β-羟基丁酸对糖氧剥夺原代神经元的保护作用及其机制研究

Protective effect of β-hydroxybutyrate on primary neurons with glucose-oxygen deprivation and related mechanism

目的探讨β-羟基丁酸(BHB)对SD大鼠神经元细胞缺氧损伤的保护作用及其机制。方法原代培养SD大鼠神经元细胞,不同浓度(2 m M、5 m M、10 m M、20 m M和50 m M)BHB预处理24 h,三气培养箱糖氧剥夺培养2 h,线粒体超氧化物探针检测细胞线粒体氧化应激状态,CCK8检测细胞活力变化,QPCR测Na+偶联单羧酸转运体1(SMCT1)、caspae3和Cytochrome C的m RNA表达,Western Blot测SMCT1、caspase3、细胞色素C(Cytochrome C)、ERK1/2和p-ERK1/2(T202/204)蛋白表达。结果 2 m M、5 m M和10 m M浓度BHB对神经元细胞活力无显著影响(P>0.05),20 m M和50m M浓度BHB可致神经元活力显著降低(P<0.05)。神经元缺氧培养后,活力降低(P<0.05),线粒体氧化应激反应增强(P<0.05),SMCT1的m RNA水平和蛋白水平显著降低(P<0.05),caspase3与Cytochrome C的m RNA水平和蛋白水平明显增强(P<0.05),ERK1/2的蛋白磷酸化水平降低(P<0.05)。BHB预处理缺氧神经元,低浓度对细胞无明显影响(P>0.05);浓度增至10 m M,相比单纯缺氧组,神经元活力显著改善(P<0.05),线粒体氧化应激状态降低(P<0.05),SMCT1的m RNA水平和蛋白水平显著增加(P<0.05),caspase3与Cytochrome C的m RNA水平和蛋白水平明显降低(P<0.05),ERK1/2的蛋白磷酸化水平升高(P<0.05)。结论高浓度(>20 m M)BHB对神经元细胞有毒性作用;低浓度(<5 m M)BHB对糖氧剥夺的神经元细胞无明显作用;10 m M浓度的BHB预处理可增加SMCT1转运体的表达,通过启动ERK1/2信号通路,降低神经元线粒体的氧化应激,减少凋亡,从而对糖氧剥夺的原代神经元细胞发挥保护作用。

Objective To investigate the protective effect of β-hydroxybutyrate （BHB） against hypoxic injury in neurons of Sprague- Dawley （SD） rats and related mechanism. Methods Primary cultured neurons of SD rats were pretreated with BHB at various concen- trations （2 mM, 5 mM, 10 raM, 20 mM, and 50 mM） for 24 hours and then put in a tri-gas incubator for glucose-oxygen deprivation for 2 hours. A mitochondrial superoxide probe was used to examine mitochondrial oxidative stress in neurons; CCK-8 assay was used to measure the change in cell viability; QPCR was used to measure the mRNA expression of sodium-coupled monocarboxylate transporter 1 （ SMCT1 ） , caspase-3, and cytochrome C, and Western blot was used to measure the protein expression of SMCT1, caspase-3, eyto- chrome C, extracellular signal-regulated kinase 1/2 （ ERK1/2 ） , and phosphorylated ERK1/2 （ p-ERK1/2 ） （ T202/204 ）. Results BHB at concentrations of 2 raM, 5 mM, and 10 mM had no significant influence on the viability of neurons （ P 〉 0.05 ） , while BHB at concentrations of 20 raM and 50 mM caused a significant reduction in the viability of neurons （ P 〈 0.05 ）. After hypoxic culture of the neurons, there was a significant reduction in viability （ P 〈 0.05 ） , a significant increase in mitochondrial oxidative stress （ P 〈 0.05 ）, significant reductions in the mRNA and protein expression of SMCT1 （ P 〈 0.05 ）, significant increases in the mRNA and protein expression of caspase-3 and cytochrome C （ P 〈 0.05 ）, and a significant reduction in the phosphorylation level of ERK1/2 （ P 〈 O. 05）. As for the hypoxic neurons pretreated with BHB, a low concentration of BHB had no significant influence on neurons （P 〉 O. 05 ） ; compared with the simple hypoxia group, the 10 mM-BHB group had a significant improvement in the viability of neurons （P 〈 0.05 ） , a significant reduction in mitochondrial oxidative stress （P 〈 O. 05）, significant increases in the mRNA and protein expres- sion of SMCT1 （ P 〈 0.05 ）, significant reductions in the mRNA and protein expression of caspase-3 and cytochrome C （ P 〈 0.05 ）, and a significant increase in the phosphorylation level of ERK1/2 （P 〈 0.05）. Conclusions High concentrations （ 〉 20 mM） of BHB have a toxic effect on neurons; low concentrations （ 〈 5 mM） of BHB have no significant influence on neurons with glucose-oxygen deprivation ; BHB at a concentration of 10 mM can increase the expression of SMCT1, reduce mitochondrial oxidative stress and apopto- sis by activating the ERK1/2 signaling pathway, and thus exert a protective effect on primary neurons with glucose-oxygen deprivation.