| Summary: | 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.
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