Mitochondrial integrity and antioxidative enzyme efficiency in fischer rats effects of ageing and epigallocatechin 3 gallate intervention 6

However, young and old HDF which were pre-treated with 25 and 50 μM EGCG significantly increased antioxidative enzyme activities and their gene expressions Figure 18A and B, with the exc

Finally, the SA-βG activity of HDF from the experimental group was compared with that from the control group HDF long-term incubated with 3 and 12.5μM EGCG significantly delayed the aging process demonstrated by less FDG fluorescence signals in the treated HDF Quantification of the X-Gal positively stained HDF also revealed the lower SA-βG activity in the EGCG-treated HDF as

a result of more juvenile cell status (Figure 20)

Figure 20 Effects of EGCG on cellular senescence of the middle-aged HDF

HDF (PDL35) incubated with 3 and 12.5μM EGCG for long term was evaluated for its cellular senescence status based on the SA-βG activity using FDG and X-Gal as substrates Fluorescence signals from FDG staining and blue colored products from X-Gal staining were quantified and expressed in the unit of RFU (relative fluorescence unit) and relative area occupied by positive (blue) cells, respectively; *p<0.05, compared to the untreated group as determined by one-way ANOVA

4.2.3 Discussions

The anti-cancer effect of EGCG has been reported in several studies However, so

far as we know, this research is the first in vitro study where EGCG is used to

understand aging and aging-related problems Initially, the dosages used in the study were optimized based on the principle that at an appropriate concentration, EGCG should offer the maximum antioxidant activity but with the minimum cytotoxicity for the following studies In the present study, it was found that the concentrations of EGCG from 1 to 50 μM did not show much cell growth inhibition within 24 hours (Figure 15A) However, 100 μM of EGCG efficiently inhibited the growth of both young and old HDF, and the cytotoxicity became increasingly significant as the incubation time was prolonged (Figure 15A) Several of the previous studies reported that the growth of the SV40 transformed human lung fibroblast (WI38VA) was completely inhibited at 40 μM of EGCG, but it did not have much inhibitory effect on the growth of the normal human lung fibroblast, WI38 EGCG also inhibited the human colorectal cancer cell line (Caco-2) and the breast cancer cell line (Hs578T), but had little inhibitory effects

on their respective normal counterparts [118]

In addition, we also investigated the dose dependent radical scavenging activity of EGCG in MEM growth medium As shown in Figure 16A, there was a dose dependent increase of EGCG’s ability to scavenge ROS, but this ability decreased over time Furthermore, EGCG was also found to induce H2O2 in dose dependent manner immediately after it was dissolved in MEM The highest H2O2 level was observed around 110 μM in response to 100 μM EGCG in the first hour and this concentration started to decrease after 2 hours (Figure 16B) In the presence of

HDF, however, the H2O2 production was largely suppressed (Figure 16C) The lower levels of H2O2 could largely attribute to either the presence of antioxidative enzymes or catabolic process of H2O2 in the cells [162]

H2O2 is well-known for causing pre-senescence or permanent cell cycle arrest of HDF [101], thus the H2O2-treated HDF was widely employed as an in vitro model

to investigate the anti-aging effect of antioxidant [163] In our short term studies, H2O2 was used as an oxidant to induce oxidative stress to HDF From Figure 17A,

it can be inferred that HDF pretreated with EGCG distinctly increased cell viability when compared to the untreated HDF after exposure to 100 and 200 μM H2O2 As it is well-known, ROS accumulation and depolarization of mitochondria membrane are positively correlated with aging [155], the protective effect of EGCG against H2O2-induced oxidative stress could largely be due to the decrease

of intracellular ROS level (Figure 17E) and the increase in mitochondrial potential (Figure 17F)

Furthermore, the decrease in intracellular ROS level upon EGCG treatment could result from the direct radical scavenging activity of EGCG, or perhaps due to the EGCG induced increase of antioxidative enzyme activity In this study, it was found that in the natural process of aging, there was a general decline of the antioxidative enzyme activities and gene expressions in HDF The mRNA levels

of CAT, SOD2 and GPx in old HDF were generally down-regulated (Figure 18A) Similarly, much lower enzyme activities of SOD1 and GPx were observed in the senescent cells, whereas there was an age-related increase in SOD2 activity (Figure 18B) The phenomenon of increased SOD2 activity is consistent with another observation that senescent keratinocytes also showed an increase of SOD2

activity [164] Such SOD2 activation is probably under the regulation of

Rel/NF-κB, a redox-sensitive transcription factor By reason of uprising ROS, Rel/NF-κB might be stimulated and in turn activate SOD2 Another possibility is that since SOD2 is predominantly present in the mitochondria, it is the first-line defence against ROS formed in mitochondria Thus, SOD2 might have adaptively increased its activity in response to more ROS in the senescent cells However, young and old HDF which were pre-treated with 25 and 50 μM EGCG significantly increased antioxidative enzyme activities and their gene expressions (Figure 18A and B), with the exceptions that SOD1 activity from the old HDF and GPx activity from the young HDF were inhibited instead of being boosted We hypothesize that this phenomenon could be largely attributed to the antioxidative effect from EGCG by a cross talk with the enzymatic defense system, namely CAT, SOD1, SOD2 and GPx It is also reported that the expression of phase II

enzymes and endogenous antioxidants that detoxify ROS can be up-regulated via

the NF-E2-related factor-2 (Nrf2) pathway [165] When the cells are exposed to oxidative stress, electrophiles or chemotherapeutic agents, Nrf2 could escape from Keap1-mediated repression and activate antioxidant responsive element (ARE)-dependent gene expressions to maintain cellular redox homeostasis [166] Taken together, in the short term studies, EGCG considerably protected HDF from H2O2-induced oxidative stress by decreasing ROS level due to the elevation of both antioxidative enzyme activities and gene expressions

Long term culturing of HDF till they reach replicative senescence is a better mimic of the natural aging process as demonstrated by the accumulation of ROS, depolarization of mitochondrial membrane and deterioration of antioxidative

enzyme activities [88] In virtue of exogenous antioxidant EGCG, however, the intracellular ROS level was reduced significantly in HDF when compared to the untreated control (Figure 19A) When looked into the mitochondrial integrity, we found that both mitochondrial membrane potential and mtDNA were better protected in the long term treatment (Figure 19B and C) The functional intact mitochondria might in turn produce less ROS As observed in the short term study, EGCG was found to cross-react with the enzymatic antioxidative system and boost its defense against oxidative stress as well Figure 19D and E show that HDF treated with 12.5 μM EGCG largely increased the gene expressions of CAT, SOD1 and SOD2 compared with the untreated group, and their corresponding enzyme activities dramatically increased in response to 12.5 μM EGCG as well Surprisingly, GPx activity dropped though its gene expression increased the most among the four enzymes Such discrepancy between gene expressions and enzyme activities have been reported in other studies as well [167] which could probably be explained by a translational block of the synthesis of GPx and the following activation of the enzyme [168] Alternatively, GPx activity is suppressed physiologically so that its transcription is up-regulated to compensate Besides, EGCG did successfully delay the aging process and kept the pre-senescence HDF at a more juvenile status (Figure 20)

the first in vitro study directly linking the mitochondrial integrity to the efficiency

of antioxidant defense system in the aging process, which could probably demonstrate the effects of EGCG well and at the same time serve as a support for

the free radical theory of aging

However, till now, there are still no conclusive answers as to whether the decrease

of ROS level in the short-term and long-term treatment is due to the direct radical scavenging activity of EGCG or due to the indirect effect of EGCG in boosting the antioxidative enzymes defense We anticipate that the gene knock-out or over expression techniques will be helpful to answer this question, and further study is needed to confirm our hypothesis In addition, it is also possible that as mitochondria are the main sources of ROS production, their integrity has been well maintained upon EGCG treatment, thus enabling them to function better and produce less ROS

Finally, although the in vitro system is useful for addressing the mechanisms of aging, this study is only at the beginning stage Whether the results getting from in vitro can also be applied on human beings or at least on experimental animals is

still in need of validation and in depth study

4.3 Part three: Regulation of age-related oxidative damage, mitochondrial integrity and antioxidative enzyme activity in Fischer 344 rats by the supplementation of the antioxidant epigallocatechin-3-gallate (Paper III)

4.3.1 Introduction

As demonstrated in our previous study in Part one, mitochondrial dysfunction in old Fischer 344 rats is associated with the decrease in antioxidative enzyme activities of CAT, GPx, SOD1 and SOD2 [88] In order to compensate for the deficiency of antioxidative enzymes, ameliorate the imbalance of redox status, and possibly maintain the mitochondrial functional integrity, the regimen of long-term supplementation of antioxidants is recommended, and so far several constructive observations have been published on these aspects [16, 86, 169] EGCG, the main component of the green tea extract, is an antioxidant well-known for its radical and oxidant scavenging activities In the Part two study, we have proved the

effects of EGCG in the in vitro model that long-term incubation of human diploid

fibroblast with EGCG results in more juvenile cell status along with the reduction

of intracellular ROS, higher mitochondrial membrane potential and less point mutation in mtDNA [170] Moreover, through a cross talk with the enzymatic defense system, EGCG considerably increases gene expressions of CAT, SOD1, SOD2 and GPx as well as their enzyme activities [170]

To extend the observations from the in vitro study, we further launch the in vivo

model to investigate the antioxidant and anti-aging effects of EGCG on the middle-aged male Fischer 344 rats through long-term dietary supplementation of EGCG The dose selected (50 mg/kg/day) for rats in the low dose group, is approximately equivalent to the consumption of 2 cups of green tea (185 mg of EGCG per cup) by a 70 kg person [93] We also extend this range by 10-fold to

500 mg/kg/day for rats in the high dose group Considering that to date, the only established nutritional intervention that slows aging process is CR [69], an additional CR group is included in this study to assess the impact of EGCG

supplementation Herein, we systematically investigate the effects of EGCG on Fischer 344 rats after 6 months of dietary supplementation, including pharmacological and histopathological studies of liver and kidney functions, biochemical analysis of the aging-related oxidative stress and activities of antioxidant enzymes as well as their gene expressions in major organs including the liver, kidney, heart and brain Besides, global gene expression profiling by using high density oligonucleotide microarray further enable us to identify the possible molecular mechanisms mediating the protection effects of EGCG in the aging process

4.3.2 Results

Weights of animal and major organs, as well as food consumption

There were no significant differences in body weight and food intake among all the four groups during the first 3 weeks From the fourth week onwards, the average body weight of CR rats was significantly (p<0.05) lower than the rats in the other 3 groups in which the body weight of rats did not show much difference

at any time in this study (Table 5) Based on the food intake, the actual EGCG consumption calculated was 40.47 and 417.1 mg/kg/day in the low and high dose groups, respectively, which was approximately 20 % lower than the original design as stated in materials and methods However, the food intake of rats in the high dose group was slightly higher than that of the rats in the low dose and control groups, which indicates that EGCG might positively affect the food intake (Table 5) On the day of sacrificing, the major organs such as liver, kidney, heart, brain and spleen were taken out and weighed It was found that there were no

EGCG-related effects on the weight of organs in the low dose, high dose and control groups but the weights of liver, kidney, heart and spleen in the CR rats

were significantly (p<0.05) lower (Table 5)

Table 5 The weights of animals, the major organs, and the food consumptions

Table 6 The histopathology observations in the liver and kidney

Animal Diagnosis Comments

Ctrl

(#1)

Liver: There was mild multifocal portal bile ductular proliferation and lymphocytic and less frequently plasmacytic

infiltrates within the portal areas There was no evidence of hepatocyte vacuolation with glycogen, lipid or water

The reticulum stain indicated no change to hepatocyte lobular structure Nor was there evidence of hepatocyte necrosis

There was mild variation in hepatocyte nuclear size

Kidney: There was mild dilation of low numbers of proximal renal tubules which are filled proteinaceous casts but no

evidence of renal tubular necrosis Small multifocal lymphocytic interstitial infiltrates of lymphocytes and macrophages

Interstitial nephritis is a common and non-specific inflammatory change in the kidney High

(#4)

Liver: Within portal areas there were bile ductular proliferation and fibroplasias Some mild infiltrate of lymphocytes and

plasma cells within portal triads There was no evidence of hepatocyte necrosis There was mild centrilobular vacuolation

of hepatocytes with clear single lipid droplets, but these were mild and low in number

Reticulum stain indicated normal hepatocyte lobular architecture

Kidney: There was fine to amorphous light brown cytoplasm pigment within the cytoplasm of renal cortical epithelial

cells This is consistent with lipofuscin and aging pigment

Lipofuscin is

a wear and tear pigment common in older rats and other animals

Low

(#3)

Liver: There was minimal bile ductular proliferation within portal triads and no evidence of fibrosis There was no

evidence of hepatocyte necrosis nor was there evidence of hepatocyte vacuolation with glycogen, lipid or water There

was no significant evidence of karyomegaly or variation in nuclear size of hepatocytes

The reticulum stain demonstrates normal hepatic lobules

Kidney: No evidence of renal tubular necrosis or other lesion

CR

(#1)

Liver: Mild bile ductular proliferation with no evidence of fibrosis in portal areas There was no significant variation in

nuclear size of hepatocytes There was no evidence of hepatocyte necrosis or cytoplasmic vacuolation containing lipid,

glycogen or water

The reticulin stain demonstrated normal morphology to the hepatocyte lobules

Kidney: No significant histological changes within the kidney

Bile ductular proliferation

is a common change in older rats

Histopathology diagnose

Histopathology was performed in order to further assess if long-term supplementation of EGCG at low and high doses causes any damages in the tissue Report shows that the morphology of liver and kidney tissues from the randomly selected rats appeared normal except for some common age-related morphological changes such as lipofuscin and bile ductular proliferation (Table 6) Such observations indicated that the concentrations of EGCG used in this study did not induce any damages in the liver and kidney tissues

Biochemical analysis of liver and kidney functions

ALT and AST are the common markers of liver disease or damage The increase

of ALT and AST activities in the serum is typically associated with hepatocellular membrane damage or distortion as well as leakage of the enzymes from hepatocytes Besides, the elevated AP activity is always associated with cirrhosis, hepatitis, fatty liver, liver tumor, liver metastases as well as drug intoxication In this study, there were no statistical differences in the activities of ALT, AST and

AP among the rats with EGCG supplementation as well as the control rats (Table 7) However, ALT and AST levels noticeably decreased by 34.08 and 32.09 % respectively in the CR rats when compared to the control, probably indicating

better liver functions as a result of caloric restriction (Table 7)

The determination of urea level is useful in assessing kidney functions In general, increased urea level is associated with nephritis, renal ischemia, urinary tract obstruction, and certain extrarenal diseases such as congestive heart failure, liver diseases and diabetes In our study, there was no significant difference in the

urinary urea among all the experimental groups while in the CR group, the decrease of urea was 16.71 % of the control (Table 7) Similarly, there was no obvious change of urea level in the plasma of rats in both EGCG supplementation groups, whereas its level in the CR rats was significantly (p<0.05) lower than that

in the control rats (Table 7) In general, the kidney function can be determined from the glomerular filtration rate by testing the creatinine clearance In this study, the creatinine clearance increased by 9.45 and 24.63 % in the rats fed with low and high dose EGCG respectively, and increased by 38.22 % in the CR rats, when compared to the control (Table 7) Thus, long-term EGCG supplementation

appears to improve the kidney function while it is more apparent in the CR rats

From the results above, we conclude that both low dose and high dose EGCG are well tolerated in the male Fischer 344 rats for a period of 6 month without any detectable damaging effect in the liver or kidney

Table 7 Liver and kidney functions of the male Fischer 344 rats

Figure 21 Concentration of H 2 O 2 in the plasma of middle-aged male Fischer

344 rats

H2O2 concentration was examined as an indicator of oxidative stress in the plasma after daily dietary supplementation with EGCG at doses of 50 and 500 mg/kg/day respectively, to the rats in the low and high dose groups, as well as 40% caloric

restriction in the CR rats, for 6 months Data were presented as mean ± SE (n=9)

*, p<0.05 compared to the control group (Ctrl) as determined by one-way ANOVA

Figure 22 Mitochondrial membrane potential of lymphocytes of middle-aged male Fischer 344 rats

Mitochondrial potential was evaluated to assess oxidative stress in the peripheral lymphocytes after daily dietary supplementation with EGCG at doses of 50 and

500 mg/kg/day respectively, to the rats in the low and high dose groups, as well as

40% caloric restriction in the CR rats, for 6 months Data were presented as mean

± SE (n=9) *, p<0.05 compared to the control group (Ctrl) as determined by way ANOVA

one-Furthermore, the aging-related oxidative damages including lipid peroxidation and DNA modification were evaluated in the plasma MDA measured as an indicator

of lipid peroxidation was considerably reduced in the rats from high dose group and CR group as compared to the control (Figure 23) 8-OHdG, the product of DNA oxidative modification, was excreted into the plasma upon the process of DNA repair The rats fed with high dose EGCG contained significantly (p<0.05) less 8-OHdG in the plasma than the control while the reduction of DNA damage was moderate in the rats from low dose and CR groups (Figure 23)

Figure 23 Lipid peroxidation and 8-OHdG in the plasma of middle-aged male Fischer 344 rats

Aging related oxidative damages including lipid peroxidation (A) and DNA modification (B) were checked in the plasma by examining their respective biomarkers of MDA and 8-OHdG, after daily dietary supplementation with EGCG

at doses of 50 and 500 mg/kg/day respectively, to the rats in the low and high dose

groups, as well as 40% caloric restriction in the CR rats, for 6 months Data were

presented as mean ± SE (n=9) *, p<0.05 compared to the control group (Ctrl) as determined by one-way ANOVA

(A)

(B)

Apart from the production of 8-OHdG, the large scale deletion encompassing the ND4 region in mtDNA is also witnessed commonly due to the oxidative damages

in the aging process The results from real-time PCR performed with the primers designed to specifically amplify the ND4 region revealed that there were relatively more ND4 templates in the liver of rats fed with low and high dose EGCG, indicating that EGCG dose dependently protected mtDNA against deletion (Figure 24) However, there was little effect of CR on mtDNA deletion as observed

Figure 24 Mitochondrial DNA deletion in the liver of middle-aged male Fischer 344 rats

ND4 region in mtDNA encompassing the common aging-related large scale deletion was amplified by quantitative real-time PCR after daily dietary supplementation with EGCG at doses of 50 and 500 mg/kg/day respectively, to the rats in the low and high dose groups, as well as 40% caloric restriction in the

CR rats, for 6 months Data were presented as mean ± SE (n=9) *, p<0.05

compared to the control group (Ctrl) as determined by one-way ANOVA

Antioxidative enzyme activities and gene expressions

In order to figure out the reason that EGCG considerably reduced the oxidative damages in the treated rats, we looked into the changes in antioxidative enzyme activities as well as the gene expressions in response to long-term EGCG supplementation, since oxidative stress could be closely regulated by the antioxidative enzymes Antioxidative enzyme activities were investigated in the liver, kidney, heart and brain of rats fed with low and high dose EGCG as well as

CR rats In the liver, CAT activity significantly declined in the rats from the high dose group, and GPx and SOD1 activity also decreased in a dose dependent manner (Figure 25A) SOD2 activity increased to some extent without any statistical differences In the liver of CR rats, CAT and SOD1 activities decreased considerably, but not much change was observed in GPx and SOD2 activities (Figure 25A) In the kidney, the only difference found was the increase of CAT activity in the low dose group when compared with the control (Figure 25B) In the heart of the rats, high dose EGCG significantly increased the activities of CAT, GPx and SOD2, and low dose EGCG considerably increased SOD2 activity but decreased SOD1 activity (Figure 25C) CR intervention greatly elevated CAT activity, but resulted in a decline in SOD2 activity (Figure 25C) In the brain, the effect of EGCG was attributed to the reduction of SOD1 activity in the low dose group and the augmentation of SOD2 activity in the high dose group CR rats, however, showed decreased SOD2 activity compared to the control (Figure 25D)

Figure 25 Antioxidative enzyme activities in the liver, kidney, heart and brain of middle-aged male Fischer 344 rats

The rats dietary supplemented with EGCG at doses of 50 and 500 mg/kg/day respectively, in the low and high dose groups, as well as the CR rats with 40% caloric restriction, for 6 months were examined the enzyme activities of CAT, GPx, SOD1 and SOD2 in the liver (A), kidney (B), heart (C) and brain (D) One unit of CAT is defined as the amount of enzyme that causes the formation of 1.0 nmol of formaldehyde per min at 25°C One unit of GPx is defined as the amount

of enzyme that will cause the oxidation of 1.0 nmol of NADPH to NADP+ per min at 25°C One unit of SOD is defined as the amount of enzyme needed to exhibit 50% dismutation of the superoxide radical at 25°C Data were presented as mean ± SE (n=9) *, p<0.05 compared to the control group (Ctrl) as determined by one-way ANOVA

Besides the enzyme activities, the antioxidant system was also evaluated from the antioxidative enzyme gene expression levels In the liver, there was not much change found in the gene expressions as a result of EGCG supplementation except that GPx expression in the rats from low dose group increased moderately by 45.45 % compared to the control (Figure 26A) Caloric restriction enhanced the SOD1 expression significantly (p<0.05), but considerably repressed the CAT expression compared to the control (Figure 26A) Similarly, in the kidney, decreased expression of GPx was found in the CR rats, but little change was observed in all the other gene expressions (Figure 26B) In the heart, CR intervention greatly increased the gene expressions of CAT, GPx, SOD1 and SOD2 (Figure 26C) In the brain, however, no significant difference was found among the groups, although low dose and high dose EGCG increased SOD2 expression by 41.59 and 41.20 % respectively compared to the control (Figure 26D)

Figure 26 Antioxidative enzyme gene expressions in the liver, kidney, heart and brain of middle-aged male Fischer 344 rats

The rats daily dietary supplemented with EGCG at doses of 50 and 500 mg/kg/day respectively, in the low and high dose groups, as well as the CR rats with 40% caloric restriction, for 6 months were investigated the relative gene expressions of CAT, GPx, SOD1 and SOD2 in the liver (A), kidney (B), heart (C) and brain (D) Results were normalized to the endogenous control nuclear β-actin Data were presented as mean ± SE (n=9) *, p<0.05 compared to the control group (Ctrl) as determined by one-way ANOVA

Microarray analysis

In view of the finding that high dose EGCG effectively attenuated aging-related oxidative damages both in the plasma and liver, though with limited impact on the antioxidative defense system, we hypothesize that EGCG should also be able to control the oxidative damage possibly by regulating other responsive genes Analyzing the gene expression profile with a total number of 22,227 genes featured in the microarray revealed that 76 (0.34%) and 162 (0.73%) genes displayed a change in expression levels greater than 1.5-fold as an effect of EGCG supplementation and CR, respectively (Figure 27) In the low dose group, 11 out

of 12 genes showed the same trend of regulation as in the high dose group with an exception of Onecut1, which was up regulated in the rats from the low dose group but down regulated in the rats from the high dose group (Figure 27) The 75 genes (47 genes annotated) altered by high dose EGCG are mostly involved in the metabolism of lipid, glutathione, hexose biosynthesis and response to stress (Table 8) Noticeably, among these 75 genes, there were only 33 overlapping with CR group (Figure 27), including the 10 genes regulated in diverse directions by EGCG and CR interventions Such observations indicate that EGCG and CR interventions might attenuate aging and aging-related oxidative damages through different mechanisms The complete list of well annotated genes with more than 1.5-fold change in response to low dose and high dose EGCG interventions compared to the control is shown in Table 9

Figure 27 Venn diagram analysis of gene profiling in the middle-aged male Fischer 344 rats

The numbers in the figure represent the differentially expressed genes, which are regulated with more than 1.5-fold changes compared to the control (Ctrl) in the middle-aged male Fischer 344 rats after dietary supplemented with EGCG at daily doses of 50 and 500 mg/kg/day respectively, in the low and high dose groups, as well as rats with 40% caloric restriction, for 6 months Total RNA from the liver was used in triplicates as start material for each group

Table 8 Global views of transcriptional changes induced by EGCG

supplementation and caloric restriction

Sensory perception of sound & mechanical stimulus ↑ —

* Only obviously detected in the low dose EGCG intervention

Table 9 Genes expression with more than 1.5-fold change vs control in response to low dose and high dose EGCG interventions*

ILMN_55995e

ILMN_48157e

1.219 2.576 1.008 Slc16a1 Rat solute carrier family 16 (monocarboxylic acid transporters), member 1 1.162 2.397 -1.385 Insig2 Rat insulin induced gene 2

ILMN_69716a 1.75 2.206 2.967 F11r Rat junctional adhesion molecule 1

ILMN_58352a 1.519 2.135 1.874 Aadac Rat arylacetamide deacetylase (esterase)

ILMN_68954e 1.007 2.052 -1.259 Ccnd1 Rat cyclin D1

ILMN_50589e 1.267 2.007 -1.113 Gjb2 Rat gap junction membrane channel protein beta 2

ILMN_66976e 1.004 1.962 -1.115 Col6a3 Rat procollagen, type VI, alpha 3

ILMN_53536c 1.353 1.946 4.601 Dhcr7 Rat 7-dehydrocholesterol reductase

ILMN_65382a 1.589 1.944 3.869 Fads1 Rat fatty acid desaturase 1

ILMN_58547c -1.073 1.916 -2.888 Crot Rat carnitine O-octanoyltransferase

ILMN_66375a 1.577 1.864 1.932 Slc25a3 Rat solute carrier family 25 (mitochondrial carrier; adenine nucleotide translocator), member 3 ILMN_55276e 1.110 1.815 1.122 Chd4 Rat chromodomain helicase DNA binding protein 4

ILMN_54327e 1.259 1.784 1.079 Slc22a7 Rat solute carrier family 22 (organic anion transporter), member 7

ILMN_61732e 1.066 1.698 -1.128 Svil Rat supervillin

ILMN_65726e -1.085 1.689 1.028 Nudt7 Rat nudix (nucleoside diphosphate linked moiety X)-type motif 7

ILMN_49450a 1.709 1.675 1.79 Skp1a Rat S-phase kinase-associated protein 1A

ILMN_52136e 1.069 1.666 -1.177 Psen2 Rat presenilin 2

ILMN_57630a 1.699 1.662 10.07 Sc4mol Rat sterol-C4-methyl oxidase-like

ILMN_65390c -1.237 1.645 1.848 Gclm Rat glutamate cysteine ligase, modifier subunit

ILMN_53785e 1.025 1.637 1.012 Abcc6 Rat ATP-binding cassette, sub-family C (CFTR/MRP), member 6

ILMN_64064e 1.197 1.630 1.061 Prss11 Rat protease, serine, 11 (Igf binding)

ILMN_52686c 1.380 1.577 3.789 Fdft1 Rat farnesyl diphosphate farnesyl transferase 1

ILMN_67373c 1.213 1.567 1.503 Acbd3 Rat DMT1-associated protein

ILMN_58243e -1.156 1.558 1.093 Thrb Rat thyroid hormone receptor beta

ILMN_66382e 1.258 1.554 1.252 Tdg Rat thymine-DNA glycosylase

ILMN_51069e 1.097 1.540 1.256 Map4k3 Rat mitogen-activated protein kinase kinase kinase kinase 3

ILMN_59474e -1.142 1.520 -1.476 Cxcl10 Rat chemokine (C-X-C motif) ligand 10

ILMN_68951c -1.240 1.518 1.659 Gclc Rat glutamate-cysteine ligase, catalytic subunit

ILMN_56091e -1.031 1.501 1.498 Cat Rat catalase

ILMN_69627d 2.211 1.05 1.582 MGC108747 Rat similar to alpha-1 major acute phase protein prepeptide

ILMN_63583e 1.285 -1.516 -1.107 Trim36 Rat tripartite motif-containing 36

ILMN_67182c -1.232 -2.47 2.713 Pfkfb1 Rat 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 1

ILMN_66196c -1.018 -2.505 2.225 Por Rat P450 (cytochrome) oxidoreductase

ILMN_54767b 1.929 -3.137 1.118 Onecut1 Rat one cut domain, family member 1

ILMN_51207b -2.204 -5.792 -1 Hsd17b2 Rat hydroxysteroid (17-beta) dehydrogenase 2

*, Only well annotated genes identified by microarray analysis were listed in the table Table is sorted according to the fold change of gene expressions in the high dose intervention; genes with more than 1.5-fold change were detected in: a, low dose, high dose and CR interventions; b, both low and high dose interventions; c, high and CR interventions; d, low dose and CR interventions; e, high dose intervention only

4.3.3 Discussions

We have previously observed that the mitochondrial functional integrity and antioxidative enzyme activities decreased along with the increase in oxidative damages to lipid and DNA in the old Fischer 344 rats when compared to the young rats [88, 133] In order to ameliorate the aging-related disorders and probably delay the aging process, the ROS produced from mitochondria needs to

be greatly reduced and consequently the ROS released should be removed in time Gene manipulation and long-term caloric restriction show effects in retarding aging mostly because of the reduction of ROS accumulation [80, 157] However, the feasibility of following these methods for human beings is uncertain, where the dietary supplementation of antioxidants becomes a more reasonable and practical approach EGCG, which has long been used for anti-cancer treatment, is well-known for its radical and oxidant scavenging activities [159] Based on its antioxidant property, we propose that EGCG might also have anti-aging effects as

evidenced in our preliminary in vitro study [170] Herein, we extend the in vitro study to a more comprehensive in vivo animal study, which offers a unique

opportunity to assess the contribution of the anti-aging property of EGCG to the physiological confines of the rats after long-term supplementation of EGCG EGCG has been examined for its genetic, acute and short-term toxicities in rats in

a 13-week study and the no-observed adverse effect level (NOAEL) was reported

as 500 mg/kg/day for rats [171, 172] In another study which was extended to a two-generation period to examine the teratogenicity and reproductive toxicity of EGCG, the NOAEL was conservatively defined as 200 mg/kg/day for female rats, since F1 generation weight-gain was reduced at 600 mg/kg/day supplementation

[173] In this study, the average EGCG consumption was 40.47 and 417.1 mg/kg/day for low and high dose groups, respectively These concentrations of EGCG were still well tolerated by rats without causing any damages to tissues as demonstrated in the histopathological observations and functional assays, even though the experimental period was extended to 6 months Based on the creatinine clearance, there was a little improvement found in the kidney function in rats from the high dosage group However, the onsets of aging-related traits such as lipofuscin appeared almost at the same level in the treated and control groups

In fact, after EGCG is taken into the gastrointestinal tract, it undergoes generally extensive biotransformation including methylation, glucuronidation, sulfation and ring-fission metabolism, enabling the metabolites to enter the blood stream with higher bioavailability [174] In this study, H2O2 level was much reduced in the plasma of rats in the low and high dose groups than that in the control group, which indicates the effect of EGCG on scavenging free radicals and other forms

of ROS Consequently, such reduction of H2O2 level in the plasma probably led to the well-maintained mitochondrial integrity in the peripheral lymphocytes, as demonstrated by the higher mitochondrial membrane potential Besides, MDA and 8-OHdG, the products of lipid peroxidation and DNA oxidative modification respectively, were less detected in the plasma of rats fed with EGCG, indicating that macromolecules like lipid and DNA exposed to the potential oxidative stress were also protected by EGCG in a dose dependent manner Some investigations claimed that 8-OHdG was more often formed in mtDNA than in nuclear DNA, whereas others pointed out that the formation of more 8-OHdG in mtDNA is because of the artifact caused during DNA preparation [49] However, the large

scale deletion in mtDNA was indeed commonly found in the old rats probably as a result of the base mismatching caused by point mutation in mtDNA due to the formation of 8-OHdG and other oxidative adducts [51] In this study, the large scale deletion in mtDNA was less happened in the liver of rats from high dose group than that from the control group, which is also well correlated with the level

of plasma 8-OHdG Meanwhile, the results from mitochondrial respiratory control ratio using succinate as substrate revealed that there was no difference in the mitochondrial functional integrity between the treated and un-treated groups (data not shown) Nakada and his colleagues explained this phenomenon through a mitochondrial complementation effect as mitochondria are able to merge with each other and exchange their genomic contents Therefore, the differences in mitochondrial functional integrity will only display when the mtDNA deletion reaches a threshold [175]

Furthermore, we are interested in understanding if the protection effect of EGCG against oxidative damages could be accounted for from its influence on the antioxidative enzymes In this study, we carried out the enzyme activity assay as well as the gene expression analysis for CAT, GPx, SOD1 and SOD2 in the four major organs, namely the liver, kidney, heart and brain To our surprise, although EGCG noticeably repressed the aging-related oxidative stress and the corresponding oxidative damages, it only has limited effects on the activities of the antioxidative enzymes or their gene expressions We proposed that besides the tentative interaction with the antioxidative enzymes, EGCG might also take effect through several other putative pathways Firstly, EGCG may directly act as antioxidant by scavenging ROS and chelating redox-active transition metal ions

[159] Alternatively, it may function indirectly through inhibiting the sensitive transcription factors, such as NF-κB, AP-1 and their downstream pro-oxidant enzymes, such as iNOS, lipoxygenases, COX-2 and xanthine oxidase [94],

redox-or through inducing of phase II antioxidant enzymes via the Nrf2 pathway [166]

Indeed, the results from microarray data support this hypothesis as CAT was the only antioxidative enzyme detected with more than 1.5-fold change in gene expression in response to EGCG treatment in the high dose group However, the corresponding gene expression of CAT as demonstrated in the results of real-time PCR was only 1.14 times compared to the control In fact, all of the 76 genes (with 47 genes annotated) altered in rats from low and high dose groups were mostly involved in lipid metabolism (9 genes); hexose, ribonucleoside triphosphate and protein metabolism (5 genes); glutathione metabolism (2 genes); and response to stress (6 genes) Such changes in the gene expressions suggest that EGCG might protect aging process from oxidative damage by other

mechanisms such as regulating energy metabolism, biosynthesis, redox status etc.,

which are fairly distinct from regulating antioxidative enzymes [176] On the other hand, considering that the aging process is not evident with widespread alterations in gene expressions [69], nutrition interventions that try to counteract the aging effects are unlikely to cause fundamental changes in gene expression profile

Moreover, caloric restriction, which is the only intervention that appears to slow down the intrinsic rate of aging [69], was used as a positive control in our study Besides the direct physical changes as a result of long-term caloric restriction such

as reduced body weight and organ weight, rats in the CR groups also showed