丙氨酸抑制PKM2表达对缺血性脑损伤保护作用

Alanine exerts neuroprotective effects in cerebral ischemia/reperfusion injury through inhibition of PKM2

目的研究丙氨酸(Ala)在缺血性脑损伤中的神经保护作用以及Ala对M2型丙酮酸激酶(PKM2)表达水平的调节。方法体外培养原代神经元并构建氧-糖剥夺/再灌注(OGD/R)模型,将神经元随机分为对照组、OGD/R模型组以及OGD/R后Ala干预组(OGD/R+Ala),应用CCK-8方法检测Ala干预对神经元存活率的影响,Western blot方法检测Ala对OGD/R模型神经元内PKM2蛋白表达水平的影响。构建大鼠大脑中动脉栓塞(MCAO)模型,随机将36只SD大鼠分为假手术组、MCAO组以及MCAO后Ala干预组(MCAO+Ala),每组12只,采用免疫荧光方法检测脑缺血/再灌注损伤后神经元内Ala含量的变化,2,3,5-氯化三苯基四氮唑(TTC)染色观察各组脑梗死面积,Western blot方法检测Ala干预对缺血损伤脑组织中PKM2蛋白表达水平的影响。结果CCK-8与Western blot检测结果显示,各组神经元存活率及PKM2蛋白表达差异有显著性(F=86.88、20.83,P<0.05),OGD/R组细胞存活率较对照组显著降低,PKM2蛋白表达较对照组升高(t_(LSD)=4.65、10.95,P<0.05);OGD/R+Ala组细胞存活率较OGD/R组显著增加,PKM2蛋白表达较OGD/R组明显下降(t_(LSD)=4.98、4.69,P<0.05)。免疫荧光染色结果显示,MCAO组大鼠受损神经元内Ala含量较假手术组明显降低。TTC结果显示,Ala干预使MCAO模型大鼠缺血面积减少(F=83.90,t_(LSD)=3.41,P<0.05)。Western blot结果显示,MCAO模型组大鼠脑组织中PKM2表达水平增高,与假手术组相比差异有显著性(F=3.60,t_(LSD)=5.10,P<0.05),Ala干预后脑组织中PKM2表达水平明显下降(t_(LSD)=6.20,P<0.05)。结论Ala可能通过抑制PKM2蛋白表达减轻脑缺血/再灌注损伤从而发挥神经保护作用。

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.Immunofluorescence 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 intervention 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(t_(LSD)=4.65,10.95;P<0.05).Compared with OGD/R,OGD/R+Ala significantly increased neuronal survival and reduced PKM2 expression(t_(LSD)=4.98,4.69;P<0.05).Immunofluorescence staining showed that Ala content in damaged neurons was significantly lower in the MCAO group compared with the Sham group.TTC showed that Ala intervention reduced the ischemic area in MCAO model rats(F=83.90,t_(LSD)=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,t_(LSD)=5.10,P<0.05).PKM2 expression levels in brain tissue decreased significantly after Ala intervention(t_(LSD)=6.20,P<0.05).Conclusion Ala can reduce cerebral ischemia/reperfusion injury and thus exert neuroprotective effects by inhibiting PKM2 protein expression.