphorata extracts. Cancer Lett. 243: 109-119, 2006
29. Hseu YC, Wu FY, Wu JJ, Chen JY, Chang WH, Lu FJ, Lai YC, Yang HL. Anti-inflammatory potential of Antrodia Camphorata through inhibition of iNOS, COX-2 and cytokines via the NF-kappaB pathway. Int Immunopharmacol. 13-14: 1914-1925, 2005.
Final Report
Robert M. Levin Ph.D. and Alpha D. Lin M.D.
Albany College of Pharmacy and Stratton VA Medical Center
Urology Division, National Yang-Ming University, and Taichung Poah-Ai hospital Taiwan
Taiwan Well Shine Biotech Company, Taiwan
Introduction
There is growing evidence that ischemia (reduced blood flow to an organ system) and reperfusion following ischemia (the generation of free radicals and subsequent oxidative damage to tissues and organs) are directly involved in several degenerative diseases associated with ageing including heart disease, kidney dysfunction, liver dysfunction, CNS degeneration, and urinary bladder dysfunction (1-8). In general antioxidant therapies have proven to be effective in both reducing the progression of ischemia / reperfusion injury, and in the treatment of ischemic / reperfusion damage (9-16). The major targets for ischemic / reperfusion injury are mitochondria, sarcoplasmic reticulum, and nerves (1,7,8,11,14-16).
We have found the urinary bladder to be an excellent model organ system to study ischemia / reperfusion injury (1,14-18). Urinary bladder dysfunction is a major affliction of aging men (19-21); more than 80% of men over the age of 50 require medical attention for bladder dysfunction. In men, it is a progressive disease that results from a slowly increasing prostate mass compressing the urethra, eventually causing the characteristic bladder symptoms of urgency, frequency and poor urine flow (19-21). As the prostate gradually enlarges, bladder function can remain relatively "normal" for many years as the organ compensates for the progressive increase in urethral resistance through hypertrophy (an increase in bladder wall thickness and net increase in bladder mass) (22,23). It is not until the bladder shifts to decompensated function that moderate to severe alterations occur and the patient seeks medical attention.
The progression of obstructed bladder dysfunction is related directly to the the increased wall thickness resulting in ischemia / reperfusion injury characterized by damage to nerves, synapses and smooth muscle cells within the bladder wall, and due, in part, to generation of reactive oxygen and nitrogen species that functionally damage plasma and subcellular membranes (24,25).
It is clear from published studies that Antrodia camphorate has significant cell-protective properties (26-29), which would make it an excellent candidate for the prevention and / or treatment of ischemic / reperfusion damage
In conclusion, we believe that if we reduce the level of ischemia / reperfusion, we would significantly reduce the progression of the pathogenesis of ischemic bladder dysfunction described above. From the studies in the literature, Antrodia camphorate extracts may be very effective in the treatment of ischemic bladder dysfunction. On a broader scale, the demonstration that Antrodia camphorate protects the bladder against ischemia / reperfusion injury would indicate that this agent would probably be very effective in the protection and treatment of other organs against ischemia / reperfusion damage; ie congestive heart failure, alzheimers, kidney disease, etc.
Specific Aims
The specific aim of our proposal is to show that orally administered Antrodia camphorata can protect rabbit bladders from the progressive dysfunctions induced by experimental bilateral ischemia / reperfusion. The preparation will be administered daily by gastric lavage to insure that uniform amounts of extracts are ingested.
Experimental Design
Antrodia camphoata (AC) preparation:
Each pellet of AC manufactured by Taiwan Well Shine Biotech Company contains 250 mg powder. For man, the daily dose is 6 capsules / day = 1500 mg/day. For a 4 kg rabbit, the dose would be 100 mg / day suspended in 10 ml room temperature water and given to the rabbit daily by oral gavage.
Specifically, we have removed the powder from the commercial capsules, weighed out the proper amount and stirred the powder in room temperature water slowly so we did not destroy the cell wall of AC.
Animal Procedures: Thirty six male New Zealand White rabbits (3-4 kg) were separated into 6 groups of 6 animals each. Rabbits in groups 1 - 3 were fed 100 mg Antrodia camphoata suspensions daily as described above; those in groups 4 - 6 received vehicle. All rabbits received their respective treatments daily for 3 weeks.
After 3 weeks, all rabbits were anesthetized and cystometries performed. After cystometry each rabbit in groups 1,2,4,and 5 were subjected to bilateral ischemia, created by placing mini vascular clamps on both vesical arteries for a period of 2 hours. At the end of the two hours, the clamps were removed and the rabbits in groups 1 and 4 allowed to recover for 2 hours in order to recover the cellular levels of ATP; the rabbits in groups 2 and 5 were allowed to recover for one week (reperfusion period). The rabbits in groups 3 and 6 were sham operated and allowed to recover for 1 week. Feeding was continue during the recovery period.
Upon completion of the experiment, each rabbit was anesthetized and cystometry repeated. Immediately after cystometry the bladder was excised rapidly and weighed.
Isolated muscle strip pharmacology: Then, three isolated strips from each bladder were mounted in separate baths and the contractile responses to FS, carbachol, ATP and KCl were determined by standard methods.
Biochemistry: The balance of each bladder was separated into its muscle and mucosal compartments, frozen in liquid nitrogen and stored at -70oC for biochemical analyses. We measured citrate synthase (CS) activity as a marker for mitochondrial function, SR Ca+2-ATPase (SERCA) as a marker for SR function and choline acetyltransferase (ChAT) activity as a marker for cholinergic nerve function.
These studies will answer the following questions:
1. Does Antrodia camphorate protect the urinary bladder from ischemia and ischemia followed by reperfusion?
2. Does Antrodia camphorate protect bladder nerves from ischemia and ischemia followed by reperfusion?
3. Does Antrodia camphorate protect detrusor muscle mitochondrial and SR membranes from ischemia and ischemia followed by reperfusion?
Positive results from these studies would strongly support the usefulness of Antrodia camphorate for the prevention of progressive lower urinary tract dysfunction in the aging male population.
Progress Report:
Figure 1 At this time, all physiological studies have been completed. The ischemia group followed by 2 hours recovery showed no positive (or negative) effects of Antrodia, and have been eliminated from the report and subsequent manuscript. Ischemia / Reperfusion in the following figures represent ischemia followed by 1 week reperfusion.
Figure 1 shows that ischemia reperfusion (I/R) results in a mild but significant increase in bladder weight. No significant increase in bladder weight was noted for the I/R Antrodia group.
B
A
Figure 2 Figure 2 shows the responses to field stimulation. Panel A displays the maximal contractile response while panel B displays the maximal rate of tension generation. For both parameters, Antrodia mediated a significant increase in the response in the control bladders to all frequencies of stimulation. I/R resulted in significant decreases in the responses to all frequencies of stimulation. In the presence of Antrodia, the responses to all frequencies of stimulation were significantly greater than the responses in the absence of Antrodia.
Figure 3Figure 3 displays the response to ATP, carbachol, and KCl. Antrodia resulted in a significant increase in the responses to all forms of stimulation in the control rabbits. I/R resulted in significant decreases in the responses to all forms stimulation; although the magnitude of the decrease was less than the magnitude of the decreased responses to FS. Antrodia resulted in completely inhibiting the reduced contractile responses to ATP and KCl, and a significantly reduced decrease in the magnitude of the decreased response to carbachol.
Figure 4 AFigure 4 shows the cystometric curves. For both control and I/R, the cystometric curves for the Antrodia treated rabbits were significantly below those of the no treatment group. This indicates that compliance of the control and the I/R bladders were significantly reduced under both conditions by Antrodia.
Figure 4 B
Figure 5Figure 5 shows the compliance as intravesical pressure change over the first 20% capacity. I/R resulted in a significant decrease in compliance indicating a stiffer bladder. Antrodia treatment resulted in a significantly more compliant bladder under both control and I/R conditions. In regard to the IR bladder, there was no change in compliance for the Antrodia treated bladder.
Figure 6Figure 6 shows the micturition pressure and the volume at micturition derived from the cystometry studies. Antrodia treatment had no effect on either parameter for the control bladders. I/R resulted in no change in micturition pressure and a significant decrease in the volume at micturition. Antrodia treatment of the I/R bladders showed a significantly decreased volume at micturition compared to the no treatment groups.
Figure 7 shows the effect pf Amtrodia on citrate synthase activity. I/R mediated a significant decrease in citrate synthase activity of the mucosa. No change was noted in the activity of the smooth muscle. The rabbits treated with Antrodia showed a significantly higher citrate synthase activity of the mucosa after I/R when compared to the activity of the rabbits receiving vehicle.
Figure 8 shows that I/R resulted in a significant decrease in the choline acetyltransferase (Chat) activity of both treatment groups. Antrodia had no significant effect on the Chat activity of control or I/R groups.
Figure 9 shows the calcium ATPase activity. I/R had no significant effect on the total clcium ATPase activity; nor did treatment with Antrodia.
Figure 10 shows the SERCA activity (thapsigargin-sensitive calcium ATPase activity). I/R had no effect on SERCA activity of muscle, however, I?R resulted in an increased activity of the mucosa. Antrodia treatment resulted in a significant increase in the SERCA activity of the muscle but no effect on the activity of the mucosa.
Discussion and Conclusions:
One of the most striking results of this study is the demonstration that orally feeding the rabbits Antrodia Camphorata improves urinary bladder function in the control animals. The bladders have increased contractile responses to all forms of stimulation tested, and improved bladder compliance. At this point we do not have a mechanism of action for the increased contractile responses nor for the increased compliance. The biochemical studies have demonstrated that Antrodia had no effect on citrate synthase activity, choline acetyltransferase activity, total calcium ATPase activity, nor SERCA activity. Future studies will look at the smooth muscle collagen ratio. It is possible that Antrodia may result in an improved smooth muscle to collagen ratio in the bladder body which may be involved in the beneficial effects observed in the control condition.
I/R mediated the following changes in the no treatment group: increased bladder weight, decreased contractile responses to all forms of stimulation, decreased compliance (stiffer bladder), and decreased volume at micturition. In addition, I/R mediated a decreased activity for citrate synthase and Chat; but no change in total calcium ATPase activity. Antrodia treatment of the I/R bladders resulted in the following: decreased bladder weight back to control values, increased contractile responses to all forms of stimulation; the responses to ATP and KCl returning to control values while the responses to FS and carbachol were still significantly below control levels; increased (improved) compliance and decreased volume at micturition. In addition, Antrodia treatment resulted in an increased citrate synthase activity, although the activity did not get back to control levels. Antrodia had no effect on choline acetyltransferase activity or on total calcium ATPase activity of the I/R bladders; however, Antrodia resulted in a significant increase in the SERCA activity of the smooth muscle above control activity. There was no effect of Antrodia on the SERCA activity of the mucosa. The increased SERCA activity of the smooth muscle is consistent with the increased contractile responses; since the mucosa does not have a contractile function, it was not supriosing there was no effect on mucosal SERCA activity.
These all are beneficial effects which demonstrate that Antrodia treatment both improved normal bladder function, and protected the bladder against the detrimental changes mediated by ischemian / reperfusion.
This completes the studies; however, we are continuing with additional studies to try to determine the mechanisms by which Antrodia has it’s beneficial action. These additional studies include histological studies. No additional funds are requested.
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