遠紅外線照射對於大鼠睪丸缺血再灌流傷害保護機制的探討

The protective mechanism of far-infrared irradiation(FIR) against ischemia-reperfusion injury on the testes in rats

杜元博(Yuan-Po Tu)

指導教授 ： 陳朝峯

國立臺灣大學/醫學院/生理學研究所/博士(2013年)

https://doi.org/10.6342/NTU.2013.03075

摘要

缺血再灌流傷害的前置保護角色(IPC) 的探討，對許多的器官保護機制中都有廣泛的探討。在心臟、肝、腎的前置保護的動物模式中，一般而言有三種成熟的遠端模式(RPC)，包含有遠端肢短暫缺血前置保護模式、低氣壓缺氧前置保護模式及藥物誘發前置保護模式。而此前置保護的處置，透過某些特殊的機轉因子，確實發揮了明確的器官保護。而這些特殊的機轉因子中，多篇文獻指出原血紅素氧化酶-1扮演了極重要的角色。
而從1996年提出缺血再灌流傷害的前置保護角色的觀念後，多篇文献也指出前置保護屬於保護時效較短的機制。所以Zhi-Qing Zhao在2003年提出了傷害後立即後置訓練保護的機制，它在傷害後立即多次重覆短暫的缺血再灌流處置，提供了相似前置保護角色器官保護的結果。在其後眾多的文献研究中，多種的機轉因子被提出，基於原血紅素氧化酶-1在前置保護訓練中扮演了極重要的角色，我們也提出了推論: 是否原血紅素氧化酶-1也在傷害後立即後置保護訓練機制中占有類似其在前置保護訓練中之極重要的保護角色。
在前述的實驗中，我們知道了原血紅素氧化酶-1確實對保護細胞組織及構造，及抑止細肥凋亡有極其重要之地位，且在文獻探討中，知道遠紅外線可誘發原血紅素氧化酶-1之產生。故我們設計了一種比缺血傷害後立即後置訓練保護機制更簡單應用之以遠紅外線照射之傷害後立即後置訓練保護機制，探究其是否能誘發原血紅素氧化酶-1之產生，且進一步能產生保護細胞組織及構造及抑止細胞凋亡之傷害後立即後置訓練保護角色的研究探討。
研究首部，利用大鼠睪丸缺血再灌流(I/R)傷害模式-完全缺血40分鐘後再灌流24小時之前24小時注射藥物誘發前置保護。實驗顯示大鼠腹腔內注射辛伐他汀藥物(Simvastatin)後，收集睪丸組織研磨萃取液，發現原血紅素氧化酶-1濃度在24小時至48小時表現最高。Tin protoporphyrin (Snpp)是原血紅素氧化酶的完全阻斷劑。過氧化物酶eNOS(內皮一氧化氮合成酶)是細胞缺血再灌流(I/R)傷害指標的表現。我們發現在起始接受單純辛伐他汀藥物誘發前置保護訓練的大鼠睪丸切片，在生殖細胞區有明顯的原血紅素氧化酶-1的免疫組織化學染色組織切片及西方墨點的檢測表現；對照單純缺血再灌流對照組及起始辛伐他汀藥物合併Snpp阻斷劑注射實驗組中，其eNOS的免疫組織化學染色組織切片及西方墨點檢測分析在生殖細胞區中也有很明顯的表現。在eNOS-細胞缺血再灌流傷害指標過氧化物酶表現的實驗組中，其不論HE細胞組織構造表現分級或TUNEL細胞組織凋亡表現都呈現明顯的細胞組織構造破壞及細胞凋亡；而在原血紅素氧化酶-1明顯表現的大鼠切片組織，則呈現保存較完好的HE細胞組織構造分級表現及低的TUNEL細胞組織凋亡呈現。且進一步的細胞凋亡因子caspases-3的西方墨點檢測也支持其明顯的對照表現。由以上的實驗結果我們推論辛伐他汀藥物誘發前置保護模式極有可能是藉由原血紅素氧化酶-1此機轉因子達到保護細胞組織構造避免細胞凋亡的前置訓練保護。
相似於心臟器官缺血再灌流的傷害，其睪丸器官的缺血再灌流的傷害也有高比率的不確定發生比率。而前置訓練保護機制屬較短暫時效性的保護機制，我們嘗試在接下來的實驗中採用傷害後立即後置訓練保護的機制，藉由睪丸器官的缺血再灌流後馬上給予保護訓練，在實驗操作後將大鼠睪丸切除，接受免疫組織化學染色組織切片、西方墨點檢測以及細胞凋亡TUNEL染色分析及細胞凋亡caspases-3因子檢測。
在我們的實驗的第二部份，我們設計了以遠紅外線照射之傷害後立即後置訓練保護機制。我們知道原血紅素氧化酶-1確實有保護細胞組織及構造，及抑止細肥凋亡之作用，在此次實驗中，我們探討遠紅外線是否可藉著誘發原血紅素氧化酶-1之產生，進而達到保護大鼠睪丸對抗缺血再灌流的傷害。實驗方法我們採用了大鼠睪丸完全缺血1、2及4小時三個群組，之後再接受灌流24小時之缺血再灌流(I/R)傷害模式。同時在接受灌流起始的30分鐘時間內，我們將三個群組各分成兩個實驗組：第一組同時一起接受遠紅外線的照射；第二組同時一起接受光源(heat light )的照射。實驗結果顯示，在缺血2小時群組中，接受遠紅外線照射的實驗組在免疫組織化學染色組織切片及細胞凋亡TUNEL的分析中都呈現明顯較低的組織構造破壞分級及低的TUNEL細胞凋亡染色表現。且同時在接受遠紅外線照射的實驗組中呈現明顯原血紅素氧化酶-1的濃度表現。而接受同環境同條件控制下的光源照射組，則呈現低的原血紅素氧化酶-1濃度表現及高濃度的細胞凋亡caspases-3因子西方墨點檢測，其對應也顯示，在免疫組織化學染色組織切片及細胞凋亡TUNEL的分析中都呈現明顯高的組織構造破壞分級及TUNEL細胞凋亡染色表現。實驗結果支持了我們的推論：遠紅外線照射碓實能藉由誘發原血紅素氧化酶-1之產生達到傷害後立即後置訓練之保護機制。
而應用在我們臨床治療男性慢性攝護腺炎的「遠紅外線療法」初步經驗中，運用遠紅外線來治療，不僅方便舒服無侵犯性，病患接受度高，治療後的效果評估良好。12位接受「遠紅外線療法」的病患，在2週「遠紅外線療法」照射治療前及治療後三項評估結果之變化：(1). IPSS；(2). PS；(3). 前列腺液白血球數等臨床數據，都顯示明顯的好轉治療效果。且12位病患在接受過2週照射治療後，在同一個門診，同一位治療醫師追蹤一至三個月後，臨床症狀也顯著改善，甚至有4位病患在照射治療後追蹤三個月至無任何症狀復發。
由以上之實驗結果，我們證實了不論是藉由藥物誘發的前置保護模式或是新的傷害後立即後置訓練保護機制的遠紅外線照射，其誘發的原血紅素氧化酶-1確實是可明顯的保護細胞組織構造及保護細胞凋亡的作用機轉因子之一。且應用遠紅外線照射傷害後立即後置訓練保護機制藉由活化引致原血紅素氧化酶-1來消減大鼠睪丸缺血再灌流I/R的氧化傷害，其照射遠紅外線射能源屬物理性治療方式，可随時施予治療，其施予的方法又比傷害後立即缺血後置訓練保護( ischemic post-conditioning, IPostC)來得簡單。而在我們應用「遠紅外線療法」治療臨床男性慢性攝護腺炎的初步經驗中，也證實了遠紅外線療法有其明確的臨床治療效果。我們推論對缺血再灌流I/R的生物體睪丸傷害，遠紅外線照射傷害後立即後置訓練保護(FIR postconditioning)確實可在未來做為一個新的標竿治療趨勢。

關鍵字

遠紅外線 ； 缺血再灌流傷害 ； 原血紅素氧化酶-1 ； 前置保護訓練 ； 後置保護訓練

並列摘要

The protective role of remote preconditioning(IPC) against ischemia–reperfusion(IR) injury of organismic organs was widely investigated in literature. The experimental animal model of IPC against I/R injury on heart, liver and kidney organs etc. involved three generally mature types: (1). Ischemic preconditioning; (2). Hypoxic preconditioning; and (3).Pharmacological preconditioning. In the IPC experiment model, the protection mechanism against ischemia/reperfusion injury was elaborated through with many biochemical factors. In that,　hemeoxygenase-1 (HO-1) play a important agent.
Since 1996, the revolutionary concept of ischemic preconditioning brought up had been proved with the powerful protective effects against I/R explored by numerous studies. Nevertheless, the major issue has been that the protective function provided by ischemic preconditioning is a relatively short-lived phenomenon. It is also proposed that postcondi-
tioning as asimilar thinking mimicked the application of ischemic preconditioning. The postconditioning is a tactics that can modify reperfusion induced adversities by applied the preconditioning stimulus to the beginning of reperfusion with repeated rapidly transient occlusion-reperfusion cycle.
We inferred the subject that used the protective mechanism postconditioning to induce the HO-1 against ischemia/reperfusion injury. Far-infrared radiation (FIR) heater may active the protective role of HO-1. On the basic concept, we design the FIR postconditioning and investigate the protective role against ischemia/reperfusion injury by the activation of HO-1 protein expression.
Testicular ischemia/reperfusion injury due to testicular torsion was a serious urological condition. Delayed treatment of testicular I/R injury can result in irreversible damage, infertility, and loss of the testis. I/R injury can induce the overexpression of reactive oxygen species (ROS) and reactive nitrogen species (RNS), and the similar mechanism to other organs such as brain, heart and kidneys. Thus, it is an important issue to attenuate the severe effects of testicular I/R injury.
First, we evaluate the protective role of simvastatin-induced HO-1 in remote preconditioning against testis ischemia–reperfusion injury in vivo. Simvastatin was intraperitoneally (i.p.) injected 24 h before I/R injury. Testis was occluded in the right testis for 40 min and followed by 24hours of reperfusion to induce I/R injury. Tin protoporphyrin (Snpp), a competitive inhibitor of hemeoxygenase, was i.p. injected 1 h before the I/R injury in separate groups of rats. The rat testes were harvested 24 h later. Induction of HO-1 expression by simvastatin was significantly increased at 24 and 48 h. Rats pre-treated with simvastatin showed higher expression of HO-1 protein by Western blotting and immunohistochemistry (IHC), and presented lower caspases-3 activity by caspase-3 activity assay. TUNEL staining analysis revealed simvastatin pretreatment significantly reduced I/R induced cellular apoptosis. Contrarily, the simvastatin induced cytoprotective effect was entirely abolished by administrations of Snpp. Further, lower caspase-3 activities were also noted in simvastatin plus Snpp (SS) group than the control plus Snpp (CS) group. AfterI/R injury, eNOS immunoreactivity was markedly increased in the germ cell and Leydig cell of testicular tissues. Pretreatment of simvastatin significantly decreased eNOS immunereactivity in the germ cell of the tubules in the rat testes. In conclusion, we suggest HO-1 plays a protective role in IR-induced injury in the testes of rats.
Secondly, Studies have shown that heme oxygenase-1 has a protective role in the mechanism underlying hypoxic preconditioning. We used a far-infrared radiation heater as a postconditioning to enhance HO-1 expression against ischemia/reperfusion injury in rat testes. Forty rats were used. Testis ischemia was mimicked by total obstructive clamping of testis vessels for 1, 2, or 4 h, and concomitant postconditioning with 30 min FIR or heat light during initially 30 min reperfusion. HO-1 expression and apoptosis of testis tissues were examined by immunohistochemistry and in situ terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL)assay, respectively. HO-1 protein level and caspase-3 activity were analyzed by Western blotting. There was less apoptotic activity in rat testis after FIR, as determined by TUNEL assay. Higher HO-1 protein expression was observed by immunohistochemistry and Western blotting (p<0.01) in testis cells after FIR postconditioning. In contrast, caspase-3 activity was significantly higher in heat light groups, as compared with FIR groups (p<0.01). FIR postconditioning attenuated I/R injury in rat testis by inducing HO-1 expression, which might have a protective role in testis apoptosis after I/R injury.
As the protective role of FIR postconditioning, we applied it to the therapy of chronic prostatitis. In our prelimited report, the FIR therapy had marked curative and protective effect to the patients sufferrred from chronic prostatitis.
According to the above studies, we propose that the hemeoxygenase-1 play a important protective role induced by Pharmacological preconditioning and FIR postconditioning against I/R injury on rat testis. And the new-designed postconditioning-FIR postconditioning may be a potential novel therapy against testes I/R injury and clinic illnesses.

並列關鍵字

far-infrared irradiation ； ischemia-reperfusion injury ； hemeoxygenase-1 ； preconditioning ； postconditioning

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