Objective To investigate the neuroprotective role of alanine(Ala)in ischemic brain injury and the regulation of M2-type pyruvate kinase(PKM2)expression levels by Ala.Methods Primary neurons were cultured in vitro and an oxygen-glucose deprivation/reperfusion(OGD/R)model was constructed.Neurons were randomly divided into the sham-operated group(Sham),oxygen-glucose deprivation/reperfusion-treated group(OGD/R),and post-OGD/R Ala intervention group(OGD/R+Ala).CCK-8 method was used to detect the effect of Ala intervention on neuronal survival rate.Western blot was used to detect the effect of Ala on PKM2 protein expression level in neurons of the OGD/R model.A middle cerebral artery embolization(MCAO)model was constructed in rats,and 36 SD rats were randomly divided into the sham-operated group(Sham),middle cerebral artery embolization group(MCAO),and post-MCAO Ala intervention group(MCAO+Ala),with 12 rats in each group.Immunofluo-rescence assay was used to detect the changes of Ala content in OGD/R injured neurons.The changes in the cerebral infarct area were observed after 2,3,5-triphenyl tetrazolium chloride(TTC)staining.Western blot was used to detect the effect of Ala inter-vention on the expression level of PKM2 protein in ischemically injured brain tissues.Results CCK-8 and Western blot showed significant differences in neuronal survival rate and PKM2 expression(F=86.88,20.83;P＜0.05).Cell survival was significantly lower and PKM2 expression was higher in the OGD/R group compared with the Sham group(tLSD=4.65,10.95;P＜0.05).Com-pared with OGD/R,OGD/R+Ala significantly increased neuronal survival and reduced PKM2 expression(tisD=4.98,4.69;P＜0.05).Immunofluorescence staining showed that Ala content in damaged neurons was significantly lower in the MCAO group com-pared with the Sham group.TTC showed that Ala intervention reduced the ischemic area in MCAO model rats(F=83.90,tLSD=3.41,P＜0.05).Western blot showed that PKM2 expression levels were significantly increased in the brain tissue of the MCAO model rats compared with the sham-operated group(F=3.60,tLSD=5.10,P＜0.05).PKM2 expression levels in brain tissue decreased significantly after Ala intervention(tLSD=6.20,P＜0.05).Conclu-sion Ala can reduce cerebral ischemia/reperfusion injury and thus exert neuroprotective effects by inhibiting PKM2 protein ex-pression.
维普
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