Mitochondrial integrity and antioxidative enzyme efficiency in fischer rats effects of ageing and epigallocatechin 3 gallate intervention 4

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Figure 17. Protective effects of EGCG against H
2
O
2
induced oxidative stress
on young HDF.

Young (PDL20-30) HDF grown in 96 well plates was pre-treated with 25 and 50
μM EGCG for 24 hours and then challenged by 100 and 200 μM H
2
O
2
for 2 hours.
Cell viability was determined on the following day (A), and the representative
pictures show the cell viability (B); Cell proliferation for 5 continuous days after
exposure to 100 μM (C) and 200 μM (D) H
2
O
2
was monitored; intracellular ROS
(E) and mitochondrial potential (F) were also evaluated using H
2
DCF-DA and JC-
1 staining respectively as mentioned in Section 3.2.5 and 3.2.6 on the following
day of H
2
O

2
exposure; RFU, Relative Fluorescence Unit. The data shown are the
mean from 3 independent experiments, #p<0.05, compared to the H
2
O
2
untreated
and EGCG untreated group, *p<0.05, compared to the EGCG untreated group as
determined by the Student’s t-test.
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After exposure to 100 and 200 μM H
2
O
2
for 2 hours, HDF grown in 96 well plates
was also looked into its intracellular ROS level in both of the EGCG pre-treated
and untreated control groups. Results shows that both 100 and 200 μM H
2
O
2

resulted in a high ROS level in the EGCG untreated HDF, whereas HDF pre-
treated with 25 and 50 μM EGCG for 24 hours moderately reduced the ROS level
by 36.9 and 28.1% after exposure to 100 μM H
2
O
2
and by 32.0 and 44.6 % after
exposure to 200 μM H

2
O
2
when compared to their respective control (Figure 17E).
Furthermore, the corresponding mitochondrial membrane potential was also
determined under the influence of both EGCG and H
2
O
2
. It was observed that
without EGCG, mitochondria integrity was significantly (p<0.05) impaired upon
200 μM H
2
O
2
exposure demonstrated by a much reduced JC-1 red/green ratio
compared to the control group (Figure 17F). HDF pre-treated with 25 and 50 μM
EGCG increased the mitochondrial potential moderately against 100 and 200 μM
H
2
O
2
when compared with their respective control (Figure 17F).

Antioxidative enzyme activity and gene expression
We also examined the changes of gene expressions of CAT, SOD1, SOD2 and
GPx in both young (PDL20-30) and old (PDL>45) HDF after 24 hours of
treatment with EGCG, since these enzymes are mostly influenced by oxidative
stress. Figure 18A shows that in the untreated group, the gene expressions of CAT,
SOD2 and GPx in the young HDF were much higher than that in the old HDF.

After 24 hours of incubation with EGCG, all the enzymes increased their gene
expressions both in the young and the old HDF. In the young HDF, the gene
expressions of CAT, SOD1, SOD2 and GPx increased in response to 25 μM
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EGCG by 94.1, 61.9, 44.6 and 139.2 % respectively (Figure 18A). The gene
expressions of CAT and GPx were also significantly enhanced in the young HDF
by 88.8 and 80.0 % when treated with 50 μM EGCG, respectively (Figure 18A).
In the old HDF, 25 μM of EGCG seemed to be more effective than 50 μM of
EGCG for SOD1, SOD2 and GPx, but there was a dose dependent increase of
CAT activity (Figure 18A).
Besides the gene expressions, the antioxidant system was also evaluated from the
antioxidative enzyme activity levels in both young and old HDF. The results show
that in untreated groups, GPx and SOD1 activities were much lower in the old
than that in the young HDF, but SOD2 activity was higher in the old HDF, while
there was no significant difference in CAT activity between the young and old
HDF (Figure 18B). Young HDF treated with EGCG for 24 hours significantly
increased CAT, SOD1 and SOD2 activities, particularly, compared to the
untreated control, SOD1 activity was 2.4 and 2.1 times higher in the 25 and 50
μM EGCG treated group, respectively (Figure 18B). However, GPx activity
dropped in young HDF upon EGCG treatment. For old HDF, EGCG distinctly
elevated CAT, SOD2 and GPx activities, with an exception of SOD1, where
EGCG’s effect seemed to be inhibiting instead of boosting.