Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/13520
標題: 免疫調控對綠膿桿菌外毒素A誘發大鼠肝毒性之研究
Study of immunomodulation on Pseudomonas aeruginosa exotoxin A induced hepatotoxicity in rats
作者: 邱健昭
Chiu, Chien-Chao
關鍵字: Pseudomonas aeruginosa exotoxin A;綠膿桿菌外毒素A;cytokine;hepatotoxicity;細胞素;肝毒性
出版社: 獸醫學系暨研究所
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摘要: 
在醫院經常發生綠膿桿菌合併其他細菌性病原共同感染,尤其以細菌性腸炎、膽管炎與肝炎等為主。之前研究指出綠膿桿菌產生之毒素中最具毒性為綠膿桿菌外毒素A (Pseudomonas aeruginosa exotoxin A, PEA)。PEA造成實驗動物肝受傷主要是過度活化宿主免疫系統與抑制細胞蛋白質合成所導致。本研究嘗試使用Salmonella來源LPS活化大鼠免疫系統,以釐清臨床上沙門氏菌如何協同PEA致肝毒性的可能機制。另外,本研究藉由不同免疫抑制劑,進一步釐清PEA誘發肝毒性可能機制。再者,在許多免疫過度活化的肝受傷模式,可發現性別差異在致病機制上扮演著重要的角色,因此釐清PEA誘發大鼠肝毒性模式在不同性別造成的差異也是本試驗標的之ㄧ。綜合以上研究目的,本研究將設計以調控免疫反應來探討PEA致大鼠肝受傷之致病機轉。
第一部份為LPS協同PEA誘發多重器官傷害 (multiple organ injury, MOI):使用七至八週齡SPF Wistar雄性大鼠,分別投與對大鼠不具毒性之劑量如10、20或40 μg/kg Salmonella enterica serotype abortus equi來源的Lipopolysaccharide (LPS),於LPS i.v.給予後再立即注射sub-toxic劑量的10 μg/kg PEA。分別在不同時間分析血清生化值、組織病理切片及血清細胞素 (TNF-α、IFN-γ及IL-2)含量。結果顯示:1死亡率:不同劑量的LPS協同PEA誘發大鼠死亡率具有劑量上明顯的差異。2. 血清生化與組織病理:LPS/PEA共同投與可造成ALT、AST、BUN與CRE濃度增加,切片則可觀察到肝臟多發局部壞死灶和門脈區周圍壞死、腎臟近端腎小管核濃縮與肺臟間質增厚與炎症細胞浸潤。3. 細胞素:在TNF-α方面,可發現不同劑量LPS/PEA組TNF-α於攻毒後3小時開始揚昇,攻毒後6小時後開始下降,且揚升之濃度有劑量上之不同。在IFN-γ方面,相同為3小時開始至6小時,隨著不同劑量LPS/PEA其揚升之幅度有正相關性。在IL-2方面,由6小時揚升而持續至12小時為最高點而18小時後下降,各組揚升之濃度有顯著性差異。以上實驗結果發現,細胞素濃度之增減與動物死亡率、肝腎肺組織病理變化與血清生化值濃度呈正相關性。由於上述細胞素與Kupffer細胞與T細胞相關,在LPS/PEA攻毒前給予GdCl3將巨噬細胞剔除或給予FK506抑制T細胞功能,再進行血清生化及組織切片的檢查,發現無論將巨噬細胞剔除或抑制T細胞功能皆能夠有效降低MOI的嚴重度。由此證明LPS在強化PEA所致MOI的程度上,確實與免疫促進作用有相關。
第二部份不同免疫抑制劑對PEA誘發大鼠肝毒性的影響:本試驗使用七至八週齡SPF Wistar雄性大鼠,分別於致死劑量40 μg/kg PEA攻毒前不同時間點,投與全面性免疫抑制劑Dexamethasone、Kupffer細胞剔除劑GdCl3、TNF-α抑制劑PTX與T細胞抑制劑Cyclosporin與FK-506,分別在不同時間點觀察並犧牲動物。使用組織切片、血清生化、TUNEL染色及細胞素ELISA檢測評估並分析結果。結果顯示:1.動物死亡率:先投予Dexamethasone、FK-506、GdCl3或投與GdCl3+FK-506都可預防致死劑量PEA對動物所造成的死亡,而Cyclosporin與PTX則無法降低動物死亡情形。2.血清生化:投與Dexamethasone可完全抑制PEA造成肝傷害指標ALT及AST濃度上升,而投與FK-506或GdCl3可減輕PEA誘發大鼠肝毒性,而投與GdCl3+FK-506後之ALT及AST資料顯示,二者合用具有相加的抑制作用。3.組織病理:投予Dexamethasone可預防PEA造成之廣泛性大區域壞死灶,而FK-506與GdCl3則可減輕PEA引起之大區域壞死灶,二者合用可大幅減輕組織的病變,而Cyclosporin與PTX皆無法抑制PEA造成之肝病變。4.TUNEL染色:肝細胞陽性數經計數後與組織病理嚴重度之結果相符合。5.給予anti-TNF-α抗體與anti-IL-2抗體:以上兩種抗體皆可有效降低PEA誘發之肝傷害。綜合上述結果,發現無論是全面抑制免疫系統,或是抑制Kupffer細胞與T細胞,或是中和其主要分泌之細胞素都能有效降低PEA所引起的肝受傷。
第三部份不同性別對PEA誘發大鼠肝毒性的影響:本試驗使用七至八週齡SPF Wistar雄性與雌性大鼠,分別給予致死劑量40 μg/kg PEA,在攻毒後48小時進行臨床觀察。而在血清生化值、組織病理與細胞素濃度的評估上,於12與24小時犧牲動物並採樣。結果顯示:臨床觀察發現,給予PEA後可造成100%雄性大鼠於21至34小時內死亡,而雌性大鼠死亡時間則為30至39小時。而肝臟組織病理變化與血清生化值顯示:雄性大鼠試驗組傷害程度皆高於雌性試驗組。血清中細胞素濃度顯示:雄性大鼠在TNF-α、IL-2與IL-6皆高於雌性試驗組,但是在IL-10則雌雄試驗組無顯著性差異。進ㄧ步將雄性與雌性大鼠進行去勢,則發現PEA誘發之肝毒性無論在血清生化與組織切片等分析上,在兩組間皆無顯著性差異。由以上結果得知,PEA誘發不同性別大鼠肝毒性之差異,推測為不同性荷爾蒙影響免疫反應所導致。未來需進一步分析性荷爾蒙的種類及濃度變化,以釐清其在PEA誘發致肝毒性的角色為何。
結論:本實驗發現PEA誘發大鼠之肝毒性,確實與免疫功能過度活化有相關性,其肝毒性之增減,確實可受到免疫調節相關藥物的調控。並証明抑制巨噬細胞與T細胞,或中和主要分泌的細胞素,皆能降低PEA致肝傷害,甚或前述細胞素在雄雌大鼠濃度不同,而使PEA致肝毒性有性別差異。因此,本研究證明PEA造成大鼠的肝毒性應與免疫調控有關,並推測巨噬細胞與T細胞可能扮演重要角色。

Pseudomonas aeruginosa and Salmonella spp. are gram-negative bacteria that are two most frequently identified microorganisms in hosts suffering bacterial enteritis, cholangitis and hepatitis. P. aeruginosa is an opportunistic microorganism. Exotoxin A (PEA) is the most potent virulence factor produced by the P. aeruginosa. It was used in these studies. In the first experimentation, we investigated the role of inflammatory mediators in synergistic reaction to coexposure of non-injurious doses Salmonella Lipopolysaccharide (LPS) plus sub-hepatotoxic PEA that lead to multiple organ injury (MOI). In the second experimentation, we applied different immuno-suppressors to investigate the mechanism of PEA-induced hepatotoxicity in rats. In the third experimentation, gender-related difference was investigated in the PEA-induced liver injury.
First experiment: Coexposure of LPS and PEA for induction of MOI in rats. We examined the combination of non-injurious doses of LPS and sub-hepatotoxic PEA for the induction of MOI. Rats treated with 10, 20 or 40 μg/kg LPS plus 10 μg/kg PEA developed severe liver, kidney, and lung injury; elevation of TNF-α, IFN-γ, and IL-2; and high mortality. Attenuation of MOI was observed in rats by pretreatment with Gadolinium Chloride or FK506 for the depletion of Kupffer cells or inhibition T-cells, respectively. In summary, LPS+PEA acted synergistically on Kupffer cells and T-cells to induce proinflammatory cytokines that contributed to MOI.
Second experiment: Treatment of different immuno-suppressor for reduction the PEA-induced hepatotoxicity in rats. This study was designed to investigate the playing role of T-cells and Kupffer cells in 40 μg/kg PEA-induced hepatotoxicity model. The pretreatment of Dexamethasone, Gadolinium Chloride, cyclosporin or FK506 at different times before PEA challenge showed that Dexamethasone, Gadolinium Chloride and FK506 prevented animal death, inhibited pathologic alteration and decreased clinical chemistry levels in the PEA-treated rats. Moreover, rats pretreated with anti-TNF-α and anti-IFN-γ antibody also prevented PEA-induced liver injury. In summary, the study demonstrated that inhibition of immune system, T-cells and Kupffer cells, or neutralized cytokine reduced the PEA-induced liver injury.
Third experiment: To investigate the presence of gender-related differences on the PEA-induced liver injury and cytokine production in rats. Male and female Wistar rats were given PEA (40 μg/kg) intravenously at 7 weeks of age. Results showed that the serum ALT and AST levels in males were significantly higher at 24 hours after PEA administration than those of females. The serum levels of TNF-α, IL-2 and IL-6 also significantly elevated in males. Those makers had no different effects seen in rats by an orchiectomy in comparison with ovariectomy. These findings indicated that PEA induced more sever liver injury in male rats than in female rats that suggested the effect of sex hormones on cytokine production may play a role in gender-related difference on PEA-induced liver injury.
In conclusion, these findings demonstrated that PEA-induced liver injury was correlated with hyper-immune reaction, and regulated by immuno-modulation drugs. We also demonstrated that depletion of Kupffer cells, inhibition of T-cells or neutralized inflammatory cytokines could prevented PEA-induced liver injury. In addition, we clarified gender-difference in PEA-induced liver injury. Therefore, we suggested that the immune reactions play an important role in PEA-induced hepatotoxicity in rats.
URI: http://hdl.handle.net/11455/13520
其他識別: U0005-1001200910483200
Appears in Collections:獸醫學系所

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