Senescence

Abstract: The effects of Yukmi (Decoction of six plants including rehmannia), an herbal formula, were studied on liver oxidant damage induced by paraquat (PQ) administered intravenously in the senescence accelerated mice (SAM-P/8). The activities of superoxide dismutase (SOD) and catalase as two major antioxidant enzymes and lipid peroxidation levels were determined for six days. Data show that the activities of hepatic SODs and catalase were increased by oral administration of Yukmi extracts following PQ pretreatment. Herbal medicine effectively blocked the PQ-induced effects on liver malondialdehyde (MDA) levels. For the histopathological changes in SAM-P/8 liver, Yukmi extracts inhibited PQ-induced damage to the hepatic mitochondria and their membranes. Data suggest that Yukmi extracts may be useful in protecting against oxidative damage.

The oxyradicals pose a threat to macromolecules and cell organelles. This toxic damage to the living cells results in genomic disruption (Adelman et al., 1988; Collins, 1999), thereby causes mutation (Storz et al., 1987) and potential carcinogenesis (Holzinger et al., 1999) leading to cellular aging processes (Ashok and Ali, 1999). Antioxidant enzymes can, however, minimize noxious damage to a limited degree. These enzymes are mainly responsible for the primary defense by superoxide dismutase (SOD) (McCord and Fridovich, 1969), catalase (hydroperoxidase)(Claiborne and Fridovich, 1979) as well as small molecules such as GSH (Bast and Haenen, 1984), ascorbic acid (Frei and Ames, 1989), tocopherol (Aruroma et al., 1990) and other natural or synthetic antioxidants (Kim, 1998; Yang and Kim, 1999). Defense against free radicals can therefore be viewed as a compendium of those enzyme functions that results in overall cellular sensitivity to oxidative stress.

Present in living organisms, natural antioxidants show their potential by catalyzing the reduction of oxidants (Stocker and Frei, 1991), scavenging of chain-breaking enzymes (Packer et al., 1979) and inhibiting the formation of oxygen radicals (Grootveld and Halliwell, 1987). Among those natural antioxidants, oriental herbal medicines have been attracting much attention recently. Yukmi (YM), an herbal formula, is known to be used to replenish the vital substance of both the liver and the kidney for treatment in chronic diseases with symptoms of dizziness, tinnitus and sore throat (Yuwen et al., 1996). Therefore, YM was used in this study to prove further its effect as a potential antioxidant.

The senescence accelerated mouse (SAM) is a murine model of accelerated aging (Takeda et al., 1981). Among two strains, a senescence accelerated prone mouse (SAM-P/8) was utilized since it possesses many signs of aging such as loss of skin glossiness, a short life span (Takeda et al., 1981) and enhanced systemic amyloidosis (Miyamoto et al., 1986). Furthermore, biochemical parameters such as hepatic SOD and malondialdehyde (MDA) were measured in this strain (Nomura et al., 1989). At present, the antioxidant potential of Yukmi in SAM strains is not known. Also, paraquat has been most widely employed as one of the best characterized artificial sources of superoxide redox cycling compounds (Moody and Hassan, 1982; Minakata et al., 1995). Therefore, potential antioxidant effects of YM against paraquat-mediated oxidative stress were tested in the present study by measuring SOD, catalase and MDA in the liver.

Materials and Methods

Preparation of Yukmi

The natural herbal medicine was used in this study. Yukmi is a mixture of extract solution derived from six medicinal plants, consisting of Rehmannia Root (rootstock of Radix rehmanniae, 16 g), Dogwood Fruit (fruit of Fructus corni, 8 g), Chinese Yam (rootstock of Rhizoma dioscoreae, 8 g), Alismatis Rhizome (rootstock of Rhizoma alismatis, 6 g), Tuckahoe (sclerotium of the Poria cocos, 6 g) and Moutan Bark (root cortex of Cortex Moutan radicis, 6 g). It was extracted with distilled water (300 ml) at 100 [degrees] C for 1 hr. The extract solution was filtered and used as indicated below.

Animals

Senescence accelerated mice (SAM-P/8) were purchased from Clea Japan Inc. (Osaka, Japan). They were housed under constant environmental conditions at a temperature of 23 [+ or -] 1 [degrees] C, relative humidity of 60 [+ or -] 5% and an illumination cycle with 12 hr light/12 hr dark. They were fed with standard food and water ad libitum.

Paraquat (PQ, 2 x [10.sup.-7] mg/kg, Minakata et al., 1995) that does show the apparent effect of such symptoms as diarrhea or anorexia at this dosage (unpublished data) was dissolved in water and injected intravenously into the tail of eight week-old male mice (25 [+ or -] 3 g). After one day, the animals were fed orally with Yukmi extracts (2.7 ml/kg) for 1 (n=5), 3 (n=7) and 6 (n=7) days using a zonde needle (Natsume, Japan). For the control group, physiological saline was used.

Sample Collection and Enzymatic Assay

Liver samples were obtained and an enzymatic assay for total SOD was performed based on the published method (Kim et al., 1997). Two grams of samples were homogenized using an ultra-turrax T-25 homogenizer (Germany) in 3 ml of 0.05 M phosphate buffer with 0.1 mM EDTA (pH 7.8) at 0 [degrees] C and the homogenates were stored at -80 [degrees] C if necessary. Each homogenized liver sample (4 ml) was treated with Triton-X 100 (0.1%, v/v) and centrifuged at 15,000 g for 5 min to remove nuclear fractions. Cytoplasmic and mitochondrial enzyme fractions could not be isolated simultaneously, therefore these fractions were isolated separately and, for simplicity, later combined and run on the gels as described below. To obtain total enzyme activity, the supernatant was recentrifuged at 60,000 g for 30 min at 4 [degrees] C. The supernatant, which corresponded to the cytosolic fraction, was collected, immediately frozen and stored at -80 [degrees] C until assay. The pellets were resuspended in fresh 0.05 M phosphate buffer (pH 7.4) containing 0.25 M sucrose and 0.1 mM EDTA and then centrifuged at 60,000 g for 60 min at 4 [degrees] C. The second supernatant corresponded to the mitochondrial fraction. The protein concentration was determined by the published method (Lowry et al., 1951).

SOD activity in 7.8% non-denaturing polyacrylamide gels was visualized by using the method of Beauchamp and Fridovich (1971). One hundred [micro]g of the recombined cytoplasmic:mitochondrial fraction (1:1) was loaded on the same lane of the gels. Activities of CuZnSOD and MnSOD were measured by quantifying the bands using a densitometer (Pharmacia, USA). One unit of SODs was defined as the quantity required to produce 50% inhibition of the rate of reduction of cytochrome c under specified conditions at 25 [degrees] C. Catalase activity was determined spectrophotometrically by measuring the rate of hydrogen peroxide decomposition at 240 nm as described by Beers and Sizer (1952). A unit of catalase was defined as the amount decomposing one micromole of hydrogen peroxide per minute under specified conditions at 25 [degrees] C.

Lipid peroxidation was determined by the formation of thiobarbituric acid-reactive substances (TBARS) in a 10 min period and expressed as a concentration of malondialdehyde (MDA). The reaction mixture containing 8% sodium dodecyl sulfate, 20% acetic acid (pH 3.5) and 0.8% thiobarbituric acid was heated at 90 [degrees] C for 60 min. After cooling, n-butanol/pyridine mixture (15:1, v/v) was added, then shaken vigorously and centrifuged at 1,000 g for 10 min. The absorbance of the supernatant was measured at 532 nm at room temperature. 1,1,3,3-Tetramethoxypropane was used as an external standard, and the amounts of lipid peroxide were expressed as nmol of MDA produced. The details were described by Okawa et al. (1979).

Electron Microscopic Study

The liver specimens (1 [mm.sup.3]) were sliced and placed in fixative, 1% Os[O.sub.4] buffer with 0.1 M collidine, 2.5% glutaraldehyde buffered with 0.1 M cacodylate followed by post-fixation in Os[O.sub.4]. The tissues were dehydrated with increasing concentrations of ethanol, then placed in propylene oxide and embedded in araldite. Thin sections were stained with uranyl acetate and lead citrate, and were examined using an electron microscope (JEM 100 CX-II).

Statistical Analysis

Data were expressed as the mean [+ or -] SD and analyzed using a one-way analysis of variance (ANOVA). The significance of the differences between groups was tested using a paired Student's t-test. The criterion for significance was p [is less than] 0.05.

Results

Hepatic Effects on Antioxidants

The MnSOD activity of the mouse liver treated with Yukmi extracts following PQ pretreatment was approximately 7-fold higher than PQ-only-treated animal in each group. Likewise, the CuZnSOD activity was consistently increased compared to the PQ treated groups at different time intervals (Figures 1 and 2). The activities of catalase were consistently two-fold higher in herbal medicine treated animals than those in PQ pretreatment. With parallel trends to SOD activity, herbal medicine gradually increased the catalase activities for a one-week period (Figure 2). When rats in the control group received the herbal medicine (Control + YM), the amount of both SODs and catalase was gradually increased compared to the control which was fed only with saline (data not shown).

[Figures 1-2 ILLUSTRATION OMITTED]

Treatment with PQ caused a marked increase in liver TBARS (converted to MDA concentration) with a maximal value expressed on the sixth day after administration (Figure 3). The level of MDA in the Control + YM was expressed with a minimal amount in a one-week period. However, the PQ-induced lipid peroxidation levels were inhibited by half after six days with herbal medicine.

[Figure 3 ILLUSTRATION OMITTED]

Pathological Changes

Electron micrographs of SAM liver treated with PQ showed several distinct changes compared with the control (Figure 4a). The nuclear envelope was separated into two prominent membranes (Figure 4b). The smooth and rough endoplasmic reticula contained dilated cisternae. Most of the mitochondrial membranes showed high electron density with PQ treatment. This is a sign of mitochondrial degeneration since their sizes can be affected by the physiological and pathological states of hepatocytes as shown with large vacuoles in the matrix. However, by treatment with Yukmi extracts, those signs were recovered to an almost normal state (Figure 4c). On the other hand, Control + YM was not much different from the normal group (data not shown).

[Figure 4 ILLUSTRATION OMITTED]

Discussion

The toxic effect of paraquat on a host cell causes not only cellular membrane damage but intracellular production of superoxide anion at a sufficient level. Therefore, it has been reasonably expected in this study that oxygen radicals should be produced and involved in this kind of noxious damage. A previous study has shown that hepatic activity of SOD and its MDA content differ significantly in SAM strains (Nomura et al., 1989).

Results of the present study showed the increased SOD activities in the liver cells of herbal medicine-treated animals compared with paraquat-treated ones. Paraquat itself caused CuZnSOD to be increased whereas MnSOD was decreased. The therapeutic effect of YM during a one-week period seemed to be carried out mainly by the combination of MnSOD and CuZnSOD since they both showed gradual increases. When control was treated with YM, total SOD and catalase activities were slightly increased whereas MDA levels were conversely decreased. It suggested that Yukmi extracts contain antioxidants.

Moreover, the herbal medicine also caused an increase of catalase under the paraquat environment. With this trend, however, an inverse relationship with the level of MDA was seen when the paraquat was present for six days. In the case of PQ + YM, the decreased difference of MDA was larger than that of Control + YM and it implies that YM has antioxidant properties against oxidative stress. Overall, these findings suggest that the superoxide redox cycling compound led to increased SOD activity and increased lipid peroxidation. The significantly increased lipid peroxidation was attenuated during an herbal medicine treatment. It shows that YM was effective in inhibiting PQ-induced hepatic lipid peroxidation in this study. The rise in SOD can be suggested to decrease lipid peroxidation in the liver at least temporarily, which may delay the cellular damage.

The disturbance of membrane permeability was confirmed by electron microscope. The dilation in the nuclear envelope, which permits certain macromolecules to pass into and out of the nucleus, looked obvious since two membranes were separated. The mitochondrial matrix appeared to be damaged and shown as vacuoles. It resulted from the damage of membrane phospholipids (Fabisiak et al., 1997). The effect of lipid peroxidation was further illustrated by the disruption of endoplasmic reticular cisternae. It might be related to a certain enzyme formation as a part of the detoxification process with the paraquat injection. The manifest membranes of mitochondria, consistently expressed with PQ treatment, could have something to do with their damage. Since mitochondria are considered to be the main physiological generator of oxygen radicals in the liver, it might cause breakage in the electron transport chain, resulting in respiratory malfunction (Halliwell and Gutteridge, 1986).

The present data suggest that herbal medicine used in this study may exert an anti-aging action in a senile animal model. Regarding dose, duration and purity of compound, further studies need to be done.

Acknowledgments

This work was supported by a grant (No. HMP-99-O-11-0010-B) from the Korean Ministry of Health and Welfare.

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Jeong Sang Kim(1), Chang Su Na(1), Sok Cheon Pak(1) and Young Gon Kim(2)(*).

(1) Department of Acupuncture and Anatomy, Oriental Medical School, Dongshin University, Naju, Chonnam, 520-714, Korea,

(2) Department of Biological Science, College of Natural Sciences, Chosun University. 375 Seosuk-dong, Dong-ku, Kwangju, 501-759, Korea

(*) Corresponding author

(Accepted for publication June 14, 2000)

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