血管紧张素1-7对氧糖剥夺神经胶质瘤细胞U373的保护作用

Protection effects of angiotensin 1-7 on glioma cell line U373 after oxygen and sugar deprivation

目的研究血管紧张素1-7(Ang1-7)对氧糖剥夺神经细胞凋亡的影响。方法用厌氧培养箱模拟缺氧环境,用EBSS(Na Cl 116,KCl 5.36,Mg SO40.81,Na H2PO41.0,hydroxyethyl piperazine ethanesulfonic acid(HEPES)10,Ca Cl21.8,Na HCO314.7,单位均mmol·L^(-1))培养基制造缺糖环境,对培养的U373细胞制作氧糖剥夺模型。实验分4组:正常组未经氧糖剥夺(OGD)处理,模型组以OGD处理3 h;单用实验组和联用实验组以OGD处理的同时,分别加入终浓度为1×10-7mol·L^(-1)血管紧张素1-7处理、终浓度为1×10-5mol·L^(-1)肽类激素(A779)和1×10-7mol·L^(-1)Ang1-7联合处理。用显微镜观察细胞形态,以噻唑蓝(MTT)法研究细胞活力,以Annexin V-FITC法结合流式细胞术研究细胞凋亡情况。结果与模型组相比,单用实验组改善了氧糖剥夺后U373细胞的形态,突触细长,联用实验组的细胞损伤比较严重,其形态接近模型组。正常组细胞的活力设定为1.00,则模型组细胞活力为0.45,组间比较差异有统计学意义(P<0.01)。单用实验组的细胞活力为0.70,与模型组相比,组间比较差异有统计学意义(P<0.05)。在联用实验组中,由于加入了A779,使得细胞活力降低为0.51,接近模型组水平,说明A779破坏了Ang1-7的保护作用,Ang1-7的保护作用是通过Mas受体介导的。正常组的细胞凋亡率为43.01%,模型组的细胞凋亡率为83.24%,组间比较差异有统计学意义(P<0.01)。与模型组相比,单用实验组的细胞凋亡率为58.46%,组间比较差异有统计学意义(P<0.05),说明Ang1-7对OGD后的U373细胞起到保护作用。联用实验组的细胞凋亡率为76.78%,接近模型组水平,说明A779能够拮抗Ang1-7的保护作用。结论 Ang1-7通过Mas受体减轻细胞凋亡,对氧糖剥夺的U373细胞有保护作用。

Objective To investigate the protection function of angiotensin 1-7（Ang1-7） on oxygen and sugar deprivation in glioma cell line U373.Methods Oxygen and sugar deprivation model was made by culturing U373 cells in EBSS（Na Cl 116,KCl 5.36,Mg SO40.81,Na H2PO41.0,hydroxyethyl piperazine ethanesulfonic acid（HEPES） 10,Ca Cl21.8,Na HCO314.7,unit of all mmol·L^（-1）） medium without sugar and in anaerobic incubator.Experiment were divided into four groups as follow：Normal group：without oxygen and glucose deprivation（OGD） treatment;Model group：OGD treatment for 3 h;Single-test group：a final concentration of 1 × 10-7mol·L^（-1）Ang1-7 was added at the same time of OGD;United-test group：a final concentration of 1 × 10-5mol·L^（-1）A779（Mas receptor antagonist,a peptide hormone consisting of seven amino acids with the last one amino acid different from Ang1-7） and 1 × 10-7mol·L^（-1）Ang1-7 were added at the same time of OGD.Microscope was used to observe cell morphology.MTT method was used to study the cell vitality.Flow cytometry was used to study cell apoptosis.Results Compared with model group,U373 cell morphology in single-test group was improved with longer synaptic.Cells in united-test group were seriously damaged,with cell morphology close to that in model group.Cell viability in normal group were set as 1.00.Relative cell viability in model group was 0.45,which was statistically significant different from that in normal group（P〈0.01）.Relative cell viability in single-test group was 0.70,which was statistically significant different from that in model group（P〈0.05）.Because of the addition of A779,relative cell viability in united-test group was 0.51,which was close to model group and show A779 destroy the protective effect of Ang1-7 mediated through Mas receptor.Cell apoptosis rate in model group were 83.24%,which was statistically significant different from that in normal group with the apoptosis rate of 43.01%（P〈0.01）.Cell apoptosis rate in single-test group was 58.46%,which was statistically significant different from that in model group（P〈0.05） and show the protective effect of Ang1-7 on OGD U373.Cell apoptosis rate in united-test group was 76.78%,which was close to that in model group and show A779 could damage the protective effect of Ang1-7.Conclusion Ang1-7 have protective effects through Mas receptor on U373 cells after oxygen and sugar deprivation by reducing apoptosis.