Kaohsiung Medical University Institutional Repository:Item 310902000/6786

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3c~r~lof

Biomedical

Science

Original Paper

J Biomed Sci 2002;9:299-302 Received: October 1, 2001 _{Accepted: December 28, 2001}

P r o t e c t i v e Effect of

T e t r a m e t h y l p y r a z i n e on A b s o l u t e

Ethanol-induced Renal T o x i c i t y in Mice

C h i - F e n g L i u a M e i - H s i u L i n b C h u n - C h i n g L i n e H u i - W e n C h a n g d

S o n g - C h o w L i n c

aNational Taipei College of Nursing, bMedical Research Institute, CDepartment of Pharmacology,

Taipei Medical University, Department of Experimental Diagnosis, Taipei Medical University Hospital, Taipei, d eGraduate Institute of Natural Products, Kaohsiung Medical University, Kaohsiung, Taiwan, ROC

Key Words

Antioxidant • Absolute ethanol • Tetramethylpyrazine • Lipid peroxidation • Malonic dialdehyde • Cytochrome C

Abstract

Acute administration of absolute ethanol (10 ml/kg) per os to fasted mice produced extensive renal failure

as

measured by a rise in blood urea nitrogen and creatinine. Pretreatment with oral administration of tetramethylpyrazine (TMP) prevented such failure. The maximal effect

against

absolute ethanol-induced renal failure could be observed 1 h after TMP administration. In order to further investigate the renal protective mechanism of TMP, ex- periments on lipid peroxidation and superoxide scavenging activity were conducted. Renal homogenates made from mice treated with ethanol showed that TMP pretreatment had an antioxidant effect. Mice in acute renal failure had higher malonic dialdehyde concentrations than those pretreated with TMP. The renal protective mechanism of TMP was attributed, in part, to its prominent superoxide scavenging effect, which protects the kidney from superoxide-induced renal damage.

Copyright @ 2002 National Science Council, ROC and S, Karger AG, Basel

Reactive oxygen species have been implicated in the pathogenesis of a variety of acute injury models, including ischemia-reperfusion injury [1, 17, 22] and ethanol- induced renal failure [ 19]. Acute ingestion of absolute ethanol (5 g/kg) has been reported to lead to an accelerated increase in lipid peroxidation, an index of oxidative stress [9]. Protection against renal injury can be achieved by a variety of agents, including scavengers of hydroxylation [2, 3, 8, 16] and superoxide dismutase, which converts superoxide to hydrogen peroxide [7]. Previous studies have shown that renal failure is often associated with ischemic injury [12] and nephrectomy [14]. Absolute ethanol- induced renal failure is a useful model in mice [9], but the detailed mechanism of its pathogenesis is still not fully clear.

Oxygen is essential for life, but it may also be danger- ous. Reduction of oxygen in tissue produces a number of oxygen free radicals which may induce cellular damage and even cell death. Oxygen-handling cells have different systems, e.g. superoxide dismutase, peroxidases and cata- lases, which protect them against the toxic effects of oxygen free radicals [24].

Tetramethylpyrazine (TMP) is a constituent of

Ligus-

ticum wallichii

French [ 18]. It not only blocks the entry of extracellular calcium through calcium channels but also inhibits the release of intracellular stored calcium in v a s -

K A R G

E R

Fax+41 61 306 1234 E-Mail karger@karger, ch um~.karger.cem

1021-7770/02/0094-0299518.50/0 Accessible online at:

www. karger.com/journals/jbs

Prof. Song-Chow Lin, PhD Professor of Pharmacology

Department of Medicine, Taipei Medical University 250, Wu-hsing Street, Taipel, Taiwan (ROC)

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cular s m o o t h muscle cells [4, 13, 14, 18]. In 1997, Feng et al. [4] reported that p r e t r e a t m e n t with T M P in hypoxic isolated rat heart enhances prostaglandin I2 outflow a n d attenuates the release o f t h r o m b o x a n e A2 in rat heart during n o r m o x i a , h y p o x i a a n d reoxygenation, a n d hence could significantly protect the m y o c a r d i u m f r o m h y p o x i c injury. Actually, T M P could be useful as a therapeutic agent in ischemic heart disease with c o r o n a r y artery disease b y suppressing c o r o n a r y vasoconstriction a n d ischemic changes in the tissues p r o d u c e d b y endothelin-1 [6,

19, 29].

Since T M P has been c o n f i r m e d to be a true calcium antagonist, it m a y play very i m p o r t a n t roles in the area o f tissue protection a n d preservation. In addition, in its use as a highly potent antihypertensive drug, it m a y exert favor- able effects on renal h e m o d y n a m i c s related to the reversal of renal vasoconstrictors [ 13]. Although the m e c h a n i s m s o f action of T M P in the setting of chronic renal failure are not yet fully established, its beneficial effects m a y be related to protective actions such as the reduction o f renal hypertro- phy, m o d u l a t i o n of mesangial cell uptake of m a c r o m o l e - cules, changes in the permselectivity o f the glomerulus a n d decreased free radical formation.

Acute a d m i n i s t r a t i o n o f absolute ethanol often leads to tissue damage, especially in the renal system [26]. T h e a i m s o f the present study were to investigate w h e t h e r T M P a d m i n i s t r a t i o n p e r os in m i c e could protect the kidney f r o m absolute ethanol-induced lesions, a n d if T M P did offer protection, what its m e c h a n i s m o f action might be.

M e t h o d s

Animals and Treatment

Male ICR mice (about 20-25 g) were purchased t¥om the animal center, College of Medicine, National Yang-Ming University, Tai- wan. They were kept for at least 1 week on commercial diets (Fu-So Co., Taipei, Taiwan) under controlled environmental conditions (25 -+ 1 °C, 55 -+ 5% humidity) with free access to food and water. ICR mice were randomly divided into eight groups of 10 animals each. Group 1 (control) received saline (0.9% sodium chloride solution, 10 ml/kg p.o.), group 2 received absolute ethanol (10 ml/kg p.o.) and groups 3-5 received ethanol mid TMP at doses of 10, 25 and 50 mg/ kg p.o., respectively. TMP was administrated orally 30 min before oral administration of 10 ml/kg absolute ethanol. The animals were killed 1 h after administration of absolute ethanol. The procedure was described in detail in a previous report of Zhang et al. [30].

Assessment of Renal Failure Index

All blood samples were collected by cutting the carotid artery and were allowed to coagulate at room temperature for 1 h. A serum sam- ple was used in the determination of blood urea nitrogen (BUN) and creatinine levels [26].

Determination of Lipid Peroxidation by Measurement of Thiobarbituric Acid Reactive Substance in vivo

The effect of TMP on mice renal homogenate with lipid peroxidation was determined using malonic dialdehyde (MDA)-thiobarbituric acid according to the modified method described by Yuda et al. [28]. Both the kidneys of the animals were removed and placed in an incubator for 1 h at 37°C for homeostasis. After incubation, 9 ml of distilled water and 2 mI of 0.6 % thiobarbituric acid were added to the incubated mixture, which was then subjected to vigorous shaking. The mixture was heated for 30 rain in a boiling water bath. After cooling, 5 ml of n-butanol was added and the mixture was again shak- en vigorously. The n-butanol layer was separated by centrifngation at 1,000 g for 10 min, and MDA production was measured at 532 nm [27].

Cytochrome C Test in vitro

Superoxide anions were assayed spectrophotometricatly according to the reduction method described by McCord and Fridovich [11]. Xanthine oxidase converts uric acid to yield superoxide anions, followed by direct reduction of ferricytochrome C to ferrocyto- chrome C, which has a specific UV absorbance at a wavelength of 550 nm. When a compound shows superoxide scavenging activity, there is a decrease of the UV absorbance spectra in the reduction of ferricytochrome C.

Drugs and Chemicals

Absolute ethanol, BUN kit, creatinine kit, thiobarbituric acid, sodium dodecyl sulfate, ferric chloride, n-butanol, xanthine oxidase and cytochrome C were all purchased from Sigma Chemical Compa- ny (St. Louis, Mo., USA). Acetic acid was obtained from a local com- pany in Taipei, Taiwan. TMP was a gift of the Institute of Chinese Materia Medica, China Academy of Traditional Chinese Medicine, Beijing, PROC.

Statistical Analysis

All data are shown as mean -+ SE (n = 10). Statistical analysis was assessed by one-way analysis of variance coupled with Dunnett's test or the Newman-Keuls test. The level of significance was chosen as p < 0.05.

Results

Renal Protective Effect o f T M P on Absolute Ethanol-Induced A cute Renal Toxicity

T h e results show a rise in B U N a n d creatinine values p r o d u c e d by ethanol (table 1); however, w h e n various doses (10, 25 a n d 50 mg/kg) o f T M P were a d m i n i s t e r e d as p r e t r e a t m e n t , these values were significantly reduced.

Positive Control by T M P

T M P at 10, 25 a n d 50 mg/kg was able to significantly i m p r o v e renal function. It is interesting to find that T M P could inhibit lipid p e r o x i d a t i o n - i n d u c e d M D A f o r m a - tion, e v e n in n o r m a l control kidney (table 2).

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T a b l e 1. Effect of T M P on absolute ethanol-induced serum B U N T a b l e 3. Inhibitory effect of T M P on absolute ethanol (10 ml/kg)-

and creatinine increases in mice induced lipid peroxidation in mice renal homogenate in vivo

N o r m a l saline p.o. 17.9 + 1.4 0.07 -+ 0.04 AE p.o. 2 7 . 9 + 1 . 7 " * * 0 . 2 1 + 0 . 0 1 " * * N o r m a l control - 0 . 1 9 4 + 0 . 0 3 0 - AE + T M P (10 mg/kg) p.o. 18.1_+1.3 ## 0 . 2 0 + 0 . 0 1 ## AE - 0.248-+0.010" - AE + T M P (25 mg/kg) p.o. 13.6-+1.2 ### 0.06_+0.03 ## A E + T M P 10 0.156_+0.020 ## 37 AE + T M P (50 mg/kg) p.o. 13.4-+ 1.3 ### 0.06 _+ 0.03 ## AE + T M P 25 0.149 _+ 0.004 ## 40 A E + T M P 50 0.119-+0.006 ## 52

Each value represents m e a n -+ SE (n = 10). AE = Absolute ethanol. *** p < 0.001 compared to the n o r m a l control group; ## p < 0.05, ###p < 0.001 c o m p a r e d to the absolute ethanol group (Newman- Keuls test).

Each value represents m e a n + SE (n = 10). PEE = Propolis ethanol extract; AE = absolute ethanol. * p < 0.05 compared to the n o r m a l control group; ##p < 0.05 c o m p a r e d to the absolute ethanol group (Newman-Keuls test).

T a b l e 2. Effect of various doses of T M P (10, 25, 50 mg/kg) on lipid T a b l e 4. ICs0 of three different concentrations of T M P in the in vitro

peroxidation in vivo assay of the i n h i b i t i o n of lipid peroxidation

N o r m a l control 0.194-+ 0.030 T M P (0.01 mg/ml) 0.114 + 0.001

T M P (10 mg/kg) 0.197 + 0.007 ### T M P (0.1 mg/ml) 0.112 + 0.002

T M P (25 mg/kg) 0.179 -+ 0.005 ### T M P (1.0 mg/ml) 0.102 -+ 0.001

T M P (50 mg/kg) 0.133 + 0.004 ###

Each value represents m e a n -+ SE (n = 10). ###p < 0.001 compared to the n o r m a l control group (one-way analysis of variance coupled with D u n n e t t ' s test).

The data shown are those derived from a concentration response tested with three different concentrations of TMP. Each value represents the m e a n + SE of three i n d e p e n d e n t assays in concentration d e t e r m i n a t i o n studies; each assay was done in triplicate.

Inhibitory Effect o f TMP on Tissue Lipid Peroxidation in Absolute Ethanol-Induced Acute Renal Toxicity in vivo

It has been reported that absolute ethanol stimulates lipid peroxidation in the kidney [9]. Ethanol treatment in the present study caused a rise in the MDA value (table 3). Various doses of TMP (10, 25, 50 mg/kg) inhibited absolute ethanol-stimulated lipid peroxidation in mice kidney (table 3).

Cytochrome C Test in vitro

In the cytochrome C test, the ICs0 of three different concentrations of TMP in the in vitro assay of the inhibition of lipid peroxidation ranged from 0.114 _+ 0.001 to 0.102 +- 0.002 ~tM. TMP at 1.0 mg/ml exhibited the strongest superoxide scavenging activity (table 4).

Discussion

In the present study, the renal protective effect of TMP on absolute ethanol-induced renal injury was investigated in ICR mice. Serum levels of BUN and creatinine and superoxide scavenging activity were used as indicators of renal protection. As one might suspect, an increase in the serum BUN level may also be induced by muscle protein breakdown [ 10, 21 ], but muscle protein breakdown could not lead to an increase in the creatinine level [15]. As many authors have reported that an increase in both serum BUN and creatinine levels could be used clinically to imply renal insufficiency [5, 20], we decided to choose the tests of serum BUN and creatinine levels as the indica- tion of renal damage.

The acute administration of absolute ethanol (10 ml/ kg) to mice leads to a marked elevation of serum BUN and creatinine levels. This elevation of BUN and creatinine levels reflects the degree of renal injury. In the

Protective Effect of Tetramethylpyrazine on Renal Toxicity

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present study, pretreatment of absolute ethanol-intoxi- cated mice with TMP significantly decreased renal toxicity and the serum BUN and creatinine levels (table 1). It can be concluded from these results that TMP possesses a remarkable protective effect on absolute ethanol-induced renal injuries.

With respect to the lipid peroxidative product MDA, many researchers have reported that overproduction of MDA may also lead to renal damage [23, 25]. Compared to the positive control, TMP, administered orally at various concentrations (10, 25 and 50 mg/kg), exhibited significant inhibition of lipid peroxidation and hence significantly decreased MDA formation in vivo (table 2). From table 3, it can be seen that TMP also exhibited significant

inhibition of absolute ethanol-induced lipid peroxidation in vivo. These findings indicate that the decrease in MDA formation is likely to play an important role in the pre- vention of renal injuries induced by absolute ethanol.

Taken together, the results of the present study indicate that the production of free radicals may be involved in the pathogenesis of renal injuries induced by absolute ethanol. They also show that TMP significantly inhibits the formation of renal injuries induced by absolute ethanol, probably through its inhibitory effect on membrane lipid peroxidation and free radical formation or due to its free radical scavenging ability, and that it can thereby simultaneously decrease BUN and creatinine increases induced by absolute ethanol.

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