摘要目的 探讨氟西汀(FL)对甲基乙二醛(MG)诱导的海马神经元毒性损伤的保护作用.方法 取新生24 h Sprague-Dawley大鼠海马神经元原代培养至第7天,予MG和(或)FL干预24 h,分为5组,每组样本数均为6.(1)MG组:培养液中加入MG;(2)FL组:培养液中加入FL;(3)MG+FL组:培养液中加入MG和FL;(4)预处理(pre)FL+MG组:海马神经元原代培养第6天加入FL,干预24 h后加MG再培养24 h;(5)对照组:仅加相应体积完全培养液.异硫氰酸荧光素标记的膜联蛋白V(AnnexinV-FITC)联合碘化丙啶(PI)法检测海马神经元凋亡率,以2,7-二氢二氯荧光素(DCFH)染色,流式细胞仪测定细胞内活性氧(ROS)水平;采用荧光实时定量聚合酶链反应及Western印迹法检测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 MG组海马神经元凋亡率(8.83±0.31)%高于对照组(1.63±0.15)%及MG+FL组(3.20±0.30)%;ROS水平比值(10 229±946)高于FL组(3076±41)、对照组(4265±82)、MG+FL组(6058±179)及pre FL+MG组(6076 ±281);BDNF mRNA比值及蛋白水平比值(2.37±0.33;0.625±0.008)分别高于对照组(1.00±0.27;0.582±0.003)而低于>FL组(3.88±0.32;0.855±0.007)、MG+FL[(7.66±0.34;1.113±0.023)和pre FL+MG组(6.96 ± 0.54;0.689±0.014)];TrkB mRNA及蛋白水平比值(0.50±0.06;0.133±0.006)则低于对照组(1.00 ± 0.06;0.328±0.000)、FL组(5.45±0.42;0.460±0.005)、MG+FL组(4.21±0.32;0.414±0.006)和pre FL+MG组(3.75±0.72;0.373±0.008).上述差异均具有统计学意义,P均<0.01.结论 FL可部分抑制MG诱导的海马神经元内ROS水平的上升,同时激活BDNF-TrkB信号通路,减少细胞凋亡,发挥神经保护作用.

abstractsObjective To investigate the protective effects of fluoxetine (FL) on hippocampal neurons damaged by methylglyoxal (MG). Methods Primary cultured of hippocampal neurons from 1-day-old Sprague-Dawley rat were incubated with MG and/or fluoxetine for 24 h respectively. The apoptosis was quantified by flow cytometer using annexin V-FITC and propidium iodide (PI) staining. The level of intracellular reactive oxygen species (ROS) was measured by an oxidant sensitive dye 2,7-dichorofluoresin diacetate (DCFH). The protein and mRNA levels of BDNF and TrkB were assayed with Western Blotting and real-time reverse-transcription polymerase chain reaction (PCR) respectively. Results After incubated the cells with 100 μmol/L MG for 24 h, the ratio of apoptotic cells in MG group (8.83 ±0.31)% significantly increased compared with the control group ( 1.63 ± 0. 15 ) % and MG + FL group ( 3.20 ±0. 30)% respectively. The level of intracellular oxidation of MG group (10 229 ± 946) also significantly increased in comparison with the FL group ( 3076 ± 41 ), control group (4265 ± 82 ), MG + FL group(6058 ±179) and pre FL+MG group (6076 ±281). The levels of BDNF mRNA and protein in the MG group [(2.37±0.33), (0.625 ±0.008)] were higher than the control group[( 1.00 ±0.27), (0.582 ±0. 003)], but lower than the FL group [(3.88 ±0.32), (0. 855 ±0. 007)], MG + FL group [(7.66 ±0. 34), ( 1.113 ± 0. 023) 1 and pre FL + MG group [(6.96± 0.54), (0.689 ± 0.014)]. The MG group showed declined levels of TrkB mRNA and protein [(0.50 ±0.06), (0. 133 ±0. 006)], compared to the control group [( 1.00 ± 0.06), ( 0. 328 ± 0. 000)], FL group [(5.45± 0.42), (0. 460 ± 0. 005 )],MG+ FL group [(4.21 ±0.32), (0.414 ±0.006)] and pre FL+MG group [(3.75 ±0.72), (0.373 ±0. 008 )]. All these differences were statistically significant (P <0.01 ). Conclusion EL has neuroprotective effect against MG induced neurotoxicity in hippocampal neurons which possible via its anti-apoptosis activity by activating BDNF/TrkB signal pathway, and partly by its antioxidant effect.