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

Effect of Electroacupuncture on Inflammation Response and Ferroptosis in Rats Modeling Postoperative Cognitive Dysfunction

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尚不明确。目的观察电针对老年POCD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穴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α(TNF-α)的水平,海马组织脂质过氧化物(LPO)、Fe^(2+),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、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),组别对逃避潜伏期主效应均不显著(P_(组间)>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-α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-α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-α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-α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-α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-α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Fe^(2+)、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Fe^(2+)、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、7 d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。

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-α)in serum and hippocampus were detected by enzyme-linked immunosorbent assay,and lipid peroxides(LPO)and Fe^(2+)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 7 d postoperatively(P_(interaction)>0.05),the main effect of training time on the escape latency of cognitive function training was significant(P_(time)<0.05),and the main effect of group on the escape latency was not significant(P_(group)>0.05).The escape latency of 3 d postoperative subgroup in model group was higher than that of 3 d postoperative subgroup in control group and 3 d postoperative subgroup in electroacupuncture group.The frequency of crossing platform and target quadrant residence time of 3 d postoperative subgroup in model group were lower than those of 3 d postoperative subgroup in control group and 3 d postoperative subgroup in electroacupuncture group;the frequency of crossing platform in 3 d postoperative subgroup in electroacupuncture group was lower than that of 3 d postoperative subgroup in control group(P<0.05).The escape latency of 7 d postoperative subgroup in model group was higher than that of 7 d postoperative subgroup in control group and 7 d postoperative subgroup in electroacupuncture group,the frequency of crossing platform was lower than that of 7 d postoperative subgroup in control group,and the target quadrant residence time was lower than that of 7 d postoperative subgroup in control group and 7 d postoperative subgroup in electroacupuncture group(P<0.05).Serum IL-6 and TNF-αin 3 d postoperative subgroup in model group were higher than those in 3 d postoperative subgroup in control group and 3 d postoperative subgroup in electroacupuncture group(P<0.05).TNF-αin 3 d postoperative subgroup in electroacupuncture group was higher than that in 3 d postoperative subgroup in control group(P<0.05),and IL-10 was higher than that in 3 d postoperative subgroup in control group and 3 d postoperative subgroup in model group(P<0.05).Serum IL-6 was higher in 7 d postoperative subgroup in model group than in 7 d postoperative subgroup in control group,and TNF-αwas higher than 7 d postoperative subgroup in control group and 7 d postoperatively subgroup in electroacupuncture group,and IL-10 was higher in 7 d postoperative subgroup in electroacupuncture group than the 7 d postoperative subgroup in control group and 7 d postoperatively subgroup in model group(P<0.05).The levels of hippocampal IL-6 and TNF-αin 3 d postoperative subgroup in model group were higher than those of 3 d postoperative subgroup in control group and 3 d postoperative subgroup in electroacupuncture group,and the level of IL-10 was higher than that of 3 d postoperative subgroup in control group and 3 d postoperative subgroup in model group(P<0.05).Hippocampal IL-6 and TNF-αlevels in 7 d postoperative subgroup in model group were higher than those in 7 d postoperative subgroup in control group and 7 d postoperative subgroup in electroacupuncture group,and IL-10 in 7 d postoperative subgroup in electroacupuncture group was higher than that in 7 d postoperative subgroup in contol group and 7 d postoperative subgroup in model group(P<0.05).Fe^(2+),LPO,ACSL4 and LPCAT3 in 3 d postoperative subgroup in model group were higher than those in 3 d postoperative subgroup in control group and 3 d postoperative subgroup in electroacupuncture group,Fe^(2+),LPO,ACSL4 and LPCAT3 in 3 d postoperative subgroup in electroacupuncture group were higher than those in 3 d postoperative subgroup in control group;FTH1 in 3 d postoperative subgroup in model group was lower than that in 3 d postoperative subgroup in control group and 3 d postoperative subgroup in electroacupuncture group,FTH1 in 3 d postoperative subgroup in electroacupuncture group was lower than that in 3 d postoperative subgroup in control group(P<0.05).Fe^(2+),LPO,ACSL4 and LPCAT3 in 7 d postoperative subgroup in model group were higher than those in 7 d postoperative subgroup in control group and 7 d postoperative subgroup in electroacupuncture group,FTH1 in 7 d postoperative subgroup in model group was lower than that in 7 d postoperative subgroup in control group and 7 d postoperative subgroup in electroacupuncture group(P<0.05).In 3 d and 7 d 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 3 d and 7 d postoperative subgroups in electroacupuncture group improved significantly compared with 3 d and 7 d 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.