MicroRNA-34a regulates the CaMKⅡ/CREB pathway to improve isoflurane anesthesia-induced cognitive dysfunction in aged rats
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摘要:目的
探讨微小RNA(miR)-34a对异氟烷麻醉引起的老年大鼠认知功能障碍的影响及其作用机制。
方法将40只20月龄大鼠随机分为正常(Con)组、模型(Model)组、miR-34a抑制剂(miR-34a inhibitor)组和miR-34a激动剂(miR-34a mimics)组，每组10只。miR-34a inhibitor组和miR-34a mimics组大鼠尾静脉注射100 nmol/kg对应药物, Con组和Model组大鼠尾静脉注射等剂量生理盐水, 1次/d, 连续5 d。第6天，除正常组外，其余组大鼠均进行单次异氟烷麻醉6 h, 构建术后认知功能障碍(POCD)模型。建模完成后12 h, 采用Morris水迷宫实验观察大鼠逃避潜伏期和目标象限停留时间。采用免疫荧光染色观察大鼠海马组织中离子钙结合衔接分子1(Iba-1)阳性表达率。采用实时荧光定量聚合酶链反应(qRT-PCR)检测大鼠海马组织中miR-34a以及B细胞淋巴瘤-2(Bcl-2)和Bcl-2相关X蛋白(Bax)的mRNA相对表达量。采用酶联免疫吸附实验(ELISA)检测大鼠血清中白细胞介素-6(IL-6)、白细胞介素-1β(IL-1β)、活性氧(ROS)和谷胱甘肽过氧化物酶(GSH-Px)水平以及海马组织中谷氨酸(Glu)、Ca2+和N-甲基-D-天冬氨酸受体2B(NMDAR2B)含量。采用蛋白质印迹(Western blotting)检测大鼠海马组织中钙-钙调蛋白依赖性蛋白激酶Ⅱ(CaMKⅡ)、pCaMKⅡ、环磷腺苷效应元件结合蛋白(CREB)、磷酸化CREB(pCREB)蛋白相对表达量。
结果与Con组比较, Model组大鼠逃避潜伏期、血清中IL-6、IL-1β、ROS水平以及海马组织中Iba-1阳性表达率、miR-34a、Bax mRNA相对表达水平和Glu、Ca2+、NMDAR2B含量延长或升高, 目标象限停留时间、血清中GSH-Px水平、海马组织中Bcl-2 mRNA相对表达水平、Bcl-2/Bax以及pCaMKⅡ/CaMKⅡ和PCREB/CREB缩短或降低，差异有统计学意义(P < 0.05)。下调miR-34a表达可缩短模型大鼠逃避潜伏期，以及降低血清中IL-6、IL-1β、ROS水平和海马组织中Iba-1阳性表达率、miR-34a、Bax mRNA相对表达水平和Glu、Ca2+、NMDAR2B含量(P < 0.05), 还可延长目标象限停留时间，以及血清中GSH-Px水平、海马组织中Bcl-2 mRNA相对表达水平、Bcl-2/Bax以及pCaMKⅡ/CaMKⅡ和PCREB/CREB(P < 0.05)。上调miR-34a表达可通过促进小胶质细胞的异常激活以及炎症、氧化应激和凋亡，抑制CaMKⅡ/CREB信号通路活化，加重模型大鼠认知障碍。
结论miR-34a在POCD老年大鼠中高表达，抑制miR-34a表达可通过激活CaMKⅡ/CREB信号通路改善老年大鼠因异氟烷麻醉引起的认知功能障碍。
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关键词:
- 微小RNA-34a /
- 异氟烷 /
- 认知功能障碍 /
- 炎症 /
- 钙-钙调蛋白依赖性蛋白激酶Ⅱ /
- 环磷腺苷效应元件结合蛋白
Abstract:ObjectiveTo investigate the effects and underlying mechanisms of microRNA (miR)-34a on isoflurane anesthesia-induced cognitive dysfunction in aged rats.
MethodsForty rats at 20 months of age were randomly divided into control (Con) group, model group, miR-34a inhibitor group and miR-34a mimics group, with 10 rats in each group. Rats in the miR-34a inhibitor and miR-34a mimics groups received a tail vein injection of 100 nmoL/kg of the corresponding drug, while those in the Con and model groups received an equal volume of saline, once daily for 5 consecutive days. At the 6th day, all groups except the Con group underwent a single 6-hour isoflurane anesthesia to establish a postoperative cognitive dysfunction (POCD) model. Twelve hours after modeling, the Morris water maze test was used to assess the escape latency and time spent in the target quadrant. Immunofluorescence staining was performed to observe the positive expression rate of ionized calcium-binding adapter molecule 1 (Iba-1) in the hippocampal tissue. Real-time quantitative polymerase chain reaction (qRT-PCR) was used to measure the relative expression levels of miR-34a, B-cell lymphoma-2 (Bcl-2) and Bcl-2-associated X-protein (Bax) mRNA in the hippocampal tissue. Enzyme-linked immunosorbent assay (ELISA) was conducted to detect serum levels of interleukin-6 (IL-6), interleukin-1β (IL-1β), reactive oxygen species (ROS) and glutathione peroxidase (GSH-Px), as well as the content of glutamate (Glu), Ca2+ and N-methyl-D-aspartate receptor 2B (NMDAR2B) in the hippocampal tissue. Western blotting was used to determine the relative expression levels of calcium-calmodulin-dependent protein kinase Ⅱ (CaMKⅡ), phosphorylated CaMKⅡ (pCaMKⅡ), cyclophosphoadenosine effector-binding protein (CREB), and phosphorylated CREB (pCREB) in the hippocampal tissue.
ResultsCompared with the Con group, the Model group exhibited significantly prolonged escape latency, elevated serum levels of IL-6 and IL-1β and ROS, as well as increased positive expression rate of Iba-1, relative expression levels of miR-34a and Bax mRNA, contents of Glu, Ca2+ and NMDAR2B in the hippocampal tissue; in contrast, the time spent in the target quadrant, serum GSH-Px levels, Bcl-2 mRNA relative expression levels, Bcl-2/Bax, as well as pCaMKⅡ/CaMKⅡ and pCREB/CREB in the hippocampal tissue were significantly reduced (P < 0.05). Downregulation of miR-34a expression shortened the escape latency and decreased serum levels of IL-6, IL-1β and ROS, as well as Iba-1 positive expression rate, relative expression levels of miR-34a and Bax mRNA, Glu, Ca2+ and NMDAR2B content in the hippocampal tissue (P < 0.05). It also extended the time spent in the target quadrant and increased serum GSH-Px levels, Bcl-2 mRNA expression levels, Bcl-2/Bax, as well as pCaMKⅡ/CaMKⅡ and pCREB/CREB in the hippocampal tissue (P < 0.05). Upregulation of miR-34a expression promoted abnormal activation of microglia, inflammation, oxidative stress and apoptosis, inhibited the activation of the CaMKⅡ/CREB signaling pathway, and exacerbated cognitive dysfunction in the model rats.
ConclusionMiR-34a is highly expressed in POCD in aged rats. Inhibition of miR-34a expression can improve isoflurane anesthesia-induced cognitive dysfunction in aged rats by activating the CaMKⅡ/CREB signaling pathway.
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图 1 各组大鼠海马CA1区Iba-1阳性表达情况(免疫荧光，放大200倍)
绿色为Iba-1阳性表达。与Con组比较, *P＜0.05; 与Model组比较, #P＜0.05; 与miR-34a inhibitor组比较, △P＜0.05。
表 1 各组大鼠血气分析指标及血糖比较(x±s)(n=10)
组别 pH值 pa(O2)/mmHg pa(CO2)/mmHg 血糖/(mmol/L) Con组 7.38±0.05 119.30±8.99 39.33±4.01 5.12±0.53 Model组 7.42±0.08 118.45±11.04 41.77±6.28 4.89±0.60 miR-34a inhibitor组 7.40±0.07 120.68±8.32 39.90±4.57 5.00±0.45 miR-34a组mimics 7.41±0.08 117.97±10.11 40.80±5.63 4.97±0.58 pa(O2): 动脉血氧分压; pa(CO2): 动脉血二氧化碳分压。 
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表 2 各组大鼠Morris水迷宫实验结果(x±s)(n=10)
s 组别 逃避潜伏期 目标象限停留时间 Con组 22.70±3.12 46.31±8.10 Model组 39.45±4.19* 28.57±5.34* miR-34a inhibitor组 30.02±2.96# 41.16±7.20# miR-34a mimics组 45.24±4.60#△ 23.87±4.51#△ 与Con组比较, * P＜0.05; 与Model组比较, #P＜0.05;
与miR-34a inhibitor组比较, △P＜0.05。
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表 3 各组大鼠海马组织中miR-34a及Bcl-2和Bax mRNA的相对表达水平比较(x±s)(n=10)
组别 miR-34a Bcl-2 mRNA Bax mRNA Bcl-2/Bax Con组 1.00±0.00 1.00±0.00 1.00±0.01 1.00±0.02 Model组 1.87±0.10* 0.40±0.03* 1.73±0.12* 0.23±0.09* miR-34a inhibitor组 0.26±0.04# 0.87±0.15# 1.24±0.11# 0.70±0.11# miR-34a mimics组 7.35±0.51#△ 0.31±0.05#△ 2.34±0.20#△ 0.13±0.10#△ 与Con组比较, * P＜0.05; 与Model组比较, #P＜0.05; 与miR-34a inhibitor组比较, △P＜0.05。
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表 4 各组大鼠血清中IL-6、IL-1β、ROS、GSH-Px水平比较(x±s)(n=10)
组别 IL-6/(ng/L) IL-1β/(ng/L) ROS/(ng/L) GSH-Px/(U/mL) Con组 9.20±0.62 3.38±0.24 44.68±3.35 76.59±11.21 Model组 14.93±1.05* 7.25±0.36* 81.52±5.17* 39.96±5.73* miR-34a inhibitor组 10.80±0.78# 4.81±0.35# 54.43±4.22# 62.87±8.08# miR-34a mimics组 19.97±1.33#△ 9.11±0.44#△ 96.64±5.60#△ 30.05±4.32#△ IL-6: 白细胞介素-6; IL-1β: 白细胞介素-1β; ROS: 活性氧; GSH-Px: 谷胱甘肽过氧化物酶。
与Con组比较, * P＜0.05; 与Model组比较, #P＜0.05; 与miR-34a inhibitor组比较, △P＜0.05。
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表 5 各组大鼠海马组织中Glu、Ca2+和NMDAR2B含量以及CaMKⅡ/CREB信号通路相关蛋白表达量比较(x±s)(n=10)
组别 Glu/(μg/g pro) Ca2+/(μmoL/g pro) NMDAR2B/(ng/g pro) pCaMKⅡ/CaMKⅡ PCREB/CREB Con组 483.94±14.62 50.06±9.88 14.62±6.49 0.93±0.07 0.89±0.08 Model组 630.91±21.04* 81.43±16.91* 27.97±9.23* 0.41±0.03* 0.43±0.04* miR-34a inhibitor组 527.85±17.27# 62.34±12.06# 19.78±7.14# 0.80±0.06# 0.77±0.06# miR-34a mimics组 750.18±26.56#△ 149.99±18.67#△ 39.90±10.06#△ 0.19±0.02#△ 0.22±0.04#△ Glu: 谷氨酸; NMDAR2B: N-甲基-D-天冬氨酸受体2B; CaMKⅡ: 钙-钙调蛋白依赖性蛋白激酶Ⅱ;
CREB: 环磷腺苷效应元件结合蛋白。与Con组比较, * P＜0.05; 与Model组比较, #P＜0.05; 与miR-34a inhibitor组比较, △P＜0.05。
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