Protection of Oxidative Brain Injury

29 Protection of Oxidative Brain Injury

by Chinese Herbal Medicine Shengmai San as a Model Formula for Antioxidant-Based-Compounds Therapy of Oxidative Stress-Related Diseases

Tetsuya Konishi, Haruyo Ichikawa, and Hiroshi Nishida

Niigata University of Pharmacy and Applied Life Sciences Niigata, Japan

XueJiang Wang Capital University of Medical Sciences

Beijing, China

I. INTRODUCTION Oxidative stress is implicated in many diseases related to lifestyle and aging.

Therefore, antioxidant protection attracts much attention against these diseases (1–3). The brain is essentially vulnerable against oxidative abuse because of its poor anitoxidant defense including low level of antioxidant

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small molecules and also antioxidant enzymes (4). Moreover, the tissue is rich in sensitive cellular components toward oxidative abuse such as polyunsat- urated fatty acids and catecholamine. Indeed accumulated oxidative injury of cellular components results in many brain disorders including Alzheimer’s and Parkinson’s diseases. Therefore, protection of cerebral oxidative injury is important for the quality of life in our society. The antioxidant approach is attractive for the treatment of cerebral disorders including dementia, and many trials have been reported to prevent cerebral oxidative tissue injury using natural and artificial small antioxidants such as vitamins E, lazaroid, and lipoic acid, as well as antioxidant enzymes (5–7).

However, this approach is complicated, because the tissue-damaging process initiated by free radicals is not simple but involves several complex steps such as Ca 2+ release in cytoplasm, membrane potential dissipation, free iron release, etc. (1). At the same time, the cellular repair system operates to prevent further progression of tissue injury. In this sense, combination therapy would be a more promising approach to treat these complex pathological conditions in that prevention and repair of radical-initiated tissue damages are modulated by the combined effects of antioxidants and other components in a complex formula that modulates cellular function.

II. TRADITIONAL CHINESE HERBAL MEDICINE AS A MODEL OF ANTIOXIDANT-BASED-COMPOUNDS FORMULA

In this sense, traditional Chinese herbal medicine (TCM) is interesting because herbs have been used to treat many complex disorders such as diabete mellitus, cancer, and others for which Western medicine may not have an appropriate diagnostic name. TCM is usually prescribed with several herbal constituents having different functions and the synergistic action exhibited by the multicomponent formula is effective for the management of many complicated diseases (8). Moreover, their therapeutic strategy is modulation of the inherent potential to recover the distorted balance of the physical condition (disease condition) by either suppressing or stimulating physiolog- ical reactions with a multifunctional-compound formula comprising several herbal components. It is also known that free-radical or reactive oxygen species (ROS) are exclusively involved in the pathogenesis of such disorders for which the Chinese herbal medicines are effectively applied. Therefore, antioxidant TCM is an attractive model for study of the antioxidant-based- compounds therapy of disorders related to oxidative stress. In this chapter, we discuss results obtained in the study of the preventive effect of Shengmai San (SMS), a traditional Chinese herbal prescription, on cerebral oxidative injury in rats.

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III. ANTIOXIDANT POTENTIAL OF SMS DETERMINED IN VITRO

SMS, a famous Chinese medicinal formula that has been used for more than 800 years in China, is comprised of three herbal components, Panax ginseng, Shisandra chinensis , and Ophiopogon japonicus (9). Traditionally, SMS is used for the treatment of excess loss of essence qi and body fluid that threatens heart failure. It can restore blood volume and prevent myocardial infarction. SMS is also prescribed for patients with coronary heart disease and various cardiovascular disorders.

Ancient TCM theory states that the physical condition of the human body is controlled by the interaction of five elemental organs, heart, liver, kidney, lung, and spleen, although the nature of these organs is not the same as understood in Western medicine. The brain was not classified in the five elemental organs but its function is considered strictly related to liver, kidney, and heart functions. Therefore, it is worth examining protective effects on cerebral oxidative damage by TCM prescriptions that have been used for treating complex diseases, especially related to heart, lung, or kidney failure. In this sense, it is interesting to study the effect of SMS on cerebral oxidative injury.

We first examined the antioxidant property of SMS by five different antioxidant assays in vitro and the antioxidant activity was compared using Trolox as the reference antioxidant in each antioxidant assays. (Table 1). Interestingly, SMS was found to have extremely strong hydroxyl-radical- scavenging activity. This characteristic was emphasized when the antioxidant activity was compared with those determined for four typical TCM or Kampo (Japanese traditional herbal prescriptions) formulations that have been tested for post stroke treatment or dementia: Cho-To-San, Zokumei-To, Ryou-Kei- Jutsu-Kan-To, and Keishi-Bukuryo-Gan+Yokuinin. All these prescriptions commonly had stronger scavenging activity toward superoxide radical than

T ABLE 1 Antioxidant Potential of SMS as Trolox Equivalent Determined by Several In Vitro Assay Systems

SMS IC 20 as Antioxidant assay

Trolox equivalent (AM) TBARS formation

DPPH quenching 3.2 Crocin bleaching test

1.1 Superoxide radical scavenging

Hydroxyl radical scavenging 0.4

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F IGURE 1 Relative antioxidant activity of brain-directed TCM formulations. SMS, Shengmai San; DTS, Cho-To-San; LGSGT, Rei-Kei-Jutsu-Kan-To; XMT, Zokumei-To; GZFL+YY, Keishi-Bukuryou-Gan+Yokuinin.

to other radicals but SMS did not. In contrast, SMS had strong hydroxyl- radical-scavenging potential (Fig. 1).

IV. PREVENTION OF CEREBRAL OXIDATIVE INJURY PRODUCED BY ISCHEMIA REPERFUSION BY SMS

When the SMS was administered to rats either before or after cerebral ischemia reperfusion, oxidative damage in the brain was markedly prevented as evaluated by two biochemical indications, thiobarbiturate reactive sub- stance (TBARS) formation and glutathione peroxidase (GPX) activity (10,11).

The same protective effect of SMS was shown against brain oxidative damage in mice as well as in rats. Figure 2 shows the preventive effect of SMS on brain oxidative injury induced by ischemia reperfusion in C57BL/6 mice. The mice were administered SMS directly into the duodenum 2 hr before ischemia produced by the occlusion of both right and left common carotid arteries exposed through a middle skin incision for 85 min. At the end of the ischemic period, carotid arteries were declamped to allow blood reperfusion for 45 min. The both TBARS formation and GPX activity were measured in isolated brain homogenate. After the ischemia reperfusion, TBARS forma- tion was increased to approximately 260% of untreated control but the formation was almost completely inhibited by the administration of SMS

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F IGURE 2 Inhibitory effects of SMS on TBARS formation and GPX activity loss in mouse brain after ischemia-reperfusion.

prior to ischemia treatment. GPX activity, on the other hand, decreased to approximately 46% of vehicle control after ischemia reperfusion but this decline was also effectively prevented by SMS preadministration (recovered to 84% of control). This SMS effect was completely dose-dependent for both the inhibition of TBARS formation and GPX activity loss; furthermore, TTC (2,3,5-trimethyl tetrazolium chloride) staining of the brain slices confirmed the preventive effect of SMS on cerebral oxidative injury caused by ischemia reperfusion (11) (Fig. 3). Recently, we also studied the effect of SMS on

F IGURE 3 Protective effect of SMS against cerebral ischemia-reperfusion injury in rats evaluated by TTC staining.

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cerebral oxidative damage caused by MPTP (1-methyl-4-phenyl-1,2,3,6- tetrahydropyridine) administration in a Parkinson’s disease model in mice and found that the oxidative injury in the substantia nigra was sufficiently inhibited by orally administered SMS (12).

V. SMS MODULATES CELLULAR ANTIOXIDANT POTENTIAL THROUGH INDUCTION OF GPX

GPX is an enzyme participating in the capture of H 2 O 2 and hydroperoxides produced in the cell. Therefore, the change in the activity reflects more or less overall tissue damage or cellular potential of antioxidant defense. Thus the preventive effect of SMS on GPX activity loss was further studied by the immunohistchemical method in culture cells using anti-GPX monoclonal antibody conjugated with FITC fluoroprobe. As shown in Figure 4, GPX

activity was significantly induced by incubating cells with SMS prior to H 2 O 2 stress, although H 2 O 2 also induced GPX, as was reported elsewhere (13). This SMS-dependent enhancement of GPX activity was also confirmed by RT-

F IGURE 4 SMS and H 2 O 2 -dependent expression of GPX. PI stain for nucleus; a- GPX stain for GPX.

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PCR at the transcriptional level (unpublished data), indicating that the protective action of SMS against oxidative abuse in the brain is due not only to its strong hydroxyl-radical-scavenging activity but also to its role as cell functional modulator.

VI. CONCLUSION Our recent studies reveled that the component herbs in SMS contributing to

the antioxidant property and GPX preservation are Shisandra and Ophiopo- gon , respectively. Ginseng seems to act as organizer in this formula (11,14). Although the importance of Shisandra as antioxidant has been suggested elesewhere (15,16) as well as in our study, the antioxidant property of other components in SMS are still in discussion. On the other hand, previous studies indicated that ginseng saponins improve brain functions such as learning ability or dementia (17). Thus it is not unexpected that SMS was active in preventing oxidative damage in the brain, as shown above, although it was primarily prescribed for treating coronal heart failure by replenishing the qi (vital energy), stimulating the pulse, and stimulating the circulation of body fluids. Further analysis of the functional roles of each component herb and their synergism will help our understanding of the basic principle underlying the mechanism of action of the complex herbal formula suitable for prevention and repair of cerebral oxidative injury.

We have also reported the similar protective effect of another TCM formula, Qizhu Tang (QZT), on cerebral oxidative injury in rats, which comprises four herbal components, Rhizoma atractyloidis, Poria, Radix notoginseng , and Radix astragali (18). QZT was traditionally prescribed for treating syndromes related to qi depression in the spleen and stomach, and is also used for enhancing the immune system of the body. Although the herbal composition of QZT is completely different from SMS, QZT was found to have the same strong hydroxyl-radical-scavenging activity as SMS (18). It is thus accurate to say that reevaluation of hydroxyl-radical-scavenging activity of the TCM formulations prescribed for qi stimulation will be important to find the model formula for antioxidant-based-compounds therapy of brain oxidative stress.

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