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Effect of Wen-Pi-Tang Extract on Lung Damage By Influenza Virus Infection

Posted on: Sunday, 30 January 2005, 03:00 CST

Abstract

The effect of Wen-Pi-Tang extract on influenza virus infection in mice was investigated. The administration of WenPi-Tang extract at a dose of 100mg/kg body wt. for 8 consecutive days to influenza virus- infected mice reversed the lack of body wt. gain and prevented the increase in lung weight caused by the infection in comparison with uninfected mice, while allopurinol, a xanthine oxidase (XOD) inhibitor, did not show these effects. The serum levels of uric acid and allantoin in influenza virus-infected mice were reduced by Wen- Pi-Tang extract administration. Moreover, WenPi-Tang extract reduced the uric acid level more as the dose increased, although it exerted lower activity than allopurinol. The XOD activity of the lungs was elevated by influenza virus infection, but Wen-Pi-Tang extract administration inhibited this activity, indicating prevention of lung damage by oxygen free radicals generated by XOD. After the administration of Wen-Pi-Tang extract to influenza virus-infected mice, the lung Superoxide dismutase activity was not significantly different from that of uninfected mice, whereas lung catalase activity was lower in the former than the latter, but slightly higher than that of influenza virus-infected mice, suggesting that Wen-Pi-Tang extract may prevent the generation of highly toxic hydroxyl radicals in the lung. In addition, the administration of both Wen-Pi-Tang extract and allopurinol reduced the degree of lung consolidation caused by influenza virus infection. In particular, Wen-Pi-Tang extract reduced the consolidation score in a dose- dependent manner and more markedly than allopurinol did. This study suggests that Wen-Pi-Tang extract could improve pathological conditions of the lungs induced by influenza virus infection.

2004 Elsevier GmbH. All rights reserved.

Keywords: Wen-Pi-Tang; Influenza virus; Uric acid; Allantoin; Xanthine oxidase; Superoxide dismutase; Catalase; Lung; Consolidation; Allopurinol

Introduction

The role of oxygen free radicals in the pathological damage of influenza virus-infected lungs has been reported by several workers (Oda et al., 1989; Akaike et al., 1996; Knobil et al., 1998; Akaike, 2001; Suliman et al., 2001). In particular, the Superoxide anion (O^sup -^^sub 2^) produced by xanthine oxidase (XOD) is considered to enhance oxidative stress after viral infection. It has been established that elevated XOD activity and enhancement of purine metabolism cause O^sup -^^sub 2^ generation, which could produce the highly toxic hydroxyl radical (OH) during the pathogenesis of influenza virus infection (Saugstad and Aasen, 1980; Akaike et al., 1990). Activated oxygen can also cause tissue injuries, such as lung damage in adult respiratory distress syndrome and other inflammatory diseases (Johnson et al., 1981; Tate and Repine, 1983). Therefore, therapeutic approaches to the elimination of oxygen radicals generated by XOD and catabolic supply from adenosine metabolism seem necessary.

Antioxidant agents with radical-scavenging activity would be expected to improve pathological conditions of free radical-related diseases, including viral diseases. Recently, traditional Chinese medicines have attracted a great deal of attention as promising new antioxidants and antioxidant therapy offers protection against a wide range of free radical-induced diseases. Wen-Pi-Tang, a Chinese traditional prescription, has been reported to have a radical- scavenging action (Yokozawa et al., 2000; Cho et al., 2000). In addition, renal failure under conditions of enhanced oxidative stress improved after the administration of Wen-Pi-Tang in both experimental and clinical settings (Yokozawa, 1996; Ninomiya et al., 1998; Mitsuma et al., 1999; Yokozawa et al., 2001). This study was carried out to investigate the effect of Wen-Pi-Tang extract on lung damage caused by influenza virusinfected pathological conditions that result in the generation of free radicals such as O^sup -^^sub 2^ and ^sup *^ OH.

Fig. 1. Three-dimension HPLC profile of Wen-Pi-Tang extract.

Materials and methods

Preparation of Wen-Pi-Tang extract

The composition of the Wen-Pi-Tang used in this study was 15 g Rhei Rhizoma (Rheum officinale BAILLON), 3 g Ginseng Radix (Panax ginseng C.A. MEYER), 9 g Aconiti Tuber (Aconitum japonicum THUNBERG), 3 g Zingiberis Rhizoma (Zingiber officinale ROSCOE) and 5 g Glycyrrhizae Radix (Glycyrrhiza glabra LINN. var. glandulifera REGEL et HERDER). Ginseng Radix was produced in Korea, Aconiti Tuber was from Japan and all the other ingredients were from China. The above crude drugs were chopped finely and extracted with distilled water at 100C for 65 min, as described previously (Oura et al., 1984). After removal of the insoluble matter by filtration, the filtrate was concentrated in vacuo and lyophilized to yield a brown residue. The yield of the extract was about 30% by weight of the original preparation. For analysis of Wen-Pi-Tang component, the aqueous extract was filtered and subjected to treatment with an Alumina cartridge (Bond Elute Co., Ltd.). HPLC equipped with a LC- 10AD pump (Shimadzu, Tokyo, Japan) and a SPD-M10AVP absorbance detector was performed using a TSK GEL ODS-80Ts column (250 4.6 mm). The solvents were (A) 0.05 M AcOH-AcONH^sub 4^ (pH 3.6), (B) 100% CH^sub 3^CN. A linear gradient of 90% A and 10% B changing over 60 min to 100% B was used. The flow rate was 1.0ml/min. The effluent from the column was monitored with a UV detector. A three-dimension HPLC profile of Wen-Pi-Tang extract is shown in Fig. 1.

Influenza virus infection model and treatment

All experiments using animals were conducted in accordance with the "Recommendations on the Establishment of Animal Experimental Guidelines" approved by Toyama Medical and Pharmaceutical University. Male ICR mice weighing about 25 g each at 5 weeks after birth, used in experiments 1 and 2, were purchased from Japan SLC Inc. (Hamamatsu, Japan). Wen-Pi-Tang extract or allopurinol (Wako Pure Chemical Industries, Ltd., Osaka, Japan) was dissolved in physiological saline and administered orally by stomach tube for one day, and then the mice were inoculated orally with influenza virus, A/PR/8 strain (1500 PFU). Subsequently, the administration of Wen- Pi-Tang extract and allopurinol were started at the day of infection with influenza virus. In experiment 1, Wen-Pi-Tang extract was administered at one dose, 100mg/kg body wt., for 6 consecutive days after infection with influenza virus and in experiment 2, groups of mice received 100, 200 or 400mg/kg body wt. for the same period. Allopurinol was administered at a dose of 20mg/kg body wt. in both experiments 1 and 2 for the same period. Six days after infection, the mice were weighed and sacrificed, and blood samples were obtained by cardiac puncture. The serum was immediately separated from the blood samples by centrifugation. The lungs were removed, weighed, rinsed with cold physiological saline and assayed.

Serum levels of uric acid and allantoin

Uric acid levels were measured using a commercial kit (Uric Acid C-Test, Wako; Wako Pure Chemical Industries, Osaka, Japan). Allantoin levels were measured according to the method of Young and Conway (1942).

Enzyme assays

The XOD activity was evaluated using the method of Bergmeyer et al. (1974). Briefly, lung tissue was homogenized in 9 volumes of 0.25 M sucrose solution and the reaction was initiated by the addition of sodium phosphate buffer (pH 7.4), EDTA and hypoxanthine. After incubation at 37C for 60 min, the XOD activity was determined by measuring the absorbance at 292 nm. To determine the activities of Superoxide dismutase (SOD) and catalase, lung tissue was homogenized with a four-fold volume of ice-cold physiological saline. The SOD activity was measured according to the nitrous acid method described by Elstner and Heupel (1976) and Oyanagui (1984), based on inhibition of nitrite formation from hydroxylamine in the presence of O^sup -^^sub 2^ generators. Catalase activity of lung tissue was determined by following the decomposition of hydrogen peroxide (H^sub 2^O^sub 2^) directly by monitoring the decrease in extinction at 240 nm (Aebi, 1974). The protein levels of lung tissues were quantified as described by Itzhaki and Gill (1964), using bovine serum albumin as a standard.

Lung consolidation

Consolidation was scored by assessing the degree of consolidation of the 5 lobes of one lung according to the method of Sidwell et al. (1998) with a slight modification. If less than 50% and 50% or more of one lobe showed consolidation, consolidation was expressed as a score of 1 and 2 respectively. Therefore, the maximum score was 10 in cases where all 5 lobes of the lung displayed [greater than over equal to]50% consolidation.

Statistical analysis

The data are presented as means S.E. Differences among groups were analyzed using Dunnett's test and those at p < 0.05 were accepted as significant.

Results

Experiment 1

Figure 2 shows the changes in body weight of influenza virus- infected mice. While the average body weight of uninfected mice increased gradually over 7 days from 25.3 to 30.8 g (5.5 g increase), that of control mice infected with influenza virus increased slightly from 24.6 to 26.9 g (2.3 g increase). In addition, mice given the XOD inhibitor allopurinol, at a dose of 20mg/kg body wt., showed a decline in body weight during the experimental pe\riod from 24.9 to 24.2 g (0.7 g decrease). In particular, their body weight decreased even more than that of control infected mice after influenza virus infection. The administration of Wen-Pi-Tang extract, however, at a dose of 100mg/ kg body wt. for the same period, increased body weight after viral infection to 28.6g.

The effects of influenza virus infection of mice on lung weights are presented in Table 1. The lungs of both control and allopurinol- treated mice (1.09 and 1.11g/ 100 g body wt., respectively) weighed significantly more than those of uninfected mice, whereas the lung weight of Wen-Pi-Tang extract-treated mice (0.92g/100g body wt.) was little different from that of uninfected mice.

Fig. 2. Body weight. ([white circle]), uninfected mice; ([white triangle up]), influenza virus-infected mice; ([white square]), influenza virus-infected mice administered 100mg Wen-Pi-Tang extract; ( ), influenza virus-infected mice administered 20 mg allopurinol.

Table 1. Lung weight

Table 2. Uric acid and allantoin in serum

Table 2 shows the effects of Wen-Pi-Tang extract on the serum levels of uric acid and allantoin in influenza virus-infected mice. The influenza virus-infected control mice had higher total serum level of uric acid and allantoin than uninfected mice. However, the administration of Wen-Pi-Tang extract to influenza virus-infected mice, at a dose of 100mg/kg body wt., reduced the uric acid level by 15% to 2.69mg/dl in comparison with the control group level of 3.18mg/dl and the total level of uric acid plus allantoin from 6.26 to 5.44mg/dl (13% decrease, p < 0.01).

The activities of XOD, SOD and catalase in the lungs after influenza virus infection are presented in Table 3. The XOD activity of mice infected with influenza virus was 391.6 pmol/mg protein/ min, while that of uninfected mice was not detectable. The administration of Wen-Pi-Tang extract, at a dose of 100mg/kg body wt. for 8 consecutive days, significantly reduced this elevated XOD activity from 391.6 to 198.8 pmol/mg protein/ min (49% decrease, p < 0.001). The SOD activity of influenza virus-infected mice given Wen- Pi-Tang extract was not significantly different from that of uninfected mice, whereas that of control mice was enhanced. The lungs of influenza virus-infected control mice showed significantly lower catalase activity than those of uninfected mice. After the administration of Wen-Pi-Tang extract, however, the catalase activity increased slightly from 29.19 to 35.18 U/mg protein, although the increase did not show significance.

Experiment 2

In experiment 1, we observed that Wen-Pi-Tang extract had a protective effect against influenza virus infection. Therefore, in experiment 2, different doses of the extract (100, 200 and 400 mg/ kg body wt.) were administered to investigate whether the effects are dosedependent. The effects of Wen-Pi-Tang extract and allopurinol on serum uric acid levels after influenza virus infection are shown in Fig. 3. The increase in the level of serum uric acid induced by influenza virus infection was attenuated significantly by Wen-Pi-Tang extract administration, although Wen- Pi-Tang extract exhibited lower activity than allopurinol. The Wen- Pi-Tang extract administration at 200 and 400 mg showed higher activity than that of 100 mg, but the activities between 200 and 400 mg oral doses did not reveal significant difference.

As shown in Fig. 4, consolidation of the lung occurred in influenza virus-infected control mice, but not in uninfected mice. Although the administration of both Wen-Pi-Tang extract and allopurinol reduced the degree of lung consolidation caused by influenza virus infection, the consolidation score of Wen-Pi-Tang extract-treated mice declined in a dose-dependent manner and to a greater extent than it did after allopurinol treatment.

Discussion

Influenza virus infection causes various physiological and pathological effects via several mechanisms. The infection has an effect on physiological parameters such as body wt. gain and tissue weight. As demonstrated by the present results, the body wt. gain was less and the lung weight, which is considered to reflect the amount of tissue damaged by the influenza virus, increased as compared with uninfected mice (Fig. 2 and Table 1). Although allopurinol is well-known to show a protective effect against influenza virus infection, it did not show protective activity against the changes in body and lung weight. In contrast, the Chinese traditional prescription Wen-Pi-Tang extract partially prevented the lack of body wt. gain, resulting in some increase in weight, and inhibited the increase in lung weight after viral infection.

Table 3. Enzyme activities in lung tissue

Fig. 3. Uric acid in serum. Statistical significance: (a)p<0.05, (b)p<0.001 vs. uninfected values, (c)p<0.05, (d)p<0.001 vs. values for influenza virus-infected control.

Generation of oxygen free radicals by XOD, coupled with hypoxanthine supplied by adenosine catabolism, is a pathogenic principle of influenza virus infection. Maeda and Akaike (1991) reported that the dramatic increase in O^sup -^^sub 2^ generation by influenza virus resulted from XOD and a highly elevated supply of hypoxanthine and xanthine, which are catabolic products located downstream of the adenosine-to-inosine conversion step. Hypoxanthine and molecular oxygen generate xanthine and O^sup -^^sub 2^ and they are converted to uric acid and O^sup -^^sub 2^ by XOD. Thus, uric acid is the excreted end product of purine metabolism in primates, birds and some other animals. In many other vertebrates, however, uric acid is degraded further to allantoin by urate oxidase. The enhancement of cellular adenosine catabolism resulting from influenza virus infection thus enhanced the serum levels of uric acid and allantoin. In this study, we confirmed that the uric acid level was elevated by this infection (Table 2 and Fig. 3), but the administration of Wen-Pi-Tang extract to influenza virus-infected mice reduced significantly the serum levels of uric acid and allantoin, suggesting that Wen-Pi-Tang extract inhibits the catabolism of adenosine to uric acid and/or the allantoin- conversion step. Furthermore, allopurinol, which is well known to be a specific inhibitor of XOD, also reduced the serum uric acid level. Akaike et al. (1990) demonstrated that allopurinol exerted a protective effect in influenza virus-infected mice: it inhibited O^sup -^^sub 2^ generation due to elevated XOD activity and eventually improved the survival rate. Our results also showed that allopurinol was effective in reduction of the uric acid level in serum and Wen-Pi-Tang extract exerted relatively low activity compared with allopurinol. Taken together with the results of changes in body weight and lung weight, however, which show the decline in body weight and the increase in lung weight by allopurinol, Wen-Pi-Tang could be expected to ameliorate the pathological conditions induced by influenza virus without toxicity.

Fig. 4. Consolidation score. Statistical significance: (a)p<0.05, (b)p<0.001 vs. values for influenza virus-infected control.

In the normal physiological state, the XOD activity in many tissues is very low, but has been shown to increase dramatically after viral infection as well as after ischemia-reperfusion. Excessive amounts of O^sup -^^sub 2^, XOD and lipid peroxidation in serum and lung tissue of mice infected with influenza virus have been demonstrated (Maeda and Akaike, 1991; Rodell et al., 1987; Peterhans, 1997; Mileva et al., 2000). The rises in adenosine deaminase and XOD activity are linked closely to the infiltration of lung tissue by inflammatory cells and later pathological manifestations in the lung. We therefore measured XOD activity in the lungs of influenza virusinfected mice (Table 3). While XOD activity was not detected in the lungs of uninfected mice, it was high in the lungs of influenza virus-infected mice. This indicates that the elevated XOD activity results in the generation of oxygen free radicals that could be responsible for tissue injuries in biological systems. It was thus considered that the inhibition of XOD was also beneficial in that it suppresses the production of active oxygen. The administration of Wen-Pi-Tang extract inhibited elevation in XOD activity, suggesting that it protected the lung from oxygen-free-radical-related pathological damage by the influenza virus.

Damage in tissues and cells induced by activated oxygen species could be prevented by SOD, catalase or both. Suliman et al. (2001) reported that enhancing the extracellular SOD levels of the lung minimized influenza-induced lung injury by ameliorating inflammation and attenuating oxidative stress. Furthermore, Oda et al. (1989) demonstrated that pyran copolymer-conjugated to copper-zinc SOD protected mice from the lethal effect of influenza virus. SOD is a metalloenzyme that effectively removes O^sup -^^sub 2^ from the system and the abundant catalase activity converts H^sub 2^O^sub 2^ into water and the less active molecular oxygen. The lungs of influenza virus-infected mice showed higher SOD activity than those of uninfected mice, while the former had lower catalase activity than the latter. This suggests that excessive generation of O^sup - ^^sub 2^ by influenza virus is liable to remove catalase and thus SOD generates H^sub 2^O^sub 2^, which is not converted to less reactive molecules because of the low catalase activity, resulting in accumulation of the more toxic and reactive ^sup *^OH responsible for pathological tissue damage. This result suggests that H^sub 2^O^sub 2^ or its metabolic products such as ^sup *^OH are central to the lung injury resulting from viral infection. It is known that O^sup -^^sub 2^ itself is not particularly toxic to some cells or tissues. Its major role in tissue injury is to reduce ferric iron to ferrous iron, which in turn catalyzes a Fenton reaction and results i\n generation of the more toxic and reactive ^sup *^OH from H^sub 2^O^sub 2^ (Halliwell and Gutteridge, 1984). Therefore, the conversion of O^sup -^^sub 2^ to ^sup *^OH may be involved in the pathogenesis of influenza virus infection in mice. After the administration of WenPi-Tang extract to influenza virus-infected mice, SOD activity was not significantly different from that of uninfected mice, whereas the catalase activity was lower than that of uninfected mice, but slightly higher than that of influenza virus- infected control mice (Table 3). Along with the much lower generation of O^sup -^^sub 2^ by the inhibition of XOD activity in Wen-Pi-Tang extract-administered mice, the oxidative damage induced by O^sup -^^sub 2^ and ^sup *^OH in the Wen-Pi-Tang extract administered mice is considered to be alleviated more efficiently than in control mice, despite the fact that catalase activity showed a slight but non-significant increase.

Another typical characteristic of influenza infection in the lung is the presence of areas of surface consolidation, which, under the microscope, show extensive hemorrhage, infiltration of lymphoid cells, including neutrophils and macrophages, and edema in the alveolar spaces (Oda et al., 1989). We also observed consolidation of lung lobes in influenza virus-infected mice, which means their lungs filled with fluids and cellular debris as the infection spread (Fig. 4). Wen-Pi-Tang extract and allopurinol, however, reduced the degree of consolidation significantly, indicating that they prevented the lung damage caused by influenza virus infection. In particular, Wen-Pi-Tang extract was even more effective in protection against lung consolidation than allopurinol (Fig. 4), even though allopurinol reduced the serum uric acid level more significantly than Wen-Pi-Tang did (Fig. 3). From these results, allopurinol is considered to inhibit purine metabolism, resulting in the decline of uric acid level; however, the lung protective activity from influenza virus is higher in Wen-Pi-Tang extract- administered mice. Although the mechanism of higher activity of Wen- Pi-Tang extract is obscure, the stronger scavenging activity of free radicals, especially OJ and ^sup *^OH, is considered to be mainly attributed to it.

Recently, among the various antioxidants available, Chinese prescriptions have attracted much attention and oriental medicines, which contain a variety of antioxidant compounds, are still in great demand despite the widespread use of conventional medicines. Of the numerous prescription Chinese medicines, Wen-PiTang, composed of Rhei Rhizoma as the main ingredient, together with Ginseng Radix, Aconiti Tuber, Zingiberis Rhizoma and Glycyrrhizae Radix, has been reported to eliminate free radicals (Yokozawa et al., 2000; Cho et al., 2000). Our previous studies demonstrated that Wen-Pi-Tang as a whole and some of its component crude drugs exerted antioxidant action on the kidney impaired by oxidative stress (Yokozawa, 1996; Ninomiya et al., 1998; Mitsuma et al., 1999; Yokozawa et al., 2001). Our present investigation demonstrated the protective activity of Wen-Pi-Tang extract against lung injury resulting from oxidative stress due to influenza virus infection. Our present results showed that the administration of Wen-Pi-Tang extract ameliorated markedly the pathological effects of influenza virus-induced conditions in mice, possibly through its protective action against oxidative stress in the lung. Thus, Wen-Pi-Tang may provide an opportunity to attenuate therapeutically the pathological events which follow influenza virus infection.

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T. Yokozawa(a),*, M. Sekiya(a), E.J. Cho(a), M. Kurokawab, K. Shiraki(b)

a Institute of Natural Medicine, Toyama Medical and Pharmaceutical University, 2630 Sugitani, Toyama 930-0194, Japan

b Department of Virology, Toyama Medical and Pharmaceutical University, Toyama, Japan

Received 4 March 2003; accepted 28 May 2003

* Corresponding author. Tel.: +81-76-434-7631; fax: +81-76-434- 4656.

E-mail address: yokozawa@ms.toyama-mpu.ac.jp (T. Yokozawa).

Copyright Urban & Fischer Verlag Nov 2004

Source: Phytomedicine

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