3K3A-APC通过抑制p53信号抑制蛛网膜下腔出血后神经元凋亡

3K3A-APC inhibits neuron apoptosis after subarachnoid hemorrhage by inhibiting p53 signaling

目的观察3K3A-APC对蛛网膜下腔出血后神经元凋亡的影响。方法原代培养SD大鼠子鼠的海马神经元细胞,接种于24孔板中,采用随机数字表法分为3组,每组8个孔:3K3A-APC组、3K3A-APC+Kevetrin hydrochloride组和空白对照组。3K3A-APC组每孔加入1.0 mg/ml的3K3A-APC,3K3A-APC+Kevetrin hydrochloride组每孔加入1.0 mg/ml的3K3A-APC和85μmol/L Kevetrin hydrochloride,空白对照组常规培养基换液,流式细胞术检测神经元细胞的凋亡率;实时定量聚合酶链反应(Real-time PCR)检测凋亡相关因子p53、半胱氨酰天冬氨酸特异性蛋白酶(Caspase)-3、B细胞淋巴瘤/白血病-2(bcl-2)的表达;采用t检验。结果通过流式细胞术检测神经元细胞的凋亡率,3K3A-APC组的凋亡率[(45.11±1.45)%]低于3K3A-APC+Kevetrin hydrochloride组[(71.99±1.70)%],差异有统计学意义(t=-41.66,P<0.05);3K3A-APC组的凋亡率[(45.11±1.45)%]低于对照组[(71.61±1.51)%],差异有统计学意义(t=-43.77,P<0.05);3K3A-APC+Kevetrin hydrochloride组的凋亡率[(71.99±1.70)%]与对照组[(71.61±1.51)%]差异无统计学意义(P>0.05);通过Real-time PCR技术检测细胞中凋亡相关因子表达,3K3A-APC组p53表达量(3.53±0.12)低于3K3A-APC+Kevetrin hydrochloride组(8.43±0.15)和对照组(8.47±0.18),差异有统计学意义(t=-87.51、-77.72,P<0.05);3K3A-APC+Kevetrin hydrochloride组p53表达量(8.43±0.15)与对照组(8.47±0.18)比较,差异无统计学意义(P>0.05);3K3A-APC组Caspase-3表达量(2.22±0.13)低于3K3A-APC+Kevetrin hydrochloride组(6.30±0.12)和对照组(6.26±0.08),差异有统计学意义(t=-79.53、-89.21,P<0.05);3K3A-APC+Kevetrin hydrochloride组Caspase-3表达量(6.30±0.12)与对照组(6.26±0.08)比较,差异无统计学意义(P>0.05);APC+Kevetrin hydrochloride组bcl-2的表达量(3.08±0.15)低于3K3A-APC组(7.22±0.13),差异有统计学意义(t=-72.03,P<0.05);对照组bcl-2的表达量(3.07±0.12)低于3K3A-APC组(7.22±0.13),差异有统计学意义(t=-83.17,P<0.05);3K3A-APC+Kevetrin hydrochloride组bcl-2表达量(3.08±0.15)与对照组(3.07±0.12)比较,差异无统计学意义(P>0.05)。结论3K3A-APC能够通过抑制p53信号抑制神经元凋亡。

Objective To observe the effect of 3K3A-APC on neuronal apoptosis after subarachnoid hemorrhage.Methods The hippocampal neuron cells of primary cultured fetal SD rats were inoculated in 24-well plates and divided into 3 groups with 8 holes in each group by the random number table:3K3A-APC group(1.0 mg/ml 3K3A-APC was added in each well),3K3A-APC+Kevetrin hydrochloride group(1.0 mg/ml 3K3A-APC and 85μmol/L Kevetrin hydrochloride were added in each well)and blank control group(routine culture medium was added in each well).The apoptosis rate of neuron cells was detected by flow cytometry.The expression levels of apoptosis-related factors p53,cysteinyl aspartate-specific protease(Caspase)-3 and B cell lymphoma/leukemia-2(bcl-2)were detected by real-time quantitative polymerase chain reaction(Real-time PCR).SPSS25.0 statistical software was used.The quantitative data were expressed as mean±standard deviation(±s).The t test was applied.Results The apoptosis rate of neurons in the 3K3A-APC group[(45.11±1.45)%]was significantly lower than that in the 3K3A-APC+Kevetrin hydrochloride group[(71.99±1.70)%](t=-41.66,P<0.05)and the control group[(71.61±1.51)%](t=-43.77,P<0.05).There was no significant difference in the apoptosis rate between the 3K3A-APC+Kevetrin hydrochloride group and the control group(P>0.05].The p53 expression level in the 3K3A-APC group(3.53±0.12)was significantly lower than that in the 3K3A-APC+Kevetrin hydrochloride group(8.43±0.15)and the control group(8.47±0.18,t=-87.51,-77.72,P<0.05),but there was no significant difference between the 3K3A-APC+Kevetrin hydrochloride group and the control group(8.47±0.18,P>0.05).The expression of Caspase-3 in the 3K3A-APC group(2.22±0.13)was significantly lower than that in the 3K3A-APC+Kevetrin hydrochloride group(6.30±0.12)and the control group(6.26±0.08,t=-79.53,-89.21,P<0.05),but there was no significant difference between the 3K3A-APC+Kevetrin hydrochloride group and the control group(6.26±0.08,P>0.05).The expression of bcl-2 in the APC+Kevetrin hydrochloride group(3.08±0.15)was significantly lower than that in the 3K3A-APC group(7.22±0.13,t=-72.03,P<0.05),and that in the control group(3.07±0.12)was significantly lower than that in the 3K3A-APC group(t=-83.17,P<0.05),but there was no significant difference between the 3K3A-APC+Kevetrin hydrochloride group and the control group(P>0.05).Conclusion 3K3A-APC can inhibit neuronal apoptosis by inhibiting p53 signal.