維生素E與氧化緊迫下老鼠初代培養肝細胞細胞形態及細胞架構蛋白變化關係之探討

氧化緊迫 (oxdative stress) 造成肝細胞傷害，甚至死亡的過程 中，我們可以從顯微鏡下觀察到細胞形態發生變化產生發泡現象 (blebbing)。讓我們感興趣的是造成細胞發泡的可能原因。除此 外，由於發泡現象發生在細胞膜上，那麼存於膜內的抗氧化分子 維生素E是否可以改善細胞膜發泡現象及維持架構形態？所以本 實驗首先即針對維生素E與氧化緊迫下，脂質過氧化情形、細胞 硫醇 (thiols) 流失、細胞發泡現象之間的關係做探討；其次再探 討細胞產生發泡是否進一步涉及本身細胞架構 (cytoskeleton) 的改變 ，維生素E是否透過保護細胞架構蛋白質免於氧化傷害來達到保 護細胞外表結構之目的。第一部份結果顯示：補充維生素E肝細胞， 在添加1.5mM或3mM t-butyl hydroperoxide (t-BuOOH) 60分鐘內，細胞 脂質過氧化 (TBARS) 受到完全抑制；至於細胞膜蛋白質硫醇 (membrane protein thiol) 則不管有無維生素E均有流失現象，但添加維 生素E組流失量僅為不添加維生素E組流失量的50%；至於細胞形態 變化方面，1.5mM t-BuOOH的處理下，未補充維生素E組肝細胞，在 1.5mM t-BuOOH處理15分鐘時，已有顯著的發泡現象，60分鐘後， 可見發泡破裂，胞內物質釋出，此時細胞死亡率 (LDH leakage) 達80% 以上。添加維生素E細胞則直到30分鐘時，才可見少數細胞膜產生發 泡。60分鐘後，細胞發泡變大，但尚未破裂，細胞死亡率也僅增加至 10%，可見發泡現象在脂質過氧化發生前即以發生；顯示過氧化緊迫 下，細胞形態受損似乎與脂質過氧化無直接關係，而和細胞膜蛋白質硫 醇的流失較有關係，並且維生素E的補充可以延緩發泡現象。第二部 份實驗利用SDS-聚丙烯醯板膠電泳法分析1.5mM t-BuOOH處理下之 細胞周邊架構蛋白-肌動蛋白 (actin) 變化情形，發現單體肌動蛋白 (monomeric actin) 在氧化緊迫時，其濃度顯著減少，並伴隨高分子量聚 合物產生，然而添加維生素E，則可減少肌動蛋白發生聚合反應。螢 光顯微鏡下，可觀察到細胞膜肌動蛋白分佈隨發泡而改變。發泡破裂 處，肌動蛋白呈現扭曲變形，更甚至形成不連續片段。另一細胞架構蛋 白-管蛋白 (tubulin)，則在細胞發泡時其變化較肌動蛋白來得較不顯 著，但在發泡破損時，也可見管蛋白纖維分佈突出破損處。肌動蛋白及 管蛋白構形發生改變的時間及程度，均可因添加維生素E而延緩。由 以上結果，我們認為維生素E並非只有抑制脂質過氧化的功能，更重 要的是其能維持細胞蛋白質硫醇及周邊架構蛋白的穩定，延緩細胞形態 改變。

A characteristic change in the surface morphology of hepatocytes, known as “plasma membrane blebbing,”occurs as an early sign of toxic injury. A relationship between the formation of surface blebs and a perturbation of normal cytoskeletal organ-ization is supported by the findings that two cytoskeleton toxins, cytochalasin B and phalloidin, cause bleb formation in hepatocytes. It is interisting us to know what cause cellular surface blebs under oxidative stress and how vitamin E protects such morphology abnormality. Primary culture rat hepatocytes were preincubated with or without 100mM vitamin E (a-tocopherol succinate, TS) for 24 hr before treating with 1.5 or 3.0mM t-butyl hydroperoxide (t-BuOOH). Samples collected at 0, 15, 30, 60 minutes following t-BuOOH treatment were used for analysis of lipid peroxidation (TBARS), cell viability (LDH leakage), membrane protein thiol deletion, and SDS-polyacryamide gel electrophoresis (SDS-PAGE). Changes of cell morphology and cytoskeleton distrubution were mointered by phase contrast inverted microscope and fluorescent miscroscope, respectively. In the first part of this study, lipid peroxidation and cell damage were inhibited in cells preincubated with TS under oxidative stress. Neither preincubated with nor without TS, the deletion of membrane protein thiol were observed, but, the deletion at 60min was 2 fold in the absence than in the presence of TS (40% vs 20%). Although, TBARs production was completely inhibited in the presence of TS, surface blebs still occurred at 30min. In contrast, bleb formation was clearly observed at 15min in the absence of TS. Results suggest that TS can delay the formation of surface blebs under oxidative stress and this morphology abnormality is associated with protein thiols rather than with membrane lipid. In the second part of this study, we further investigated how TS delys surface bleb formation and what the role of cytoskeletal proteins in this protection. By fluorescence microscopy, actin network was shown to relate to (- tocopherol status as demonstrated by the early change in actin organization in cells in the absence of TS. However, less change in tubulin network was observed. Results from SDS-polyacrylamide gel electrophores further indicated that, under oxidative stress, actin molecules (45kDa) decreased and accompanied with the formation of high molecular weight molecules, which were reversible by the addition of dithiothreitol. The loss of actin showed a time-dependent response and could be observed after 15min with t-BuOOH treatment either in the presence or absence of TS, but the extent was much more siginificant in cells with no TS. The deletion of total membrane protein thiols was also greater in cells with no TS. Cell damage, as determined by lactate dehydrogenase (LDH) leakage, was protected by TS as compared to rapid increase of LDH leakage in cells in the absence of TS over 120 min with t-BuOOH treatment. These results indicate that vitamin E protects cell morphology by inhibiting surface bleb formation is related to actin molecules and the thiol groups in actin probably play a key role in cytoskeleton protection.