Therapeutic Target Of Neuroprotection Sreening And The Underlying Mechanism Exploring

【Background】Stroke is responsible for the leading cause of long term disability and ranks as the second leading cause of death worldwide.Consequently,stroke becomes the emergency disease that should be concerned.Considerable efforts have been devoted to explore novel protective measures against stroke.However,there was no drug approved for hemorrhagic stroke.And in the past 2 decades,only tissue plasminogen activator has been proved and recommended as an effective treatment for ischemic stroke.However,the application is with strict limitations,including a narrow time window and risk of inducing intracranial hemorrhage.Experimental studies,mostly in rodent models of stroke,have overwhelmingly suggested that the brain could be acutely protected from stroke with a plethora of different therapeutic strategies.However,more than 1000 clinical trails failed to show efficacy of any of these promising strategies,which makes neuroscientists doubting stroke could be cured.Therefore,the research direction for stroke treatment needs to be rerouted.The brain has a remarkable endogenous self-protective capacity,which can be induced by ischemic and nonischemic preconditioning,such as hyperbaric oxygen preconditioning.Compared with t PA,there are lots of advantages of hyperbaric oxygen preconditioning,such as inducing multi pro-survival signaling pathway,having no risk of intracranial hemorrhage et al.However,according to our experiments in the past decade,we noticed that although HBO could benefits most of the animals and patients,not all animals or patients who received HBO preconditioning exhibited a favorable outcome after cerebral injury.It is reasonable to deduce that some intrinsic molecules are responsible for the differential response after HBO preconditioning because of differences in genetic and epigenetic.Therefore,identifying the crucial beneficial molecules may help to understand the endogenous neuroprotective mechanism and provide new insights into stroke therapy.In the other hand,as the management of ischemic stroke and hemorrhagic stroke is so different,the clinical distinction between the subtypes is one of the most important steps in stroke management.However,the distinction by CT(Computed Tomography)or MRI(Magnetic Resonance Imaging),consumes hours and would miss the golden window time to save the patients.Combining preclinical as well as clinical evidence,one can tell neuroprotectants failed to benefit patients not only because the drugs were ineffective,but rather because treatment came too late.Therefore,developing drugs that interfere with the common pathological cascades both in ischemic and hemorrhagic stroke which do not rely on the CT and MRI scanning is an appealing idea.The aim of the research is to screen the endogenous protective moleculars of HBO preconditioning and explore the underlying mechanism.Besides,according to the common pathophysiological cascades of ischemic/hemorrhagic stroke from the publication,we tried to develop therapeutic peptides that benefit against both ischemic/hemorrhagic.Part Ⅰ Endogenous protective moleculars of HBO preconditioning screening and the underlying mechanism exploringExperiment 1.Endogenous protective molecules of HBO preconditioning screening【Objectives】1.Explore the molecules responsible for the endogenous neuroprotection induced by hyperbaric oxygen preconditioning.2.Conrfirm the neuroprotective effect of the potential molecule that screened using proteomic method.【Methods】Adult male Sprague–Dawley rats were subjected to transient middle cerebral artery occlusion.To confirm the long lasting neuroprotection of HBO preconditioning,we measured the infact volume of rats at 28 days after stroke.High-throughput proteomic technique,isobaric tag for relative and absolute quantification(i TRAQ),was used to screen differentially expressed proteins in the rats that developed ischemic tolerance from hyperbaric oxygen(HBO)preconditioning.The proteomic results were verified by Western blot and ELISA.Then,short interfering RNA and gene knockout rats were used to further determine the pivotal role of candidate proteins in endogenous neuroprotection induced by HBO preconditioning.【Results】This is the first time we proved that HBO preconditioning could reduce neurological deficit and infarct at 28 days after MCAO.Compared with beneficial group,the serum level of cystatin C and mannose-binding protein C(MBLC)were significantly different from both non-beneficial and control group.To further verify the results,we validated the proteins in individual samples using Western blot and ELISA.We found changes of Cystatin C and MBLC were in accordance with i TRAQ analysis.We then analysed the correlation between protein concentration and brain infarct volume of all rats.The results showed that the concentration of Cystatin C in the serum before stroke was negatively correlated with the infarct volume.And the concentration of MBLC was positively correlated with the infarct volume.We further demonstrated that HBO preconditioning could up-regulate the expression of Cystatin C in the brain of rat.The ischemic tolerance induced by HBO preconditioning was attenuated by knocking down of Cystatin C using si RNA.Besides,the neuroprotection of HBO preconditioning disappeared in Cystatin C genetic deletion rats.In addition,we found that exogenous Cystatin C significantly reduced ischemia injury by a single intracerebroventricular injection after ischemia-reperfusion.【Conclusion】Cystatin C plays very important role in the neuroprotection of HBO preconditioning,suggesting a potential drug for ischemia stroke.Experiment 2.Cystatin C is the Key Mediator of Autophagy Induced by Hyperbaric Oxygen Preconditioning【Objectives】Hyperbaric oxygen(HBO)preconditioning has robust neuroprotective effect,but the underlying mechanism is not fully understood.The aim of the present study is to explore the mechanism of elevated Cystatin C induced by HBO in mediating neuroprotection.【Methods】Male Sprague-Dawley rats were preconditioned with HBO for five consecutive days.Twenty four hours after the last session of preconditioning,rats received 2 hours of focal cerebral ischemia.The protein level of Cystatin C in the penumbra of the brain was measured at different time point after MCAO.To test the relationship between Cystatin C and autophagy,the autophagosome related proteins LC3 Ⅱ and Beclin-1 were also measured.We then employed the si RNA of Cystatin C to knock down Cystatin C and tested its affect to autophagosome related proteins.Finally,using immunofluorescence staining and immunoelectron microscopy,we tested the role of Cystatin C in maintaining lysosomal integrity and enhancing autolysosome formation induced by HBO preconditioning.【Results】We found that Cystatin C expression elevated in the penumbra as early as 3 hours after reperfusion.This increase was further enhanced by HBO preconditioning.At the same time,changes of autophagy related proteins LC3Ⅱ/ LC3Ⅰand Beclin-1 shared the same pattern with Cystatin C,while knocked down Cys C with si RNA blocked the elevation of LC3 Ⅱ / LC3 Ⅰ and Beclin-1.Furthermore,knocking out Cystatin C diminished the beneficial effects of preserving lysosomal membrane integrity and enhancing autophagy induced by HBO preconditioning.【Conclusion】These results indicated that up-regulation of Cystatin C was pivotal for activating autophagy induced by HBO preconditioning.The present study further clarified the mechanism of HBO preconditioning and would help to facilitate its clinical use.Part Ⅱ Common pathophysiological cascades of ischemic/hemorrhagic stroke exploring and therapeutic peptides developing【Objectives】To explore the common pathophysiological cascades of ischemic/hemorrhagic stroke and develop therapeutic peptides that do not have to wait until the CT(Computed Tomography)or MRI(Magnetic Resonance Imaging)results.【Methods】According the publication,we found that MD2-TLR4 is one of the common pathophysiological cascades of ischemic/hemorrhagic stroke.To mimic stroke model in vitro,we used a NMDA-induced excitotoxicity protocol in cultured cortical neuron in vitro.In the in vitro experiment,we used a neuronal death assay,LDH(lactate dehydrogenase)release,which can allow for the nonbiased quantitative identification of both apoptotic and necrotic neuronal damage.We next designed two peptides that flank the MD2 residue with Tat-CIRP(YGRKKRRQRRRGRGFSRGGGDRGYGG)and knockdown MD2 with Tat-CIRP-CMA(YGRKKRRQRRRG RGFSRGGGDRGYGGKFERQKILDQRFFE),respectively.And then determined whether these two peptides can exert neuroprotection or not.In vivo,we used ischemic stroke and hemorrhagic stroke model,and assessed the expression of MD2 after stroke.To examine the causative link between ischemia-induced MD2 elevation and ischemic neuronal death,we inhibited or knocked down MD2 with systemic intravenous administration of Tat-CIRP and Tat-CIRP-CMA,or with saline as control.With the help of genetic deletion mice(TLR4 and MD2 deletion),we then explored the underlying mechanism of MD2 mediating cell death.【Results】Here,we report that high expression of MD2(myeloid differentiation protein 2)leading to excitotoxic(in vitro)and ischemic/hemorrhagic(in vivo)neuronal injuries,which traditionally well studied in glia cells by triggering inflammation in the brain.We found that excitotoxic stimulation of N-methyl-D-aspartate(NMDA)resulted in the expression of MD2 elevated in cultured neurons,which was TLR4(Toll like receptor 4)independent.More importantly,we demonstrated that systemic application of a small peptide Tat-CIRP,which interfered the binding between cold-inducible RNA-binding protein(CIRP)and MD2,strongly protected against excitotoxic(in vitro)and ischemic(in vivo)neuronal injuries through inhibiting both apoptosis and necroptosis.Genetic deletion of MD2 also induced robust neuroprotection against cerebral ischemic insults in mice.Even for hemorrhagic stroke,Tat-CIRP reduced cerebral injury.【Conclusion】These data indicate an unexpected function of MD2 mediating cell death in neuron,which is TLR4 independent.Our study suggests that inhibiting the function of MD2 by Tat-CIRP may represent a novel therapy against both ischemic and hemorrhagic stroke.