The Neuroprotective Effects Of Ginkgolides On Acute Injury In Rodent Model Of Cerebral Ischemia Reperfusion

Stroke is a common clinical disease of the central nervous system,which seriously harms to human health and life.Stroke can be classified into ischemic and hemorrhagic stroke.Ischemic stroke is common and accounts for about 85%of all the strokes.Ischemia-reperfusion injury pathological mechanism mainly include a series of cascade events,such as the energy failure,excitotoxicity,calcium overload,oxidative stress,inflammation and so on,which eventually lead to the death of nerve cells.A growing number of studies have found that,the adaptive control events,endoplasmic reticulum stress and autophagy,play an important role in the pathological process of cerebral ischemia.After stroke,the accumulation of unfolded proteins and the damage of cell components and organelles trigger endoplasmic reticulum stress and up-regulation of autophagy.It's generally believed that,in the early stage of ischemia or the ischemia being mild,ERS and autophagy can maintain cells under the steady condition and play a protective role,whereas prolonged periods of ERS and autophagy can be deleterious and damaging.In addition,some studies also thought that neuronal death due to autophagy is also a major consequence of ER stress.On the other side,BDNF is expressed in the central nervous system with the highest amount of all the nutritional factors.BDNF can decrease infarct volume and improve neurological outcome via either exogenously supplied or overexpression in vivo in experimental stroke.A large number of studies have shown that,BDNF protein was regulated by MicroRNAs(miRNAs),and microRNAs were involved in important physiological and pathological processes after stroke.The use of recombinant tissue plasminogen activator(rtPA)has been the only treatment of acute ischemic stroke which was approved by the United States food and drug administration(FDA).However,the shot therapeutic time window,the short half-life together with its limitation in remedy of reperfusion injury and long-term clinical symptoms greatly limit its clinical application.Therefore,seeking for drugs with neuroprotective effect is still an urgent problem to be solved.The use of traditional Chinese medicine(TCM)on cerebral ischemia-reperfusion injury treatment is drawing more and more attention.For example,Ginkgo biloba extract(GBE)is used widely in the treatment of cardiovascular and cerebrovascular disease for many years.Bilobalide(BB)and Ginkgolide B(GB)are two primary constituents of the terpenoid fraction in GBE.In the past decades,the pharmacological studies have showed that these terpenoid fractions can exert inhibition of platelet aggregation,prevention of excitatory toxicity,inhibition of oxidative stress injury and anti-inflammation and so on.However,it remains unclear whether BB and GB could affect ERS and autophagy and whether BB and GB modulate the level of BDNF after ischemic stroke.GIs is one of the newly approved drugs for the treatment of stroke,with BB and GB accounted for 48%and 34%,respectively.It was used for the later restoration in clinical treatment.There is no research about the treatment in acute phase.In the present study,we firstly evaluated the therapeutic effect of GIs in transient middle cerebral artery occlusion(tMCAO)rats,and then,Western Blot was used to evaluate the expression level of protein related to ER stress,autophagy and apoptosis in penumbra region at 24h and 72h after reperfusion.In the second part,we evaluated the therapeutic effect of BB and GB in tMCAO mice and oxygen-glucose deprivation/reoxygenation(OGD/R)-treated Neuro-2a cells and found that BB and GB modulated BDNF expression via miR-206-mediated post-transcriptional regulation in ischemic stroke.This study revealed new targets and mechanism of Ginkgo biloba for the treatment of cerebral ischemia.Part I Ginkgolides Injection Inhibits Endoplasmic Reticulum Stress and Attenuates Autophagy in Rat Brain induced by Cerebral Ischemia Re perfusionAIM:To investigate the neuroprotective effects of the Ginkgolides Injection(GIs)against ischemic stroke-induced injury and further explore the possible mechanisms concerned.METHODS:1)The modified method of Zea Longa was used to establish the transient middle cerebral artery occlusion(tMCAO)model in rats.GIs was injected intraperitoneally(i.p,bid)to rats 1h after onset of reperfusion with different doses(1.25,2.5 and 5mg/kg).The neurological deficits were assessed in each group after cerebral ischemia.Brain water content was measured by wet/dry weight method and Infarct volume was observed by TTC staining.2)Western Blot was used to evaluate the expression level of protein related to ER stress,CHOP and caspase-12;protein related to autophagy,LC3 and P62;and protein related to apoptosis,P53,Bcl-2,Bax and caspase 3 in penumbra region of rats treated with GIs(2.5 mg/kg,i.p,bid)at 24h and 72h after reperfusion.RESULTS:1)GIs(2.5 mg/kg,ip.,bid,3d)treatment at 1h after reperfusion significantly reduced neurological deficits,water content,and cerebral infarct volume.2)GIs(2.5mg/kg,ip.,bid)significantly reduced the expression of ERS related protein,CHOP and caspase-12,increased the expression of P62,reduced LC3-?/LC3-? ratio,increased Bel-2/Bax ratio and reduced the expression of P53 and caspase-3.CONCLUSIONS:1.Ginkgolides injection ameliorated acute cerebral ischemia/reperfusion injury in rats characterized by improvement of neurological deficits,water content,and cerebral infarct volume.2.The neuroprotection of Ginkgolides injection was correlated with inhibiting the ERS caused by ischemia-reperfusion,lowering the level of autophagy and apoptosis.Part ? Bilobalide and Ginkgolide B modulate miR-206-mediated BDNF repression in acute ischemic strokeAIM:To investigate the protective effects of GIs,BB and GB to neuron against ischemic stroke-induced injury in vivo and in vitro,and further explore the possible mechanisms concerned.METHODS:1)The modified method of Zea Longa was used to establish the transient middle cerebral artery occlusion(tMCAO)model in mice.GIs was injected intraperitoneally(i.p,bid,3d)lh after onset of reperfusion with different doses(2 mg/kg,3.5 mg/kg,5 mg/kg).The neurological deficit was assessed in each group after cerebral ischemia.Brain water content was measured by wet/dry weight method and Infarct volume was observed by TTC staining.2)immunohistochemical method a was used to observe the effect of GIs on neuron loss caused by ischemia-reperfusion injury.3)Western Blotting was used to evaluate the expression level of BDNF and qRT-PCR was used to evaluate the expression level of microRNAs in penumbra region of mice treated with GIs(3.5mg/kg,ip.,bid)at 24h after reperfusion.4)We cultured Neuro-2a cells,pretreated cells with GIs,BB or GB for 2h,and then deprive the oxygen and glucose for 3 h and reperfusion for 24 h.MTT assay and LDH measurement were applied to examine cell viability.5)Cell apoptosis was detected by Hoechst33342.Dyed cells with Annexin-V and PI,the apoptosis rate was detected by fllow cytometer.Expression of Caspase-3,Bax and Bcl-2 were semi-quantified by western blotting.6)Expression of pro-BDNF and BDNF were semi-quantified by western blotting.mRNA of BDNF and microRNAs were detected by qRT-PCR.7)miR-206 mimic and inhiitor was used to alter miR-206 level in Neuro-2a cells.RESULTS:1)GIs(3.5 mg/kg,ip.,bid,3d)treatment significantly reduced neurological deficits,water content,and cerebral infarct volume after stroke.2)GIs(3.5 mg/kg,ip.,bid)significantly decreases neuronal loss induced by tMCAO in the cortex of mice.3)GIs(3.5 mg/kg,ip.,bid)significantly increased the expression of BDNF and supressed the aberrant expression of miR-206,-155,-16,and-124 after stroke.4)10-40 uM GIs,BB and GB could ma:intain the cell viability of Neuro-2a cells injured by OGD.5)20uM BB and GB could reduce the number of pyknotic nuclei and the apoptosis rate of Neuro-2a cells injured by OGD.6)2uM BB and GB caused the up-regulation of BDNF protein,not BDNF mRNA,and inhibited the up-regulation of miR-206 and 155,not miR-16,induced by OGD/R.7)Down-regulation of miR-206 induced neuroprotection against OGD/R injury.Up-regulation of miR-206 partly abolished the neuroprotection of BB and GB.CONCLUSIONS:1.GIs significantly decreased neuronal loss induced by tMC AO in the cortex of mice.GIs,BB and GB protected Neuro-2a cells against OGD/R injury.2.BB and GB are neuroprotective against ischemic injury by down-regulation of miR-206 targeted BDNF transcripts.In summary,the innovations of the present study lie in:1.GIs are neuroprotective against ischemic brain injury in the acute phase of stroke,through the down regulation of ER stress and autophagy.GIs treatment significantly reduced neurological deficits,water content,and cerebral infarct volume in the acute phase of stroke,and inhibited the activation of ERS,autophagy and apoptosis induced by ischemia/reperfusion.This study provides academic foundation for the clinical application of Ginkgolides injection in the acute phase of stroke,and revealed the possible involved.2.GIs,BB and GB protected neurons injured by cerebral ischemia/reperfusion,through their modulation of miR-206-mediated BDNF repression.GIs reduced neuronal loss induced by tMCAO and protected Neuro-2a cells against OGD/R injure.We found that GIs exhibited neuroprotection due to the repression of miR-206 and up-regulation of BDNF.It is the first time to study the effect of Ginkgolides on microRNAs,which provides a new way to study the mechanism of Ginkgo lides.