Protective effects of s propargylcysteine (SPRC) on in vitro neuronal damage induced by amyloid beta (25 35 6 7

PART III: FIBRILLAR Aβ
145

CHAPTER 6: FIBRILLAR Aβ
25-35

PRE-TREATMENT WITH SPRC REDUCED DAMAGE INDUCED BY Aβ
25-35
FIBRILS
6.1 Results
6.1.1 Effects of incubation time on cell viability

Figure 42: Effects of different times of incubation with aggregated Aβ
25-35
on cell viability,
expressed as percentage of cell viability compared to the untreated control ± S.E.M. N= 3, *:
p<0.05 and **: p<0.01 when compared to the untreated control.
To test the cytotoxicity of aggregated Aβ
25-35
incubated at 37°C for 24 hours, the Aβ
25-35
was fixed at a dose of 1 µM and incubated with the cells for different lengths of time (Figure 42).
The cell viability of C6 cells decreased with increasing length of incubation. At 3 hours, 1 µM
Aβ
25-35
could already significantly reduce cell viability to 83 ± 3.5% (p<0.05). By 16 hours of
incubation, the cell viability was decreased to 61 ± 0.7% (p<0.01). After 24 hours of incubation,
the cell viability was 66 ± 0.3% (p<0.01), suggesting that the effect of the aggregated Aβ
25-35
had
reached a maximum. Since both 3- hour and 16- hour incubation both resulted in significant

toxicity, these two time points were further investigated with pre-treatment of drugs.
0
0.2
0.4
0.6
0.8
1
1.2
1 3 6 16 24
Cell viability (% control)
Time of incubation/h
Without Aβ 1 µM With Aβ 1 µM
*
*
**
**
PART III: FIBRILLAR Aβ
146

6.1.2 Effects of drugs on Aβ
25-35
-induced cytotoxicity
6.1.2.1 SPRC on cell viability
Figure 43: Effects of SPRC pre-treatment on Aβ-induced cytotoxicity. (a) 24-hour SPRC pre-
treatment on different incubation times of Aβ1 µM. (b) 24-hour pre-treatment of 0.1 µM -1 mM
SPRC restored Aβ-induced cytotoxicity significantly, but did not result in any toxicity in
untreated cells. The values are percentages of cell viability compared to the untreated control ±
S.E.M. N=6, #: p<0.05 when compared to the untreated control; **: p<0.01 when compared with
the Aβ-only control.
Since treatment with 1 µM aggregated Aβ

25-35
resulted in comparable cytotoxicity, both
time points were used to investigate the effects of SPRC on cell viability. Different doses of
SPRC were pre-treated to C6 cells for 24 hours before treating with 1 µM aggregated Aβ
25-35
for
another 3 or 16 hours (Figure 43a). Cells pre-treated with only SFM for 24 hours followed by
Aβ
25-35
treatment for 3 or 16 hours resulted in 83 ± 2.6% (p<0.01) and 61 ± 0.2% (p<0.01) cell
viability respectively. The longer length of incubation with Aβ
25-35
resulted in lower cell viability
expectedly, accounted for by the increased toxicity. Pre-treatment with SPRC slightly, but not
significantly, increased the cell viability in cells treated with Aβ
25-35
for 3 hours (F
(6,27)
= 5.556;
p<0.01). However, pre-treatment with SPRC significantly increased the cell viability in cells
treated with Aβ
25-35
for 16 hours (F
(6, 41)
= 60.668; p<0.01). The dose-dependent increase in cell
viability was steady from 0.1 µM to 1 mM. Cells pre-treated with 10 µM SPRC was effective
with Aβ 3 h or 16 h, resulting in 88 ± 1.0% and 93 ± 0.1% (p<0.01) of viable cells respectively.
50%
60%
70%

80%
90%
100%
110%
SFM SFM
SPRC
0.1 µM
SPRC
1 µM
SPRC
10 µM
SPRC
100 µM
SPRC
1 mM
Cell viability (% control)
3 h Aβ 1 µM
16 h Aβ 1 µM
1µM Aβ
25-35
#
#
**
**
*
*
**
*
*
50%

60%
70%
80%
90%
100%
110%
SFM
SPRC
0.1 µM
SPRC 1
µM
SPRC
10 µM
SPRC
100 µM
SPRC 1
mM
Cell viability (% control)
With Aβ 1 µM
Without Aβ 1 µM
#
**
**
**
**
**
(a) (b)
PART III: FIBRILLAR Aβ
147

The effects of the SPRC pre-treatment were hence only visible upon a longer incubation time
with Aβ. These doses were not toxic to the cells (Figure 43b), although very slight variations in
cell viability following SPRC treatment could be observed at 10 µM and 100 µM. Both doses
resulted in 102 ± 1.2% and 101 ± 4.1% cell viability respectively.
Hence, 24-hour pre-treatment with SPRC followed by 16-hour incubation with 1 µM
aggregated Aβ
25-35
was selected as the experimental model. Also, the dose of interest for SPRC
was fixed at 10 µM for subsequent studies.
6.1.2.2 SAC and NaHS on cell viability

Figure 44: Effects of SAC and NaHS pre-treatment on Aβ-induced cytotoxicity, expressed as
percentage of cell viability compared to the untreated control ± S.E.M. 24-hour 0.1 µM -1mM of
both drugs pre-treatment resulted in significant restoration of cell viability. N≥5, #: p<0.05 when
compared to the untreated control; *: p<0.05; **: p<0.01 when compared with the Aβ-only
control.
The cells were pre-treated for 24 hours with 0.1 µM- 1mM of either SAC or NaHS before
16 hours of Aβ insult (Figure 44). Both drugs resulted in a dose-dependent restoration of cell
viability, although the increase in viable cells was much more obvious in SAC-treated cells. SAC
treatment increased the number of viable cells significantly for all treatment doses (F
(7, 46)
=
14.027; p<0.01). Cell viability was generally restored to around 90% for all investigated doses,
50%
60%
70%
80%
90%
100%
110%

SFM SFM
0.1 µM 1 µM 10 µM 100 µM
1 mM
Cell viability (% control)
SAC NaHS
1µM Aβ
25-35
#
** **
**
**
**
**
**
**
**
*
PART III: FIBRILLAR Aβ
148

and the highest restoration was 94 ± 1.1% of control (p<0.01), observed at 100 µM. Even the
lowest dose of 0.1 µM resulted in a viability of 87 ± 1.2% of control (p<0.01), significantly
higher than that in the Aβ-only group.
Likewise, pre-treatment with 0.1 µM -1 mM NaHS increased cell viability significantly
across all doses (F
(7, 47)
= 11.02; p<0.01). However, the extent of restoration was lower than that
observed in either SPRC- or SAC-treated cells, in which generally cell viability hovered around
82%. Cells were most significantly increased to 85 ± 1.4% of control (p<0.01) after pre-
treatment with 100 µM NaHS.

6.1.2.3 Comparisons with equimolar concentrations of drugs

Figure 45: Comparison between equimolar concentrations of drugs on Aβ-induced cytotoxicity,
expressed as percentage of cell viability compared to the untreated control ± S.E.M. Pre-treating
cells with 10 µM of SPRC, SAC or NaHS restored viability significantly. N=6, #: p<0.05 when
compared to the untreated control; **: p<0.01 when compared with the Aβ control.
10 µM was used as a dose for comparison between the drugs in all ensuing studies as this
is the effective dose that resulted in restoration of cell viability following SPRC pre-treatment
(Figure 45). While pre-treatment with SPRC, SAC or NaHS resulted in significant differences in
viability (F
(4, 29)
= 28.070; p<0.01), SPRC pre-treatment could best restore Aβ-induced
cytotoxicity to 93 ± 0.6% of control (p<0.01), a large increase in viable cells. SAC pre-treatment
0%
20%
40%
60%
80%
100%
120%
SFM SFM
SPRC 10
µM
SAC 10
µM
NaHS 10
µM
Cell viability (% control)
#
**

**
**
1µM Aβ
25-35
PART III: FIBRILLAR Aβ
149

restored the cell viability slightly lower compared to the SPRC-treated cells, to 91 ± 1.1% of
control (p<0.01). Pre-treatment with 10 µM NaHS also significantly increased cell viability;
however, the extent of restoration was the weakest amongst all three drugs, about 84 ± 2.0% of
control (p<0.01).
Since pre-treatment with all three drugs could protect C6 glioma cells from subsequent
Aβ- induced injury, all three were included in following studies to further understand the
mechanisms of action.
6.1.3 Effects on H
2
S pathway
6.1.3.1 Effects on H
2
S content in cell medium

Figure 46: Effects of pre-treatment of drugs on H
2
S concentrations in cell medium. The values
are expressed as fold increase in concentration compared to the untreated control ± S.E.M. N=3,
#: p<0.05 compared to untreated control; *: p<0.05 compared to Aβ-only group.
The H
2
S concentration in the cell culture media changed significantly following Aβ
treatment (F

(4, 34)
= 5.498; p<0.01) (Figure 46). The Aβ treatment reduced the H
2
S concentration
in the culture media to 0.84 ± 0.04- fold compared to that in the untreated control (p<0.01). Pre-
treatment with 10 µM SPRC and NaHS restored the decline in H
2
S released to 1.02 ± 0.05- fold
(p<0.05) and 1.01 ± 0.06- fold (p<0.05) respectively. Contrary to that in Part II: Oligomeric Aβ,
0
0.2
0.4
0.6
0.8
1
1.2
SFM SFM
SPRC 10
µM
SAC 10
µM
NaHS 10
µM
Fold increase in H
2
S
concentration compared to
control
1µM Aβ
25-35

#
*
*
PART III: FIBRILLAR Aβ
150

pre-treatment of SAC did not change the H
2
S concentration in the culture media, maintaining the
H
2
S content at 0.79 ± 0.04- fold.
6.1.3.2 Effects on CBS expression






Figure 47: Effects of pre-treatment of drugs on CBS expression in cell lysates. (a) Representative
blots for CBS expression in treated cells. N=3. (b) Percentage fold difference in CBS expression
± S.E.M. N=3, #: p<0.05 compared to untreated control.
The Aβ
25-35
treatment significantly reduced the CBS expression (Figure 47) to 86 ± 0.4%,
from 100% in the untreated control cells and similar to that in Part II: Oligomeric Aβ. Pre-
treatment of 10 µM SPRC and 10 µM NaHS both restored the CBS expression to 92 ± 2.5% and
95 ± 4.3% respectively. SAC pre-treatment however, did not result in any observable change in
the CBS expression, remaining at 86 ± 1.8%.

50%
60%
70%
80%
90%
100%
SFM SFM
SPRC 10
µM
SAC 10
µM
NaHS 10
µM
Percentage fold difference
of CBS expression
(compared to control)
1µM Aβ
25-35
#
SFM SFM
SPRC
10 µM
SAC
10 µM
NaHS
10 µM
CBS
β-tubulin

1µM Aβ

25-35
(a)
(b)
PART III: FIBRILLAR Aβ
151

6.1.3.3 Effects of CBS inhibitor on cell viability




Figure 48: Effects of the CBS inhibitor AOAA on cell viability. (a) Dose dependence of 24h pre-
treatment of AOAA with and without 1µM Aβ
25-35
treatment. (b) Pre-treatment of 0.1-100 µM
SPRC restored the aggravated cytoxicity by AOAA on Aβ injury significantly. The values are
percentages of cell viability compared to the untreated control ± S.E.M. N>6, #: p<0.05 when
compared to the untreated control; &: p<0.05 when compared to the SFM + Aβ control; *:
p<0.05 when compared with the Aβ + 100 µM AOAA control.

Different doses of the CBS inhibitor aminoxyacetic acid (AOAA) were pre-treated to the
cells for 24 hours before the additional Aβ
25-35
insult for 16 hours (Figure 48a). Similar to Part II:
Oligomeric Aβ, there was a visible, significant dose-dependent aggravation in the cell viability
with increasing doses of AOAA. The Aβ-only control group resulted in cell viability of 64 ± 4.6%
(p<0.01) but this was aggravated to 50 ± 2.6% in the group with additional 100 µM AOAA
(p<0.05). While increasing doses of AOAA in normal cells without Aβ
25-35
did not significantly

decline in cell viability, there was a slight cytotoxicity at 100 µM AOAA with 87 ± 2.2%
viability (Figure 48a). 100 µM AOAA was again chosen as the investigation model for the CBS
inhibitor. Together with the 100 µM AOAA, increasing doses of SPRC from 0.1 µM -100 µM
were pre-incubated in the cells for 24 hours before 1 µM Aβ
25-35
was added to the cells. There
0%
20%
40%
60%
80%
100%
120%
SFM
0.1 µM 1 µM 10 µM 100 µM
Cell viability (% control)
Without Aβ1 µM
With Aβ1 µM
#
&
AOAA
0%
20%
40%
60%
80%
100%
120%
Cell viability (% control)
#

*
*
#,&
1 µM
Aβ
25
-
35

- + + + + + +
100 µM
AOAA
- - + + + + +
SPRC
(µM)
- - - 0.1 1 10 100

(a) (b)
PART III: FIBRILLAR Aβ
152

was a gradual, dose-dependent increase in cell viability following SPRC treatment (Figure 48b),
although the restoration in cell viability was not to a great extent as in Part II: Oligomeric Aβ.
The maximum restoration was observed in the pre-treatment from 10 µM SPRC onwards where
the Aβ-induced cytotoxicity was significantly increased from 50 ± 3.0% in the Aβ + AOAA-
control to 59 ± 7.6% (p<0.05). Likewise, 100 µM SPRC also increased the cell viability to 60 ±
7.2% (p<0.05) significantly. Even with SPRC pre-treatment, the cell viability could not be
restored to the levels of the Aβ-only control.
6.1.4 Effects on oxidative stress
6.1.4.1 Production of DCF-DA

Figure 49: Effects of pre-treatment of drugs on DCF production, presented as percentage control
± S.E.M. N=5, #: p<0.05 compared to untreated control; *: p<0.05, **: p<0.01 compared to Aβ-
only group.
Aβ treatment drastically increased the oxidative stress seen in the increased DCF
produced (Figure 49) to 214 ± 15% compared to the 100% in the untreated control (p<0.01).
This is similar to Part II: Oligomeric Aβ and the oxidative stress was also significantly altered (F
(4, 24)
= 10.34; p<0.01). After pre-treatment with the various drugs, the oxidative stress induced
by Aβ
25-35
in the cells was generally reduced. Pre-treatment with 10 µM SPRC significantly
0%
50%
100%
150%
200%
250%
SFM SFM
SPRC 10
µM
SAC 10
µM
NaHS 10
µM
Units of DCF produced per min
(% control)
1 µM Aβ
25-35
#

**
**
*
PART III: FIBRILLAR Aβ
153

reduced the DCF produced to 128 ± 12% (p<0.01). Likewise, the pre-treatment with 10 µM
SAC significantly reduced the DCF produced to 112 ± 13% (p<0.01). Pre-treatment with NaHS
also significantly reduced the production of DCF to 137 ± 12% (p<0.05).
6.1.4.2 DHE staining



I II
III IV
(a)
PART III: FIBRILLAR Aβ
154


Figure 50: Effects of pre-treatment of drugs on DHE fluorescence. (a) Representative photos of
DHE-stained cells under 20X magnification with a scale bar of 100 µm indicated at the lower left
of each panel. I: SFM-only control; II: 1 µM Aβ-only control; III: 10 µM SPRC + 1 µM Aβ; IV:
10 µM SAC + 1 µM Aβ; V: 10 µM NaHS + 1 µM Aβ. (b) Fold difference of fluorescence units
as calculated from the DHE staining ± S.E.M. N=4, #: p<0.05 compared to untreated control.
The free radicals levels visualized in cells were decidedly different following Aβ
treatment (F
(4, 19)
= 3.810; p<0.05) (Figure 50a). Aβ
25-35

typically increases the intracellular free
radical levels that contribute to increased oxidative stress, similarly observed using the dye DCF-
DA presented in Part 6.1.4.1. As such, the panels treated with Aβ
25-35
(Figure 50a, Panels II-V)
showed stronger red fluorescence compared to the untreated control (Figure 50a, Panel I), where
weak red fluorescence represented the background free radical levels. The Aβ-only group
recorded a significantly higher 1.26 ± 0.07- fold of red fluorescence (p<0.05) that were reversed
by pre-treatment of any drugs (Figure 50b). While all drugs slightly reduced the fold differences,
none were significant statistically. SPRC pre-treatment resulted in a 1.18 ± 0.06-fold, SAC pre-
treatment resulted in a 1.18 ± 0.05-fold (p=N.S) and NaHS pre-treatment resulted in a 1.22 ±
0.05-fold (p=N.S).


0
0.2
0.4
0.6
0.8
1
1.2
1.4
SFM SFM
SPRC 10
µM
SAC 10
µM
NaHS 10
µM
Fold difference of DHE

fluorescence units
#
1 µM Aβ
25-35
V
(b)
PART III: FIBRILLAR Aβ
155

6.1.4.3 Expression of antioxidant enzymes






Figure 51: Effects of pre-treatment of drugs on SOD-1 expression in cell lysates. (a)
Representative blots for SOD-1 expression in treated cells. (b) Percentage fold difference in
SOD-1 expression ± S.E.M. N=6. (c) Effects of pre-treatment of drugs on total SOD activity.
The values are expressed as a percentage of the activity in the untreated cells ± S.E.M. N=5, #:
p<0.05 compared to untreated control; *: p<0.05, **: p<0.01 compared to Aβ-only group.
The SOD-1 expression in the cells changed significantly after the various drug treatments
(F
(4, 20)
= 23.336; p<0.05), suggesting an alteration to the amounts of SOD-1 in the cell lysates
(Figure 51a). Aβ-only treatment significantly reduced the SOD-1 expression to 0.80 ± 0.02-fold
compared to the untreated control (p<0.01) (Figure 51b), accounting for the oxidative stress
observed. The pre-treatment of all three drugs positively up-regulated the SOD-1 expression; and
SAC restored the levels most greatly to 1.02 ± 0.01-fold (p<0.01). Pre-treatment of SPRC and
NaHS both restored the SOD-1 levels to 0.96 ± 0.02-fold (p<0.01) and 0.96 ± 0.01-fold (p<0.01)

respectively. The total SOD activity in cells was significantly reduced to 69 ± 7.8% after Aβ
25-35
treatment (p<0.05) (Figure 51c), contributing to the decrease in antioxidant protection and higher
0
0.2
0.4
0.6
0.8
1
1.2
SFM SFM
SPRC
10 µM
SAC 10
µM
NaHS
10 µM
Fold difference in SOD-1
expression
#
**
**
**
1 µM Aβ
25-35
0%
20%
40%
60%
80%

100%
120%
SFM SFM
SPRC
10 µM
SAC 10
µM
NaHS
10 µM
SOD Activity (% control)
#
*
*
*
1µM Aβ
25-35
SFM SFM
SPRC
10 µM
SAC
10 µM
NaHS
10 µM

1 µM Aβ
25-35

(a)
SOD-1
β-tubulin

(b)
(c)
PART III: FIBRILLAR Aβ
156

oxidative stress observed. The total SOD activity changed significantly after the various drug
treatments as calculated from one-way ANOVA (F
(4, 24)
=5.677; p<0.01). Pre-treatment with all
three drugs significantly restored the total SOD activity comparable to the untreated control, and
the effects were equal amongst the drugs. This trend was similar to that observed in the SOD
protein expressions. SPRC pre-treatment significantly restored the SOD activity to 91 ± 3.4%
(p<0.05) while SAC pre-treatment significantly restored the activity to 92 ± 6.7% (p<0.05).
NaHS pre-treatment also restored the activity to 93 ± 2.2% (p<0.05).





Figure 52: Effects of pre-treatment of drugs on catalase expression in cell lysates. (a)
Representative blots for catalase expression in treated cells. (b) Percentage fold difference in
catalase expression ± S.E.M. N=6. (c) Effects of pre-treatment of drugs on catalase activity. The
values are expressed as units of activity/µg protein ± S.E.M. N≥3. #: p<0.05 compared to
untreated control; *: p<0.05 compared to Aβ-only group; **: p<0.01 compared to Aβ-only
group
The catalase expression following Aβ
25-35
treatment was significantly altered within the
different drug groups (F

(4, 17)
= 7.76; p<0.01) (Figure 52a). Catalase is slightly but significantly
up-regulated after the Aβ-only treatment to 1.07 ± 0.01-fold (p<0.05) (Figure 52b). Pre-
0
0.2
0.4
0.6
0.8
1
1.2
1.4
SFM SFM
SPRC
10 µM
SAC 10
µM
NaHS
10 µM
Fold difference in
catalase expression
1 µM Aβ
25-35
#
**
*
0
0.01
0.02
0.03
0.04

0.05
0.06
SFM SFM
SPRC
10 µM
SAC 10
µM
NaHS
10 µM
Catalase activity
(U/ug protein)
#
1 µM Aβ
25
-
35
SFM SFM
SPRC
10 µM
SAC
10 µM
NaHS
10 µM
1 µM Aβ
25-35

Catalase
β-tubulin

(a)

(b) (c)
PART III: FIBRILLAR Aβ
157

treatment of 10µM SPRC significantly reversed this Aβ-induced up-regulation to 0.84 ± 0.004-
fold (p<0.01). While the same trend was observed following SAC pre-treatment, the decrease to
0.85 ± 0.12- fold was not statistically significant (p=N.S). In contrast, NaHS pre-treatment
resulted in even higher expression of catalase (1.27 ± 0.04- fold; p<0.01) that suggested a
different mode of regulation on this antioxidant enzyme compared to other drugs of interest.
Catalase activities were measured spectrophotometrically and were significantly increased after
all drug treatments (F
(4, 16)
= 8.572; p<0.01) (Figure 52c). There was significant increase after
Aβ treatment to 0.036 ± 0.001 U/µg protein (p<0.05), from the 0.019 ± 0.001 U/µg protein in the
untreated control group. Interestingly, pre-treatments with the various drugs further increased the
catalase activity, most obviously seen in the SAC pre-treated group. Pre-treatment with SPRC
increased the activity to 0.043 ± 0.004 U/µg protein (p=N.S) while SAC pre-treatment increased
the activity to 0.048 ± 0.005 U/µg protein (p=N.S). NaHS pre-treatment also very slightly
increased the activity to 0.038 ± 0.006 U/µg protein (p=N.S). Although no significance was
observed in all pre-treated groups, the increase in antioxidant catalase activity may be an
inherent nature of the drugs to counter the increase in oxidative stress from the Aβ treatment.

PART III: FIBRILLAR Aβ
158

Figure 53: Effects of pre-treatment of drugs on glutathione peroxidase (GPx) expression in cell
lysates. (a) Representative blots for GPx expression in treated cells. (b) Percentage fold
difference in GPx expression ± S.E.M. N=6. (c) Effects of pre-treatment of drugs on GPx
activity. The values of activity are expressed as µmol/NADPH/min/mg protein ± S.E.M. N=3, #:
p<0.05 compared to untreated control; *: p<0.05 compared to Aβ-only group.
Drug treatments altered the expressions of GPx significantly (F
(4, 18)
= 6.054; p<0.01)
among the groups (Figure 53a). While the fold difference in GPx expression was significantly
lowered by Aβ-only treatment to 0.82 ± 0.05-fold (p<0.05) (Figure 53b), this decline in
expression was not restored by pre-treatment of any of the tested compounds. Pre-treatment with
10 µM SPRC resulted in 0.70 ± 0.04-fold expression (p=N.S) while 10 µM SAC resulted in a
comparable 0.73 ± 0.08-fold expression (p=N.S). Interestingly, 10 µM NaHS resulted in a
further dip in expression to 0.64 ± 0.09-fold (p=N.S). GPx activity changed significantly after
treatments with Aβ
25-35
and the drugs of interest (F
(4, 14)
= 14.246; p<0.01) (Figure 53c). The
activity decreased drastically after Aβ treatment, from 3.28 ± 0.25 µmol/NADPH/min/mg
0
0.2
0.4
0.6
0.8
1
1.2
SFM SFM
SPRC
10 µM

SAC 10
µM
NaHS
10 µM
Fold difference in GPx
expression
#
1 µM Aβ
25-35
0
0.5
1
1.5
2
2.5
3
3.5
4
SFM SFM
SPRC
10 µM
SAC 10
µM
NaHS
10 µM
GPx activity (µmol
NADPH/min/mg protein)
1 µM Aβ
25-35
#

*
SFM SFM
SPRC
10 µM
SAC
10 µM
NaHS
10 µM
(a)
1 µM Aβ
25-35

GPx
β-tubulin

(b)
(c)
PART III: FIBRILLAR Aβ
159

protein in the untreated control to 1.39 ± 0.18 µmol/NADPH/min/mg protein in the Aβ-only
control. There was a stark restoration in GPx activity following 10 µM SPRC pre-treatment to
3.38 ± 0.37 µmol/NADPH/min/mg protein (p<0.05) that was not observed in other groups. Pre-
treatment with SAC remained at 1.36 ± 0.38 µmol/NADPH/min/mg protein (p=N.S) and NaHS
resulted in an activity of 1.29 ± 0.41 µmol/NADPH/min/mg protein (p=N.S).

PART III: FIBRILLAR Aβ
160

6.1.5 Effects on inflammation

6.1.5.1 Expression of pro- inflammatory IL-1β

Figure 54: Effects of pre-treatment of drugs on IL-1β expression. (a) mRNA expression of IL-1β
expressed as fold difference of the untreated control group ± S.E.M. (b) Protein expression of IL-
1β expressed as concentration (pg/ml) ± S.E.M. N≥3, #: p<0.05 compared to untreated control;
**: p<0.01 compared to Aβ-only group.
The mRNA and protein expressions of the pro-inflammatory IL-1β altered significantly
after Aβ treatment. Aβ-only treatment significantly increased the mRNA expression of IL-1β to
7.08 ± 0.73-fold (p<0.01) and this increase was reversed by pre-treating the cells with 10 µM
SPRC or SAC (Figure 54a). SPRC pre-treatment reduced IL-1β mRNA expression to 3.27 ±
0.32-fold (p<0.01), and SAC pre-treatment resulted in an even lower expression of 2.43 ± 0.20-
fold (p<0.01). 10 µM NaHS pre-treatment slightly reduced the mRNA expression to 6.40 ± 1.01-
fold (p= N.S), although it was not statistically significant.
The concentration of IL-1β in the cell lysates after Aβ treatment was also significantly
increased from 1689 ± 84.7 pg/ml in the untreated control to 1945 ± 44 pg/ml in the Aβ-only
control (p<0.05) (Figure 54b). Pre-treatment with the drugs significantly decreased the IL-1β
expressions in a similar manner. 10 µM SPRC expressed the least pro-inflammatory IL-1β to
1484 ± 49 pg/ml (p<0.01). IL-1β expressions following SAC or NaHS treatments were
0
1
2
3
4
5
6
7
8
9
SFM SFM
SPRC

10 µM
SAC 10
µM
NaHS
10 µM
Fold difference of
IL-1β expression
#
**
**
1 µM Aβ
25-35
0
500
1000
1500
2000
2500
SFM SFM
SPRC
10 µM
SAC 10
µM
NaHS
10 µM
Concentration of
IL-1β (pg/ml)
1 µM Aβ
25-35
#

**
**
**
(a)
(b)
PART III: FIBRILLAR Aβ
161

comparable; SAC resulted in an expression of 1519 ± 58 pg/ml (p<0.01) and NaHS resulted in
an expression of 1513 ± 71 pg/ml (p<0.01).
6.1.5.2 Expression of pro- inflammatory IL-6

Figure 55: Effects of pre-treatment of drugs on IL-6 expression. (a) mRNA expression of IL-6
expressed as fold difference of the untreated control group ± S.E.M. (b) Protein expression of IL-
6 expressed as concentration (pg/ml) ± S.E.M. N≥3, #: p<0.05 compared to untreated control; *:
p<0.05 compared to to Aβ-only group.
Both the mRNA and protein expressions of the pro-inflammatory IL-6 were up-regulated
after Aβ treatment. The mRNA expression of IL-6 increased to 1.69 ± 0.11-fold (p<0.01) (Figure
55a). Of note, pre-treatment with drugs enhanced the mRNA expression of IL-6, especially after
SPRC pre-treatment. The IL-6 mRNA expression was 3.71 ± 0.68- fold, although there was no
statistical significance. Likewise, SAC and NaHS pre-treatments increased the mRNA
expressions to 2.29 ± 0.12- fold (p<0.05) and 1.85 ± 0.21- fold respectively.
The protein expression of IL-6 was also augmented by Aβ treatment, from 16.8 ± 4.4
pg/ml in the untreated control to 35.2 ± 4.8 pg/ml in the Aβ-only control (p<0.05) (Figure 55b).
This increase was not changed drastically by pre-treating with the drugs, although SAC pre-
treatment slightly increased the IL-6 protein expression. SPRC and NaHS pre-treatments resulted
in a comparable 36.6 ± 5.6 pg/ml expression (p= N.S) and 31.0 ± 0.7 pg/ml expression (p= N.S)
0
0.5
1

1.5
2
2.5
3
3.5
4
4.5
SFM SFM
SPRC
10 µM
SAC 10
µM
NaHS
10 µM
Fold difference of IL-6
expression
#
1 µM Aβ
25-35
*
0
10
20
30
40
50
SFM SFM
SPRC
10 µM
SAC 10

µM
NaHS
10 µM
Concentration of IL-6
(pg/ml)
#
1 µM Aβ
25-35
(a)
(b)
PART III: FIBRILLAR Aβ
162

respectively. Though not statistically significant, SAC pre-treatment resulted in a slightly higher
expression of 43.8 ± 2.5 pg/ml.
6.1.5.3 Expression of pro- inflammatory TNF-α

Figure 56: Effects of pre-treatment of drugs on TNF-α expression. (a) mRNA expression of
TNF-α expressed as fold difference of the untreated control group ± S.E.M. (b) Protein
expression of TNF-α expressed as concentration (pg/ml) ± S.E.M. N≥3, #: p<0.05 compared to
untreated control; *: p<0.05 compared to Aβ-only group **: p<0.01 compared to Aβ-only
group.
The mRNA and protein expressions of pro-inflammatory TNF-α were significantly
increased after Aβ treatment (Figure 56). The mRNA expression was increased to 2.20 ± 0.27-
fold in the Aβ-only group (p<0.05) and further increased after pre-treatment with the various
drugs (Figure 56a). Pre-treating the cells with 10µM SPRC or SAC slightly enhanced the TNF-α
mRNA expression to 2.50 ± 0.13- fold (p=N.S) and 3.10 ± 0.34- fold (p=N.S) respectively. Pre-
treatment with 10 µM NaHS however, drastically increased the mRNA expression to 4.71 ±
0.29- fold (p<0.01).
Likewise, the protein expression of TNF-α in the cell lysates was increased following Aβ

treatment, from 53.2 ± 7.3 pg/ml in the untreated control group to 88.9 ± 5.7 pg/ml in the Aβ-
only control (Figure 56b). While both SAC and NaHS pre-treatment did not affect the protein
expression, the concentration of TNF-α in the cell lysates following SPRC pre-treatment was
0
1
2
3
4
5
6
SFM SFM
SPRC
10 µM
SAC 10
µM
NaHS
10 µM
Fold difference of TNF-α
expression
#
1 µM Aβ
25-35
**
0
20
40
60
80
100
120

SFM SFM
SPRC
10 µM
SAC 10
µM
NaHS
10 µM
Concentration of TNF-α
(pg/ml)
#
*
1 µM Aβ
25-35
(a)
(b)
PART III: FIBRILLAR Aβ
163

decreased to 59.6 ± 9.1 pg/ml (p<0.05). SAC and NaHS pre-treatment resulted in 102.0 ± 7.1
pg/ml expression (p=N.S) and 94.9 ± 7.6 pg/ml expression (p=N.S) respectively.
6.1.5.4 Expression of anti-inflammatory IL-10

Figure 57: Effects of pre-treatment of drugs on IL-10 expression. (a) mRNA expression of IL-10
expressed as fold difference of the untreated control group ± S.E.M. (b) Protein expression of IL-
10 expressed as concentration (pg/ml) ± S.E.M. N≥3, #: p<0.05 compared to untreated control; *:
p<0.05 compared to Aβ-only group **: p<0.01 compared to Aβ-only group.
The expressions of the anti-inflammatory IL-10 were reduced drastically after Aβ
treatment. The mRNA expression of IL-10 dropped significantly to 0.25 ± 0.02- fold in the Aβ-
only group (p<0.01) (Figure 57a). However, the IL-10 expression was restored after pre-
treatment with SPRC or SAC. Pre-treatment with SPRC increased the mRNA expression of IL-

10 to 0.69 ± 0.07- fold (p<0.05) while pre-treatment with SAC further increased the mRNA
expression to 0.81 ± 0.06- fold (p<0.01). In contrast, the NaHS pre-treatment had little effect on
up-regulating IL-10 expression, with only a 0.26 ± 0.07-fold expression.
IL-10 protein expression in the cell lysates significantly declined after Aβ treatment from
104.4 ± 2.3 pg/ml in the untreated control cells to 91.3 ± 2.1 pg/ml in the Aβ-only control cells
(p<0.05) (Figure 57b). SPRC pre-treatment did not alter the IL-10 protein expression,
maintaining the expression at 91.3 ± 2.1 pg/ml (p=N.S). SAC pre-treatment, however, increased
0
0.2
0.4
0.6
0.8
1
1.2
SFM SFM
SPRC
10 µM
SAC 10
µM
NaHS
10 µM
Fold difference of IL-10
expression
#
*
**
1 µM Aβ
25-35
80
85

90
95
100
105
110
SFM SFM
SPRC
10 µM
SAC 10
µM
NaHS
10 µM
Concentration of IL-10
(pg/ml)
#
*
1 µM Aβ
25-35
(a)
(b)
PART III: FIBRILLAR Aβ
164

the IL-10 expression significantly to 98.7 ± 2.4 pg/ml (p<0.05). Likewise, NaHS also increased
the IL-10 expression to 94.9 ± 0.5 pg/ml (p=N.S), although this was not statistically significant.
6.1.6 Effects on cell death mechanisms
6.1.6.1 Cell cycle analysis


Figure 58: Effects of pre-treatment of drugs on cell cycle status. (a) Pre-treatment of 10 µM of

each drug altered the cell cycle profile of the glioma cells; especially obvious declines in
percentages of cells the sub-G1 phase and a slight increase in cells at S-phase. (b) Percentage of
cells in the sub-G1 phase for each treatment group, where pre-treatment of drugs significantly
decreased the sub-G1 cell population. N=3, #: p<0.05 when compared to the untreated control; *:
p<0.05 when compared with the Aβ control; **: p<0.01 when compared with the Aβ control.
0.00%
20.00%
40.00%
60.00%
80.00%
100.00%
Sub-G1 G1 S G2/M
Percentage of total cells
SFM-only
Aβ 1 µM
Aβ 1 µM + SPRC 10 µM
Aβ 1 µM + SAC 10 µM
Aβ 1 µM + NaHS 10 µM
#
**
*
**
0.00%
0.10%
0.20%
0.30%
0.40%
0.50%
0.60%
0.70%

SFM SFM
SPRC 10 µM SAC 10 µM NaHS 10 µM
Percentage of total cells
#
**
**
*
1
µ
M A
β
(a)
(b)
PART III: FIBRILLAR Aβ
165

The observation of changes in cell viability following drug pre-treatment suggested
alterations to the cell cycle status, determining the amount of cells rescued by the pre-treatment
regimes. Cell cycle status was significantly affected by both pre-treatment and subsequent Aβ
insult (Figure 58a), where the largest variation was observed at the R1 region (F
(4, 19)
= 7.782;
p<0.01). Aβ-only control resulted in about twice the percentage of cells at the sub-G1 region
(Figure 58b) of 0.57 ± 0.01% of total cells, compared to 0.31 ± 0.05% in SFM-only control. This
increase in percentage of cells in the sub-G1 region was significantly declined by pre-treatment
of SPRC, SAC or NaHS. The largest decrease was observed in the SPRC-treated cells, where
only 0.20 ± 0.01% (p<0.01) of cells were in the sub-G1 region. Likewise, SAC could reverse the
Aβ-induced increase to 0.33 ± 0.06% (p<0.05), and NaHS resulted in 0.27 ± 0.05% (p<0.01).
Notably, even though there were fluctuations in sub-G1 cell populations, the fluctuations
were very small. There was, however, an observable alteration of the cell population in S phase;

though it was not found to be statistically different (F
(4, 19)
= 1.209; N.S). This showed a slight
shift towards cell cycle progression after pre-treatment as there were decreases in percentage of
cells in G1phase and increases in the S or G2/M phase.


PART III: FIBRILLAR Aβ
166

6.1.6.2 Apoptosis

(a)
PART III: FIBRILLAR Aβ
167


PART III: FIBRILLAR Aβ
168


Figure 59: Effects of pre-treatment of drugs on DNA fragmentation using TUNEL staining. (a) Representative photos of drug-treated
groups at 20X magnification where fragmented DNA is stained green and indicated with white arrows. A scale bar of 100 µm is
indicated at the lower left of each panel. The nucleus is counter-stained with DAPI and presented as blue fluorescence. I: Untreated
control; II: Aβ-only control; III: SPRC-treated group; IV: SAC-treated group; V: NaHS-treated group. (b) Proportion of green/blue
fluorescence ± S.E.M, normalized to the untreated control. N=3, #: p<0.05 compared to untreated control; *: p<0.05, **: p<0.01
compared to Aβ-only group.
0
0.5
1

1.5
2
2.5
3
SFM SFM
SPRC 10
µM
SAC 10 µM NaHS 10
µM
Proportion of green/blue
fluorescence
1 µM Aβ
25-35
#
**
*
(b)
PART III: FIBRILLAR Aβ
169

DNA fragmentation was detected by TUNEL staining and statistically different among
all treatment groups (F
(4,14)
= 12.448; p<0.01). The treatment period for all groups was longer
than that in Part II: Oligomeric Aβ, and that might account for a higher amount of apoptotic cells
observed in the SFM-only group (Figure 59a, Panel I). Nonetheless, Aβ treatment resulted in a
significantly higher proportion of apoptosis as indicated with a larger number of cells stained
green (Figure 59a, Panel II). This proportion was decreased when the cells were pre-treated with
drugs, where fewer cells stained green was observed (Figure 59a, Panels III- V). However, while
all pre-treatments decreased the proportion of apoptosis, SPRC and SAC pre-treatments resulted

in significantly lowered proportion which was not observed in the NaHS pre-treated group, in
which a proportion of 1.86 ± 0.22 was obtained (Figure 59b). The SPRC-treated group resulted
in a proportion of 1.29 ± 0.11 (p<0.01), slightly lower than the proportion of 1.49 ± 0.04 (p<0.05)
in the SAC-treated group. The calculated proportions of the drug- treated groups were almost
halved of that in the Aβ-only group with a proportion of 2.32 ± 0.22.








SFM SFM
SPRC
10 µM
SAC
10 µM
NaHS
10 µM
1 µM Aβ
25-35

PARP (116

kDa and 87

kDa)

Pro

-
caspase 3 (32 kDa)

β
-
tubulin

(a)