缺血性脑卒中患者外周血CBX7的表达水平变化及其通过Nrf2/HO-1通路对神经元凋亡的影响

The expression level changes of CBX7in peripheral blood of patients with ischemic stroke and its effect on neuronal apoptosis through Nrf2/HO-1pathway

目的探讨Polycomb chromobox7(CBX7)对氧糖剥夺/再灌注(Oxygen-glucose deprivation/reperfusion,OGD/R)神经元的影响及其可能的作用机制。方法收集缺血性脑卒中(Ischemic stroke,IS)组患者和体检健康人群(对照组)外周血,分离外周血单个核细胞;大鼠海马神经元OGD/R处理后细胞转染CBX7特异性siRNA(OGD/R+si-CBX7组)及其阴性对照(OGD/R+NC组)。此外,设置Zinc Protoporphyrin(ZnPP)干扰组(OGD/R+si-CBX7+ZnPP组)及对照组。逆转录-聚合酶链反应(Reverse transcription-polymerase chain reaction,RT-PCR)检测细胞中CBX7 mRNA表达水平;Western blot检测细胞中CBX7,Bcl-2关联X的蛋白(Bcl-2 associated X protein,Bax)、B淋巴细胞瘤-2(B-cell lymphoma-2,Bcl-2)和核因子E2相关因子2(Nuclear factor E2-related factor 2,Nrf2)/血红素加氧酶-1(Heme oxygenase-1,HO-1)通路相关蛋白的表达水平;CCK-8试剂盒检测细胞活力;流式细胞术检测细胞凋亡。结果与对照组比较,IS组外周血CBX7 mRNA和蛋白表达水平明显升高(P<0.05)。与对照组比较,OGD/R组细胞中CBX7 mRNA和蛋白表达水平均明显升高(P<0.05),细胞凋亡率和Bax蛋白表达水平明显升高(P<0.05),细胞增殖能力(24、48和72 h)、Bcl-2,Nrf2和HO-1蛋白表达水平明显降低(P<0.05);与OGD/R+NC组比较,OGD/R+si-CBX7组细胞中CBX7 mRNA和蛋白表达水平明显降低(P<0.05),细胞凋亡率和Bax蛋白表达水平明显降低(P<0.05),细胞增殖能力(48和72 h)、Bcl-2,Nrf2和HO-1蛋白表达水平明显升高(P<0.05);与OGD/R+si-CBX7组比较,OGD/R+si-CBX7+ZnPP组细胞增殖能力(24、48和72 h)明显降低(P<0.05),细胞凋亡率和Bax蛋白表达水平明显升高(P<0.05),Bcl-2,Nrf2和HO-1蛋白表达水平明显降低(P<0.05)。结论敲低CBX7可能是通过激活Nrf2/HO-1通路来抑制OGD/R神经元凋亡。

Objective To study the effect of CBX7 on oxygen-glucose deprivation/reperfusion(OGD/R) in neurons and its possible mechanism.Methods Peripheral blood samples were collected from patients with ischemic stroke(IS group) and healthy people(control group), and then peripheral blood mononuclear cells were separated. After OGD/R treatment, rat hippocampal neurons were transfected with CBX7 specific siRNA(OGD/R+si-CBX7 group) and its negative control(OGD/R+NC group). In addition, a ZnPP interference group(OGD/R+si-CBX7+ZnPP group) and a control group were included. RT-PCR was used to detect the mRNA expression of CBX7 in cells. Western blot was used to detect the expressions of CBX7, Bax, Bcl-2 and Nrf2/HO-1 pathway related proteins in cells. CCK-8 kit was used to detect cell viability. Flow cytometry was used to detect apoptosis.Results Compared with the control group, the levels of of CBX7 mRNA and protein in peripheral blood of the IS group were significantly increased(P<0.05). However, in OGD/R group, the levels of CBX7 mRNA and protein, the apoptosis rate and Bax protein expression were significantly increased(P<0.05) while cell proliferation ability(24 h, 48 h and 72 h), the expressions of Bcl-2, Nrf2 and HO-1 protein were significantly reduced(P<0.05) compared with control group. Compared with OGD/R+NC group, the levels of CBX7 mRNA and protein expression, cell apoptosis rate and Bax protein expression were significantly reduced(P<0.05) in OGD/R+si-CBX7 group, while cell proliferation ability(48 h and 72 h) and the levels of Bcl-2, Nrf2 and HO-1 proteins were significantly increased(P<0.05). Compared with the OGD/R+si-CBX7 group, the cell proliferation ability(24 h, 48 h and 72 h) and the expressions of Bcl-2, Nrf2 and HO-1 in OGD/R+si-CBX7+ZnPP group were significantly decreased(P<0.05), while the apoptosis rate and the expression of Bax protein were significantly increased(P<0.05).Conclusion Knocking down of CBX7 inhibits OGD/R-induced neuronal apoptosis by activating Nrf2/HO-1 pathway.