Effect of Vitamin E on the Morphology of Cultured Rat Hepatocytes Under Oxidative Stress is Related to Thiol Groups of Cytoskeletal Proteins

• Main Authors: Kuo, Jyh-Huang, 郭智宏
• Other Authors: Lii Chong-Kuei
• Format: Others
• Language: zh-TW
• Online Access: http://ndltd.ncl.edu.tw/handle/32927662025124398977

碩士 === 中山醫學院 === 營養科學研究所 === 84 === 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.