Mitochondrial integrity and antioxidative enzyme efficiency in fischer rats effects of ageing and epigallocatechin 3 gallate intervention 5

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Figure 18. Effects of EGCG in antioxidative enzyme gene expressions and
activities in short term treatment.

Young (PDL20-30) HDF was treated with 25 and 50μM EGCG for 24 hours and
then the antioxidative enzyme gene expressions (A) were measured by
quantitative real-time PCR and the enzyme activities (B) were determined by
Cayman chemical kits, as mentioned in Section 3.2.7 and 3.2.8, respecctively. The
data shown are the mean from 3 independent experiments, #p<0.05, comparing
untreated HDF-Yong (Y) with untreated HDF-Old (O); *p<0.05, comparing
treated HDF-Y with untreated HDF-Y, and +p<0.05 comparing treated HDF-O
with untreated HDF-O, respectively; as determined by one-way ANOVA.
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Effects of EGCG in long term treatment
In the long term treatment, the middle-aged HDF (PDL35) was continuously
cultured in MEM with 12.5 μM of EGCG until they reach replicative senescence.
In the process of HDF reaching the end point, samples were collected, and
intracellular ROS accumulation, mitochondrial integrity, antioxidative enzyme
activities and gene expressions were examined and compared. It was found that
middle-aged HDF treated with 12.5 μM EGCG for long term decreased the
intracellular ROS level significantly (P<0.05) (Figure 19A) and in the meantime
increased the mitochondrial membrane potential by 61.5 % compared to the
control (Figure 19B). Moreover, the long term incubation of middle-aged HDF
with 12.5 μM of EGCG significantly (P<0.05) increased mitochondrial DNA
integrity, which was demonstrated by a much higher mtDNA/nDNA ratio in the

treated group for both D310 and D-loop region (Figure 19C). In addition, upon
EGCG (12.5 μM) treatment on middle-aged HDF, all of the gene expressions of
CAT, SOD1, SOD2 and GPx extensively increased by 58.3, 86.7, 73.8 and 94.0%,
respectively (Figure 19D). Besides, the enzyme activities of CAT, SOD1 and
SOD2 also enhanced significantly by 11.6%, 58.0 and 47.9%, respectively,
whereas GPx activity dropped by about 18.9 % (Figure 19E).











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Figure 19. Effects of EGCG on the middle-aged HDF in long term treatment.

HDF (PDL35) incubated with 12.5 μM EGCG for long term was tested for
intracellular ROS (A), mitochondrial potential (B), mitochondrial DNA integrity
(C), antioxidative enzyme gene expressions (D) and enzyme activities (E) as
mentioned in Materials and methods; *p<0.05, compared to the untreated group as
determined by the Student’s t-test; MFI (median fluorescence intensity) was used

as an indicator of the ROS level. (C) and (D) show the normalized results where
the values of EGCG untreated HDF is set as ‘one’.