Neuromechanisms And Translational Studies Of Aspirin Protection Against Cerebral Ischemia

Ischemic stroke is a leading cause of death and disability worldwide.Although neuroprotective agents can improve ischemia/reperfusion injury and prolong the time window for acute stroke treatment,few have been successfully translated into clinical treatment.Aspirin,a first-line antithrombotic agent,has shown promising neuroprotective effects in cellular and animal models of ischemic stroke.However,little is known about the neuroprotective mechanisms of aspirin and its administration leads to an increased risk of bleeding.The results of previous studies have shown that the death of neurons in cerebral ischemia is related to the over-activation of signaling pathways in neuronal calcium overload.In animal models of cerebral ischemia,blocking the signaling pathway of calcium overload can exert neuroprotective effects.Aspirin has an active acetyl group that acetylates the-amino group of protein lysine for modification.Therefore,we propose the hypothesis that Aspirin can acetylate calcium signaling pathway proteins during cerebral ischemia and thus exert neuroprotective effects.To test this hypothesis,we first explored which proteins in the mouse cerebral cortex could be upregulated in acetylation levels by oral aspirin administration through quantitative acetylation protein profiling.The results showed that aspirin could increase the acetylation levels of 466 proteins in the brain.These elevated acetylation genes were found to be significantly enriched in the neuronal death-related HIF-1 and Necroptosis signaling pathways by KEGG analysis,with CaMK2α,CaMK2β,and CaMK2δappearing in both signaling pathways.We predicted the effects of acetylation on the functions of the three genes by structural simulations and found that the lysine site of CaMK2α(K42)acetylation showed the greatest alteration of protein function.The mass spectrometry results further suggested that aspirin-mediated K42AC-CaMK2α is a possible neuroprotective mechanism.To investigate the pattern of up-regulation of the CaMK2α protein acetylation(K42AC-CaMK2α)pattern by Aspirin,we prepared antibodies that specifically recognize K42AC-CaMK2α.We found by western blot experiments that administration of drinking water containing Aspirin(0.1 mg/ml)to adult mice for 2weeks resulted in significant upregulation of K42AC-CaMK2α protein levels in the cytoplasm of the cerebral cortex,while K42AC-CaMK2α protein levels in synapses were not changed significantly.We further calculated that aspirin drinking resulted in acetylation of approximately 12% of CaMK2α protein in the cytoplasm of the cerebral cortex using the method of titration of acetylated protein standards established in the pre-laboratory.To investigate the effect of acetylation on the physicochemical properties of CaMK2α protein,we performed ATP affinity assays,computer structural simulations and in vitro kinase activity assays,and found that K42 acetylation severely impaired CaMK2α and ATP binding and inhibited the kinase activity of CaMK2α,and acetylation of 12% of CaMK2α protein was sufficient to reduce the kinase activity of all CaMK2α kinase activity was reduced.To clarify whether K42AC-CaMK2α is involved in the protective effect of Aspirin against cerebral ischemia,we injected interfering peptides into the ventricles every other day during Aspirin administration to inhibit K42AC-CaMK2α,and found that inhibition of K42AC-CaMK2α significantly diminished the neuroprotective effect of Aspirin.effect.To further investigate whether K42AC-CaMK2α could exert a protective effect against cerebral ischemia,we used a codon expansion technique to overexpress K42AC-CaMK2α in the mouse cerebral cortex.K42AC-CaMK2α was overexpressed in mouse cerebral cortex using codon expansion.Compared with control mice,mice overexpressing K42AC-CaMK2α showed reduced mortality,decreased infarct size,and improved cognitive function after cerebral ischemia.The above results indicate the adequacy of K42AC-CaMK2α in the protective effects of cerebral ischemia.Since CaMK2α is specifically expressed in excitatory neurons of the cerebral cortex and is not expressed in platelets our findings reveal an anticoagulation-independent neuronal autonomic mechanism of Aspirin protection against cerebral ischemia.Based on our findings neuroautonomous mechanisms modify Aspirin by retaining the acetyl group and modifying the carboxyl group differently.We found that ethyl Oacetylsalicylate,obtained by one-step esterification of the carboxyl group,significantly reduced the affinity for cyclooxygenase 1 and had a lower anticoagulant effect compared to Aspirin,using computer structural simulations and further comparative experiments with two consecutive weeks of administration.decreased.In vitro and in vivo conditions also increased the acetylation of CaMK2α,which still reduced mortality,infarct size and improved cognitive function after cerebral ischemia in animal models.In conclusion,we propose a new proof-of-principle for Aspirin for stroke prevention: modification of Aspirin to reduce its anticoagulant effect but retain its acetylating effect on CaMK2α may be a safer and more effective strategy for cerebral ischemia protection.