Ganoderma Lucidium Extract(GLE) And Apocynum Venetum Leaf Extract (AVLE) Protect Rat Cerebral Cortical Neurons From Hypoxia/reoxygenation Injury In Vitro

Stroke is a life-threatening disease characterized by rapidly developing clinical signs of focal or global disturbance of cerebral function due to cerebral ischemia.The physiopathologic mechanisms of cerebral ischemia reperfusion injury were extremely complex.Multiple pathways are involved in the ischemic process that ultimately leads to cell death.In particular,and neuron apoptosis after ischemia/reperfusion is one of the major pathways that leads to the process of cell death.Herbal medicines possesses multiple pharmacological activities may aim directly at the cascade of damage of cerebral ischemia.Recently,Ganoderma lucidum total sterol,Ganoderma lucidum polysaccharide and the extract of Apocynum venetum have been reported to modulated the cerebral injuries in stroked animal models by inhibit the oxidative stress and inflammatory response.Therefore,we hypothesized that Ganoderma lucidum extract(GLE) or Apocynum venetum extract(AVLE) might had a neuroprotective effect via modulation of multiple pathways associated with apoptosis.PartⅠNeuroprotective effect of Ganoderma lucidium extract (GLE) against OGD-induced cortical neuron injuryObjective To investigate the neuroprotective effects of GLE and AVLE in an "in vitro" model of ischemia and the possible mechanisms involved.Methods Cultured rat brain cortical neurons were treated or not with increasing concentrations of GLE,were exposed to 2 hours combined oxygen-glucose deprivation(OGD) in an anaerobic chamber followed by reoxygenation.Assessment of cell injury was quantitatively performed by two biochemical tests measuring the release of cytoplasmic lactate dehydrogenase(LDH) and the reduction of 2,3-bis(2-methoxy-4-nitro-5-sulfopheny1)-2H-tetrazolium-5-carboxanilide(XTT) at 0,3,6,12,24,48 and 72 hours after the OGD.The rate of apoptosis was detected at 24 hours and 72 hours after OGD during the reoxygenation period using flow cytometry.Caspase-3,caspase-8,caspase-9,bcl-2 and bax activation was determined at 24 h after OGD during the reoxygenation period by Western blot.Results 1,After OGD treating in cortical neurons for 2 hours and reoxygenation treating for 3 hours the swelling of neuronal body were significant,while GLE could lessen the swelling of neuronal body.2,GLE(0.1,1,10μg/ml) group increased neuron viability following OGD/reoxygenation and also significantly reduced lactate dehydrogenase(LDH) release.Seventy-two hours after OGD,the maximal neuroprotection was afforded by 10μg/ml GLE and was similar when evaluated by LDH release or by XTT reduction.Neuroprotection by GLE is dose- and time-dependent.3,After OGD treating in cortical neurons for 2 hours and reoxygenation treating for 24,72 hours the apoptosis rate of neurons increased significantly.GLE(0.1μg/ml,0.1μg/ml and 0.1μg/ml) could significantly inhibited OGD/reoxygenation-induced apoptosis of cultured rat cortical neurons in a concentration-dependent manner(P＜0.05).4,Caspase-3,caspase-8,caspase-9 proenzyme and bax protein were activated 24 hours after OGD,we observed that GLE(0.1μg/ml,1μg/ml,10μg/ml) inhibited caspase-3,caspase-8 proenzyme and bax protein expression;GLE(10μg/ml)suppressed caspase-9 proenzyme additionally.Moreover,24 hours after reoxygenation,the reduction of bcl-2 was significant.GLE(0.1μg/ml,1μg/ml,10μg/ml)also inhibited the reduction of bcl-2.Conclusion 1,GLE exhibits remarkable protection against hypoxia/reoxygenation injury in rat cortical neurons.2,The mechanism of GLE antiapoptosis may related to its suppresssion of elevated Bax protein and caspase-3,-8,-9 proenzyme and decreased bcl-2.10μg/ml GLE inhibit the expression of caspase-3 via both extrinsic pathway and intrinsic pathway possibly.PartⅡNeuroprotective effect of Apocynum venetum leaf extract (AVLE) against OGD-induced cortical neuron injuryObjective To investigate the neuroprotective effects of AVLE in an "in vitro" model of ischemia and the possible mechanisms involved. Methods Cultured rat brain cortical neurons were treated or not with increasing concentrations of AVLE,were exposed to 2 hours combined oxygen-glucose deprivation(OGD) in an anaerobic chamber followed by reoxygenation.Assessment of cell injury was quantitatively performed by two biochemical tests measuring the release of cytoplasmic lactate dehydrogenase(LDH) and the reduction of 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide(XTT) at 0,3,6,12,24,48 and 72 hours after the OGD.The rate of apoptosis was detected at 24 hours and 72 hours after OGD during the reoxygenation period using flow cytometry.Caspase-3,caspase-8,caspase-9,bcl-2 and bax activation was determined at 24 h after OGD during the reoxygenation period by Western blot.Results 1,After OGD treating in cortical neurons for 2 hours and reoxygenation treating for 24 hours and 72 hours the injury of neurons were significant,AVLE lessened the damage of neurons.2,AVLE(500μg/ml and 5000μg/ml) group increased neuron viability following OGD/reoxygenation and also significantly reduced lactate dehydrogenase(LDH) release.Seventy-two hours after OGD,the maximal neuroprotection was afforded by 5000μg/ml AVLE and was similar when evaluated by LDH release or by XTT reduction.Neuroprotection by AVLE is dose- and time-dependent.3,After OGD treating in cortical neurons for 2 hours and reoxygenation treating for 24,72 hours the apoptosis rate of neurons increased significantly.AVLE(500μg/ml and 5000μg/ml) could significantly inhibited OGD/reoxygenation-induced apoptosis of cultured rat cortical neurons in a concentration-dependent manner(P＜0.05).4,Caspase-3,caspase-8,caspase-9 proenzyme and bax protein were activated 24 hours after OGD,we observed that AVLE(500μg/ml and 5000μg/ml) inhibited caspase-3,caspase-8 proenzyme and bax protein expression;AVLE(50μg/ml,5000μg/ml and 500μg/ml) failed to suppress caspase-9 proenzyme.Moreover,24 hours after reoxygenation,the reduction of bcl-2 was significant.AVLE(50μg/ml,5000μg/ml and 500μg/ml) were unable to inhibit the reduction of bcl-2.Conclusion 1,AVLE exhibits moderate protection against hypoxia/reoxygenation injury in rat cortical neurons.2,The mechanism of AVLE antiapoptosis may related to its suppresssion of elevated Bax protein and caspase-3,-8,-9 proenzyme and decreased bcl-2.AVLE inhibited the expression ofc aspase-3,-8 proenzyme and bax protein,but had no effect on caspase-9 and bcl-2.It indicated that the anti-apoptosis effect of AVLE may related to the supression of extrinsic pathway activated caspase-3.