實驗性敗血症大白鼠中熱休克蛋白70 的表現及麩醯胺酸對熱休克蛋白70 基因表現之影響

Expression of hsp70 gene during experimental sepsis and the effect of glutamine in rats

王淑容(Shu-Jung Wang)

指導教授 ： 楊瑞成

高雄醫學大學/醫學院/醫學研究所/博士(2007年)

摘要

熱休克蛋白質(heat shock proteins: Hsps)的合成失敗是影響宿主自我防衛及敗血症預後不良的原因之ㄧ，敗血症誘發前予以體外進行熱休克前處置使其產生熱休克蛋白質，可以明顯的減少其死亡率，這些結果顯示熱休克蛋白質維持體內代謝及發炎反應恆定效應有很大的關係。敗血症動物無法製造合成熱休克蛋白質，被認為可能和此疾病的預後不良有關。而敗血症發生的進程中，熱休克蛋白質基因(hsp gene)受到抑制的作用，機制仍不清楚，文獻指出熱休克因子-1(heat shock factor 1: HSF-1)被認為是調控誘發性熱休克蛋白質基因表現最主要的轉錄因子，其在敗血症期間的活性就顯的相當重要。最近幾年麩醯胺酸(glutamine)受到許多學者的青睞，麩醯胺酸被認為是一具潛力且安全的熱休克蛋白質誘發物質。有許多的研究將麩醯胺酸誘發熱休克蛋白質的效應應用到敗血症的動物模式中，成功的降低敗血症的死亡率、減少敗血症所誘發細胞激素的產生、組織型態上的傷害。但其參與熱休克蛋白質表現，其機制不明，但也同時發現不同的器官對於麩醯胺酸的感受性有所不同，尤其在敗血症的疾病病程中扮演重要角色的肝臟。

在這個研究中，以Sprague-Dawley(SD)大白鼠作為我們的實驗動物，熱休克前處置採用全身性電毯加熱的方式誘發熱休克蛋白質大量表現，利用盲腸結紮穿孔手術誘發敗血症的產生，敗血症鼠之麩醯胺酸給予，在敗血症手術後一小時採單一劑量尾靜脈注射的方式，針對麩醯胺酸誘發熱休克蛋白質70基因活化之探討，則在熱休克前處置24小時後給予，以作用不同的時間。另外，熱休克蛋白質抑制劑-Quercetin則在熱休克處置前6小時注射。利用這樣的動物模式及麩醯胺酸的應用方式，來探討實驗性敗血症大白鼠肝臟熱休克蛋白質70的表現及麩醯鞍酸對熱休克蛋白質70基因表現之影響。實驗內容分成三部份：第一部分：探討在實驗性敗血症發生的過程中，熱休克轉錄因子-1被活化的過程，以及調控熱休克轉錄因子-1磷酸化作用的磷酸酶活性及蛋白激酶的表現；第二部分：利用麩醯胺酸誘發熱休克蛋白質表現的特性，探討麩醯胺酸在敗血症過程中對於肝臟熱休克蛋白質70基因表現的影響；第三部分：探討熱休克蛋白質72的存在對於麩醯胺酸在實驗鼠肝臟中，誘發熱休克蛋白質70基因表現的角色。

探討在實驗性敗血症發生的過程中，熱休克因子-1被活化的過程，以及調控熱休克因子-1磷酸化作用的磷酸酶活性及蛋白激酶的表現方面，實驗結果顯示，在敗血症期間所有組別均無法偵測到hsp72 mRNA的合成，但熱休克因子-1卻會受到活化，由電泳移動偏移進行熱休克因子-1和熱休克要素之結合活性的結果發現熱休克因子-1可以接合到HSE上，而熱休克因子-1在細胞質中可隨著敗血症的進程，磷酸化的作用隨著增加，而在細胞核中的熱休克因子-1僅在熱休克前處置之加熱組可偵測到有磷酸化形式的熱休克因子-1，進一步偵測影響熱休克因子-1磷酸化作用的因子-蛋白激酶(PKC??)的表現和磷酸酶(phosphatase)的活性，結果發現，PKC?悛漯穛{隨著敗血症的進展，從早期就有明顯的下降，而磷酸酶的活性則在所有實驗組別是無差異的。由此可知在敗血症的進程中，細胞核內熱休克因子-1的磷酸化作用被抑制了，若予以加熱前處置誘發熱休克蛋白質72，則增加熱休克因子-1的磷酸化及相關之PKC?悛漯穛{，另外敗血症時磷酸化型式熱休克因子-1的存在卻無法進一步合成熱休克蛋白質72，顯示在轉錄能力上的失敗是敗血症發生時最主要的因素之一。

其次利用麩醯胺酸誘發熱休克蛋白質表現的特性，針對麩醯胺酸在實驗性敗血症鼠肝臟中，對誘發熱休克蛋白質72表現的影響所作的探討方面，實驗結果顯示，在偽手術或敗血症晚期組之實驗鼠，無論麩醯胺酸注射與否，均無法偵測到熱休克蛋白質72的表現。在加熱組可偵測到熱休克蛋白質72的表現量明顯的較沒有注射的多。且在加熱前處置之實驗鼠中，麩醯胺酸明顯誘發hsp72 mRNA的產生。並使加熱前處置所造成之磷酸化熱休克因子-1的堆積減少，進行轉錄作用合成mRNA，因此無法測得磷酸化熱休克因子-1，推測其可能進一步促使熱休克蛋白質72基因的活化。總結以上結果，顯示麩醯胺酸可能是使原來在敗血症的過程中，所造成被抑制或阻斷的熱休克蛋白質72基因再活化，而熱休克前處置所誘發之熱休克蛋白質72的表現是必需存在的。

最後針對熱休克蛋白質72對於麩醯胺酸在實驗鼠肝臟中，誘發熱休克蛋白質70基因表現的影響所作的探討方面。結果顯示，熱休克處置後肝臟中熱休克蛋白質基因72的活化反應，在24小時後就沒有新合成蛋白質，而維持大量熱休克蛋白質基因72表現的狀態，而此時加入麩醯胺酸，結果顯示，麩醯胺酸作用可增加熱休克蛋白質的產生，且於作用8小時達到一高峰，且可偵測到新合成之hsp72 mRNA的表現，顯示麩醯胺酸活化了熱休克蛋白質72基因，相較於熱休克處置前以Quercetin抑制熱休克蛋白質的產生，麩醯胺酸誘發熱休克蛋白質的能力就消失了，表示熱休克蛋白質的存在是必要的，且此活化作用和細胞內存在之熱休克蛋白質72呈現量的相關性，而熱休克因子-1的磷酸化作用及和熱休克要素之結合活性也相對的增加。實驗結果證實麩醯胺酸在熱休克前處置的條件下，可經由促進熱休克因子-1的磷酸化作用，經由活化熱休克因子-1的訊息傳導路徑而活化熱休克蛋白質72基因的表現，且其活化的效應和熱休克蛋白質72呈現量的相關性。

總結而言，本論文重新檢視熱休克蛋白質70的表現機制，及敗血症之生理病理變化過程中，影響熱休克蛋白質70基因表現的機制，並將麩醯胺酸應用於敗血症病程中，重新活化熱休克蛋白質的合成，成功的將原來受阻斷的誘發熱休克蛋白質基因再度合成，相信能對於未來敗血症的治療特別是肝臟的保護方面提供一道更確切的方向。

關鍵字

熱休克蛋白質 ； 敗血症 ； 麩醯胺酸 ； 肝臟 ； 熱休克因子-1

並列摘要

From our previous study results, it is getting more and more clear that failure in the induction of heat shock protein (Hsps) is highly associated with the poor outcome of sepsis, and their over-expression significantly do benefit to subjects facing a severe infection. Moreover, the Hsps synthesis is inhibited during sepsis, which might be a crucial factor leading to a poor prognosis of the disease. However the mechanism is still obscure. The expression of Hsps is regulated by a multi-step manner. Heat shock factor 1 (HSF-1) is recognized as a central component of the hsp gene expression. Recently, glutamine is attracted by researchers and believed to play a beneficial effect in many tissues of sepsis by inducing Hsps, although the molecular mechanism is still not clear. Unfortunately, majority of the reported data show that the liver, a central organ of metabolism of the body, seems less responsive to the treatment. Accordingly, the aims of this study were:
1) to investigate the mechanism of transcription failure of Hsp72 in sepsis, focusing on the HSF-1 turn-activation, and the possible effect of heat shock pretreatment was discussed
2) to explore the effect of glutamine in Hsp72 expression in experimental septic livers
3) to evaluate the effect of glutamine-induced activation of hsp72 gene preconditioned with heat shock treatment

Experimental sepsis was induced by cecal ligation and puncture (CLP) in adult male Sprague-Dawley (SD) rats. Heat shock treatment was applied to the rats’ whole bodily using an electric heating pad. Glutamine was administered 1 hr after initiation of sepsis via tail vein injection. To investigate the effect of glutamine in Hsp70 gene expression in heat-shock pretreated rats, glutamine was administered via tail vein injection 24 hours after heat shock treatment. Quercetin, an inhibitor of Hsps expression, was treated 6 hours before heat shock treatment.

First, the results showed that both non-phosphorylated HSF-1 and phosphorylated HSF-1 were detectable in the cytosolic protein, while non-phosphorylated HSF-1 was only detectable in nuclear protein of early and late phase of sepsis. HSF1-HSE binding activity was detectable but neither the hsp mRNA nor Hsp72 was expressed in early or late stage of sepsis. The expression of PKC?? was not different in cytosolic protein during all phases, but that in the nucleus was significantly different between non-heat and heat group. These results suggest that, in sepsis, HSF-1 was activated and translocated into the nucleus, and then bound with HSE on the promoter of hsp gene, but the subsequent transcription was inhibited. The nuclear PKC?? might be the critical role in influencing the expression of Hsp72 during sepsis.

Secondary, in the study of the effect of glutamine administration in experimental septic livers, the results showed that Hsp72 content noticeably increased in the livers of preheated rats supplied by glutamine 1 hr after sepsis. However, no further synthesis of Hsp72 was found in septic livers or sham glutamine-treated livers. Hsp72, which was induced by preheat, decreased with time while a large amount of Hsp72 could be detected by glutamine administration. RT-PCR data indicated that Hsp72 mRNA could only be detected in the group treated with preheat and glutamine administration. However, only the preheated group showed the phosphorylation in HSF-1. With the administration of glutamine, the nuclear accumulation of phosphorylated HSF-1 was observed to decline significantly 9 and 18 hr after CLP when the Hsp72 mRNA became detectable. These results demonstrated that Hsp72 could be induced by glutamine in septic liver only if the liver has been preconditioned by heat shock response. The selective facilitating effect might depend on decreasing the accumulation of intranuclear phosphorylated HSF-1 caused by previous heat shock treatment.

Finally, to evaluate the effect of glutamine-induced activation of hsp72 gene preconditioned with heat shock treatment, the results showed that glutamine administration enhanced Hsp72 induction significantly only in rats’ livers preconditioned by heat shock. Further evaluation of hsp72 mRNA synthesis and HSF-1 activation showed that glutamine induced hsp72 mRNA expression through the pathway of HSF-1 activation and that the reactivation of hsp72 mRNA expression was correlated to the amount of intracellular Hsp72 in a dose-dependent manner. In conclusion, glutamine reactivated the Hsp72 gene expression in liver preconditioned by heat shock treatment through the pathway of HSF-1 activation.

In conclusion, HSF-1 phosphorylation was failure during sepsis and heat shock treatment maintained phosphorylated HSF-1 in nucleus. Hsp72 could only be induced by glutamine in septic liver preconditioned by heat shock treatment. The effect might depend on decreasing the accumulation of intranuclear phosphorylated HSF-1 caused by previous heat shock treatment. Glutamine-induced reactivation of hepatic hsp70 gene expression is Hsp72 dependent and the activation might be through the pathway of HSF-1 activation. The present studies provide the fundamental information for exploring the therapeutic potential of glutamine-induced Hsps in sepsis, especially the protection of liver.

並列關鍵字

heat shock proetin ； sepsis ； glutamine ； liver ； heat shock factor-1

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