Philip Y. Lam

Activation of Erk1/2 and Akt in astrocytes under ischemia.

Substantial evidence has shown that extracellular signal-regulated kinases 1 and 2 (Erk1/2) and serine/threonine kinase (Akt) play important roles in regulating cell survival. We examined the activities of these kinases in astrocytes under ischemia in an anaerobic chamber. The level of phosphorylated Erk1/2 in astrocytes began to increase after 1 h ischemia, reached a maximum after 4 h ischemia, before decreasing from 5 to 6 h. Akt was activated later than Erk1/2. It was significantly increased after 4 h ischemia before declining steadily afterwards. Lactate dehydrogenase (LDH) assay and Hoechst nucleic staining indicated that U0126, which inhibits Erk1/2 phosphorylation, enhanced ischemia-induced cell death, whereas LY294002, which inhibits Akt phosphorylation, delayed cell death. These effects were dose-dependent. At 4 and 6 h ischemia, U0126-treated astrocytes expressed a lower level of Bcl-2 than controls. In contrast, LY294002-treated astrocytes expressed a higher level of Bcl-2 than controls as shown by Western blots. Bcl-x(L) expression level was not affected by either treatment. These data suggest that activation of the MAPK/Erk1/2 pathway might protect astrocytes from ischemic injury, but activation of the PI3-K/Akt pathway does not. The effect may involve Bcl-2 but not Bcl-x(L) expression.

Apoptosis and activation of Erk1/2 and Akt in astrocytes postischemia

We have shown previously that in vitro ischemia could induce apoptosis in primary culture of astrocytes. In this paper we demonstrate that astrocytes in culture could undergo apoptosis during in vitro incubation postischemia. We also measured the changes of phosphorylated Erk1/2 (p-Erk1/2) and phosphorylated Akt (p-Akt) in order to determine whether these two pathways play a role in apoptosis. After 4 h in vitro ischemic incubation of cultured astrocytes, a limited amount of nuclear condensation was demonstrated by Hoechst 33342 staining. When ischemic incubation was halted and the cultures transferred to standard normoxic incubation (postischemia) conditions, DNA fragmentation and apoptosis were demonstrated by TUNEL and DNA laddering analysis. TUNEL-positive astrocytes began to appear at 6 h postischemia and increased in number from 12 h postischemia. Western blot analysis showed that both p-Erk1/2 and p-Akt were elevated in astrocytes subjected to 4 h of ischemia. Elevated p-Erk1/2 levels were sustained during the postischemia incubation for 12 h and decreased significantly afterward, but did not return to the levels in the control cultures that did not experience ischemic insult. In contrast, the p-Akt level continued to increase at 6 and 12 h postischemia before declining significantly. The changes in p-Erk1/2 and p-Akt correlated well with the appearance of apoptotic astrocytes in the culture.

Long-Term Treatment with Danshen-Gegen Decoction Protects the Myocardium against Ischemia/Reperfusion Injury via the Redox-Sensitive Protein Kinase C-ε/mKATP 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.

Changes of ATP and ADP in Cultured Astrocytes Under and After in Vitro Ischemia

A very large body of evidence from in vivo studies has been accumulated on a link between the change of energy and cell survival/apoptosis. Using an in vitro ischemia model, we have previously shown that ischemia could induce apoptosis in astrocytes. In this study, we utilized the same in vitro model to investigate changes in ATP and ADP levels in cultured astrocytes and attempted to demonstrate an energy-cell death linkage. Astrocytes remained unaltered after 2 hr of ischemia but were moderately or severely damaged after 4 or 6-8 hr, respectively. The astrocytes that survived various lengths of in vitro ischemic incubation retained their ability to produce ATP after ischemia. Both ATP and ADP levels were increased in astrocytes that remained alive under in vitro ischemia for over 6 hr. The largest decline in the percent of viable astrocytes during ischemia corresponded well to the reduction in ATP and ADP levels in these cultures.

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.

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 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 Parkinsons 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.

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

BackgroundDanshen-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.MethodsISO 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.ResultsISO inflicted acute myocardial injury in the rats as evidenced by increased plasma enzyme activities. DG post-treatment alleviated the ISO-induced acute myocardial injury.ConclusionDG 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 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.