长链非编码RNAβ分泌酶1反义转录物在七氟醚诱发老年大鼠认知功能下降中的作用

Role and mechanism of long non-coding RNA β secretase 1 antisense transcription in the cognitive dysfunction induced by sevoflurane in rats

目的探讨长链非编码RNA(lncRNA)β分泌酶1反义转录物(BACE1-AS)在七氟醚诱发老年大鼠认知功能下降中的作用。方法SPF级健康雄性SD老年大鼠64只,18月龄,体重480~580 g。采用随机数字表法分为四组:对照组(C组)、七氟醚组(S组)、七氟醚+阴性对照质粒组(SN组)和七氟醚+BACE1-AS抑制剂(siRNA-BACE1-AS)组(ST组),每组16只。SN组和ST组分别侧脑室注射阴性对照质粒和siRNA-BACE1-AS,C组和S组注射等体积PBS溶液,注射后1 d C组吸入30%氧气6 h,S组、SN组和ST组吸入3.6%七氟醚与30%氧气的混合气体6 h。气体吸入后1 d,采用水迷宫实验测实验第1~5天逃避潜伏期和实验第6天穿越平台次数。水迷宫实验结束后1 h内处死大鼠,取海马组织,采用ELISA法检测海马组织TNF-α、IL-1β、丙二醛(MDA)和超氧化物歧化酶(SOD)浓度,可见分光光度计法检测海马组织caspase-3活性,Western blot法检测海马组织Bax、Bcl-2和BACE1蛋白含量,RT-qPCR法检测海马组织lncRNA BACE1-AS、miR-124和BACE1 mRNA表达量,TUNEL法测定海马组织神经细胞凋亡百分比,苏木素伊红染色法观察海马组织病理结构。结果与C组比较,S组、SN组和ST组水迷宫实验第3~5天逃避潜伏期明显延长(P<0.05),实验第6天穿越平台次数明显减少(P<0.05),海马组织TNF-α、IL-1β和MDA浓度、BACE1和Bax蛋白含量、lncRNA BACE1-AS和BACE1 mRNA表达量、凋亡细胞百分比明显升高(P<0.05),SOD浓度、Bcl-2蛋白含量、miR-124 mRNA表达量明显降低(P<0.05),caspase-3活性明显增强(P<0.05)。与S组比较,ST组水迷宫实验第3~5天逃避潜伏期明显缩短(P<0.05),实验第6天穿越平台次数明显增多(P<0.05),海马组织TNF-α、IL-1β和MDA浓度、BACE1和Bax蛋白含量、lncRNA BACE1-AS和BACE1 mRNA表达量、凋亡细胞百分比明显降低(P<0.05),SOD浓度、Bcl-2蛋白含量、miR-124 mRNA表达量明显升高(P<0.05),caspase-3活性明显减弱(P<0.05)。S组和SN组上述指标差异均无统计学意义。结论长链非编码RNAβ分泌酶1反义转录物参与七氟醚诱导老年大鼠认知功能下降,其机制与神经细胞炎症、氧化应激损伤、调控miR-124和BACE1表达和促进细胞凋亡有关。

Objective To investigate the role and possible mechanism of long non-coding RNA(lncRNA)βsecretase 1 antisense transcription(BACE1-AS)in sevoflurane induced cognitive dysfunction in aged rats.Methods Sixty-four healthy male SPF Sprague-Dawley rats,aged 18 months,weighing 480-580 g,were randomly divided into the control group(group C),sevoflurane group(group S),sevoflurane+negative control group(group SN)and sevoflurane+BACE1-AS inhibitor group(group ST),16 rats in each group.Rats in the groups SN and ST were injected with negative control plasmid and siRNA-BACE1-AS in the lateral ventricle,and groups C and S were treated with PBS solution in equal volume.After 1 day,group C was inhaled 30%O 2 for 6 hours and groups S,SN and ST were inhaled 3.6%sevoflurane+30%O 2 for 6 hours.After 1day,the Morris water maze experiment was used to measure the escape incubation period from day 1 to 5 and the times of crossing the platform on day 6.The rats were sacrificed within 1 hour after the water maze experiment,and the hippocampal tissue was collected.The concentrations of TNF-α,IL-1β,malondialdehyde(MDA)and superoxide dismutase(SOD)in the hippocampus were detected by ELISA,the caspase-3 activity of hippocampus was detected by visible spectrophotometry,the protein contents of Bax,Bcl-2 and BACE1 in hippocampus were detected by Western blot,the expression of lncRNA BACE1-AS,miR-124 and BACE1 mRNA were detected by RT-qPCR,the percentage of apoptotic nerve cells in hippocampus was measured by TUNEL,and the pathological structure of hippocampus was observed by hematoxylin eosin staining.Results Compared with group C,the water maze experiment escape incubation periods in day 3 to 5 in groups S,SN and ST were prolonged(P<0.05),the times of crossing the platform in the day 6 was decreased(P<0.05),the concentrations of TNF-α,IL-1βand MDA in hippocampus,the protein contents of BACE1 and Bax,the expression of lncRNA BACE1-AS and BACE1 mRNA and apoptosis rate was significantly increased(P<0.05),the SOD concentration,the Bcl-2 protein content,the mRNA expression of miR-124 were decreased,the activity of caspase-3 was reduced significantly(P<0.05).Compared with group S,the water maze experiment escape incubation periods in day 3 to 5 in group ST were shortened(P<0.05),the times of crossing the platform in the day 6 was increased(P<0.05),the concentrations of TNF-α,IL-1βand MDA in hippocampus,the protein contents of BACE1 and Bax,the expression of lncRNA BACE1-AS and BACE1 mRNA and apoptosis rate was significantly decreased(P<0.05),the SOD concentration,the Bcl-2 protein content,the mRNA expression of miR-124 were increased(P<0.05),the activity of caspase-3 was increased significantly(P<0.05).There was no statistical significance in the above indexes between groups S and SN.Conclusion LncRNAβsecretase 1 antisense transcriptase is involved in sevoflurane induced cognitive impairment in aged rats,and its mechanism may be related to the neuronal inflammation,oxidative stress injury,regulation of miR-124 and BACE1 expression and promotion of cell apoptosis.