电针对术后认知功能障碍模型大鼠炎症反应和铁死亡影响的研究

• 作者： 秦晓宇 ^1,2,3,4 张斌森 ^1,3,4 张笑佳 ^1,3,4 逯晓婷 ¹ 刘鸿鑫 ⁵ 王春爱 ⁶
• 作者单位：
  1.  730030 甘肃省兰州市，甘肃中医药大学第一临床医学院
  2.  200120 上海市，同济大学附属东方医院麻醉科
  3.  730030 甘肃省兰州市，甘肃省中医方药挖掘与创新转化重点实验室；4.
  4.  730030 甘肃省兰州市，甘肃省中药新产品创制工程实验室
  5.  730030 甘肃省兰州市，甘肃中医药大学针灸推拿学院
  6.  730050 甘肃省兰州市，甘肃省中医院麻醉疼痛医学中心
  7.  730050 甘肃省兰州市，甘肃省中西医结合麻醉临床医学研究中心
• 通讯作者： 王春爱 
• 提交时间：2023-09-22

摘要: 背景　术后认知功能障碍（POCD）是增加患者术后发病率和死亡率的重要原因之一。炎症反应和铁死亡是 POCD 发生的重要机制假说，而电针改善 POCD 患者学习和记忆功能机制不清。目的　观察电针对 POCD 老年大鼠学习记忆及炎性细胞因子和海马神经元铁死亡的影响，探讨电针改善 POCD 的作用机制。方法　2022 年 1 月2023 年 2 月选取 18~20 月龄 SD 大鼠 72 只，按照随机数字表法分为 3 组：对照组（n=24）、模型组（n=24）和电针组（n=24）。根据术后 3、7 d 两个观察时间点将每组大鼠分为 2 个亚组（对照组术后 3 d 亚组、对照组术后 7 d 亚组、模型组术后3 d 亚组、模型组术后 7 d 亚组、电针组术后 3 d 亚组、电针组术后 7 d 亚组），每组 12 只。采用剖腹探查手术建立POCD 模型，选取电针组大鼠百会和内关穴进行电针刺激。采用 Morris 水迷宫装置检测大鼠行为学表现，酶联免疫吸附试验检测血清、海马中白介素（IL）6、IL-10、肿瘤坏死因子 （TNF-）的含量，检测海马组织脂质过氧化物（LPO）、Fe2+，免疫印迹法检测海马酰基辅酶 A 合成酶长链家族成员 4（ACSL4）、铁蛋白重链 1（FTH1）、血磷脂酰胆碱酰基转移酶 3（LPCAT3）蛋白表达水平。采用透射电镜观察海马区神经细胞超微结构。结果　术后 3 d、7 d 大鼠组别与时间对大鼠术前认知功能训练逃避潜伏期均不存在交互作用（P 交互 >0.05），训练时间对逃避潜伏期主效应均显著（P 时间0.05）。模型组术后 3 d 亚组逃避潜伏期高于对照组术后 3 d 亚组、电针组术后 3 d 亚组，穿越平台次数、目标象限停留时间低于对照组术后 3 d 亚组、电针组术后 3 d 亚组，电针组术后3 d 亚组穿越平台次数低于对照组术后 3 d 亚组（P<0.05）。模型组术后 7 d 亚组逃避潜伏期高于对照组术后 7 d 亚组、电针组术后 7 d 亚组，穿越平台次数低于对照组术后 7 d 亚组，目标象限停留时间低于对照组术后 7 d 亚组、电针组术后 7 d 亚组（P<0.05）。模型组术后 3 d 亚组血清 IL-6、TNF- 高于对照组术后 3 d 亚组、电针组术后 3 d 亚组，电针组术后 3 d 亚组 TNF- 高于对照组术后 3 d 亚组，IL-10 高于对照组术后 3 d 亚组、模型组术后 3 d 亚组（P<0.05）。模型组术后 7 d 亚组血清 IL-6 高于对照组术后 7 d 亚组，TNF- 高于对照组术后 7 d 亚组、电针组术后 7 d 亚组，电针组术后 7 d 亚组 IL-10 高于对照组术后 7 d 亚组、模型组术后 7 d 亚组（P<0.05）。模型组术后 3 d 亚组海马 IL-6、TNF- 高于对照组术后 3 d 亚组、电针组术后 3 d 亚组，电针组术后 3 d 亚组 IL-6、TNF- 高于对照组术后 3 d 亚组，IL-10 高于对照组术后 3 d 亚组、模型组术后 3 d 亚组（P<0.05）。模型组术后 7 d 亚组海马 IL-6、TNF- 高于对照组术后 7 d 亚组、电针组术后 7 d 亚组，电针组术后 7 d 亚组 IL-10 高于对照组术后 7 d 亚组、模型组术后 7 d 亚组（P<0.05）。模型组术后 3 d 亚组 Fe2+、LPO、ACSL4、LPCAT3 高于对照组术后 3 d 亚组、电针组术后 3 d 亚组，电针组术后 3 d 亚组高于对照组术后 3 d 亚组，模型组术后 3 d 亚组 FTH1 低于对照组术后 3 d 亚组、电针组术后 3 d 亚组，电针组术后 3 d 亚组低于对照组术后 3 d 亚组（P<0.05）。模型组术后 7 d 亚组 Fe2+、LPO、ACSL4、LPCAT3 高于对照组术后 7 d 亚组、电针组术后 7 d 亚组，FTH1 低于术后 7 d 亚组、电针组术后 7 d 亚组（P<0.05）。模型组术后 3、7d 亚组海马组织视野内细胞核双核膜结构清晰，核周隙未见明显增宽，形态不规则，表面凹凸不平；核内染色质浓缩边集；胞质内少量线粒体膜破裂，膜结构消失；部分内质网明显扩张；并可见部分髓鞘断裂，排列紊乱；电针组术后3、7 d 亚组较模型组明显改善。结论 炎性细胞因子失衡和神经元铁死亡可能是 POCD 发生的重要病因机制；电针能够改善 POCD 老年大鼠的学习记忆能力，其发挥脑保护作用机制可能与其调控全身和中枢炎性细胞因子水平以及神经元细胞铁死亡途径有关。

Abstract: Background Postoperative cognitive dysfunction（POCD）is one of the major causes of increased postoperative morbidity and mortality in patients. The inflammatory response and ferroptosis are the principal hypotheses of the mechanism of POCD development. But the mechanism of electroacupuncture to improve learning and memory function in POCD patients is unclear. Objective To observe the effect of electroacupuncture on learning，memory，inflammatory cytokines and ferroptosis of hippocampal neurons in aged rats with POCD，so as to explore the mechanism of improving POCD by electroacupuncture. Methods From January 2022 to February 2023，72 SD rats aged 18 to 20 months were selected and divided into 3 groups according to random number table method of control group（n=24），model group（n=24）and electroacupuncture group（n=24）. According to the observation time points of 3 and 7 days postoperative，the rats in each group were divided into 2 the subgroups（3 d postoperative subgroup in control group，7 d postoperative subgroup in control group，3 d postoperative subgroup in model group，7 d postoperative subgroup in model group，3 d postoperative subgroup in electroacupuncture group，7 d postoperative subgroup in electroacupuncture group），with 12 rats in each group. POCD model was established by exploratory laparotomy，and Baihui and Neiguan points were stimulated by electroacupuncture. The Morris water maze device was used to detect the behavioral performance of rats. The contents of interleukin （IL） 6，IL-10，tumor necrosis factor alpha （TNF-） in serum and hippocampus were detected by enzyme-linked immunosorbent assay，and lipid peroxides（LPO） and Fe2+in hippocampus were detected. The protein expression levels of acyl coenzyme A synthetase long-chain family member 4（ACSL4），ferritin heavy chain 1（FTH1）and lysophosphatidylcholine acyltransferase-3（LPCAT3）in hippocampus were detected by Western Blotting. The ultrastructure of hippocampal neurons was observed by transmission electron microscopy. Results Group and time had no interaction effect on the escape latency of cognitive function training at 3 and 7d postoperatively （Pinteraction>0.05），the main effect of training time on the escape latency of cognitive function training was significant （Ptime0.05）. The escape latency of 3d postoperative subgroup in model group was higher than that of 3d postoperative subgroup in control group and 3d postoperative subgroup in electroacupuncture group. The frequency of crossing platform and target quadrant residence time of 3d postoperative subgroup in model group were lower than those of 3d postoperative subgroup in control group and 3d postoperative subgroup in electroacupuncture group；the frequency of crossing platform in 3d postoperative subgroup in electroacupuncture group was lower than that of 3d postoperative subgroup in control group（P<0.05）. The escape latency of 7d postoperative subgroup in model group was higher than that of 7d postoperative subgroup in control group and 7d postoperative subgroup in electroacupuncture group，the frequency of crossing platform was lower than that of 7d postoperative subgroup in control group，and the target quadrant residence time was lower than that of 7d postoperative subgroup in control group and 7d postoperative subgroup in electroacupuncture group（P<0.05）. Serum IL-6 and TNF- in 3d postoperative subgroup in model group were higher than those in 3d postoperative subgroup in control group and 3d postoperative subgroup in electroacupuncture group（P<0.05）. TNF- in 3d postoperative subgroup in electroacupuncture group was higher than that in 3d postoperative subgroup in control group（P<0.05），and IL-10 was higher than that in 3d postoperative subgroup in control group and 3d postoperative subgroup in model group（P<0.05）. Serum IL-6 was higher in 7d postoperative subgroup in model group than in 7d postoperative subgroup in control group，and TNF- was higher than 7d postoperative subgroup in control group and 7 d postoperatively subgroup in electroacupuncture group，and IL-10 was higher in 7d postoperative subgroup in electroacupuncture group than the 7d postoperative subgroup in control group and 7d postoperatively subgroup in model group （P<0.05）. The levels of hippocampal IL-6 and TNF- in 3d postoperative subgroup in model group were higher than those of 3d postoperative subgroup in control group and 3d postoperative subgroup in electroacupuncture group，and the level of IL-10 was higher than that of 3d postoperative subgroup in control group and 3d postoperative subgroup in model group（P<0.05）. Hippocampal IL-6 and TNF- levels in 7d postoperative subgroup in model group were higher than those in 7d postoperative subgroup in control group and 7d postoperative subgroup in electroacupuncture group，and IL-10 in 7d postoperative subgroup in electroacupuncture group was higher than that in 7d postoperative subgroup in contol group and 7d postoperative subgroup in model group（P<0.05）. Fe2+，LPO，ACSL4 and LPCAT3 in 3d postoperative subgroup in model group were higher than those in 3d postoperative subgroup in control group and 3d postoperative subgroup in electroacupuncture group，Fe2+，LPO，ACSL4 and LPCAT3 in 3d postoperative subgroup in electroacupuncture group were higher than those in 3d postoperative subgroup in control group；FTH1 in 3d postoperative subgroup in model group was lower than that in 3d postoperative subgroup in control group and 3d postoperative subgroup in electroacupuncture group，FTH1 in 3d postoperative subgroup in electroacupuncture group was lower than that in 3d postoperative subgroup in control group（P<0.05）. Fe2+，LPO，ACSL4 and LPCAT3 in 7d postoperative subgroup in model group were higher than those in 7d postoperative subgroup in control group and 7d postoperative subgroup in electroacupuncture group，FTH1 in 7d postoperative subgroup in model group was lower than that in 7d postoperative subgroup in control group and 7d postoperative subgroup in electroacupuncture group（P<0.05）. In 3d and 7d postoperative subgroups in model group，the structure of double nuclear membrane of cell nucleus was clear in the hippocampal field of vision，and the perinuclear space was not significantly widened，with irregular shape and uneven surface；the chromatin in the nucleus was concentrated and marginalized；a small amount of mitochondrial membranes in the cytoplasm were broken and the membrane structure disappeared；part of the endoplasmic reticulum expanded obviously；some myelin sheaths were broken and disorganized. The 3d and 7d postoperative subgroups in electroacupuncture group improved significantly compared with 3d and 7d postoperative subgroups in model group. Conclusion The imbalance of inflammatory cytokines and ferroptosis of neurons may be the important etiological mechanism for the development of POCD. Electroacupuncture can improve the learning and memory abilities in aged rats with POCD，and the mechanism of its brain protection may be related to the regulation of systemic and central inflammatory cytokines and the pathway of ferroptosis in neurons.

• 期刊： 中国全科医学
• 分类： 医学、药学 >> 临床医学
• 引用： ChinaXiv:202309.00186 (或此版本 ChinaXiv:202309.00186V1)
  DOI:10.12114/j.issn.1007-9572.2023.0449
  CSTR:32003.36.ChinaXiv.202309.00186.V1
• 推荐引用方式： 秦晓宇,张斌森,张笑佳,逯晓婷,刘鸿鑫,王春爱.(2023).电针对术后认知功能障碍模型大鼠炎症反应和铁死亡影响的研究.中国全科医学.doi:10.12114/j.issn.1007-9572.2023.0449 (点此复制)