电针调控 NLRP3/Caspase-1 信号通路对老年大鼠术后认知功能障碍的影响

Effects of electroacupuncture-mediated regulation of the NLRP3/Caspase-1 signaling pathway on postoperative cognitive dysfunction in aged rats

摘要

目的 探讨电针（EA）百会、合谷穴对老年大鼠术后认知功能障碍（POCD）的影响，并阐明其是否通过调控 NOD 样受体热蛋白结构域相关蛋白 3（NLRP3）/天冬氨酸蛋白水解酶 1（Caspase-1）信号通路发挥作用。方法 采用随机数表法将 40 只 20 月龄（相当于人类 60 岁老年阶段）SD 大鼠分为对照组（C 组）、电针组（E 组）、手术组（S 组）、电针+手术组（ES 组），每组 10 只。采用脾切除术联合腹腔探查术建立老年大鼠 POCD 模型。E 组与 ES 组选取百会穴（GV20）和合谷穴（LI4）进行电针干预，采用 2 Hz/15 Hz 疏密波型，1 mA 电流，30 min/次，1 次/d，连续 5 d。通过 Morris 水迷宫、Y 迷宫和新物体识别实验评估大鼠的认知功能，采用苏木精-伊红（HE）染色、Nissl 染色、dUTP 原位切口末端标记（TUNEL）染色观察海马病理变化，采用免疫荧光检测海马组织中 NLRP3 的表达，采用蛋白免疫印迹法检测 NLRP3 炎症小体相关蛋白 NLRP3、Caspase-1、凋亡相关斑点样蛋白（ASC），炎症因子白细胞介素-1β（IL-1β）、IL-18 以及抗凋亡蛋白 B 细胞淋巴瘤-2 基因（Bcl-2）的表达变化，采用实时荧光定量逆转录聚合酶链反应（RT-qPCR）检测海马中 IL-1β、IL-18 的 mRNA 表达水平。结果 手术与电针的交互效应有统计学意义。简单效应分析表明，在无电针条件下，相较于 C 组，S 组大鼠逃避潜伏期延长［（27.00±4.21）s vs. （47.68±9.00）s］，穿越平台次数［（6.20±1.64）次 vs. （0.80±0.84）次］、自发交替率［（66.10±9.94）% vs. （41.43±7.34）%］和新物体识别指数［（72.31±6.25）% vs. （46.36±8.68）%］均减少，海马神经元结构损伤，海马 CA1 区神经元数量[（61.67±17.39）个/高倍视野 vs.（20.67±4.51）个/高倍视野]减少，凋亡指数（4.65±1.09 vs. 49.38±6.85）增大，海马组织中 NLRP3 累积荧光强度（0.25±0.05 vs. 0.59±0.03）增强，NLRP3（0.80±0.05 vs. 1.12±0.04）、Caspase-1（0.59±0.03 vs. 1.23±0.03）、 ASC（0.67±0.02 vs. 1.24±0.02）、 IL-1β（0.68±0.04 vs. 1.12±0.08）及 IL-18（0.41±0.04 vs. 1.15±0.07）表达均升高，Bcl-2 表达（ 0.88±0.10 vs. 0.27±0.03）降低（均 P ＜ 0.05）。在有手术条件下，相较于 S 组，ES 组大鼠认知功能改善，海马神经元结构损伤减轻，海马 CA1 区神经元数量［（20.67±4.51）个/高倍视野 vs.（56.00±6.08）个/高倍视野］增加，凋亡指数（49.38±6.85 vs. 10.87±1.18）减少，海马组织中 NLRP3 累积荧光强度（0.59±0.03 vs. 0.32±0.05）减弱， NLRP3（1.12±0.04 vs. 0.84±0.03）、 Caspase-1（1.23±0.03 vs. 0.69±0.02）、 ASC（1.24±0.02 vs. 1.04±0.04）、 IL-1β（1.12±0.08 vs. 0.81±0.02）及 IL-18（1.15±0.07 vs. 0.78±0.05）表达均降低，Bcl-2 表达（0.27±0.03 vs. 0.84±0.03）升高（均 P ＜ 0.05）。结论 电针百会、合谷穴能够改善老年 POCD 大鼠认知功能，其作用与抑制 NLRP3/Caspase-1 信号通路、减轻海马神经炎症反应及神经元凋亡相关。

Abstract

Objective To investigate the effects of electroacupuncture (EA) at Baihui (GV20) and Hegu (LI4) on postoperative cognitive dysfunction (POCD) in aged rats, and to clarify whether these effects are exerted through regulation of the NOD-like receptor pyrin domain-containing protein 3 (NLRP3)/cysteine-aspartic protease-1 (Caspase-1) signaling pathway. Methods Forty 20-month-old SD rats, equivalent to approximately 60 years of age in humans, were divided by the random number table method into the control group (C group), electroacupuncture group (E group), surgery group (S group), and electroacupuncture plus surgery group (ES group), with 10 rats in each group. A POCD model in aged rats was established by splenectomy combined with exploratory laparotomy. Rats in the E and ES groups received electroacupuncture intervention at Baihui (GV20) and Hegu (LI4), using a 2 Hz/15 Hz disperse-dense wave, 1 mA current, 30 min per session, once daily for 5 consecutive days. Cognitive function was evaluated using the Morris water maze, Y-maze, and novel object recognition tests. Pathological changes in the hippocampus were observed by hematoxylin-eosin (HE) staining, Nissl staining, and dUTP nick-end labeling (TUNEL) staining. Immunofluorescence was used to detect the expression of NLRP3 in hippocampal tissue. Western blotting was used to detect changes in the expression of NLRP3 inflammasome-related proteins, including NLRP3, Caspase-1, and apoptosis-associated speck-like protein (ASC), the inflammatory factors IL-1β and IL-18, and the anti-apoptotic protein B-cell lymphoma/leukemia-2 (Bcl-2). Real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) was used to detect the mRNA expression levels of IL-1β and IL-18 in the hippocampus. Results The interaction effect between surgery and electroacupuncture was significant. Further simple effect analysis showed that, in the absence of electroacupuncture, compared with the C group, rats in the S group had a prolonged escape latency [(27.00±4.21) s vs. (47.68±9.00) s], reduced number of platform crossings [(6.20±1.64) times vs. (0.80±0.84) times], reduced spontaneous alternation rate [(66.10±9.94)% vs. (41.43±7.34)%], and reduced novel object recognition index [(72.31±6.25)% vs. (46.36±8.68)%]. Hippocampal neuronal structural damage was observed, the number of neurons in the hippocampal CA1 region decreased [(61.67±17.39) cells/high-power field vs. (20.67±4.51) cells/high-power field], the apoptosis index increased (4.65±1.09 vs. 49.38±6.85), and the integrated fluorescence intensity of NLRP3 in hippocampal tissue increased (0.25±0.05 vs. 0.59±0.03). The expression levels of NLRP3 (0.80±0.05 vs. 1.12±0.04), Caspase-1 (0.59±0.03 vs. 1.23±0.03), ASC (0.67±0.02 vs. 1.24±0.02), IL-1β (0.68±0.04 vs. 1.12±0.08), and IL-18 (0.41±0.04 vs. 1.15±0.07) were all increased, while Bcl-2 expression decreased (0.88±0.10 vs. 0.27±0.03) (all P ＜ 0.05). Under surgical conditions, compared with the S group, rats in the ES group showed improved cognitive function, reduced hippocampal neuronal structural damage, increased number of neurons in the hippocampal CA1 region [(20.67±4.51) cells/high-power field vs. (56.00±6.08) cells/high-power field], decreased apoptosis index (49.38±6.85 vs. 10.87±1.18), and reduced integrated fluorescence intensity of NLRP3 in hippocampal tissue (0.59±0.03 vs. 0.32±0.05). The expression levels of NLRP3 (1.12±0.04 vs. 0.84±0.03), Caspase-1 (1.23±0.03 vs. 0.69±0.02), ASC (1.24±0.02 vs. 1.04±0.04), IL-1β (1.12±0.08 vs. 0.81±0.02), and IL-18 (1.15±0.07 vs. 0.78±0.05) were all decreased, while Bcl-2 expression increased (0.27±0.03 vs. 0.84±0.03) (all P ＜ 0.05). Conclusions Electroacupuncture at Baihui and Hegu can improve cognitive function in aged rats with POCD. Its effects are associated with inhibition of the NLRP3/Caspase-1 signaling pathway and attenuation of hippocampal neuroinflammatory responses and neuronal apoptosis.

关键词

电针 / NLRP3 / Caspase-1 / 老年大鼠 / 术后认知功能障碍

Key words

Electroacupuncture / NLRP3 / Caspase-1 / Aged rats / Postoperative cognitive dysfunction

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基金资助

广西自然科学基金项目(2025GXNSFAA069393)

广西壮族自治区中医药管理局项目(GXZYZ20210352)

广西中医药大学研究生教育创新计划项目(YCSY2025087)