| Summary: | 碩士 === 國立臺灣大學 === 藥理學研究所 === 86 === Summary
1. The possible mechanisms of apoptosis-inducing effect of
saikosaponin-d, a triterpene saponin from Bupleurum falcatum L.,
were studied in human CEM lymphocytes (CEM cells) and compared
with those of dexamethasone.
2. The DNA synthesis was determined from the uptake of
tritiated thymidine. At the concentration range of 10-8 to 10-5
M, saikosaponin-d inhibited serum-stimulated [3H]thymidine
incorporation of CEM cells in a concentration-dependent manner.
The IC50 value was 3.1(0.4(10-6 M and the maximal inhibition was
100.0(0.0 % at 10-5 M. Dexamethasone (10-5 M) also inhibited
serum-stimulated [3H]thymidine incorporation (93.0(3.3 %
inhibition).
3. Cell viability, as determined by the trypan blue dye
exclusion method, was unaffected by 10-6 M saikosaponin-d in CEM
cells. However, the number of viable cells after saikosaponin-d
treatment at higher concentrations (10-5-10-4 M) was
significantly reduced to less than the basal value (2(105
cells). Dexamethasone (10-5 M, 48 h) also significantly reduced
the number of viable cells.
4. Following saikosaponin-d (10-6-10-4 M) treatment, analysis
utilizing flow cytometry of propidium iodide stained cells
revealed an increase in the percentage of cells in apoptotic
region. The percentages of CEM cells in an apoptotic region
were significantly increased to 35.0(2.5 % by 48 h treatment
with 10-5 M saikosaponin-d. Dexamethasone (10-5 M, 48 h) also
significantly increased the percentage of apoptotic cells to
24.5(1.7 %.
5. The apoptotic effect of saikosaponin-d was also demonstrated
by TdT-mediated dUTP nick end labeling (TUNEL) analysis, and DNA
laddering. After 48 h exposure, saikosaponin-d (10-6 to 10-4 M)
induced apoptosis in CEM cells. Saikosaponin-d induced cell
shrinkage, chromatin condensation, and DNA fragmentation.
Dexamethasone (10-5 M, 48 h) also induced apoptosis in CEM
cells. 6.
By TUNEL staining and methyl green-counterstaining, CEM cells
untreated were negative staining (TUNEL-negative, green-
colored). However, a large number of TUNEL-positive (brown-
colored) cells were found after treatment with 10-6, 10-5 or
10-4 M saikosaponin-d. The TUNEL-positive cells were also found
after dexamethasone (10-5 M, 48 h) treatment.
7. After treatment of CEM cells with saikosaponin-d (10-5 to
10-4 M), a typical DNA fragmentation ladder was found in DNA gel
electrophoresis. By contrast, the pattern of fragmentation was
not visible in cells untreated or treated with lower
concentration of saikosaponin-d (10-6 M). Dexamethasone (10-5
M, 48 h) also induced DNA laddering.
8. The possible mechanism of the apoptotic effect of
saikosaponin-d was further studied. The levels of c-myc, p53,
and bcl-2 mRNA were analyzed using a reverse transcription-
polymerase chain reaction (RT-PCR) technique. The levels of c-
myc and p53 mRNA were significantly increased and the level of
bcl-2 mRNA was significantly reduced by saikosaponin-d (10-5 M)
treatment. Dexamethasone (10-5 M) also increased p53 mRNA and
reduced c-myc and bcl-2 mRNA level. However, the alteration of
the p53, c-myc and l-2 mRNA by dexamethasone did not achieve
significance.
9. Our results demonstrate that saikosaponin-d induced cell
apoptosis in CEM cells. Saikosaponin-d may be useful as a
template for the development of immunosuppressive agents or
drugs to prevent the pathological changes of atherosclerosis.
Our results suggest that the apoptotic effect of saikosaponin-d
may partly be mediated through increasing c-myc and p53 mRNA
levels and reducing bcl-2 mRNA level.
Summary
1. The proliferaion-inhibitory and apoptosis-inducing effect of
saikosaponin-d, a triterpene saponin from Bupleurum falcatum L.,
were studied in rat thoracic aortic smooth muscle cells (A7r5
cells).
2. The DNA synthesis was determined from the uptake of
tritiated thymidine. At the concentration range of 10-6 to 10-4
M, saikosaponin-d inhibited serum-stimulated [3H]thymidine
incorporation of A7r5 cells in a concentration-dependent manner.
The IC50 value was 3.1(0.7(10-6 M.
3. The membranous protein tyrosine kinase activity of A7r5
cells were determined. The membranous protein tyrosine kinase
activities stimulated by 5 % FBS in A7r5 cells were reduced by
saikosaponin-d (10-5-10-4 M) in a concentration-dependent
manner.
4. Cell viability, as determined by the trypan blue dye
exclusion method, was unaffected by 10-6 M saikosaponin-d and
10-5 M dexamethasone in A7r5 cells. However, the number of
viable cells after saikosaponin-d treatment at higher
concentrations (10-5-10-4 M) was reduced to less than the basal
value (2.9(105 cells). 5.
Following saikosaponin-d (10-5-10-4 M) treatment, analysis
utilizing flow cytometry of propidium iodide stained cells
revealed an increase in the percentage of cells in apoptotic
region. The percentages of A7r5 cells in an apoptotic region
were increased to 19.0 %, 99.3 % by 48 h treatment with 10-5 M,
10-4 M saikosaponin-d, respectively. In contrast, dexamethasone
(10-5 M, 48 h) had no effect on apoptosis-induction.
6. Our results demonstrate that saikosaponin-d inhibited cell
proliferation and induced cell apoptosis in A7r5 cells. In
addition to the apoptosis-inducing effect in CEM cells,
saikosaponin-d may be useful as a template for the development
of immunosuppressive agents or drugs to prevent the pathological
changes of atherosclerosis. Our results suggest that the
proliferation-inhibitory effect of saikosaponin-d in A7r5 cells
may partly be mediated through reducing membranous protein
tyrosine kinase activity.he other possible mechanisms of
saikosaponin-d induced cell apoptosis in A7r5 cells is under
further study in our laboratory.
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