海马小胶质细胞NF-κB/NLRP3信号通路在右美托咪定改善新生小鼠多次七氟烷麻醉后远期认知功能中的作用
Role of NF-κB/NLRP3 signaling pathway in hippocampal microglia in dexmedetomidine-induced im-provement in long-term cognitive function after multiple sevoflurane anesthesia in newborn mice
朱春花 1牛志强 1王本清 1于健1
作者信息
- 1. 沧州市中心医院麻醉科,沧州 061001
- 折叠
摘要
目的 评价海马小胶质细胞NF-KB/NOD样受体热蛋白结构域相关蛋白3(NLRP3)信号通路在右美托咪定改善新生小鼠多次七氟烷麻醉后远期认知功能中的作用.方法 SPF级健康雄性C57BL/6小鼠100只,6日龄,体质量3～5 g,采用随机数字表法分为5组(n=20):对照组(C组)、多次七氟烷麻醉组(S组)、多次七氟烷麻醉+NF-κB抑制剂吡咯烷二硫代甲酸铵盐组(SP组)、多次七氟烷麻醉+右美托咪定(SD组)和多次七氟烷麻醉+NF-κB激动剂佛波酯+右美托咪定(SPD组).七氟烷麻醉方法:吸入3％七氟烷进行麻醉诱导,持续吸入60％氧气及3％七氟烷2 h进行麻醉维持,连续3 d.每次吸入七氟烷前30 min时,SP组腹腔注射吡咯烷二硫代甲酸铵盐100 mg/kg,SD组腹腔注射右美托咪定20 μg/kg.SPD组每次吸入七氟烷前1 h腹腔注射佛波酯15 μg/kg,30 min后腹腔注射右美托咪定20 μg/kg.小鼠于11日龄时处死,分离海马组织,采用Western blot法测定磷酸化NF-κB p65(p-NF-κB p65)和NLRP3的表达,采用免疫荧光法计算NLRP3与小胶质细胞特异性离子钙结合衔接分子1(Iba-1)双阳性细胞(NLRP3+-Iba-1+细胞)共染面积.于小鼠29日龄时行旷场实验和新物体识別实验,于30～34日龄时行水迷宫实验.结果 5组小鼠旷场实验各指标比较差异无统计学意义(P＞0.05).与C组比较,S组海马组织p-NF-κB p65和NLRP3表达上调,NLRP3+-Iba-1+细胞共染面积增加,新物体探索百分比和辨别指数降低,逃避潜伏期延长,穿越原平台位置次数减少(P＜0.05);与S组比较,SP组、SD组和SPD组海马组织p-NF-κB p65和NLRP3表达下调,NLRP3+-Iba-1+细胞共染面积减少,新物体探索百分比和辨别指数升高,逃避潜伏期缩短,穿越原平台位置次数增加(P＜0.05);与SD组比较,SPD组海马组织p-NF-KB p65和NLRP3表达上调,NLRP3+-Iba-1+细胞共染面积增加,新物体探索百分比和辨别指数降低,逃避潜伏期延长,穿越原平台位置次数减少(P＜0.05).结论 右美托咪定改善新生小鼠多次七氟烷麻醉后远期认知功能的机制,可能与抑制海马小胶质细胞NF-κB/NLRP3信号通路激活有关.
Abstract
Objective To evaluate the role of nuclear factor kappa B(NF-κB)/NOD-like receptor thermoprotein structural domain-associated protein 3(NLRP3)signaling pathway in hippocampal microglia in dexmedetomidine-induced improvement in long-term cognitive function after multiple sevoflurane anesthe-sia in newborn mice.Methods One hundred SPF healthy male C57BL/6 mice,aged 6 days,weighing 3-5 g,were divided into 5 groups(n=20 each)by a random number table method:control group(group C),multiple sevoflurane anesthesia group(group S),multiple sevoflurane anesthesia+NF-KB inhibitor pyrroli-dine dithiocarboxylate group(group SP),multiple sevoflurane anesthesia+dexmedetomidine group(group SD),and multiple sevoflurane anesthesia+NF-κB agonist phorbol 12-myristate 13-acetate(PMA)+dexmedetomidine group(group SPD).Anesthesia was induced by inhalation of 3％sevoflurane and main-tained by inhalation of 60％oxygen and 3％sevoflurane for 2 h for 3 consecutive days in S,SP,SD and SPD groups.At 30 min before each sevoflurane inhalation,pyrrolidine dithiocarbamate 100 mg/kg was intra-peritoneally injected in group SP,and dexmedetomidine 20 μg/kg was intraperitoneally injected in group SD.NF-κB agonist PMA 15 μg/kg was intraperitoneally injected at 1 h before each sevoflurane inhalation,and 30 min later dexmedetomidine 20 μg/kg was intraperitoneally injected in group SPD.The mice were sac-rificed at the age of 11 days,and the hippocampal tissues were isolated for determination of the expression of phosphorylated NF-κB p65(p-NF-κB p65)and NLRP3(by Western blot).The co-staining area of NLRP3 and microglia-specific ionized calcium binding adaptor molecule 1(Iba-1)double positive cells(NLRP3+-Iba-1+cells)was calculated by immunofluorescence.The open field test and novel object recognition test were performed at 29 days of age,and the Morris water maze test was performed at 30-34 days of age.Re-sults There were no significant differences in each parameter of the open field test among the five groups(P＞0.05).Compared with group C,the expression of p-NF-κB p65 and NLRP3 in hippocampal tissues was significantly up-regulated,the co-staining area of NLRP3+-Iba-1+cells was increased,the percentage of no-vel object exploration and discrimination index were decreased,the escape latency was prolonged,and the frequency of crossing the original platform was reduced in group S(P＜0.05).Compared with group S,the expression of p-NF-κB p65 and NLRP3 in hippocampal tissues was significantly down-regulated,the co-stai-ning area of NLRP3+-Iba-1+cells was reduced,the percentage of novel object exploration and discrimination index were increased,the escape latency was shortened,and the frequency of crossing the original platform was increased in SP,SD and SPD groups(P＜0.05).Compared with SD group,the expression of p-NF-κB p65 and NLRP3 was significantly up-regulated,the co-staining area of NLRP3+-Iba-1+cells was increased,the percentage of novel object exploration and discrimination index were decreased,the escape latency was prolonged,and the number of crossing the original platform location was decreased in SPD group(P＜0.05).Conclusions The mechanism by which dexmedetomidine improves long-term cognitive function after multi-ple sevoflurane anesthesia may be related to inhibiting the activation of NF-κB/NLRP3 signaling pathway in the hippocampal microglia of newborn mice.
关键词
麻醉药,吸入/婴儿,新生/NF-κB/NLR家族,热蛋白结构域包含蛋白3/认知障碍Key words
Anesthetics,inhalation/Infant,newborn/NF-kappaB/NLR family,pyrindom-ain-containing 3 protein/Cognition disorders引用本文复制引用
出版年
2024
NETL
NSTL
万方数据