After exposure to 100 and 200 μM H2O2 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 grou
Trang 1Figure 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 H2O2 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) H2O2 was monitored; intracellular ROS (E) and mitochondrial potential (F) were also evaluated using H2DCF-DA and
JC-1 staining respectively as mentioned in Section 3.2.5 and 3.2.6 on the following day of H2O2 exposure; RFU, Relative Fluorescence Unit The data shown are the mean from 3 independent experiments, #p<0.05, compared to the H2O2 untreated and EGCG untreated group, *p<0.05, compared to the EGCG untreated group as
Trang 2After exposure to 100 and 200 μM H2O2 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 H2O2
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 H2O2 and by 32.0 and 44.6 % after exposure to 200 μM H2O2 when compared to their respective control (Figure 17E) Furthermore, the corresponding mitochondrial membrane potential was also determined under the influence of both EGCG and H2O2 It was observed that without EGCG, mitochondria integrity was significantly (p<0.05) impaired upon
200 μM H2O2 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
H2O2 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
Trang 3EGCG 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