Schisandrin B as a hormetic agent for preventing age-related neurodegenerative diseases.

Oxidative stress and mitochondrial dysfunction have been implicated in the pathogenesis of neurodegenerative diseases, with the latter preceding the appearance of clinical symptoms. The energy failure resulting from mitochondrial dysfunction further impedes brain function, which demands large amounts of energy. Schisandrin B (Sch B), an active ingredient isolated from Fructus Schisandrae, has been shown to afford generalized tissue protection against oxidative damage in various organs, including the brain, of experimental animals. Recent experimental findings have further demonstrated that Sch B can protect neuronal cells against oxidative challenge, presumably by functioning as a hormetic agent to sustain cellular redox homeostasis and mitoenergetic capacity in neuronal cells. The combined actions of Sch B offer a promising prospect for preventing or possibly delaying the onset of neurodegenerative diseases, as well as enhancing brain health.

Beneficial effect of (-)schisandrin B against 3-nitropropionic acid-induced cell death in PC12 cells.

Huntington's disease (HD) is characterized by the dysfunction of mitochondrial energy metabolism, which is associated with the functional impairment of succinate dehydrogenase (mitochondrial complex II), and pyruvate dehydrogenase (PDH). Treatment with 3-nitropropionic acid (3-NP), a potent irreversible inhibitor of succinate dehydrogenase, replicates most of the pathophysiological features of HD. In the present study, we investigated the effect of (-)schisandrin B [(-)Sch B, a potent enantiomer of schisandrin B] on 3-NP-induced cell injury in rat differentiated neuronal PC12 cells. The 3-NP caused cell necrosis, as assessed by lactate dehydrogenase (LDH) leakage, and mitochondrion-dependent cell apoptosis, as assessed by caspase-3 and caspase-9 activation, in differentiated PC12 cells. The cytotoxicity induced by 3-NP was associated with a depletion of cellular reduced glutathione (GSH) as well as the activation of redox-sensitive c-Jun N-terminal kinase (JNK) pathway and the inhibition of PDH. (-)Sch B pretreatment (5 and 15 µM) significantly reduced the extent of necrotic and apoptotic cell death in 3-NP-challenged cells. The cytoprotection afforded by (-)Sch B pretreatment was associated with the attenuation of 3-NP-induced GSH depletion as well as JNK activation and PDH inhibition. (-)Sch B pretreatment enhanced cellular glutathione redox status and ameliorated the 3-NP-induced cellular energy crisis, presumably by suppressing the activated JNK-mediated PDH inhibition, thereby protecting against necrotic and apoptotic cell death in differentiated PC12 cells.

Danshen-Gegen decoction protects against hypoxia/reoxygenation-induced apoptosis by inhibiting mitochondrial permeability transition via the redox-sensitive ERK/Nrf2 and PKCε/mKATP pathways in H9c2 cardiomyocytes.

Danshen-Gegen (DG) Decoction, an herbal formulation containing Radix Salviae miltiorrhizae and Radix Puerariae lobatae, has been used for the treatment of coronary artery disease in Chinese medicine. In the present study, the involvement of ERK- and PKCε-mediated pathways in the cytoprotection against apoptosis afforded by DG pretreatment was investigated in H9c2 cardiomyocytes. Pretreatment with a methanol extract of aqueous DG decoction protected against hypoxia/reoxygenation-induced apoptosis in H9c2 cardiomyocytes. The cytoprotection was associated the enhancement of cellular reduced glutathione and a reduced sensitivity to Ca(2+)-induced mitochondrial permeability transition. DG extract increased the production of cytochrome P-450 (CYP)-dependent reactive oxygen species (ROS) in H9c2 cardiomyocytes, which was accompanied by the concomitant activation of ERK1/2 and PKCε. The DG-induced ERK1/2 activation was followed by the translocation of Nrf2 from the cytosol to the mitochondria accompanied by an increase in the expression of glutathione-related antioxidant proteins. In addition, the increased expression of hemeoxygenase-1 was associated with the activation of Akt and BAD, indicative of anti-apoptotic activity. In conclusion, DG treatment activated both ERK/Nrf2 and PKCε pathways, presumably by ROS arising from CYP-catalyzed processes, with resultant inhibition of hypoxia/reoxygenation-induced apoptosis immediately after DG treatment or even after an extended time interval following DG treatment.

Schisandrin B enhances the glutathione redox cycling and protects against oxidant injury in different types of cultured cells.

Tert-butylhydroperoxide (tBHP) challenge caused an initial depletion of cellular reduced glutathione (GSH), which was followed by a gradual restoration of cellular GSH in AML12, H9c2, and differentiated PC12 cells. The time-dependent changes in cellular GSH induced by tBHP were monitored as a measure of GSH recovery capacity (GRC), of which glutathione reductase (GR)-mediated glutathione redox cycling and γ-glutamate cysteine ligase (GCL)-mediated GSH synthesis were found to play an essential role. While glutathione redox cycling sustained the GSH level during the initial tBHP-induced depletion, GSH synthesis restores the GSH level thereafter. The effects of (-)schisandrin B [(-)Sch B] and its analogs (Sch A and Sch C) on GRC were also examined in the cells. (-)Sch B and Sch C, but not Sch A, ameliorated the extent of tBHP-induced GSH depletion, indicative of enhanced glutathione redox cycling. However, the degree of restoration of GSH post-tBHP challenge was not affected or even decreased. Pretreatment with (-)Sch B and Sch C, but not Sch A, protected against oxidant injury in the cells. The (-)Sch B afforded cytoprotection was abolished by N,N'-bis(chloroethyl)-N-nitrosourea pretreatment suggesting the enhancement of glutathione redox cycling is crucially involved in the cytoprotection afforded by (-)Sch B against oxidative stress-induced cell injury.

Acute treatment with Danshen-Gegen decoction protects the myocardium against ischemia/reperfusion injury via the redox-sensitive PKCɛ/mK(ATP) pathway in rats.

Danshen-Gegen (DG) decoction, an herbal formulation comprising Radix Salvia Miltiorrhiza and Radix Puerariae Lobatae, is prescribed for the treatment of coronary heart disease in Chinese medicine. Experimental and clinical studies have demonstrated that DG decoction can reduce the extent of atherosclerosis. In the present study, using an ex vivo rat model of myocardial ischemia/reperfusion (I/R) injury, we investigated the myocardial preconditioning effect of an aqueous DG extract prepared from an optimized weight-to-weight ratio of Danshen and Gegen. Short-term treatment with DG extract at a daily dose of 1 g/kg and 2 g/kg for 3 days protected against myocardial I/R injury in rats. The cardioprotection afforded by DG pretreatment was paralleled by enhancements in mitochondrial antioxidant status and membrane structural integrity, as well as a decrease in the sensitivity of mitochondria to Ca²âº-stimulated permeability transition in vitro, particularly under I/R conditions. Short-term treatment with the DG extract also enhanced the translocation of PKCɛ from the cytosol to mitochondria in rat myocardium, and this translocation was inhibited by α-tocopherol co-treatment with DG extract in rats. Short-term DG treatment may precondition the myocardium via a redox-sensitive PKCɛ/mK(ATP) pathway, with resultant inhibition of the mitochondrial permeability transition through the opening of mitochondrial K(ATP) channels. Our results suggest that clinical studies examining the effectiveness of DG extract given prophylactically in affording protection against myocardial I/R injury would be warranted.

Schisandrin B protects against solar irradiation-induced oxidative injury in BJ human fibroblasts.

The effects of schisandrin B (Sch B) and its analogs on solar irradiation-induced oxidative injury were examined in BJ human fibroblasts. Sch B and schisandrin C (Sch C) increased cellular reduced glutathione (GSH) level and protected against solar irradiation-induced oxidative injury. The photoprotection was paralleled by decreases in the elastases-type protease activity and matrix-metalloproteinases-1 expression in solar-irradiated fibroblasts. The cytochrome P-450-mediated metabolism of Sch B or Sch C caused ROS production. The results suggest that by virtue of its pro-oxidant action and the subsequent glutathione antioxidant response, Sch B or Sch C may offer the prospect of preventing skin photo-aging.

Myocardial post-conditioning with Danshen-Gegen decoction protects against isoproterenol-induced myocardial injury via a PKCε/mKATP-mediated pathway in rats.

BACKGROUND: Danshen-Gegen decoction (DG), a Chinese herbal formula, has been demonstrated to be effective for the treatment of coronary heart disease such as myocardial infarction. In the present study, we investigated the effect of DG post-conditioning on isoproterenol (ISO)-induced myocardial injury in rats. METHODS: ISO was injected intraperitoneally (200 mg/kg) to induce acute (2-6 hours) myocardial injury in adult female rats. DG (4 g/kg) was administered per oral immediately after the injection of ISO in the rats. Extent of myocardial injury was assessed by measurements of plasma enzyme activities. Myocardial mitochondrial glutathione antioxidant status, lipid peroxidation and mitochondrial calcium ion loading and cytochrome c release were also measured. Effects of inhibitors of protein kinase C-epsilon (PKCε) ranslocation and mitochondrial ATP-sensitive potassium channel (mKATP) on myocardial post-conditioning by DG were investigated. RESULTS: ISO inflicted acute myocardial injury in the rats as evidenced by increased plasma enzyme activities. DG post-treatment alleviated the ISO-induced acute myocardial injury. CONCLUSION: DG post-treatment protected the myocardium against ISO-induced acute injury in rats. The myocardial post-conditioning by DG is likely mediated by PKCε/mKATP signaling pathway.

(-)Schisandrin B ameliorates paraquat-induced oxidative stress by suppressing glutathione depletion and enhancing glutathione recovery in differentiated PC12 cells.

Exposure to paraquat (PQ; N,N'-dimethyl-4-4'-bipyridium), a potent herbicide, can lead to neuronal cell death and increased risk of Parkinson's disease because of oxidative stress. In this study, we investigated the effect of (-)schisandrin B [(-)Sch B, a potent enantiomer of schisandrin B] on PQ-induced cell injury in differentiated pheochromocytoma cells (PC12). PQ treatment caused cell injury in PC12 cells, as indicated by the significant increase in lactate dehydrogenase (LDH) leakage. Pretreatment with (-)Sch B (5 µM) protected against PQ-induced toxicity in PC12 cells, as evidenced by the significant decrease in LDH leakage. (-)Sch B induced the cytochrome P-450-mediated reactive oxygen species generation in differentiated PC12 cells. The cytoprotection afforded by (-)Sch B pretreatment was associated with an increase in cellular reduced glutathione (GSH) level as well as the enhancement of γ-glutamylcysteine ligase (GCL) and glutathione reductase (GR) activity in PQ-challenged cells. Both GCL and GR inhibitors abrogated the cytoprotective effect of (-)Sch B in PQ-challenged cells. The biochemical mechanism underlying the GSH-enhancing effect of (-)Sch B was further investigated in PC12 cells subjected to an acute peroxide challenge. Although the initial GSH depletion induced by peroxide was reduced through GR-catalyzed regeneration of GSH in (-)Sch B-pretreated cells, the later enhanced GSH recovery was mainly mediated by GCL-catalyzed GSH synthesis. The results suggest that (-)Sch B treatment may increase the resistance of dopaminergic cells against PQ-induced oxidative stress through reducing the extent of oxidant-induced GSH depletion and enhancing the subsequent GSH recovery.

Co-treatment with Shengmai San-derived herbal product ameliorates chronic ethanol-induced liver damage in rats.

Wei Kang Su (WKS) is an antioxidant-enriched herbal product manufactured on the basis of Shengmai San, a well-known traditional Chinese herbal formula. In the present study, we investigated the effects of WKS co-treatment on chronic ethanol toxicity in rats. WKS co-treatment protected against chronic ethanol-induced hepatotoxicity, as evidenced by the suppression of plasma enzyme activities and reactive oxygen metabolite levels, as well as the inhibition of hepatic mitochondrial malondialdehyde production in chronic ethanol-intoxicated rats. The hepatoprotection afforded by WKS co-treatment in chronic ethanol-intoxicated rats was associated with a reversal of altered hepatic mitochondrial antioxidant status and adenosine triphosphate (ATP) generation capacity, as well as heat shock protein 25/70 production. Therefore, WKS may offer the prospect of preventing ethanol-associated liver damage by increasing the resistance of mitochondria to oxidative stress.

Long-term treatment with danshen-gegen decoction protects the myocardium against ischemia/reperfusion injury via the redox-sensitive protein kinase C-ε/mK(ATP) pathway in rats.

Danshen-Gegen (DG) decoction, an herbal formulation comprised of radix Salvia Miltiorrhiza and radix Puerariae Lobata, is prescribed for the treatment of coronary heart disease in Chinese medicine. Experimental and clinical studies have indicated that DG decoction can reduce the extent of atherosclerosis. In the present study, using an ex vivo rat model of myocardial ischemia/reperfusion (I/R) injury, we investigated the myocardial preconditioning effect of an aqueous DG extract prepared from an optimized weight-to-weight ratio of danshen and gegen. Long-term treatment with DG extract at increasing doses (including the equivalent of a human dose) protected against myocardial I/R injury in rats. The cardioprotection afforded by DG pretreatment was paralleled by enhancements in mitochondrial antioxidant status and membrane integrity, as well as a decrease in the sensitivity of mitochondria to Ca(2+)-stimulated permeability transition in vitro, particularly under I/R conditions. Long-term treatment with the DG extract enhanced the translocation of protein kinase C-epsilon (PKCε) from the cytosol to mitochondria in rat myocardium, and this translocation was inhibited by α-tocopherol co-treatment with DG extract in rats. Long-term DG treatment may precondition the myocardium via a redox-sensitive PKCε/mK(ATP) pathway, with resultant inhibition of the mitochondrial permeability transition. The results suggest that clinical studies examining the effectiveness of DG extract given prophylactically in affording protection against myocardial I/R injury would be warranted.

Schisandrin B protects against solar irradiation-induced oxidative stress in rat skin tissue.

Schisandrin B (Sch B) and schisandrin C (Sch C), but not schisandrin A and dimethyl diphenyl bicarboxylate, protected rat skin tissue against solar irradiation-induced oxidative injury, as evidenced by a reversal of solar irradiation-induced changes in cellular reduced glutathione and α-tocopherol levels, as well as antioxidant enzyme activities and malondialdehyde production. The cytochrome P-450-mediated metabolism of Sch B or Sch C caused ROS production in rat skin microsomes. Taken together, Sch B or Sch C, by virtue of its pro-oxidant action and the subsequent eliciting of a glutathione antioxidant response, may prevent photo-aging of skin.

Schisandrin B co-treatment ameliorates the impairment on mitochondrial antioxidant status in various tissues of long-term ethanol treated rats.

The effect of schisandrin B (Sch B) on long-term ethanol-induced oxidative stress in various rat tissues was investigated. Long-term ethanol treatment increased reactive oxygen metabolites (ROM) level in plasma. The ethanol-induced oxidative stress was assessed by mitochondrial glutathione and α-tocopherol levels, antioxidant enzyme activities, malondialdehyde (mtMDA) production and heat shock protein (Hsp) 25/70 levels. Liver was most susceptible to oxidative stress with a significant increase in mtMDA production. Long-term Sch B treatment enhanced mitochondrial antioxidant status in a tissue non-specific manner. Sch B co-treatment ameliorated the alterations in plasma ROM levels, mtMDA production and Hsp 25/70 expression in rat tissues.

Akt1 intramitochondrial cycling is a crucial step in the redox modulation of cell cycle progression.

Akt is a serine/threonine kinase involved in cell proliferation, apoptosis, and glucose metabolism. Akt is differentially activated by growth factors and oxidative stress by sequential phosphorylation of Ser(473) by mTORC2 and Thr(308) by PDK1. On these bases, we investigated the mechanistic connection of H(2)O(2) yield, mitochondrial activation of Akt1 and cell cycle progression in NIH/3T3 cell line with confocal microscopy, in vivo imaging, and directed mutagenesis. We demonstrate that modulation by H(2)O(2) entails the entrance of cytosolic P-Akt1 Ser(473) to mitochondria, where it is further phosphorylated at Thr(308) by constitutive PDK1. Phosphorylation of Thr(308) in mitochondria determines Akt1 passage to nuclei and triggers genomic post-translational mechanisms for cell proliferation. At high H(2)O(2), Akt1-PDK1 association is disrupted and P-Akt1 Ser(473) accumulates in mitochondria in detriment to nuclear translocation; accordingly, Akt1 T308A is retained in mitochondria. Low Akt1 activity increases cytochrome c release to cytosol leading to apoptosis. As assessed by mass spectra, differential H(2)O(2) effects on Akt1-PDK interaction depend on the selective oxidation of Cys(310) to sulfenic or cysteic acids. These results indicate that Akt1 intramitochondrial-cycling is central for redox modulation of cell fate.

Elevated neuronal nitric oxide synthase expression during ageing and mitochondrial energy production.

This study evaluated the effect of ageing on brain mitochondrial function mediated through protein post-translational modifications. Neuronal nitric oxide synthase increased with age and this led to a discreet pattern of nitration of mitochondrial proteins. LC/MS/MS analyses identified the nitrated mitochondrial proteins as succinyl-CoA-transferase and F1-ATPase; the latter was nitrated at Tyr269, suggesting deficient ADP binding to the active site. Activities of succinyl-CoA-transferase, F1-ATPase and cytochrome oxidase decreased with age. The decreased activity of the latter cannot be ascribed to protein modifications and is most likely due to a decreased expression and assembly of complex IV. Mitochondrial protein post-translational modifications were associated with a moderately impaired mitochondrial function, as indicated by the decreased respiratory control ratios as a function of age and by the release of mitochondrial cytochrome c to the cytosol, thus supporting the amplification of apoptotic cascades.

Activation of c-Jun-N-terminal kinase and decline of mitochondrial pyruvate dehydrogenase activity during brain aging.

Mitochondrial dysfunction is often associated with aging and neurodegeneration. c-Jun-N-terminal kinase (JNK) phosphorylation and its translocation to mitochondria increased as a function of age in rat brain. This was associated with a decrease of pyruvate dehydrogenase (PDH) activity upon phosphorylation of the E(1alpha) subunit of PDH. Phosphorylation of PDH is likely mediated by PDH kinase, the protein levels and activity of which increased with age. ATP levels were diminished, whereas lactic acid levels increased, thus indicating a shift toward anaerobic glycolysis. The energy transduction deficit due to impairment of PDH activity during aging may be associated with JNK signaling.

Green tea polyphenols block the anticancer effects of bortezomib and other boronic acid-based proteasome inhibitors.

The anticancer potency of green tea and its individual components is being intensely investigated, and some cancer patients already self-medicate with this "miracle herb" in hopes of augmenting the anticancer outcome of their chemotherapy. Bortezomib (BZM) is a proteasome inhibitor in clinical use for multiple myeloma. Here, we investigated whether the combination of these compounds would yield increased antitumor efficacy in multiple myeloma and glioblastoma cell lines in vitro and in vivo. Unexpectedly, we discovered that various green tea constituents, in particular (-)-epigallocatechin gallate (EGCG) and other polyphenols with 1,2-benzenediol moieties, effectively prevented tumor cell death induced by BZM in vitro and in vivo. This pronounced antagonistic function of EGCG was evident only with boronic acid-based proteasome inhibitors (BZM, MG-262, PS-IX), but not with several non-boronic acid proteasome inhibitors (MG-132, PS-I, nelfinavir). EGCG directly reacted with BZM and blocked its proteasome inhibitory function; as a consequence, BZM could not trigger endoplasmic reticulum stress or caspase-7 activation, and did not induce tumor cell death. Taken together, our results indicate that green tea polyphenols may have the potential to negate the therapeutic efficacy of BZM and suggest that consumption of green tea products may be contraindicated during cancer therapy with BZM.

Compromised proteasome degradation elevates neuronal nitric oxide synthase levels and induces apoptotic cell death.

The significance of impairment of proteasome activity in PC12 cells was examined in connection with nitrative/nitrosative stress and apoptotic cell death. Treatment of differentiated PC12 cells with MG132, a proteasome inhibitor, elicited a dose- and time-dependent increase in neuronal nitric oxide synthase (nNOS) protein levels, decreased cell viability, and increased cytotoxicity. Viability and cytotoxicity were ameliorated by L-NAME (a broad NOS inhibitor). Nitric oxide/peroxynitrite formation was increased upon treatment of PC12 cells with MG132 and decreased upon treatment with the combination of MG132 and 7-NI (a specific inhibitor of nNOS). The decreases in cell viability appeared to be effected by an activation of JNK and its effect on mitochondrial Bcl-x(L) phosphorylation. These effects are strengthened by the activation of caspase-9 along with increased caspase-3 activity upon treatment of PC12 cells with MG132. These results suggest that impairment of proteasome activity and consequent increases in nNOS levels lead to a nitrative stress that involves the coordinated response of JNK cytosolic signaling and mitochondrion-driven apoptotic pathways.

c-Jun N-terminal kinase regulates mitochondrial bioenergetics by modulating pyruvate dehydrogenase activity in primary cortical neurons.

This study examines the role of c-jun N-terminal kinase (JNK) in mitochondrial signaling and bioenergetics in primary cortical neurons and isolated rat brain mitochondria. Exposure of neurons to either anisomycin (an activator of JNK/p38 mitogen-activated protein kinases) or H2O2 resulted in activation (phosphorylation) of JNK (mostly p46(JNK1)) and its translocation to mitochondria. Experiments with mitochondria isolated from either rat brain or primary cortical neurons and incubated with proteinase K revealed that phosphorylated JNK was associated with the outer mitochondrial membrane; this association resulted in the phosphorylation of the E(1alpha) subunit of pyruvate dehydrogenase, a key enzyme that catalyzes the oxidative decarboxylation of pyruvate and that links two major metabolic pathways: glycolysis and the tricarboxylic acid cycle. JNK-mediated phosphorylation of pyruvate dehydrogenase was not observed in experiments carried out with mitoplasts, thus suggesting the requirement of intact, functional mitochondria for this effect. JNK-mediated phosphorylation of pyruvate dehydrogenase was associated with a decline in its activity and, consequently, a shift to anaerobic pyruvate metabolism: the latter was confirmed by increased accumulation of lactic acid and decreased overall energy production (ATP levels). Pyruvate dehydrogenase appears to be a specific phosphorylation target for JNK, for other kinases, such as protein kinase A and protein kinase C did not elicit pyruvate dehydrogenase phosphorylation and did not decrease the activity of the complex. These results suggest that JNK mediates a signaling pathway that regulates metabolic functions in mitochondria as part of a network that coordinates cytosolic and mitochondrial processes relevant for cell function.