探討熱休克前處置對於敗血症誘發腎上腺功能缺失之影響

Effect of Heat Shock Pretreatment in Sepsis-induced Adrenal Dysfunction

許子涵(Tzu-Han Hsu)

指導教授 ： 楊瑞成

高雄醫學大學/醫學院/醫學研究所/碩士(2006年)

摘要

中文摘要
敗血症晚期誘發腎上腺功能缺失的機制仍未清楚的瞭解，目前只知道可能與荷爾蒙、細胞激素及一些神經性胜肽的產生及失調有關聯；其中，文獻指出介質中的腫瘤壞死因子 (TNF)會干擾固醇類荷爾蒙的生成路徑，並可能為造成腎上腺功能缺失的因素之一。
熱休克蛋白質，一種存在於各種生物細胞體內具有伴隨者保護角色的蛋白質。有文獻指出，熱休克前處置及熱休克蛋白質能影響TNF的生合成，故我們的假設為熱休克前處置以及產生的熱休克蛋白質對於敗血症晚期誘發腎上腺功能缺失的現象有緩解的功效。
實驗過程中，我們將實驗大鼠分為加熱及非加熱兩大組再利用盲腸結紥及穿孔術 (cecal ligation and puncture；CLP)來誘發大鼠產生敗血症，另用偽手術大鼠作為控制組。我們將實驗組別分成四組：1) Sham組表示給予剖腹縫合的動作但不進行盲腸結紥及穿孔術的偽手術；2) CLP組表示僅給予盲腸結紥及穿孔術；3) HCLP組表示加熱後24小時進行盲腸結紥及穿孔術；及4) HS組表示加熱後24小時施行與Sham組相同的偽手術。於敗血症動物模式晚期 (CLP手術後18小時)予以犧牲並取其血清及腎上腺用以實驗的材料。統計結果顯示敗血症中有熱休克蛋白質存在的HCLP組相較於CLP組，確實有意義的降低了敗血症的死亡率。我們利用酵素免疫分析試劑偵測血清中ACTH及corticosterone，實驗結果顯示HCLP組中有最低的血清ACTH含量及僅次於CLP組的血清corticosterone含量。另外我們採用corticosterone與ACTH的比值來表示腎上腺的反應性，結果發現HCLP組有最好的腎上腺反應性。免疫組織化學染色及西方墨點法分析腎上腺組織中P450scc (cytochrome P450 side chain cleavage) 酵素蛋白質含量，結果指出P450scc主要表現在腎上腺皮質區且蛋白質含量在四組並無統計上顯著的差異。TUNEL分析結果顯示，無論敗血症的發生與否，四組組織切片中並無明顯細胞凋亡的現象發生。因此我們合理推斷，造成敗血症誘發腎上腺功能缺失並不是因腎上腺組織細胞凋亡所引起。
具有熱休克蛋白質72存在的HCLP組對於內因性ACTH有最好的反應性並且有效的降低了敗血症的死亡率，我們認為是熱休克前處置後誘發熱休克蛋白質72在敗血症病程中扮演著保護腎上腺功能的角色。

關鍵字

熱休克前處置 ； 熱休克蛋白質 ； 敗血症 ； 盲腸結紮及穿孔手術 ； 腎上腺功能缺失 ； 腎上腺 ； 腫瘤壞死因子 ； 類固醇荷爾蒙新生 ； 皮質固酮 ； 促腎上腺皮質荷爾蒙 ； 酵素免疫分析 ； 免疫組織化學

並列摘要

Abstract
Introduction: The mechanism of sepsis-induced adrenal insufficiency in late sepsis is still unclear. Recent studies indicated that the mechanism may involve in hormones, cytokines, and neuropeptides. In cytokines, tumor necrosis factor alpha (TNF-α) has been shown to reduce the synthesis of steroid hormones by inhibiting the stimulatory effect of ACTH on adrenal cells. The role of TNF-α seems to be evident in causing adrenal insufficiency during sepsis. Heat shock proteins (HSPs) play the role of chaperone to protect the cell from the subsequent stress. Our previous studies have indicated that the synthesis of HSP72, a member of 70kD family, was inhibited in cecal ligation and puncture (CLP)-induced sepsis model. However, in the sepsis with previous heat shock pretreatment, HSP72 could be detected and bind to NFκB and thereby decrease the production of TNF-α and the mortality rate. Hypothesis: We hypothesized that heat shock pretreatment may attenuate the severity of adrenal dysfunction induced by CLP. Materials and Methods: We used CLP-induced sepsis animal model to simulate the clinical sepsis in human. Sprague-Dawley rats were divided into 4 groups: 1) sham-operated rats (Sham group, n=18); 2) CLP-induced sepsis rats (CLP group, n=41); 3) heat shock pretreatment and then CLP-induced sepsis rats (HCLP group, n=39); 4) heat shock pretreatment and then sham-operated rats (HS group, n=22). Rats were sacrificed and the serum and adrenal glands were harvested at 18 hours after CLP. We used enzyme immunoassay (EIA) commercial kit to detect levels of serum adrenocorticotropin (ACTH) and corticosterone. Western blot analysis was used for evaluating the HSP72 and cytochrome P450 side chain cleavage (P450scc), immunohistochemistry was used for detecting P450scc, and TUNEL assay was used for evaluating apoptosis in the adrenal gland. Results: The mortality rate of HCLP group was significant lower than CLP group. Expression of P450scc detected by immunohistochemistry and western blot were not significantly different among four groups. TUNEL assay showed whether CLP operated or not, there were no apoptotic happened in adrenal glands significantly. Although the HCLP group had lower level of serum ACTH and higher level of serum corticosterone, it revealed the highest corticosterone/ACTH ratio, indicating a better adrenal responsiveness. Conclusions: We found that the HCLP group, which had expression of HSP72, had lower mortality rate compared with CLP only group and had a better adrenal responsiveness for endogenous ACTH. However, the adrenal dysfunction dose not associated with apoptosis. We suggest that heat shock pretreatment, inducing HSP72 during sepsis, could protect the adrenal function through enhancement of the adrenal responsiveness.

並列關鍵字

heat shock pretreatment ； heat shock protein ； HSP ； sepsis ； cecal ligation and puncture ； CLP ； adrenal dysfunction ； adrenal ； tumor necrosis factor ； TNF ； steroidogenesis ； corticosterone ； adrenocorticotropin hormone ； ACTH ； enzyme immunoassay ； EIA ； immunohistochemistry ； IHC

參考文獻

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