Mitochondrial integrity and antioxidative enzyme efficiency in fischer rats effects of ageing and epigallocatechin 3 gallate intervention 1 3

Epigallocatechin-3-gallate EGCG, the main component of the green tea extract, is well-known for its radical and oxidant scavenging activity [159] as well as its chemotherapeutic properti

4.2 Part two: Effects of epigallocatechin-3-gallate on mitochondrial integrity and antioxidative enzyme activity in aging process of human fibroblast (Paper II)

4.2.1 Introduction

As stated in Section 1.3 the free radical theory of aging, the oxidative damages to many biological macromolecules as a result of ROS attack is probably a direct cause of cell senescence [89, 155] And as justified in Section 3.1 Part one, mitochondrial dysfunction is probably a major underlying event in aging Mitochondria are the main resource of intracellular ROS, and at the same time they are subjected to ROS attack themselves A number of age-related oxidative damages have been identified in mtDNA, especially within the mtDNA control region for replication [156] ROS also causes damage to mitochondrial membrane, resulting in a decreased mitochondrial membrane potential [146] The impairment

of mitochondria concomitantly causes even greater leakage of ROS, leading to the formation of a vicious cycle [136] On the other hand, the antioxidant defense system is considered to involve in the scavenging of ROS and thus protect organisms against oxidative damage Generally, the primary enzymatic antioxidant defense system, including CAT, GPx, SOD1 and SOD2, is the first-line defense to detoxify ROS Meanwhile, components from the non-enzymatic antioxidant defense system such as vitamin C and E, co-enzyme Q10 and other small molecules and compounds which are better known as ‘nutrition supplements’, not only participate in radical scavenging directly, but also serve as essential cofactors for various enzymes that decrease oxidative stress Researches

aim to delay the aging process are usually based on three principles The first one

is to control the ROS production, which can be achieved by calorie restriction [157] or manipulating the oxygen concentration [158] The second is to reinforce the enzymatic antioxidant system in transgenic organisms or over-expression of

antioxidative enzymes [80] A third alternative is to enhance the non-enzymatic

antioxidant system through pharmacological administration or dietary supplementation, so that the non-enzymatic antioxidant system could compensate for the deficiency of the enzymatic system, or both the non-enzymatic and enzymatic systems could work synergically and effectively

Epigallocatechin-3-gallate (EGCG), the main component of the green tea extract,

is well-known for its radical and oxidant scavenging activity [159] as well as its chemotherapeutic properties [87, 88], whilst its anti-aging effect has little been known yet In this study, therefore, we attempt to fill up this gap by extrapolating the anti-aging effect of EGCG on the human diploid fibroblast (HDF), a well-established model for cellular aging studies [160, 161] The LC50 value of EGCG and its anti- and pro-oxidant effects are examined initially Then in the short term study, HDF is exposed to H2O2 induced oxidative stress and pre-senescence, and

in the long term study, HDF is continuously cultured till they reach replicative senescence In both of the approaches, ROS accumulation, mitochondrial integrity and antioxidative enzyme regulation are studied in HDF in the presence or absence of EGCG

4.2.2 Results

Cytotoxicity of EGCG

In order to determine the cytotoxicity of EGCG, both young (PDL20-30) and old (PDL>45) HDF grown in 96 well plates were treated with 0, 1, 2, 3, 6.25, 12.5, 25,

50 and 100 μM of EGCG for up to 7 days Cell viability result shows that for the young HDF, 50 μM EGCG significantly (p<0.05) inhibited cell growth on the 3rd day of cell culture, while 100 μM EGCG obviously (p<0.05) inhibited cell growth from the 3rd day onwards There were no obvious killing effects from 1 to 50 μM

of EGCG (Figure 15A) The old HDF showed the similar results as in the young HDF Given an incubation period of 7 days, LC50 of EGCG was determined as 78.0 and 84.4 μM for young and old HDF respectively, in which test the concentrations of EGCG were used up to 400 μM (Figure 15B)

Figure 15 Cytotoxicity of EGCG on HDF

Young (PDL20-30) and old (PDL>45) HDF grown in 96 well plates were treated with various concentrations of EGCG for up to 7 days (A) Cell viability of young HDF was examined using Alamar Blue as mentioned in Section 3.2.2; RFU,

Relative Fluorescence Unit, is positively related to the cell viability (B) LC50 for

Anti- and Pro- oxidant effects of EGCG

In order to understand the ability of EGCG to scavenge free radicals in MEM, various concentrations of EGCG such as 0, 6.25, 12.5, 25, 50 and 100 μM were added into the MEM For the first 30min, 100 μM of EGCG cleared about 32 % free radicals of DPPH but its radical scavenging activity declined over time On day 3, the radical scavenging activity of EGCG remained only 7% in MEM (Figure 16A) At the same time, we noted that similar to many other antioxidants such as vitamin C and E, EGCG also possessed the pro-oxidative activity to induce H2O2 in the MEM After adding 100 μM of EGCG into the MEM for an hour, H2O2 concentration was detected as high as 110 μM, while the lower levels

of H2O2 were induced as a result of adding lower concentrations of EGCG correspondingly (Figure 16B) After 2 hours, however, all the induced H2O2 began

to diminish and reached the lowest levels on the 3rd day It is further interested to determine the ability of EGCG to produce H2O2 in MEM with the presence of HDF Results show that there was a dose dependent production of H2O2 with the maximum of 35 μM H2O2 in response to 100 μM of EGCG in the first hour (Figure 16C) However, the absolute amount of H2O2 was much less Especially after 4 hours, the maximum concentration of H2O2 in response to 100 μM EGCG was only 6.4 μM, while all the other induced H2O2 were below 5 μM in response

to lower concentrations of EGCG (Figure 16C)

Figure 16 Anti- and pro-oxidant effects of EGCG in MEM in the presence and absence of HDF

(A) Various concentrations of EGCG were evaluated for the ability to scavenge the stable radical DPPH in MEM at different time points EGCG-induced H2O2

Based on the above observations, in the following short term studies, we utilized

25 and 50 μM of EGCG after 4 hours of preparation not only to obtain the maximum antioxidant effects of EGCG but also to minimize its cytotoxicity as well as H2O2 production In the long term studies which lasted for more than one month, considering that the prolonged incubation of cells might increase the cytotoxicity of EGCG significantly, we further reduced the EGCG concentration

to 12.5 μM but the medium was changed every other day

Effects of EGCG in short term treatment

Protection against H2O2

H2O2 induced oxidative stress is able to cause pre-senescence or permanent cell cycle arrest of HDF In the short term studies, young (PDL20-30) HDF grown in

96 well plates was pre-treated with 25 and 50 μM of EGCG for 24 hours and then challenged with 100 and 200 μM H2O2 for 2 hours The viability of cells obviously decreased after exposure to 100 and 200 μM H2O2 in the absence of EGCG(Figure 17A) But HDF pre-treated with EGCG showed a dose dependent protective effect against H2O2 In particular, in the 200 μM H2O2 treated group, 25 and 50 μM of EGCG increased the cell viability by 52.2 and 60.9 %, respectively, which was almost back to the same level as H2O2 untreated group (Figure 17A) This observation was also confirmed by photography as shown in Figure 17B HDF pre-treated with EGCG and challenged with 100 and 200 μM H2O2 was allowed to grow for another 5 days in normal growth medium, MEM Results show that HDF pre-treated with 25 and 50 μM EGCG were generally more viable than their untreated counterparts (Figure 17C and 17D)