| Stroke is a central nervous system disease with high morbidity,disability and mortality.According to etiology,stroke is mainly classified into ischemic stroke and hemorrhagic stroke,in which ischemic stroke accounts for approximately 85% of all cases.Current studies show that the progressive injury following stroke associates with complex pathologic mechanisms,such as excitotoxicity,calcium overloading,peri-infarct depolarisation,Oxidative or nitrative stress,inflammatory reaction and neuron apoptosis,etc.Though progress has been made in pathologic mechanisms of stroke in animal experiments,there is no effective drugs available for patients apart from recombinant tissue plasminogen activator(rt PA)approved by the United States food and drug administration(FDA).AS the strict therapeutic time window and adverse reaction of hemorrhage,rt PA cannot be widely used clinically.So,it is urgent to find effective drugs and clarify its targets exactly.Inflammation acts as a double-edged sword in stroke.At early stage,TNF-?,IL-1?,IL-6 and IL-18 released from neurons,astrocytes,microglia and macrophages accelerate the death of neurons and glia,while at later stage,inflammation will promote the recovery of ischemia injury.Recently,more and more researches have shown inflammasomes,acts as a switch of inflammation in cytosolic play an important role in stroke.Evidences showed that inflammasomes widely expressed in ischemic neurons,astrocytes,microglia and macrophages,among which NLRP1 and NLRP3 were researched thoroughly.Inflammasomes activation aggravated brain ischemia injury and drugs inhibited inflammasomes activation performed a protective effect on ischemia injury.Thus,developing drugs targeting on inflammasomes is the important therapeutic strategy of stroke.Ginkgo biloba extracts(GBE)is one of the drugs to treat stroke in clinic,terpene lactones are active ingredients to exert the neuroprotection effect in GBE.Ginkgolides(produced by Chengdu Bai Yu technology co.,LTD)is one of the drugs for stroke,the total effective constituents is more than 99%,mainly containing bilobalide,ginkgolide B and ginkgolide A.In clinic,ginkgolides is used for convalescence of mild-to-moderate stroke patients.Researchers have shown that the main components of ginkgolides,bilobalide and ginkgolide B alleviated cerebral I/R injury in acute phase of stroke.Can ginkgolides work in acute phase of stroke? What is the target and mechanism? There is no answer so far.Therefore,this topic is to investigate the effect of ginkgolides on acute cerebral I/R injury,and to find the target and mechanism.Our data will provide academic foundation for widening the clinical use of ginkgolides.So,in part I,we subjected both SD(Sprague Dawley)rats and C57BL/6 mice to cerebral ischemia/reperfusion(I/R)injury with middle cerebral artery occlusion(MCAO)model,and to investigate the impacts of ginkgolides on acute ischemic stroke.Our data reveal that ginkgolides exerts neuroprotective effects during cerebral I/R injury which is related to its protection of neurovascular unit and inhibition of neuroinflammation.Then in part II,we focused on the roles of ginkgolide B(GB)and bilobalide(BB)which are the main components of ginkgolides used in part I.We investigated their impacts on t MCAO mice and LPS+ATP,oxygen-glucose deprivation/reperfusion(OGD/R)BV2 cell lines induced inflammation.Our results show that GB and BB can inhibit microglia activation during ischemia/reperfusion injury and inhibit NLRP3 inflammasome activation induced by LPS+ATP and OGD/R in BV2 cell lines.These results suggested that inhibition of inflammasomes activation in ischemic stroke would be the target of ginkgolides for the treatment of stroke.Part I Effects of ginkgolides on acute cerebral I/R injury in rats and miceAIM: To investigate the effects of ginkgolides on acute injuries of t MCAO model in rats and mice.METHODS: 1)Two to three months old male Sprague Dawley(SD)rats or C57BL/6 mice were subjected to ischemic stroke using the advanced intraluminal transient middle cerebral artery occlusion(t MCAO)model.To rats,ginkgolides(0.625,1.25,2.5 mg/kg,ip.,bid)and positive drug edaravone(3.0 mg/kg,ip.,bid)were administrated 1 h after 1.5 h occlusion.72 h after reperfusion,infarct volume was measured by TTC staining,and the brain water content and neurological deficits were measured in each group.Then,ginkgolides(2.5 mg/kg,ip.,bid)and edaravone(3.0 mg/kg,ip.,bid)were administrated 1 h after 1.5 h occlusion,immunohistochemistry was used to investigate the impacts of drugs on neurovascular unit such as neuron,astrocyte,microglia and vascular.2)According to the previous results,we chose ginkgolides(2.5 mg/kg,ip.,bid)to investigate its therapeutic window.Ginkgolides was administrated 1,3,6,9 h after 1.5 h occlusion,and 72 h after reperfusion,infarct volume was measured by TTC staining,and the brain water content and neurological deficits were measured in each group.3)To mice,ginkgolides(3.5 mg/kg,ip.,bid)and positive drug edaravone(8.0 mg/kg,ip.,bid)were administrated 1 h after 1 h occlusion.72 h after reperfusion,the infarct volume was measured by TTC staining,the brain water content and neurological deficits were measured in each group.Immunohistochemistry was used to investigate the impacts of drugs on microglia activation,and immunofluorescent double staining was used to investigate the location and expression of NLRP3.Pro-inflammatory cytokines IL-1?,TNF-?,IL-18,IL-6,IL-12,and inflammasomes NLRP1,NLRP2,NLRP3,and AIM2 were detected by Real-time PCR and ELISA.Western blotting was used to detect inflammsome related proteins including NLRP1,NLRP3,NLRP2,AIM2,pro-Caspase 1,Caspase 1,pro-IL-1? and IL-1?.RESULTS: 1)Ginkgolides alleviated acute cerebral I/R injury and protected neurovascular unit in rats.Ginkgolides(2.5mg/kg,ip.,bid)alleviated acute neural injury induced by cerebral I/R including improving neurological deficient,reducing infarct volume and brain edema.Meanwhile,ginkgolides reduced neuronal death and loss,inhibited astrocytes and microglia proliferation and activation,attenuated collagen IV degradation.2)The therapeutic window of ginkgolides was over 3 h but less than 6 h after reperfusion.Ginkgolides(2.5 mg/kg,ip.,bid)administerd 1 h or 3 h after reperfusion alleviated acute neural injury induced by cerebral I/R including improving neurological deficient,reducing infarct volume and brain edema.However,ginkgolides administerd 6 h or 9 h did not work.Thus,the therapeutic window of ginkgolides was over 3 h but less than 6 h after reperfusion.3)Ginkgolides ameliorated acute cerebral I/R injury and inhibited ischemic stroke induced inflammation in mice.Ginkgolides(3.5 mg/kg,ip.,bid,3 d)alleviated acute neural injury induced by cerebral I/R including improving neurological deficient,reducing infarct volume and brain edema.Meanwhile,ginkgolides inhibited ischemic stroke induced inflammation,such as inhibiting microglia proliferation and activation,reducing NLRP3 expression in microglia,inhibiting proinflammatory cytokines including IL-1?,TNF-?,IL-18,IL-6,IL-12 and inflammasome related proteins including NLRP3,pro-Caspase 1,Caspase 1,pro-IL-1?,IL-1?.CONCLUSIONS: 1.Ginkgolides ameliorates acute cerebral ischemia/reperfusion injury in rats and mice.2.The therapeutic window of ginkgolides is over 3 h but less than 6 h after reperfusion.3.The underlying mechanism is attributed to that ginkgolides protects neurovascular unit,inhibits microglia mediated inflammation.Part II Roles of ginkgolide B and bilobalide in NLRP3 inflammasome activation induced by ischemic strokeAIM: To investigate the effects of ginkgolide B and bilobalide on inflammation induced by cerebral ischemia/reperfusion injury.METHODS: 1)Two to three months C57BL/6 mice were subjected to t MCAO model,ginkgolide B(GB,1.75,3.5,7.0 mg/kg,ip.,bid,3 d),bilobalide(BB,3.5,7.0,14.0 mg/kg,ip.,bid,3 d)and positive drug edaravone(Eda,8.0 mg/kg,ip.,bid,3 d)were administrated 1 h after 1 h occlusion.72 h after reperfusion,the brain water content and neurological deficits were measured in each group,infarct volume was measured by TTC staining.Then,we chose GB and BB(7.0 mg/kg,ip.,bid,3 d)to do a further investigation on microglia activation by immunohistochemistry and NLRP3 expression in microglia by immunofluorescent double staining.2)Mouse microglia cell lines BV2 cells were cultured and ELISA was used to screen the effective concentrations of GB and BB(0.01 ?M-100 ?M)on LPS induced upregulation of inflammatory cytokines such as IL-1?,TNF-? and IL-6.Western blotting was used to evaluate the effects of GB and BB on NLRP3 inflammasome activation induced by LPS+ATP and OGD/R.Finally,BV2 cells incubated with GB and BB(1 ?M),and then stimulated by LPS+ATP,culture mediums were collected as conditioned mediums,and mouse neuroblastoma Neuro2 a cells were stimulated by the conditioned mediums.24 h after stimulation,Hoechst and Western blotting were used to evaluate the neuronal apoptosis.RESULTS: 1)Ginkgolide B and bilobalide attenuated cerebral I/R injury,inhibited microglia activation and NLRP3 expression in mice.GB(3.5,7.0 mg/kg,ip.,bid,3 d)and BB(7.0,14.0 mg/kg,ip.,bid,3 d)alleviated acute neural injury induced by cerebral I/R including improving neurological deficient,reducing infarct volume and brain edema.Meanwhile,GB and BB(7.0 mg/kg,ip.,bid,3 d)inhibited microglia activation and NLRP3 expression induced by ischemic stroke model in mice.2)Ginkgolide B and bilobalide inhibited NLRP3 inflammasome activation induced by LPS+ATP and OGD/R,and reduced neuronal apoptosis.GB and BB(1 ?M)suppressed inflammatory cytokines such as IL-1?,TNF-? and IL-6 upregulation induced by LPS in BV2 cells.In addition,GB and BB inhibited NLRP3 inflammasome activation induced by LPS+ATP and OGD/R,including inhibiting NLRP3,pro-Caspase1,pro-IL-1? expression,reducing NF-?B nuclear translocation and Caspase1,IL-1? secretion.GB and BB reduced Neuro2 a apoptosis induced by BV2 cells condition mediums detected by Hoechst and Western blotting.CONCLUSIONS: 1.Ginkgolide B and bilobalide attenuate cerebral ischemia/reperfusion injury and inhibit inflammation in t MCAO mice.2.Ginkgolide B and bilobalide reduce neuronal apoptosis via inhibiting NLRP3 inflammasome activation.IN SUMMARY: 1.Ginkgolides ameliorates acute cerebral ischemia/reperfusion injury.Ginkgolides alleviated acute neural injury induced by cerebral I/R including improving neurological deficient,reducing infarct volume and brain edema.Meanwhile,ginkgolides reduced neuronal death and loss,inhibited astrocytes and microglia proliferation and activation,attenuated collagen IV degradation,the therapeutic window was over 3 h but less than 6 h.These results indicate that ginkgolides ameliorates acute cerebral I/R injury,which expands the use of ginkgolides in clinic.2.Ginkgolides alleviates ischemic stroke via inhibiting NLRP3 inflammasome activation.Inflammatory cytokines such as IL-1?,TNF-?,IL-6 were upregulated after ischemic stroke,and inflammasomes such as NLRP1,NLRP3 were activated.GB and BB reduced the secretion of IL-1?,TNF-?,IL-6 and suppressed the activation of NLRP3 inflammasome.These data suggest that NLRP3 inflammasome is one of the targets of ginkgolides in regulating post-ischemic inflammation. |
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