PRMT6抑制剂调控Bnip3选择性剪切改善氧糖剥夺/再灌注诱导的神经元细胞凋亡  

PRMT6 inhibitor attenuated oxygen-glucose deprivation/reoxygenation-induced neuronal apoptosis through mediating the alternative splicing of Bnip3

作　　者：东方玥 罗士元 李银娇 罗艳[2] DONGFANG Yue;LUO Shi-Yuan;LI Yin-Jiao(School of Anesthesiology,Weifang Medical University,Weifang 261053,Shandong,China)

机构地区：[1]潍坊医学院麻醉学院,山东潍坊261053 [2]上海交通大学医学院附属瑞金医院麻醉科

出　　处：《中国老年学杂志》2024年第14期3458-3464,共7页Chinese Journal of Gerontology

基　　金：国家自然科学基金面上项目(81871101);上海市科学技术委员会“扬帆计划”项目(19YF1430600)。

摘　　要：目的 观察蛋白精氨酸甲基转移酶(PRMT)6抑制剂EPZ020411对氧糖剥夺/再灌注(OGD/R)诱导的小鼠海马神经元细胞系HT22细胞凋亡的作用,并探讨其可能的机制。方法 体外培养HT22神经元,构建OGD/R诱导的HT22细胞损伤模型。利用PRMT6抑制剂EPZ020411抑制PRMT6酶活性,将对数期生长的HT22神经元随机分为对照组、EPZ组、OGD/R组、OGD/R+EPZ组,CCK-8法检测细胞存活率,乳酸脱氢酶(LDH)法检测细胞毒性,Western印迹检测凋亡相关蛋白Cleaved-胱天蛋白酶(Caspase)3表达,JC-1染色检测线粒体膜电位改变,活性氧检测氧化应激水平,实时荧光定量聚合酶链反应(qRT-PCR)检测Bnip3不同剪切异构体水平。结果 OGD/R处理后,HT22神经元细胞活率显著降低,细胞毒性显著加重(P<0.05);同时,Cleaved-Caspase3显著升高(P<0.05),活性氧水平升高,线粒体膜电位下降。EPZ020411改善OGD/R诱导的HT22细胞损伤,抑制Cleaved-Caspase3蛋白表达,提高线粒体膜电位水平并减少活性氧生成。EPZ020411显著升高Bnip3剪切异构体Bnip3ΔExon3表达水平(P<0.05)。结论 PRMT6抑制剂EPZ020411改善OGD/R诱导的HT22神经元损伤,其机制可能是通过调控Bnip3 mRNA前体的选择性剪切过程,上调Bnip3剪切异构体Bnip3ΔExon3表达水平,抑制氧化应激,从而减轻OGD/R诱导的细胞凋亡。Objective To investigate the effects of the protein arginine methyltransferase(PRMT)6 inhibitor EPZ020411 on oxygen-glucose deprivation/reoxygenation(OGD/R)-induced apoptosis in the mouse hippocampal neuronal cell line HT22,and to ex-plore its possible mechanisms.Methods HT22 neurons were cultured in vitro,and an OGD/R-induced cell injury model was estab-lished.PRMT6 enzyme activity was inhibited using the PRMT6 inhibitor EPZ020411.Logarithmically growing HT22 neurons were ran-domly divided into control group,EPZ group,OGD/Rgroup and OGD/R+EPZ group.Cell viability was assessed using the CCK-8 as-say,while cytotoxicity was evaluated by measuring lactate dehydrogenase(LDH)release.Western blotting was used to detect the ex-pression of the apoptosis-related protein Cleaved-caspase3.Changes in mitochondrial membrane potential were assessed by JC-1 stai-ning,and oxidative stress levels were measured by detecting reactive oxygen species.Real-time polymerase chain reaction(qRT-PCR)was employed to quantify the levels of different Bnip3 splice isoforms.Results After OGD/Rtreatment,the survival rate of HT22 neurons was significantly decreased,and the cytotoxicity was increased(P<0.05).At the same time,Cleaved-Caspase3 level was sig-nificantly increased,reactive oxygen species level was increased and mitochondrial membrane potential was decreased.Treatment with EPZ020411 attenuated OGD/R-induced HT22 cell damage,Cleaved-Caspase 3 protein expression was suppressed,mitochondrial mem-brane potential level and reduced reactive oxygen species generation were enhanced.EPZ020411 significantly increased the expression level of the Bnip3 splice isoform Bnip3ΔExon3(P<0.05).Conclusions The PRMT6 inhibitor EPZ020411 could ameliorate OGD/R-induced HT22 neuronal damage,its mechanism potentially through the modulation of alternative splicing of the Bnip3 mRNA precur-sor,leading to upregulated expression level of the Bnip3ΔExon3 splice isoform,inhibiting oxidative stress and ultimately alleviating OGD/R-induced apoptosis.

关 键 词：蛋白精氨酸甲基转移酶(PRMT)6 神经元 氧糖剥夺/再灌注 选择性剪切 细胞凋亡 

分 类 号：R743.3[医药卫生—神经病学与精神病学]