Schisandrin B protects against menadione-induced hepatotoxicity by enhancing DT-diaphorase activity

Pretreating mice with schisandrin B (Sch B), a dibenzocyclooctadiene derivative isolated from the fruit of Schisandra chinensis, at a daily dose of 1 mmol/kg for 3 days protected against menadione-induced hepatic oxidative damage in mice, as evidenced by decreases in plasma alanine aminotransferase activity (78%) and hepatic malondialdehyde level (70%), when compared with the menadione intoxicated control. In order to define the biochemical mechanism involved in the hepatoprotection afforded by Sch B pretreatment, we examined the activity of DT-diaphorase (DTD) in hepatocytes isolated from Sch B pretreated rats. Hepatocytes isolated from Sch B pretreated (a daily dose of 1 mmol/kg for 3 days) rats showed a significant increase (25%) in DTD activity. The increase in DTD activity was associated with the enhanced rate of menadione elimination in the hepatocyte culture. The ensemble of results suggests that the ability of Sch B pretreatment to enhance hepatocellular DTD activity may at least in part be attributed to the protection against menadione hepatotoxicity.     

Role of cytochrome P-450 in schisandrin B-induced antioxidant and heat shock responses in mouse liver

In order to explore the role of cytochrome P-450 (P-450) in schisandrin B (Sch B)-induced antioxidant and heat shock responses, the effect of 1-aminobenzotriazole (ABT, a broad spectrum inhibitor of P-450) on hepatic mitochondrial glutathione antioxidant status (mtGAS) and heat shock protein (Hsp)25/70 expression was examined in Sch B-treated mice. The non-specific and partial inhibition of cytochrome P-450 (P-450) by ABT pretreatment significantly caused a protraction in the time-course of Sch B-induced enhancement in hepatic mitGAS and Hsp25/70 expression in mice. Using mouse liver microsomes as a source of P-450, Sch B, but not dimethyl diphenyl bicarboxylate (a non-hepatoprotective analog of Sch B), was found to serve as a co-substrate for the P-450-catalyzed NADPH oxidation reaction, with a concomitant production of oxidant species. Taken together, the results suggest that oxidant species generated from P-450-catalyzed reaction with Sch B may trigger the antioxidant and heat shock responses in mouse liver.     

Schisandrin B induced antioxidant response is partly mediated by cytochrome P-4502E1 catalyzed reaction in mouse liver

In order to explore the role of cytochrome P-450 (CYP) 2E1 in schisandrin B (Sch B)-induced antioxidant and heat shock responses, the effects of Sch B treatment on hepatic mitochondrial glutathione antioxidant status (mtGAS) and heat shock protein (Hsp)25/70 expression were compared between wild-type and cyp2e1 knock-out C57B/6N mice. Cyp2e1 knock-out mice exhibited a significantly smaller degree of Sch B-induced enhancement in hepatic mtGAS when compared with the wild-type counterpart. But Hsp25/70 expression induced by Sch B was not affected. Sch B-induced enhancement of mtGAS was corroborated by the increase in hepatic mitochondrial antioxidant capacity, as assessed by in vitro measurement of oxidant production, with the enhancing effect being slightly reduced in the knock-out mice. Using liver microsomes prepared from wild-type and knock-out mice as a source of CYP, Sch B was found to be a good co-substrate for the CYP-catalyzed reaction, with the rate of NADPH oxidation observable in microsomes prepared from knock-out mice being slower. The CYP-catalyzed reaction with Sch B was associated with a concomitant production of oxidant species, with the extent of oxidant production being reduced in cyp2e1 knock-out mouse microsomes. Taken together, the results indicate that CYP2E1 is partly responsible for the hepatic metabolism of Sch B that may trigger the antioxidant response in vivo.     

Hepatoprotective mechanism of schisandrin B: role of mitochondrial glutathione antioxidant status and heat shock proteins

In this study, the time course of schisandrin B- (Sch B-) induced changes in hepatic mitochondrial glutathione antioxidant status (mtGAS) and heat shock protein (HSP) 25/70 induction was examined to study their differential roles in the hepatoprotection afforded by Sch B pretreatment against carbon tetrachloride (CCl(4)) toxicity in mice. Dimethyl diphenyl bicarboxylate (DDB), a nonhepatoprotective analog of Sch B, was also included for comparison. The results indicate that Sch B treatment (2 mmol/kg) produced maximum enhancement in hepatic mtGAS and increases in both hepatic HSP 25 and HSP 70 levels at 24 h after dosing. While the extent of hepatoprotection afforded by Sch B pretreatment against CCl(4) was found to correlate inversely with the elapsed time postdosing, the protective effect was associated with the ability to sustain mtGAS and/or HSP 70 levels in a CCl(4)-intoxicated condition. On the other hand, DDB (2 mmol/kg) treatment, which did not sustain mtGAS and HSP 70 level, could not protect against CCl(4) toxicity. Abolition of the Sch B-mediated enhancement of mtGAS by buthionine sulfoximine/phorone did not completely abrogate the hepatoprotective action of Sch B. The results indicate that Sch B pretreatment independently enhances mtGAS and induces HSP 25/70 production, particularly under conditions of oxidative stress, thereby protecting against CCl(4) hepatotoxicity.     

Hepatoprotective action of schisandrin B against carbon tetrachloride toxicity was mediated by both enhancement of mitochondrial glutathione status and induction of heat shock proteins in mice

In the present study, we investigated the differential role of the mitochondrial glutathione status and induction of heat shock proteins (HSPs) 25/70 in protecting against carbon tetrachloride (CCl_4) hepatotoxicity in schisandrin B (Sch B)-pretreated mice. The time-course of Sch B-induced changes in these hepatic parameters were examined. Dimethyl diphenyl bicarboxylate (DDB), a non-hepatoprotective analog of Sch B, was studied for comparison. Sch B treatment (2 mmol/kg) produced maximal enhancement in hepatic mitochondrial glutathione status as well as increases in hepatic HSP 25/70 levels at 24 h post-dosing. The stimulatory effect of Sch B then gradually subsided, but the activities of hepatic mitochondrial glutathione reductase (GR) and glutathione S-transferases (GST) as well as the level of HSP 25 remained relatively high even at 72 h post-dosing. CCl_4 challenge caused significant impairment in mitochondrial glutathione status and a decrease in HSP 70 level, but the HSP 25 level was significantly elevated. While the extent of hepatoprotection afforded by Sch B pretreatment against CCl_4 was found to inversely correlate with the time elapsed after the dosing, the protective effect was associated with the ability of Sch B to maintain the mitochondrial glutathione status and/or induce further production of HSP 25 in CCl_4-intoxicated condition. On the other hand, DDB treatment (2 mmol/kg), which did not increase mitochondrial GSH level and GST activity or induce further production of HSP 25 after CCl_4 challenge, could not protect against CCl_4 toxicity. The results suggest that the enhancement of mitochondrial glutathione status and induction of HSP 25/70 may contribute independently to the hepatoprotection afforded by Sch B pretreatment.     

The essential role of PKCalpha in the protective effect of heat-shock pretreatment on TNFalpha-induced apoptosis in hepatic epithelial cell line

During sepsis, hepatic apoptosis occurred, which is associated with inactivation of PKCalpha and elevation of tumor necrosis factor-alpha (TNFalpha), an apoptosis trigger. Heat shock, accompanied by the increase of heat-shock protein (Hsp72), has been shown to exhibit a protective role on cell survival. However, Hsp72 was unable to express during sepsis when the apoptosis was markedly increased. We hypothesized that hepatic apoptosis during sepsis may be due to the failure to induce expression of Hsp72, which is activated by PKC-phosphorylated HSF. This study was designed to examine the role of PKCalpha in Hsp72 expression and the anti-apoptotic effect of Hsp72 on hepatic epithelial cells by analyzing a TNFalpha-induced apoptosis system. The following results were observed: (1) Hsp72 was highly expressed at 8 h after heat-shock treatment in a clone 9 hepatic epithelial cell line; (2) the protein expression of PKCalpha in membrane-associated fraction was decreased by TNFalpha treatment; (3) the TNFalpha-induced cell death, especially apoptosis, was diminished by heat-shock pretreatment; (4) in the presence of PKCalpha antisense, which blocks the PKCalpha resynthesis, no protective effect of heat-shock pretreatment was observed, and the protein expression of Hsp72 was significantly suppressed. These results suggest that PKCalpha plays a critical role in the expression of Hsp72, which subsequently protects against TNFalpha-induced hepatic apoptosis.     

Cytochrome P450-catalysed reactive oxygen species production mediates the (-)schisandrin B-induced glutathione and heat shock responses in AML12 hepatocytes

Sch B (schisandrin B), the most abundant dibenzocyclooctadiene lignan in Fructus schisandrae, can induce glutathione antioxidant and heat shock responses, as well as protect against oxidant-induced injury in various tissues, including the liver in rodents and AML12 (alpha mouse liver 12) hepatocytes. (-)Sch B is the most potent stereoisomer of Sch B in its cytoprotective action on AML12 hepatocytes. To define the role of ROS (reactive oxygen species) arising from CYP (cytochrome P450)-catalysed metabolism of (-)Sch B in triggering glutathione antioxidant and heat shock responses, the effects of a CYP inhibitor [ABT (aminobenzotriazole)] and antioxidants [DMTU (dimethylthiouracil) and TRX (trolox)] on (-)Sch B-induced ROS production and associated increases in cellular GSH level, as well as Hsp25/70 (heat-shock protein 25/70) production, were investigated in AML12 hepatocytes. The results indicated that (-)Sch B causes a dose dependent and sustained increase in ROS production over 6 h in AML12 hepatocytes, which was completely suppressed by pre-/co-treatment with ABT or DTMU/TRX. Incubation with (-)Sch B for 6 h caused optimal and dose-dependent increases in cellular GSH level and Hsp25/70 production at 16 h post-drug exposure in AML12 hepatocytes. These cellular responses were associated with protection against menadione-induced apoptosis. Pre-/co-treatment with ABT or antioxidants completely abrogated the (-)Sch B-induced glutathione antioxidant and heat shock responses, as well as protection against menadione-induced apoptosis. Experimental evidence obtained thus far supports the causal role of ROS arising from the CYP-catalysed metabolism of (-)Sch B in eliciting glutathione antioxidant and heat shock responses in AML12 hepatocytes.     

Schisandrin B protects against tert-butylhydroperoxide induced cerebral toxicity by enhancing glutathione antioxidant status in mouse brain

Schisandrin B (Sch B), a dibenzocyclooctadiene derivative isolated from Fructus Schisandrae, has been shown to produce antioxidant effect on rodent liver and heart. A mouse model of tert-butylhydroperoxide (t-BHP) induced cerebral toxicity was adopted for examining the antioxidant potential of Sch B in the brain. Intracerebroventricular injection of t-BHP caused a time-dependent increase in mortality rate in mice. The t-BHP toxicity was associated with an increase in the extent of cerebral lipid peroxidation and an impairment in cerebral glutathione antioxidant status, as evidenced by the abrupt decrease in reduced glutathione (GSH) level and the inhibition of Se-glutathione peroxidase activity at 5 min following t-BHP challenge. Sch B pretreatment (1 or 2 mmol/kg/day × 3) produced a dose-dependent protection against t-BHP induced mortality. The protection was associated with a decrease in the extent of lipid peroxidation and an enhancement in glutathione antioxidant status in brain tissue detectable at 5 min post t-BHP challenge, with the assessed biochemical parameters being returned to normal values at 60 min in Sch B pretreated mice at a dose of 2 mmol/kg. The ensemble of results suggests the antioxidant potential of Sch B pretreatment in protecting against cerebral oxidative stress.     

Splice isoform of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-4: Expression and hypoxic regulation

Using an ex vivo model of isolated–perfused rat hearts and cultured H9c2 cells, the structure–activity relationships of schisandrin B (Sch B), and analogs lacking either the methylendioxy group or cyclooctadiene ring, schisandrin A (Sch A) and dimethyl diphenyl bicarboxylate (DDB), respectively, were investigated. Pretreatment with Sch B, but not with Sch A or DDB, protected against myocardial ischemia–reperfusion (I-R) injury in rats. Although Sch B pretreatment largely prevented H9c2 cells from menadione-induced cytotoxicity, Sch A pretreatment produced only a marginal protection. However, DDB pretreatment did not cause any detectable effect. The myocardial and cellular protection afforded by Sch B pretreatment correlated with increases in mitochondrial ATP generation capacity and/or reduced glutathione level as well as heat shock protein (Hsp)25/70 expression, under both control and oxidative stress conditions. The results indicate that the methylenedioxy group and the cyclooctadiene ring are important structural determinants of Sch B in enhancing mitochondrial functional ability and glutathione status, as well as tissue Hsp25/70 expression, thereby protecting the myocardium against I-R injury.     

Schisandrin B protects myocardial ischemia-reperfusion injury partly by inducing Hsp25 and Hsp70 expression in rats

Schisandrin B (Sch B) is a hepato- and cardioprotective ingredient isolated from the fruit of Schisandra chinensis, a traditional Chinese herb clinically used to treat viral and chemical hepatitis. In order to investigate whether the induction of heat shock protein (Hsp)25 and Hsp70 expression plays a role in the cardioprotection afforded by Sch B pre-treatment against ischemia-reperfusion (I-R) injury, the time-course of myocardial Hsp25 and Hsp70 expression was examined in Sch B-pre-treated rats. Sch B pre-treatment (1.2 mmol/kg) produced time-dependent increases in Hsp25 and Hsp70 expression in rat hearts, with the maximum enhancement observable at 48 and 72 h post-dosing, respectively. Buthionine sulfoximine/phorone treatment, while abolishing the beneficial effect of Sch B on mitochondrial glutathione redox status, did not completely abrogate the cardioprotection against I-R injury. Heat shock treatment could increase myocardial Hsp25 and Hsp70 expression and protect against I-R injury under the present experimental conditions. The results indicate that the induction of Hsp25 and Hsp70 expression contributes at least partly to the cardioprotection afforded by Sch B pre-treatment against I-R injury.     

Differential effect of schisandrin B and dimethyl diphenyl bicarboxylate (DDB) on hepatic mitochondrial glutathione redox status in carbon tetrachloride intoxicated mice

The effects of schisandrin B (Sch B), a dibenzocyclooctadiene derivative isolated from the fruit of Schisandra chinensis, and dimethyl diphenyl bicarboxylate (DDB), a synthetic intermediate of schisandrin C (also a dibenzocyclooctadiene derivative), on hepatic mitochondrial glutathione redox status in control and carbon tetrachloride (CCl₄)-intoxicated mice were examined. Treating mice with Sch B or DDB at a daily oral dose of 1 mmol/kg for 3 d did not produce any significant alterations in plasma alanine aminotransferase (ALT) and sorbital dehydrogenase (SDH) activities. CCl₄ treatment caused drastic increases in both plasma ALT and SDH activities in mice. Pretreating mice with Sch B or DDB at the same dosage regimen significantly suppressed the CCl₄-induced increase in plasma ALT activity, with the inhibitory effect of Sch B being much more potent. Sch B, but not DDB, pretreatment could also decrease the plasma SDH activity in CCl₄-intoxicated mice. The lowering of plasma SDH activity, indicative of hepatoprotection against CCl₄ toxicity, by Sch B pretreatment was associated with an enhancement in hepatic mitochondrial glutathione redox status as well as an increase in mitochondrial glutathione reductase (mtGRD) activity in both non-CCl₄ and CCl₄-treated mice. DDB pretreatment, though enhancing both hepatic mitochondrial glutathione redox status and mtGRD activity in control animals, did not produce any beneficial effect in CCl₄-treated mice. The difference in hepatoprotective action against CCl₄ toxicity between Sch B and DDB may therefore be related to their ability to maintain hepatic mitochondrial glutathione redox status under oxidative stress condition.     

Tumor necrosis factor alpha-mediated activation of c-Jun NH(2)-terminal kinase as a mechanism for fumonisin B(1) induced apoptosis in murine primary hepatocytes

Fumonisin B(1) is a mycotoxin produced by Fusarium verticillioides, frequently associated with corn. It produces species-specific and organ-specific toxicity, including equine leukoencephalomalacia, porcine pulmonary edema, and hepatic or renal damage in most animal species. Fumonisin B(1) perturbs sphingolipid metabolism by inhibiting ceramide synthase. Our previous studies indicated that fumonisin B(1) caused localized activation of cytokines in liver produced by macrophages and other cell types that modulate fumonisin B(1) induced hepatic apoptosis in mice. The role of tumor necrosis factor alpha (TNFalpha) in fumonisin B(1) mediated hepatocyte apoptosis has been established; not much is known about the downstream events leading to apoptosis. In the current study, fumonisin B(1) induced apoptosis in primary culture of liver cells. In consistence with previous reports, fumonisin B(1) caused accumulation of sphingoid bases and led to increase in TNFalpha expression. Phosphorylated and total c-Jun NH(2)-terminal kinase (JNK) activities were increased after 24 h fumonisin B(1) treatment. JNK inhibitor (SP600125) and anti-TNFalpha reduced the apoptosis induced by fumonisin B(1). The role of JNK signaling in fumonisin B(1) induced apoptosis is downstream of TNFalpha production, as fumonisin B(1)-mediated activation of JNK was reduced by the presence of anti-TNFalpha in the medium, whereas the presence of JNK inhibitor did not change the fumonisin B(1) induced TNFalpha expression. Results of this study imply that generation of fumonisin B(1) induced TNFalpha results in modulation of mitogen activated protein kinases, particularly of JNK, and provides a possible mechanism for apoptosis in murine hepatocytes.     

Schisandrin B Prevents Doxorubicin Induced Cardiac Dysfunction by Modulation of DNA Damage,Oxidative Stress and Inflammation through Inhibition of MAPK/p53 Signaling

Doxorubicin (Dox) is a highly effective antineoplastic drug. However, Dox-induced apoptosis in cardiomyocytes leads to irreversible degenerative cardiomyopathy, which limits Dox clinical application. Schisandrin B (Sch B), a dibenzocyclooctadiene derivative isolated from the fruit of Schisandra chinensis, has been shown to protect against oxidative damage in liver, heart and brain tissues in rodents. In current study, we investigated possible protective effects of Sch B against Dox-induced cardiomyopathy in mice. Mice received a single injection of Dox (20 mg/kg IP). Five days after Dox administration, left ventricular (LV) performance was significantly depressed and was improved by Sch B treatment. Sch B prevented the Dox-induced increase in lipid peroxidation, nitrotyrosine formation, and metalloproteinase activation in the heart. In addition, the increased expression of phospho-p38 MAPK and phospho-MAPK activated mitogen kinase 2 levels by Dox were significantly suppressed by Sch B treatment. Sch B also attenuated Dox-induced higher expression of LV proinflammatory cytokines, cardiomyocyte DNA damage, myocardial apoptosis, caspase-3 positive cells and phopho-p53 levels in mice. Moreover, LV expression of NADPH oxidase subunits and reactive oxygen species were significantly less in Sch B treatment mice after Dox injection. These findings suggest that Sch B attenuates Dox-induced cardiotoxicity via antioxidative and anti-inflammatory effects.     

Cell surface-bound heat shock protein 70 (Hsp70) mediates perforin-independent apoptosis by specific binding and uptake of granzyme B

Cell surface-bound heat shock protein 70 (Hsp70) renders tumor cells more sensitive to the cytolytic attack mediated by natural killer (NK) cells. A 14-amino acid Hsp70 sequence, termed TKD (TKDNNLLGRFELSG, aa450-463) could be identified as the extracellular localized recognition site for NK cells. Here, we show by affinity chromatography that both, full-length Hsp70-protein and Hsp70-peptide TKD, specifically bind a 32-kDa protein derived from NK cell lysates. The serine protease granzyme B was uncovered as the 32-kDa Hsp70-interacting protein using matrix-assisted laser desorption ionization time-of-flight mass peptide fingerprinting. Incubation of tumor cells with increasing concentrations of perforin-free, isolated granzyme B shows specific binding and uptake in a dose-dependent manner and results in initiation of apoptosis selectively in tumor cells presenting Hsp70 on the cell surface. Remarkably, Hsp70 cation channel activity was also determined selectively in purified phospholipid membranes of Hsp70 membrane-positive but not in membrane-negative tumor cells. The physiological role of our findings was demonstrated in primary NK cells showing elevated cytoplasmic granzyme B levels following contact with TKD. Furthermore, an increased lytic activity of Hsp70 membrane-positive tumor cells could be associated with granzyme B release by NK cells. Taken together we propose a novel perforin-independent, granzyme B-mediated apoptosis pathway for Hsp70 membrane-positive tumor cells.     

Alterations in susceptibility to carbon tetrachloride toxicity and hepatic antioxidant/detoxification system in streptozetocin-induced short-term diabetic rats: Effects of insulin and Schisandrin B treatment

The streptozotocin-induced short-term (2 week) diabetic rats showed an increase in susceptibility to carbon tetrachloride (CCl4)-induced hepatocellular damage. This diabetes-induced change was associated with a marked impairment in the hepatic glutathione antioxidant/detoxification response to CCl₄ challenge, as indicated by the abrogation of the increases in hepatic reduced glutathione (GSH) level, glucose-6-phosphate dehydrogenase and microsomal glutathione S-transferases (GST) activities upon challenge with increasing doses of CCl₄. While the hepatic GSH level was increased in diabetic rats, the hepatic mitochondrial GSH level and Se-glutathione peroxidase activity were significantly reduced. Insulin treatment could reverse most of the biochemical alterations induced by diabetes. Both insulin and schisandrin B (Sch B) pretreatments protected against the CCl₄ hepatotoxicity in diabetic rats. The hepatoprotection was associated with improvement in hepatic glutathione redox status in both cytosolic and mitochondrial compartments, as well as the increases in hepatic ascorbic acid level and microsomal GST activity. The ensemble of results suggests that the diabetes-induced impairment in hepatic mitochondrial glutathione redox status may at least in part be attributed to the enhanced susceptibility to CCl4 hepatotoxicity. Sch B may be a useful hepatoprotective agent against xenobiotics-induced toxicity under the diabetic conditions. (Mol Cell Biochem 175: 225–232, 1997)     

Heat shock protein 70, heat shock protein 32, and vascular endothelial growth factor production and their effects on lipopolysaccharide-induced apoptosis in porcine aortic endothelial cells

Lipopolysaccharide (LPS) is a highly proactive molecule that causes in vivo a systemic inflammatory response syndrome and activates in vitro the inflammatory pathway in different cellular types, including endothelial cells (EC). Because the proinflammatory status could lead to EC injury and apoptosis, the expression of proinflammatory genes must be finely regulated through the induction of protective genes. This study aimed at determining whether an LPS exposure is effective in inducing apoptosis in primary cultures of porcine aortic endothelial cells and in stimulating heat shock protein (Hsp)70 and Hsp32 production as well as vascular endothelial growth factor (VEGF) secretion. Cells between third and eighth passage were exposed to 10 microg/mL LPS for 1, 7, 15, and 24 hours (time-course experiments) or to 1, 10, and 100 microg/mL LPS for 7 and 15 hours (dose-response experiments). Apoptosis was not affected by 1 microg/mL LPS but significantly increased in a dose-dependent manner with the highest LPS doses. Furthermore, apoptosis rate increased only till 15 hours of LPS exposure. LPS stimulated VEGF secretion in a dose-dependent manner; its effect became significant after 7 hours and reached a plateau after 15 hours. Both Hsp70 and Hsp32 expressions were induced by LPS in a dose-dependent manner after 7 hours. Subsequent studies were addressed to evaluate the protective role of Hsp32, Hsp70, and VEGF. Hemin, an Hsp32 inducer (5, 20, 50 microM), and recombinant VEGF (100 and 200 ng/mL), were added to the culture 2 hours before LPS (10 microg/mL for 24 hours); to induce Hsp70 expression, cells were heat shocked (42 degrees C for 1 hour) 15 hours before LPS (10 microg/mL for 24 hours). Hemin exposure upregulated Hsp32 expression in a dose-dependent manner and protected cells against LPS-induced apoptosis. Heat shock (HS) stimulated Hsp70 expression but failed to reduce LPS-induced apoptosis; VEGF addition did not protect cells against LPS-induced apoptosis at any dose tested. Nevertheless, when treatments were associated, a reduction of LPS-induced apoptosis was always observed; the reduction was maximal when all the treatments (HS + Hemin + VEGF) were associated. In conclusion, this study demonstrates that LPS is effective in evoking "the heat shock response" with an increase of nonspecific protective molecules (namely Hsp70 and Hsp32) and of VEGF, a specific EC growth factor. The protective role of Hsp32 was also demonstrated. Further investigations are required to clarify the synergic effect of Hsp32, Hsp70, and VEGF, thus elucidating the possible interaction between these molecules.     

Schisandrin B stereoisomers protect against hypoxia/reoxygenation-induced apoptosis and inhibit associated changes in Ca2+-induced mitochondrial permeability transition and mitochondrial membrane potential in H9c2 cardiomyocytes

The effects of schisandrin B stereoisomers, (+/-)gamma-schisandrin [(+/-)gamma-Sch] and (-)schisandrin B [(-)Sch B], on hypoxia/reoxygenation-induced apoptosis were investigated in H9c2 cardiomyocytes. Changes in cellular reduced glutathione (GSH) levels, Ca(2+)-induced mitochondrial permeability transition (MPT), and mitochondrial membrane potential (Deltapsi(m)) values, were examined in (+/-)gamma-Sch-pretreated and (-)Sch B-pretreated cells, without or with hypoxia/reoxygenation challenge. The (+/-)gamma-Sch and (-)Sch B (2.5-5.0 microM) pretreatments protected against hypoxia/reoxygenation-induced apoptosis of H9c2 cells in a concentration-dependent manner, with (-)Sch B being more potent. The degrees of protection decreased, however, at the higher drug concentrations of 7.5 microM in both (+/-)gamma-Sch-pretreated and (-)Sch B-pretreated cells. The anti-apoptotic effects of the drugs were further evidenced by the suppression of hypoxia/reoxygenation-induced mitochondrial cytochrome c release and the subsequent cleavage of caspase 3 and poly-ADP-ribose polymerase after (-)Sch B pretreatment. Both (+/-)gamma-Sch and (-)Sch B pretreatments increased GSH levels in H9c2 cells, with (-)Sch B being more potent. Hypoxia/reoxygenation challenge caused a depletion in cellular GSH and the cytoprotection afforded by (+/-)gamma-Sch/(-)Sch B was associated with enhancement of cellular GSH in H9c2 cells, as compared to the drug-unpretreated control. Whereas hypoxia/reoxygenation challenge increased the extent of Ca(2+)-induced MPT pore opening and decreased Deltapsi(m) in H9c2 cardiomyocytes, cytoprotection against hypoxia/reoxygenation-induced apoptosis afforded by (+/-)gamma-Sch/(-)Sch B pretreatments was associated with a decreased sensitivity to Ca(2+)-induced MPT and an increased Deltapsi(m) in both unchallenged and challenged cells, as compared to the respective drug-unpretreated controls. The degrees of protection against apoptosis correlated negatively with the extents of Ca(2+)-induced MPT (r=-0.615, P<0.01) and positively with the values of Deltapsi(m) (r=0.703, P<0.01) in (+/-)gamma-Sch/(-)Sch B-pretreated and hypoxia/reoxygenation challenged cells. The results indicate that (+/-)gamma-Sch/(-)Sch B pretreatment protected against hypoxia/reoxygenation-induced apoptosis in H9c2 cardiomyocytes and that the cytoprotection afforded by (+/-)gamma-Sch/(-)Sch B may at least in part be mediated by a decrease in cellular sensitivity to Ca(2+)-induced MPT, which may in turn result from enhancement of cellular GSH levels by drug pretreatments.     

Induction of Heat-Shock Protein 70 Expression by Geranylgeranylacetone Shows Cytoprotective Effects in Cardiomyocytes of Mice under Humid Heat Stress

Background

Increasing evidence has revealed that humid heat stress (HHS) causes considerable damage to human health. The cardiovascular system has been suggested to be the primary target of heat stress, which results in serious cardiovascular diseases. However, there is still a lack of effective approaches for the prevention and treatment of cardiovascular diseases induced by HHS.

Objective

Heat-shock proteins (Hsps), especially Hsp70, are reported to provide effective cytoprotection under various stress stimuli. In the present study, we evaluated the cytoprotective effect of geranylgeranylacetone (GGA), which was previously been reported to induce Hsp70 expression in cardiomyocytes under HHS.

Methods and Principal Findings

Using a mouse model of HHS, we showed that the pretreatment of GGA enhanced Hsp70 expression under HHS, as examined by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot. We then examined the effect of GGA pretreatment on the cardiomyocyte apoptosis induced by HHS using terminal-deoxynucleoitidyl transferase mediated nick end labeling (TUNEL) staining, and found that GGA pretreatment inhibited mitochondria-mediated apoptosis. GGA pretreatment could reverse the effect of HHS on cell apoptosis by increasing expression of Bcl-2, decreasing cytochrome c in cytosol, and increasing cytochrome c in mitochondria. However, GGA pretreatment had no effect on the oxidative stress induced by HHS as determined by levels of superoxide dismutase (SOD), malondialdehyde (MDA), and glutathione (GSH).

Conclusion

We have demonstrated that GGA pretreatment suppressed HHS-induced apoptosis of cardiomyocytes through the induction of Hsp70 overexpression.     

Antiproliferative and apoptosis-inducing activity of schisandrin B against human glioma cells

Background

Malignant glioma is the most devastating and aggressive tumour in the brain and is characterised by high morbidity, high mortality and extremely poor prognosis. The main purpose of the present study was to investigate the effects of schisandrin B (Sch B) on glioma cells both in vitro and in vivo and to explore the possible anticancer mechanism underlying Sch B-induced apoptosis and cell cycle arrest.

Methods

The anti-proliferative ability of Sch B on glioma cells were assessed by MTT and clony formation assays. Flow cytometric analysis was used to detect cell cycle changes. Apoptosis was determined by Hoechst 33342 staining and annexin V/PI double-staining assays. The mitochondrial membrane potential was detected by Rhodamine 123 staining. The in vivo efficacy of Sch B was measured using a U87 xenograft model in nude mice. The expressions of the apoptosis-related and cell cycle-related proteins were analysed by western blot. Student’s t-test was used to compare differences between treated groups and their controls.

Results

We found that Sch B inhibited growth in a dose- and time-dependent manner as assessed by MTT assay. In U87 and U251 cells, the number of clones was strongly suppressed by Sch B. Flow cytometric analysis revealed that Sch B induced cell cycle arrest in glioma cells at the G0/G1 phase. In addition, Sch B induced glioma cell apoptosis and reduced mitochondrial membrane potential (ΔΨm) in a dose-dependent manner. Mechanically, western blot analysis indicated that Sch B induced apoptosis by caspase-3, caspase-9, PARP, and Bcl-2 activation. Moreover, Sch B significantly inhibited tumour growth in vivo following the subcutaneous inoculation of U87 cells in athymic nude mice.

Coclusions

In summary, Sch B can reduce cell proliferation and induce apoptosis in glioma cells and has potential as a novel anti-tumour therapy to treat gliomas.