从PI3K/Akt和MAPK/Erk信号通路探讨复方当归注射液对拟缺血神经元的作用

Exploration of the mechanism of Compound Angelica Injection on mimicischemic neurons from PI3K/Akt and MAPK/Erk signal pathways

[目的]探讨复方当归注射液(CAI)干预拟缺血损伤脑微血管内皮细胞(BMECs)后的条件培养液对拟缺血损伤神经元的影响及作用机制。[方法]原代培养SD大鼠BMECs及皮层神经元,免疫荧光鉴定两种细胞,收集正常BMECs条件培养液(N-CM)、拟缺血损伤BMECs条件培养液(I-CM)及CAI低、中、高剂量干预后的拟缺血损伤BMECs条件培养液(IC-CM,1.25、2.5、5μL/mL),分别作用于正常神经元和拟缺血损伤神经元,用CCK8法检测各组神经元活性、蛋白免疫印迹(Western Blot)法检测磷脂酰肌醇-3激酶(PI3K)/丝氨酸/苏氨酸激酶(Akt)信号通路Akt的磷酸化水平[磷酸化Akt(p-Akt)/Akt]、丝裂原活化蛋白激酶(MAPK)/细胞外信号调节激酶(Erk)信号通路Erk的磷酸化水平[磷酸化Erk(p-Erk)/Erk]。[结果]与N-CM处理组相比,I-CM处理组神经元活性显著降低(P<0.01)、Akt磷酸化水平下降(P<0.05)、Erk磷酸化水平显著上升(P<0.01);CAI干预后IC-CM低剂量处理组神经元活性、Akt磷酸化水平高于I-CM处理组(P<0.05),IC-CM中、高剂量处理组神经元活性、Akt磷酸化水平显著高于I-CM处理组(P<0.01),各IC-CM处理组较I-CM处理组的Erk磷酸化水平变化不明显(P>0.05)。[结论]CAI可通过干预拟缺血损伤内皮细胞进而对拟缺血损伤神经元达到保护作用,该保护作用可能与活化PI3K/Akt信号通路存在相关性,与MAPK/Erk信号通路关联不大。

[Objective]To investigate the effect and mechanism of Compound Angelica Injection(CAI)on conditioned medium after mimic ischemic brain microvascular endothelial cells(BMECs)on mimic ischemic injured neurons.[Methods]Primitive culture SD rats’BMECs and cortex neuron were identifiedby immunofluorescence.Normal BMECs conditioned medium(N-CM),mimic ischemic injury BMECs conditioned medium(I-CM)andmimic ischemic injury BMECs with different doses of CAI intervention conditioned medium(IC-CM,L=1.25μL/mL,M=2.5μL/mL,H=5μL/mL)were collected,which applied to normal neurons and mimic ischemic neurons respectively.The activity of neurons in each group was detected by CCK-8 method and the phosphorylation level of Akt in PI3K/Akt signaling pathway(p-Akt/Akt)and Erk in MAPK/Erk signaling pathway(p-Erk/Erk)were detected by western blotting.[Results]Compared with N-CM treatment group,the neuronal activity of I-CM treatment group was significantly decreased(P<0.01).Akt phosphorylation level was decreased(P<0.05),Erk phosphorylation level increased significantly(P<0.01).After CAI intervention,the neuronal activity and Akt phosphorylation level in IC-CML group were higher than that in I-CM group(P<0.05).The activity of neurons and the phosphorylation level of Akt in the IC-CML and IC-CMH groups were significantly higher than those in the I-CM group(P<0.01).The phosphorylation level of Erk in each IC-CM group was not significantly different from that in the I-CM group(P>0.05).[Conclusion]CAI can protect the neurons of mimic ischemic injury by interfering with the endothelial cells of ischemic injury.The protective effect may be correlated with the activation of PI3K/Akt signaling pathway,but has little correlation with MAPK/Erk signaling pathway.