摘要
目的评价长链非编码RNA NORAD在氯胺酮诱发小鼠海马神经元毒性中的作用及其与内质网应激的关系。方法分离并培养原代小鼠海马神经元,采用随机数字表法分为5组(n=36):对照组(C组)、氯胺酮组(K组)、氯胺酮+pcDNA3.1-NORAD质粒组(K+NORAD组)、氯胺酮+对照质粒组(K+NC组)和氯胺酮+NORAD+衣霉素组(K+NORAD+TM组)。C组使用正常培养基培养24 h;K组加入40μmol/L氯胺酮孵育24 h;K+NORAD组先转染pcDNA3.1-NORAD质粒至神经元中,48 h后加入氯胺酮40μmol/L孵育24 h;K+NC组先转染pcDNA3.1(+)质粒至神经元中,48 h后加入氯胺酮40μmol/L孵育24 h;K+NORAD+TM组先转染pcDNA3.1-NORAD质粒,24 h后加入内质网应激激活剂衣霉素1μg/ml孵育24 h,然后再加入氯胺酮40μmol/L孵育24 h。采用CCK-8法检测神经元活力,检测乳酸脱氢酶(LDH)释放量,采用TUNEL法和流式细胞术检测细胞凋亡情况,Real-time PCR法测定NORAD表达,Western blot法检测蛋白激酶R样内质网激酶(PERK)、磷酸化PERK(p-PERK)和C/EBP同源蛋白(CHOP)的表达。结果与C组相比,K组神经元活力降低,LDH释放量增加,凋亡神经元百分比和细胞凋亡率升高,NORAD表达下调,CHOP表达上调,p-PERK/PERK升高(P<0.05);与K组相比,K+NORAD组神经元活力升高,LDH释放量减少,凋亡神经元百分比和细胞凋亡率降低,NORAD表达上调,CHOP表达下调,p-PERK/PERK降低(P<0.05),K+NC组上述指标差异无统计学意义(P>0.05);与K+NORAD组相比,K+NORAD+TM组神经元活力降低,LDH释放量增加,凋亡神经元百分比和细胞凋亡率升高,CHOP表达上调,p-PERK/PERK升高(P<0.05),NORAD表达差异无统计学意义(P>0.05)。结论过表达NORAD可通过抑制内质网应激,减轻氯胺酮诱发的小鼠海马神经元毒性。
Objective To evaluate the role of long non-coding RNA(lncRNA)NORAD in ketamine-induced neurotoxicity in mouse hippocampal neurons and the relationship with endoplasmic reticulum stress.Methods Primary mouse hippocampal neurons were isolated and cultured and then divided into 5 groups(n=36 each)using a random number table method:control group(group C),ketamine group(group K),ketamine+pcDNA3.1-NORAD plasmid group(group K+NORAD),ketamine+control plasmid group(group K+NC),and ketamine+NORAD+tunicamycin group(group K+NORAD+TM).Group C was cultured with normal medium for 24 h.Group K was cultured with 40μmol/L ketamine for 24 h.Group K+NORAD was transfected with pcDNA3.1-NORAD overexpressing plasmid for 48 h,followed by treatment with 40μmol/L ketamine for 24 h.Group K+NC was transfected with pcDNA3.1(+)plasmid for 48 h,followed by treatment with 40μmol/L ketamine for 24 h.Group K+NORAD+TM was transfected with pcDNA3.1-NORAD overexpressing plasmid,24 h later endoplasmic reticulum stress activator tunicamycin 1μg/ml was added and the neurons were cultured for 24 h,and then ketamine 40μmol/L was added and the neurons were cultured for another 24 h.Cell viability was detected by CCK-8 assay.The amount of lactate dehydrogenase(LDH)released was analyzed.Cell apoptosis was determined by TUNEL and flow cytometry methods.The NORAD expression was detected by real-time polymerase chain reaction.The expression of endoplasmic reticulum stress-related proteins protein kinase R-like ER kinase(PERK),phosphorylated PERK(p-PERK)and C/EBP homologous protein(CHOP)was detected by Western blot.Results Compared with group C,the cell viability was significantly decreased,the amount of LDH released,percentage of apoptotic neurons and apoptosis rate were increased,NORAD expression was down-regulated,CHOP expression was up-regulated,and p-PERK/PERK was increased in group K(P<0.05).Compared with group K,the cell viability was significantly increased,the amount of LDH released,percentage of apoptotic neurons and apoptosis rate were decreased,NORAD expression was up-regulated,CHOP expression was down-regulated,and p-PERK/PERK was decreased in group K+NORAD(P<0.05),and no significant change was found in the parameters mentioned above in group K+NC(P>0.05).Compared with group K+NORAD,the cell viability was significantly decreased,the amount of LDH released,percentage of apoptotic neurons and apoptosis rate were increased,CHOP expression was up-regulated,and p-PERK/PERK was increased(P<0.05),and no significant change was found in the NORAD expression in group K+NORAD+TM(P>0.05).Conclusions Over-expressed NORAD can alleviate ketamine-induced neurotoxicity in mouse hippocampal neurons via inhibition of the endoplasmic reticulum stress.
作者
张琳
陈文超
王静瑞
Zhang Lin;Chen Wenchao;Wang Jingrui(Department of Anesthesiology and Perioperative Medicine,Henan Provincial People′s Hospital,Zhengzhou University People′s Hospital,Henan University People′s Hospital,Zhengzhou 450003,China;Department of Gastrointestinal Surgery,Henan Provincial People′s Hospital,Zhengzhou University People′s Hospital,Henan University People′s Hospital,Zhengzhou 450003,China)
出处
《中华麻醉学杂志》
CAS
CSCD
北大核心
2023年第7期814-818,共5页
Chinese Journal of Anesthesiology
基金
河南省医学科技攻关联合共建项目(LHGJ20220065,LHGJ20220021)。
