Schisandrin B (Sch B) is the most abundant, active dibenzocyclooctadiene derivative isolated from the fruit of Schisandra chinensis (FS), a traditional Chinese herb clinically used for the treatment of viral and chemical hepatitis. 1)A recent study from our laboratory has demonstrated that long-term treatment with Sch B was able to enhance mitochondrial antioxidant status and the resistance to Ca 2ϩ -induced mitochondrial permeability transition (PT) in an ageindependent manner in various rat organs including the heart and brain.2) The Sch B-induced enhancement of mitochondrial protective parameters in the heart was associated with the protection against myocardial ischemia/reperfusion (I/R) injury in both young and middle-aged rats.2) Given that the maintenance of mitochondrial antioxidant status and structural integrity is crucial for cell survival, 3) Sch B has been proposed to be used as a universal cell protectant against tissue damage caused by endogenous and exogenous oxidants. 2)While the protection of Sch B against I/R injury has been demonstrated in the heart, 2,4) it is still unclear whether Sch B treatment can produce any beneficial effect on cerebral I/R injury.In the present study, we investigated the effect of longterm treatment with Sch B on cerebral I/R injury in rats. To elucidate the biochemical mechanism involved in the cerebroprotection against I/R injury, cerebral mitochondrial antioxidant status as well as mitochondrial structural integrity were assessed in control and Sch B-treated rats, without or with I/R challenge. MATERIALS AND METHODS ChemicalsReduced glutathione (GSH), oxidized glutathione, glutathione reductase, cytochrome c, a-tocopherol (a-TOC), cyclosporin A (Cs A) and 2,3,5-triphenyl tetrazolium chloride (TTC) were purchased from Sigma Chemical Co. (St. Louis, MO, U.S.A.). All other chemicals were of analytical grade. Solvents used for HPLC were of HPLC grade.Herbal Material Dried FS was imported from mainland China. It was authenticated and supplied by a commercial dealer (Lee Hoong Kee Ltd.) in Hong Kong. Sch B was purified from the petroleum ether extract of FS, with the purity being higher than 95% as determined by HPLC analysis. 5)Animal Care Adult female Sprague-Dawley rats (8-10 weeks; 200-250 g) were maintained under a 12-h dark/light cycle at about 22°C, and allowed food and water ad libitum. Experimental protocols were approved by the Research Practice Committee at the Hong Kong University of Science & Technology.Drug Treatment Animals were randomly divided into groups, with five animals in each. In the Sch B treatment groups, rats were intragastrically administered with Sch B (dissolved/suspended in olive oil) at a daily dose of 1, 10 or 30 mg/kg from day 1 to day 15 of the experiment. This dosage regimen was found to be effective in protecting against myocardial ischemia/reperfusion injury in rats.2) Sch B-untreated animals received the vehicle (i.e. olive oil) only. The rat model of cerebral I/R injury was modified from that of Ischikawa and Konishi.6) Phenobarbita...

This study examined the effects of (-)schisandrin B [(-)Sch B] on MAPK and Nrf2 activation and the subsequent induction of glutathione antioxidant response and cytoprotection against apoptosis in AML12 hepatocytes. Pharmacological tools, such as cytochrome P-450 (CYP) inhibitor, antioxidant, MAPK inhibitors and Nrf2 RNAi, were used to delineate the signalling pathway. (-)Sch B caused a time-dependent activation of MAPK in AML12 cells, particularly the ERK1/2. The MAPK activation was followed by an enhancement in Nrf2 nuclear translocation and the eliciting of a glutathione antioxidant response. Reactive oxygen species arising from a CYP-catalysed reaction with (-)Sch B seemed to be causally related to the activation of MAPK and Nrf2. ERK inhibition by U0126 or Nrf2 suppression by Nrf2 RNAi transfection almost completely abrogated the cytoprotection against menadione-induced apoptosis in (-)Sch B-pre-treated cells. (-)Sch B pre-treatment potentiated the menadione-induced ERK activation, whereas both p38 and JNK activations were suppressed. Under the condition of ERK inhibition, Sch B treatment did not protect against carbon tetrachloride-hepatotoxicity in an in vivo mouse model. In conclusion, (-)Sch B triggers a redox-sensitive ERK/Nrf2 signalling, which then elicits a cellular glutathione antioxidant response and protects against oxidant-induced apoptosis in AML12 cells.

Herba Cistanches (Cistanche deserticola Y. C. Ma) is a 'Yang-invigorating' tonic herb in Chinese medicine. Preliminary chemical analysis indicated that β-sitosterol (BS) is one of the chemical constituents in an active fraction of Herba Cistanches. To investigate whether BS is an active ingredient of Herba Cistanches, the effects of BS on H9c2 cells and rat hearts were examined. The results indicated that BS stimulated the mitochondrial ATP generation capacity in H9c2 cells, which was associated with the increased production of mitochondrial reactive oxygen species. BS also stimulated mitochondrial state 3 and state 4 respiration, with the resultant decrease in coupling efficiency. BS produced an up-regulation of cellular glutathione redox cycling and protected against hypoxia/reoxygenation-induced apoptosis in H9c2 cells. However, the protective effect of BS against myocardial ischemia/reperfusion injury was seen in female but not male rats ex vivo. The cardioprotection afforded by BS was likely mediated by an up-regulation of mitochondrial glutathione redox cycling in female rat hearts. In conclusion, the ensemble of results suggests that BS is an active ingredient of Herba Cistanches. The gender-dependent effect of BS on myocardial protection will further be investigated.

This study investigated the signal transduction pathway involved in the cytoprotective action of (-)schisandrin B [(-)Sch B, a stereoisomer of Sch B]. Using H9c2 cells, the authors examined the effects of (-)Sch B on MAPK and Nrf2 activation, as well as the subsequent eliciting of glutathione response and protection against apoptosis. Pharmacological tools, such as cytochrome P-450 (CYP) inhibitor, antioxidant, MAPK inhibitor, and Nrf2 RNAi, were used to delineate the signaling pathway. (-)Sch B caused a time-dependent activation of MAPK in H9c2 cells, with the degree of ERK activation being much larger than that of p38 or JNK. The MAPK activation was followed by an increase in the level of nuclear Nrf2, an indirect measure of Nrf2 activation, and the eliciting of a glutathione antioxidant response. The activation of MAPK and Nrf2 seemed to involve oxidants generated from a CYP-catalyzed reaction with (-)Sch B. Both ERK inhibition by U0126 and Nrf2 suppression by Nrf2 RNAi transfection largely abolished the cytoprotection against hypoxia/reoxygenation-induced apoptosis in (-)Sch B-pretreated cells. (-)Sch B pretreatment potentiated the reoxygenation-induced ERK activation, whereas both p38 and JNK activations were suppressed. Under the condition of ERK inhibition, Sch B treatment did not protect against ischemia/reperfusion injury in an ex vivo rat heart model. The results indicate that (-)Sch B triggers a redox-sensitive ERK/Nrf2 signaling, which then elicits a cellular glutathione antioxidant response and protects against hypoxia/reoxygenation-induced apoptosis in H9c2 cells. The ERK-mediated signaling is also likely involved in the cardioprotection afforded by Sch B in vivo.

Mitochondrial decay is a major cause of aging, leading to the subsequent death of aerobic organisms including humans. In the present study, we examined the effects of supplementation with schisandrin B (Sch B, a dibenzocyclooctadiene derivative isolated from the fruit of Schisandra chinensis), administered at 0.012% (w/w) of diet, starting from the age of 36 weeks, on age-dependent changes in mouse mitochondrial antioxidant status and functional ability in various tissues (brain, heart, liver, and kidney) up to the age of 120 weeks. We also monitored survival of male and female C57BL/6J mice. Aging caused progressive impairment in mitochondrial antioxidant status in various tissues, as evidenced by decreases in reduced glutathione and alpha-tocopherol levels, and Mn-superoxide dismutase activity. Impairments in mitochondrial antioxidant status were invariably associated with increases in mitochondria-driven reactive oxygen species (ROS) production in tissue homogenates, as well as decreased mitochondrial ATP-generation capacities (ATP-GCs), in all tested tissues. Diet supplementation with Sch B ameliorated impairment in mitochondrial antioxidant status during aging. The effects were more pronounced in younger than in older mice, when compared to age-matched non-supplemented controls. Sch B supplementation also suppressed mitochondria-driven ROS production and enhanced mitochondrial ATP-GC in various tissues during aging. The beneficial effects of Sch B supplementation on mitochondrial antioxidant status and functional ability were paralleled by survival improvement in aging male mice, when compared with controls. Sch B supplementation also improved the survival in female mice. In conclusion, long-term Sch B supplementation mitigated age-dependent impairments in mitochondrial antioxidant capacity and functional ability, thereby retarding the aging process in mice, particularly during early aging.

Schisandrin B (Sch B) ( Fig. 1) is the most abundant active dibenzocyclooctadiene derivative isolated from the fruit of Schisandra chinensis (FS), a traditional Chinese herb commonly used as astringent and clinically used for the treatment of viral and chemical hepatitis.1) Previous studies in our laboratory have demonstrated the ability of Sch B to protect against myocardial ischemia/reperfusion (I/R) injury.2) The cardioprotection afforded by Sch B pretreatment was associated with enhancement in tissue glutathione antioxidant status, particularly in the mitochondrion.2-4) Recent studies also showed that Sch B protected against myocardial I/R injury partly by inducing heat shock protein (Hsp) 25 and Hsp 70 expression in rats. 5) In regard to the action on mitochondria, Sch B treatment was found to decrease the sensitivity of myocardial mitochondria to Ca 2ϩ -induced permeability transition, particularly under I/R condition. 6) However, the biochemical mechanism(s) underlying the Sch B-induced glutathione antioxidant and/or heat shock responses in the myocardium remains unclear. In this connection, it has been suggested that reactive oxidant species (ROS) generated from cytochrome P-450 (CYP)-catalyzed reaction with Sch B may trigger the glutathione antioxidant and heat shock responses in mouse livers. 7,8) CYP are a superfamily of hemoproteins that catalyze the oxidative metabolism of many endogenous (endobiotic) and xenobiotic compounds including Sch B.9) To explore the role of CYP in Sch B-induced glutathione antioxidant response in the myocardium, we first examined CYP-catalyzed reaction with Sch B and associated ROS production in rat heart microsomes. Sch B analogs [schisandrin A (Sch A), schisandrin C (Sch C), and dimethyl diphenyl bicarboxylate (DDB)] (see also Fig. 1), which were found to produce differential cardioprotective effects, 10) were also studied for comparison. The in vitro studies were then paralleled by in vivo investigations on Sch B or its analogs-induced changes in myocardial mitochondrial reduced glutathione (GSH) and susceptibility to I/R injury in rats. To confirm the determinant role of ROS arising from Sch B metabolism in cardioprotection, we also examined the effect of antioxidant pretreatment on the cardioprotective action of Sch B in rats. MATERIALS AND METHODS Chemicals

Previous work in our laboratory has shown that long-term treatment with Vigconic 28 (VI-28), a Yang-invigorating Chinese herbal formula used for the promotion of overall wellness in Chinese medicine, can enhance the mitochondrial functional ability and antioxidant capacity in various tissues of both male and female rats. To investigate whether the VI-28 treatment regimen could afford tissue protection against oxidative injury, the effects of long-term VI-28 treatment (80 or 240 mg/kg/d x 30) on oxidative stress-induced tissue damage in various organs (brain, heart, liver, and kidney) were examined in female rats. The results indicated that long-term VI-28 treatment invariably protected against oxidative tissue damage in the rat models of cerebral/myocardial ischemia-reperfusion injury, CCl4 hepatotoxicity, and gentamicin nephrotoxicity. The tissue protection was associated with increases in the levels and activities of mitochondrial antioxidant components as well as with the preservation of mitochondrial structural integrity. This was evidenced by decreases in the sensitivity of mitochondria to Ca2+-induced permeability transition, and in the levels of mitochondrial malondialdehyde production, Ca2+ loading, and cytochrome c release in the tissues examined. Interestingly, the VI-28 treatment increased red cell CuZn-superoxide dismutase (CuZn-SOD) levels, and these levels correlated positively with the degree of tissue protection afforded by long-term VI-28 treatment in rats. The generalized tissue protection afforded by long-term VI-28 treatment may have clinical implications in the prevention of age-related diseases, and VI-28 treatment may possibly delay the aging process.

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