甲基乙二醛对大鼠海马神经元的毒性作用及脑源性神经营养因子、TrKB表达的影响

目的 探讨葡萄糖降解产物甲基乙二醛(MG)对海马神经元的毒性作用及可能机制.方法取新生24h Sprague-Dawley大鼠海马神经元原代培养至第7天,给予MG干预24h,四甲基偶氮唑蓝(MTT)法检测海马神经元存活率,异硫氰酸荧光素标记的膜联蛋白V联合碘化丙啶(PI)法检测海马神经元的凋亡率,采用实时RT-PCR法及Western印迹法测定脑源性神经营养因子(BDNF)及其受体酪氨酸蛋白激酶(TrkB)mRNA和蛋白表达水平.结果 1.随着MG浓度的增加及干预时间延长,海马神经元存活率逐渐降低,呈浓度依赖性(r=0.946,P<0.01)和时间依赖性(r=0.993,P<0.01).与0h组海马神经元凋亡率(1.633±0.153)%相比,100μM MG干预2h、6h、12h和24h后,其凋亡率逐渐增加[分别为(2.833±0.153)%、(3.367±0.153)%、(4.433±0.404)%和(8.833±0.306)%;均P<0.01];2.与同时间对照组相比,MG干预12h组及24h组海马神经元BDNF mRNA及蛋白表达增加(P<0.05或P<0.01);而MG干预6h组、12 h及24h组TrkB mRNA及蛋白表达减少(P<0.05或P<0.01).结论 MG对海马神经元具有直接毒性作用,可能首先抑制海马神经元TrKB表达,导致BDNF表达代偿性增高,损伤BDNF-TrKB通路,诱导神经元凋亡增加.

The cytotoxic effect of methylglyoxal on BDNF and TrkB expression in rat hippocampal neurons

Objective To investigate the mechanisms of methylglyoxal(MG)-induced injury of hippocam-pal neurons. Methods Primary cultured of hippocampal neurons from 1-day-old Sprague-Dawley rats were incuba-ted with MG for different time and dose period. Cells proliferation were assayed by methyl thiazolyl tetrazolium (MTT),and apoptosis was quantified by flow cytometer using annexin V-FITC and propidium iodide (PI) stai-ning. The protein and mRNA levels of brain-derived neurotrophie factor (BDNF) and tyrosine kinase B(TrkB) were assayed with Western Blotting and real-time PCR. Results Treatment with MG resulted in a concentration-dependent (r=0.946, P < 0.01) and time-dependent (r=0.993, P < 0.01) decreasing neurons viability. Com-pared with Oh group(1. 633±0. 153)%, 100 μM MG treatment for 2h,6h, 12h and 24h,the cellular apeptosis rate were significantly increased ((2. 833±0. 153)%, (3. 367±0. 153)%, (4. 433±0. 404)% and (8. 833± 0. 306)% respectivdy,all P<0.01). MG also increased the BDNF mRNA and protein expression after 12h treat-ment (P<0.05 or P<0.01),but decreased the TrkB mRNA and protein expression in the cells after 6h treatment (P<0.05 or P < 0.01). Conclusion MG has direct toxic effect on hippocampal neurons and can impaire the BD-NF-TrkB signal pathway by inhibiting the expression of TrkB,and increasing the apoptosis of hippocampal neurons.