miR–138–5p/FOXO3信号轴在七氟醚麻醉诱导的老龄小鼠术后谵妄中的作用及分子机制

秦君; 王海萍; 师秋茹

miR–138–5p/FOXO3信号轴在七氟醚麻醉诱导的老龄小鼠术后谵妄中的作用及分子机制

The role and molecular mechanism of miR-138-5p/FOXO3 signaling axis in sevoflurane anesthesia-induced postoperative delirium in aged mice

摘要

摘要:
目的： 探讨miR–138–5p/FOXO3信号轴在七氟醚（Sev）麻醉诱导的老龄小鼠术后谵妄（POD）中的作用及分子机制。
方法： 将70只18月龄SPF级C57BL/6J雄性小鼠分为7组：对照组（对照组不进行任何操作）、手术组（1%戊巴比妥麻醉后实施剖腹探查术）、Sev组（4%七氟醚诱导麻醉2 min后，通过3%七氟醚维持麻醉4 h，实施剖腹探查术）、Sev + NC antagomir组（术前侧脑室注射NC antagomir，七氟醚维持麻醉4 h，实施剖腹探查术）、Sev + miR–138–5p antagomir组（术前侧脑室注射miR–138–5p antagomir，七氟醚维持麻醉4 h，实施剖腹探查术）、Sev + miR–138–5p antagomir + sh–NC组（术前侧脑室注射miR–138–5p antagomir和sh–NC，七氟醚维持麻醉4 h，实施剖腹探查术）、Sev + miR–138–5p antagomir + sh–FOXO3组（术前侧脑室注射miR–138–5p antagomir和sh–FOXO3，七氟醚维持麻醉4 h，实施剖腹探查术），每组10只。采用旷场实验与觅食实验评估谵妄样行为。通过TUNEL染色观察海马神经元凋亡水平，Western blotting检测Bax、Bcl–2、PSD–95、BDNF及FOXO3的蛋白表达，RT–qPCR分析miR–138–5p与FOXO3的mRNA表达。使用试剂盒测定海马神经元炎症因子与氧化应激指标，并利用双荧光素酶报告实验验证miR–138–5p与FOXO3之间的靶向关系。
结果： 与对照组和手术组比较，Sev组小鼠进入中心区域潜伏期、静止时间、进食潜伏期、TUNEL阳性细胞比、Bax、miR–138–5p表达水平、IL–6、IL–1β、TNF–α和MDA水平增加（P ＜ 0.05），在中心区域停留的时间、Bcl–2、PSD–95、BDNF和FOXO3表达水平、SOD水平均降低（P ＜ 0.05）；与Sev组和Sev + NC antagomir组比较，Sev + miR–138–5p antagomir组小鼠进入中心区域潜伏期、静止时间、进食潜伏期、TUNEL阳性细胞比、Bax、miR–138–5p表达水平、IL–6、IL–1β、TNF–α和MDA水平降低（P ＜ 0.05），在中心区域停留的时间、Bcl–2、PSD–95、BDNF和FOXO3表达水平、SOD水平增加（P ＜ 0.05）；与Sev + miR–138–5p antagomir组和Sev + miR–138–5p antagomir + sh–NC组比较，Sev + miR–138–5p antagomir + sh–FOXO3组小鼠进入中心区域潜伏期、静止时间、进食潜伏期、TUNEL阳性细胞比、Bax、miR–138–5p表达水平、IL–6、IL–1β、TNF–α和MDA水平增加（P ＜ 0.05），在中心区域停留的时间、Bcl–2、PSD–95、BDNF和FOXO3表达水平、SOD水平降低（P ＜ 0.05）。双荧光素酶报告实验显示，与NC mimic组比较，miR–138–5p mimic组WT–FOXO3荧光素酶活性降低（P ＜ 0.05），而MUT–FOXO3组活性变化无统计学意义（P ＞ 0.05）。
结论： 在七氟醚麻醉诱导的老龄小鼠POD中，miR–138–5p表达上调，可通过靶向抑制FOXO3，促进神经元凋亡、炎症反应和氧化应激，并引起突触功能障碍，从而诱导术后谵妄的发生。

Abstract:
Objective To investigate the role and molecular mechanism of the miR-138-5p/FOXO3 signaling axis in sevoflurane (Sev) anesthesia-induced postoperative delirium (POD) in aged mice.
Methods Seventy 18-month-old SPF-grade male C57BL/6J mice were divided into seven groups (n = 10 per group): Control group (no procedure), Surgery group (exploratory laparotomy under 1% pentobarbital anesthesia), Sev group (exploratory laparotomy under anesthesia induced with 4% sevoflurane for 2 min and maintained with 3% sevoflurane for 4 h), Sev + NC antagomir group (intracerebroventricular injection of NC antagomir prior to anesthesia and surgery as in Sev group), Sev + miR-138-5p antagomir group (intracerebroventricular injection of miR-138-5p antagomir prior to anesthesia and surgery as in Sev group), Sev + miR-138-5p antagomir + sh-NC group (intracerebroventricular injection of miR-138-5p antagomir and sh-NC prior to anesthesia and surgery as in Sev group), Sev + miR-138-5p antagomir + sh-FOXO3 group (intracerebroventricular injection of miR-138-5p antagomir and sh-FOXO3 prior to anesthesia and surgery as in Sev group). Delirium-like behavior was assessed using the open field test and buried food test. Hippocampal neuronal apoptosis was assessed by TUNEL staining. Western blotting was performed to determine the protein expression levels of Bax, Bcl-2, PSD-95, BDNF, and FOXO3. RT-qPCR was used to measure miR-138-5p expression and FOXO3 mRNA expression. Levels of inflammatory cytokines and oxidative stress markers in hippocampal tissue were determined using commercial kits. The targeting relationship between miR-138-5p and FOXO3 was verified by dual-luciferase reporter assay.
Results Compared with the Control and Surgery groups, the Sev group showed significant increases in latency to enter the center zone, immobility time, feeding latency, percentage of TUNEL-positive cells, expression levels of Bax and miR-138-5p, and levels of IL-6, IL-1β, TNF-α, and MDA (P ＜ 0.05), while significant decreases were observed in the time spent in the center zone, expression levels of Bcl-2, PSD-95, BDNF, and FOXO3, and SOD levels (P ＜ 0.05). Compared with the Sev and Sev + NC antagomir groups, the Sev + miR-138-5p antagomir group exhibited significant reductions in latency to enter the center zone, immobility time, feeding latency, percentage of TUNEL-positive cells, Bax and miR-138-5p expression, and levels of IL-6, IL-1β, TNF-α, and MDA (P ＜ 0.05), alongside significant increases in time spent in the center zone, expression of Bcl-2, PSD-95, BDNF, and FOXO3, and SOD levels (P ＜ 0.05). Compared with the Sev + miR-138-5p antagomir and Sev + miR-138-5p antagomir + sh-NC groups, the Sev + miR-138-5p antagomir + sh-FOXO3 group demonstrated significant increases in latency to enter the center zone, immobility time, feeding latency, percentage of TUNEL-positive cells, expression of Bax and miR-138-5p, and levels of IL-6, IL-1β, TNF-α, and MDA (P ＜ 0.05), and significant decreases in time spent in the center zone, expression of Bcl-2, PSD-95, BDNF, and FOXO3, and SOD levels (P ＜ 0.05). The dual-luciferase reporter assay showed that, compared with the NC mimic group, the miR-138-5p mimic group had significantly reduced luciferase activity of WT-FOXO3 (P ＜ 0.05), while the activity of MUT-FOXO3 remained unchanged (P ＞ 0.05).
Conclusions In sevoflurane anesthesia-induced POD in aged mice, upregulated miR-138-5p expression may promote neuronal apoptosis, inflammatory responses, and oxidative stress and induce synaptic dysfunction by targeting and suppressing FOXO3, thereby contributing to the development of postoperative delirium.

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