The Neuroprotective Effects And Mechanisms Of 5-(4-Hydroxy-3-dimethoxybenzylidene)-rhodanine On Cerebral Ischemia-reperfusion Injuries

Ischemic stroke is one of the leading causes of disability and mortality in the world,which exerts a profoundly negative impact on both patients and society.Thrombolysis with tissue plasminogen activator is the only effective therapeutic drug approved by the European Medicines Agency and the US Food and Drug Administration for the treatment of ischemic stroke.But the reperfusion injury after thrombolysis treatment is closely related with prognosis of ischemic stroke and the formation of sequelae.Therefore,it is urgent to develop safe and effective drugs for ischemia reperfusion therapy.We previously synthesized and screened a derivative of rhodanine via chemical modifications,named 5-(4-hydroxy-3-dimethoxybenzylidene)-2-thioxo-4-thiazolidinone(RD-1).Further study showed that RD-1 possesses protective effects on movement disorder and mitochondrial dysfunction in Parkinson’s disease mice model induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine treatment.Deficits in mitochondrial function play vital roles in the major pathways that drive neuronal cell death in the ischemic process.Considering the similarities in the pathologic mechanisms underlying both cerebral ischemia and Parkinson’s disease,we investigated the potential protective effect of RD-1 on focal cerebral ischemia reperfusion injury both in rats with middle cerebral artery occlusion(MCAO)model in vivo and in primary cortical neurons(PCNs)with oxygen-glucose deprivation and reoxygenation(OGD/R)injury in vitro.We also further revealed the underlying mechanisms whereby RD-1 alleviates mitochondrial damage caused by cerebral ischemia reperfusion injury.Part Ⅰ:Studies on the neuroprotective effects of 5-(4-hydroxy-3-dimethoxybenzylidene)-rhodanine on cerebral ischemia-reperfusion injuries1.OGD/R model was applied to mimic the ischemia-reperfusion injury in vitro followed by CCK-8 cell viability assay.Cell survival was suppressed when PCNs were subjected to OGD/R injury.However,treatment with different concentrations of RD-1(1,5 and 10 μmol/L)for 48 h exerted a protective effect against OGD/R induced damage(P<0.05).2.In MCAO rats,treatment with RD-1 for 14 days remarkably decreased neurological deficit score(P<0.05),accelerated the recovery of somatosensory function(P<0.05)and improved mental disorder(P<0.05).At the same time,RD-1 could also shorten the time of beam-walking to some extent.The ischemia reperfusion injury had no influence on the ability of spontaneous movement and working memory.3.Results of HE staining showed that infarct volume in MCAO rats was significantly increased(P<0.01).Treatment with RD-1 significantly decreased the infarct volume(P<0.05).4.Immunohistochemistry analysis indicted that ischemia reperfusion injury significantly decreased the number of NeuN positive cells in the ischemic cortex(P<0.001),and treatment with RD-1 obviously prevented the loss of NeuN-positive neurons(P<0.01).There had no differences in ipsilateral cortex between each group.Part Ⅱ:Studies on the mechanisms of 5-(4-hydroxy-3-dimethoxybenzylidene)-rhodanine on cerebral ischemia-reperfusion injuries1.In primary cortical neurons,OGD/R injury significantly induced the outburst of reactive oxygen species(ROS),declined the mitochondrial membrane potential(MMP)and caused the excessive influx of calcium.However,RD-1 protected the neurons against mitochondrial damage as evidenced by reducing the overproduction of ROS,stabilizing the MMP and alleviating the Ca2+ overload intracellular(P<0.001,P<0.05).2.The expression of DRP1 in neurons subjected with OGD/R injury had no changes,but OPA1 was decreased obviously(P<0.01).RD-1 treatment significantly increased the expression of OPA1(P<0.05).3.In ischemic cortex of MCAO rats,the western blot results demonstrated that mitochondrial fission and fusion protein(DRP1,OPA1 and MFN2)were decreased at 14 d after ischemia reperfusion(P<0.05,P<0.01,P<0.001).RD-1 treatment significantly elevated these three proteins and defended the mitochondrial fission-fusion dysfunction.4.We also found that mitophagy in ischemic cortex was inhibited.The expression of Beclinl was significantly decreased and the portion of LC3-Ⅱ declined.Treatment with RD-1 could facilitate mitophagy via activating the PINK1/Parkin pathway and Nix pathway.5.In the mitochondrial axonal transport test,we found OGD/R injury significantly increased the mitochondrial retrograde and decreased the anterograde velocity(P<0.001).By contrast,this damage was reversed dramatically by 5 μmol/L RD-1 treatment,which significantly improved the mitochondrial anterograde transport and slowed down the retrograde transport(P<0.001).Additionally,western blot results showed that OGD/R injury remarkably decreased KIF1B and Miro2 level,but RD-1 treatment alleviated these pathological changes(P<0.05).Similar data were obtained in in vivo experiments.6.OGD/R injury significantly reduced the efficacy of neurotransmission(P<0.01),as more than 60%neurotransmitter of model cells were not released in 2 min,while RD-1 treatment markedly elevated the efficacy of neurotransmission and improved neuronal terminal activities(P<0.05).Concurrently,the expression of SNAP25 and Complexin-1/2 were down-regulated by ischemia reperfusion while significantly enhanced by RD-1 treatment(P<0.05).The western blot results of our in vivo experiments were in accordance with the in vitro study.7.TUNEL staining in the primary cortical neurons showed that,the ratio of TUNEL-positive cells significantly increased after OGD/R exposure.RD-1 treatment inhibited the apoptosis.8.Meanwhile,ischemia reperfusion injury decreased the Bcl-2(P<0.05),increased the Bax(P<0.01),hence declined the ratio of Bcl-2/Bax(P<0.05).RD-1 treatment significantly increased the Bcl-2/Bax level both in vivo and in vitro(P<0.05,P<0.01).In summary,the present study firstly demonstrated the neuroprotective effects of RD-1 on cerebral ischemia/reperfusion induced brain injury both in vivo and in vitro.And the effect at least partially due to the improvement ability of attenuating mitochondrial dysfunctions and promoting the synaptic activity.Our research showed that RD-1 may be a candidate drug for cerebral stroke therapy,however other potential involved signaling pathways should be illustrated in the following research.