Paraquat 和 Diquat 對老鼠肝中脂質過氧化及蛋白質glutathionation的影響

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Title:	Paraquat 和 Diquat 對老鼠肝中脂質過氧化及蛋白質glutathionation的影響 The effect of paraquat and diquat on rat liver lipid peroxidation and protein S-glutationation
Authors:	王世宗
Contributors:	中山醫學大學：醫學研究所;李宗貴
Keywords:	rats, glutathione, protein mixed-disulfides, paraquat, diquat rats, glutathione, protein mixed-disulfides, paraquat, diquat
Date:	1995
Issue Date:	2010-07-20T02:58:03Z (UTC)
Abstract:	為應用一可準確分析蛋白質與GSH發生mixed-disulfides(glutathionation)的方法於動物實驗(in vivo)中，來分析當老鼠處於過氧化緊迫(oxidative stress)時蛋白質的變化。本計畫利用等電點電泳法(isoelectric focusing)可分離不同等電點蛋白質的特性，結合免疫西方墨點法(Immuno-Western blotting)的敏感性及專一性，提供一較傳統方法準確且省時的選擇。Carbonic anhydrase III (CA III)是依在雄性老鼠肝細胞中含量高且可發生glutathionation的細胞質蛋白質，所以在本實驗中被選為指標分子。過氧化緊迫分別以兩種廣效性殺草劑paraqauat(PQ)和diquat(DQ)經腹腔注射誘發。首先，CA III 利用兩步驟的DAEA-triscaryl M ionexchange chromatography 純化出，其純度最少95%。其次，CA III 抗血清則經由兔子來生成。經測試，利用此方法偵測CA III的靈敏度可達20ng。老鼠在經PQ以20或40mg/ng bw或DQ以85或170mg/kg bw的劑量腹腔注射5、15、40、120、240分後分別犧牲取樣。Thiobarbituric acid-reactive substances(TBARS)均於PQ或DQ注射後顯著增加並呈現time-dependent response。血漿glutamate pyruvate transaminase(PGPT)活性，則分別在低劑量DQ注射後240分鐘或高劑量DQ注射後40分鐘開始顯著上升(p<0.05)，至於接受PQ處理老鼠，其PGPT則僅於高劑量PQ(40mg/kg bw)處理240分鐘後，顯著較對照組為高(p<0.05)。肝細胞內GSSG濃度則不受PQ或DQ影響。還原態GSH及total GSH濃度在PQ高劑量處理240分鐘後顯著較對照組為低(p<0.05)，在高劑量DQ處理組則經120分鐘後，肝細胞內還原態GSH及total GSH濃度也顯著較低(p<0.05)。CA III glutathionation在所有處理組中均無顯著差異。由TBARS增加結果顯示是，PQ和DQ確實在肝細胞中誘發氧化傷害，至於無法偵測到CA III glutathionation的發生，根據我們先前實驗結果(Lii,1993)，可能是由於glutathionation CA III 發生dethiolation的緣故，導致glutathionation CA III 在實驗中無法偵測到有增加情形。 A newly developed method which combining isoelectric focusing (IEF) and immunostaining is successful in analyzing protein-GSH mixed disulfides formation (protein glutathionation) in rat hepatocytes under oxidative stress. So far, several methods are widely used in determining protein mixed disulfide formation, such as radioisotope-based method and sodium borohydride reduction method, but they may limit in sensitivity, specificity, and/or be a time consuming work. Therefore, this new method provides us an alternative and a powerful tool in this area. In the present syudy, we try to apply this technique to mrature carbonic anhydrase III (CA III)-GSH mixed disulfide formation in rat liver by administratung PQ and DQ to induce oxidative stress. Firstly, CA III was purified from rat liver by a two steps DEAE-trisacyl M ion-exchange chromatotography and the finl purity was at least 95%. Then, CA III antiserum was produced by immunization of rabbits with this purified CA III . By this method, as little as 20ng of CA III could be clearly identified. Rat were given intraperitoneally either prarquat at 20 or 40 mg/kg bw or diquat at 85 or 170 mg/kg bw and sacrifice was performed 5,15,40,120 and 240 mins, respectively, after administration. PQ and DQ significantly increased the thiobarbituric acid- reactive substances (TBARS) production and showed a time-dependent response. The significant effect on plasma glutamate-pyruvate transaminase(GPT) activity was also obtained and depeded on tiome and dosage. GPT leakage was significantly increased (p<0.05) at 240 min in rats dosed with 85 mg/kg bw of DQ or begining at 40 min in those dosed with 170mg/kg of DQ. However, this significant increase of GPT by PQ was only seen at treated with 40mg/kg bw at 240 min. Hepatic cellular GSSH content was tended to decrease with PQ and DQ treatments. A significant decrease of reduced GSH and total GSH were observed in rats dosed with high dosage of DQ at 120 min and dosed with high dosage of PQ after 40 mon. In spite of the fact of increasing oxyradicals generation as indicated by TBARS. No detectable change of CA III glutathionation was observed. Based on the dethiolatable characteristic of S-glutathionated CA III(Lii and Hendich, 1993), we, thus, propose that the occurance of S-glutathionation of CA III was probably offseted by this reversible reaction and resulted in this protein modification nondetectable.
URI:	https://ir.csmu.edu.tw:8080/handle/310902500/1799
Appears in Collections:	[Institute of Medicine] Electronic Theses of Dissertations

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