E2F1 siRNA通过抑制血红素诱导的神经元凋亡发挥保护作用

E2F1 siRNA attenuates hemin-induced primary cortical neuronal injuries

目的探讨E2F转录因子1(E2F1)在血红素诱导的大鼠原代皮层神经元损伤中的作用及机制。方法体外培养大鼠原代皮层神经元并鉴定,采用不同浓度血红素处理神经元,12 h后LDH实验及MTT实验检测细胞损伤;免疫荧光双染观察神经元E2F1定位;Western blot检测神经元E2F1表达;将神经元分为溶剂组(DMSO组)、损伤组(Hemin组)、阴性对照组(Hemin+Control siRNA组)、治疗组(Hemin+E2F1siRNA组)进行TUNEL染色及caspase活性检测。结果神经元培养至第7天,NeuN染色阳性率达95%;血红素(50、100、200μmol/L)处理后神经元LDH释放率增加、细胞生存率降低、E2F1表达上调(F=22.9,P<0.05);与阴性对照组相比,治疗组神经元LDH释放率降低([(73.6±5.2)%vs(42±4.1)%,F=343.4,P<0.05]、细胞生存率升高[(28.7±3.4)%vs(61±2.9)%,F=127.7,P<0.05];TUNEL结果显示血红素诱导神经元凋亡增加,治疗组凋亡减少(F=67.2,P<0.05);caspase活性检测显示血红素损伤组神经元caspase-3、caspase-8、caspase-9活性升高(t=15.7,t=9.7,t=7.8,P<0.05),治疗组caspase-3、caspase-8、caspase-9活性降低(t=8.6,t=7.3,t=5.7,P<0.05)。结论 E2F1 siRNA可能通过抑制血红素诱导的神经元凋亡而发挥神经保护作用。

Objective To investigate the role of E2 F transcription factor 1(E2 F1)in hemin-induced primary cortical neuron injuries and the possible mechanisms in rats.Methods Rat primary cortical neurons were cultured,identified and treated with different concentrations of hemin in vitro.LDH and MTT assays were performed to detect cell damage when neurons were treated for 12 h.The location and the expression of E2 F1 in neurons were detected by immunofluorescence double staining and Western blot respectively.Rats were divided into vehicle group(DMSO),injury group(Hemin),negative control group(Hemin+Control siRNA)and treatment group(Hemin+E2 F1 siRNA).TUNEL staining and caspase activity were performed in four groups.Results The positive rate of NeuN staining was 95%on the 7 d.With the treatment of hemin(50,100,200μmol/L),the LDH release of neurons increased,cell viability decreased and E2 F1 expression was up-regulated(F=22.9,P<0.05).Compared with the negative control group,LDH release decreased[(73.6±5.2)%vs(42±4.1)%,F=343.4,P<0.05]and the cell viability increased[(28.7±3.4)%vs(61±2.9)%,F=127.7,P<0.05]in the E2 F1 siRNA-treated group.The TUNEL results showed that hemin treatments increased neuronal apoptosis,and E2 F1 siRNA treatments reduced the apoptosis(F=67.2,P<0.05).The caspase activity assay showed that the activities of caspase-3,caspase-8 and caspase-9 in the hemin group increased(t=15.7,t=9.7,t=7.8,P<0.05),but E2 F1 siRNA treatment decreased the activities of caspase-3,caspase-8 and caspase-9(t=8.6,t=7.3,t=5.7,P<0.05).Conclusion E2 F1 siRNA may play a neuroprotective role by inhibiting hemin-induced neuronal apoptosis.