研究背景：

心脏骤停后综合征（Post-Cardiac Arrest Syndrome, PCAS）是指心脏骤停复苏后心肌功能障碍、脑损伤和全身缺血-再灌注损伤等病理生理过程为特征的一组综合征。PCAS是重大的临床问题，其致死率高，预后极差。心脏骤停（Cardiac Arrest, CA）和心肺复苏（Cardiopulmonary Resuscitation, CPR）本质上是一种全身性的严重缺血再灌注事件，大脑与心脏是主要靶器官；复苏后严重心肌功能障碍和脑损伤是初始心脏骤停患者成功复苏后早期死亡的主要原因。尽管复苏后心脏和脑损伤的潜在病理机制不同，但它们有共同的发病机制，如缺血-再灌注损伤、氧化应激、炎症免疫反应和线粒体损伤等。探索PCAS的病理生理机制和寻找心脑损伤的潜在共同靶点和干预措施对于临床治疗PCAS和改善远期预后具有重要意义。

CCL7（Chemokine C-C motif Ligand 7, CCL7）编码单核细胞趋化蛋白3，是一种在炎症反应时招募巨噬细胞分泌炎症因子的趋化因子。研究表明CCL7是心肌缺血再灌注损伤中的潜在治疗靶基因，干预CCL2（Chemokine C-C motif Ligand 2）/CCL7信号通路可以改善心肌梗死后的心脏重构。同时，早期的生物学信息分析提示CCL7是卒中后脑缺血再灌注损伤的关键免疫分子标志物；最新的研究也表明调节CCL7信号转导可以改善小鼠脑缺血模型小胶质细胞的炎症反应和脑损伤。我们在第一部分验证了Ccl7可能是是大鼠心脏骤停复苏模型早期心脏和脑损伤的关键基因，且炎症免疫反应可能在复苏后早期脑损伤发挥重要作用。因此，CCL7可能在心脏骤停复苏后心脏和脑损伤同时发挥作用。吡斯的明（Pyridostigmine, PYR）是一种胆碱酯酶抑制剂，临床主要用于治疗重症肌无力，也可用于逆转非去极化肌松药的作用或改善某些自主神经功能障碍。先前的研究表明吡斯的明（PYR）可以通过非神经元胆碱能心脏系统作用于CCL2/CCL7信号通路从而减轻心肌损害和炎症。同时，PYR还可以激活副交感神经减轻心肌梗死后心肌缺血-再灌注损伤；最新的动物实验研究表明，迷走神经刺激可以提高窒息心脏骤停模型中复苏大鼠的存活率，改善脑血流量和神经功能的预后。然而，由于PCAS复杂的病理生理特征，关于PYR在心脏骤停复苏模型中使用的证据尚欠缺。

研究目的：

1.探索CA/CPR大鼠模型中复苏后心脑损伤的共同关键基因和潜在机制；

2.观察低剂量PYR对大鼠心脏骤停复苏早期心脏及脑损伤的影响；

3.探究PYR是否通过调节CCL7相关信号传导从而改善心脏骤停复苏后心脏和脑损伤。

研究方法：

第一部分：将400~500g雄性SD大鼠分组随机分为2组，Con组和CA/CPR组。Con组行假手术，不给予右心室电刺激;CA/CRP组给予右心室电刺激诱发室颤，并行心肺复苏术，存活到24小时的大鼠留取左心室（Left Ventricle , LV）和海马组织用于RNAseq检测和后续其他检测，并留取整个心脏和大脑组织用于组织病理学检测。

第二部分：将400~500g雄性SD大鼠随机分为4组：Con组，Con-PYR组，CA组和CA-PYR。Con组和CA组术前30分钟一次性腹腔注射生理盐水（0.25mg/kg），Con-PYR和CA-PYR组术前30分钟一次性腹腔注射PYR（0.25mg/kg, Sigma），24小时后处死，取材做后续指标检测。Con组和Con-PYR组行假手术，不给予右心室电刺激；CA组和CA-PYR组给予右心室电刺激诱发室颤，并行心肺复苏术，存活到24小时的大鼠用于后续检测。24小时后检测大鼠心脏超声，记录15分钟心电图分析心率变异性（Heart Rate Variability, HRV），并进行有创血流动力学的检测，采集腹主动脉血检测动脉血气，留取心脏和大脑组织等组织标本进行组织病理学和分子生物学相关指标检测。

研究结果：

第一部分：复苏后24小时取大鼠海马组织RNAseq检测分析发现，与对照组相比，CA/CPR组有159个基因上调，58个基因下调；GO富集分析强调了肿瘤坏死因子（Tumor Necrosis Factor, TNF）产生的正向调节是最重要的生物过程调节途径，可能在VF诱导CA/CPR大鼠早期脑损伤中可能至关重要。此外，脑组织差异基因的GO富集分析表明，这217个差异表达基因参与多种免疫炎症途径，包括免疫系统过程、炎症反应、整合素介导的信号传导、细胞因子反应和吞噬。大鼠心肌组织RNAseq分析发现，与Con组相比，CA/CPR组共筛选出79个差异基因，其中32个下调和47个基因上调；GO富集提示最显著改变的生物过程是淀粉样蛋白β的清除，也同时存在于海马组织的富集分析中。CA/CRP模型中的大鼠LV和海马组织中诱导的差异基因通过韦恩图相交得到11个共同基因。通过RT-PCR验证后，发现Ccl7、Timp1、Apln和Lgals3同时在LV和海马组织中的mRNA相对表达量存在显著差异，与转录组分析趋势一致。

第二部分：PYR预处理改善心脏骤停复苏大鼠的缺氧状态，降低血清乳酸脱氢酶（Lactate Dehydrogenase, LDH）、肌酸激酶(Creatine Kinase , CK）、肌酸激酶同工酶MB（Creatine Kinase-MB, CK-MB）的含量，减少心肌细胞TUNEL阳性细胞；PYR预处理增加心脏骤停复苏大鼠增加左心室射血分数（Left Ventricular Ejection Fraction, LVEF）和左心室短轴缩短率（Left Ventricular Fractional Shortening, LVFS），还升高CA大鼠的左心室收缩压（Left Ventricular Systolic Pressure, LVSP）、左室内压上升最大速率（maximum rate of the decrease of LV pressure, LV dP/dt_max）和左室内压下降最大速率（maximum rate of the decrease of LV pressure , LV dP/dt_min）；PYR降低心脏骤停复苏大鼠LV组织的CCL7相对蛋白表达量，降低CD68阳性细胞、CCL2和CCL7的相对mRNA水平，还降低LV中IL-6的mRNA表达水平；PYR预处理逆转了CA大鼠血清丙二醛（Malondialdehyde , MDA）的升高和谷胱甘肽（Glutathione, GSH）和超氧化物歧化酶（Superoxide Dismutase, SOD）的降低，降低LV组织的活性氧（Reactive Oxygen Species, ROS）含量；PYR改善CA大鼠线粒体功能障碍，增加LV组织的ATP含量，降低线粒体相关蛋白动力相关蛋白（Dynamin-Related Protein 1,DRP1）和线粒体融合蛋白（Mitofusin 1, MFN1）的相对表达； PYR还降低心脏骤停复苏大鼠LV中M2毒蕈碱型乙酰胆碱受体（M2 Muscarinic Acetylcholine Receptor, m2AchR）的蛋白表达，增加HRV，恢复了部分自主神经功能。PYR预处理减少心脏骤停复苏后大鼠的海马区域TUNEL阳性细胞、CD68阳性细胞以及CCL2的相对mRNA水平，抑制CA大鼠血清神经元特异性烯醇化酶（Neuron-Specific Enolase, NSE）水平的增高。PYR还激活心脏骤停复苏大鼠的LV和海马组织的磷脂酰肌醇3-激酶/蛋白激酶B（Phosphoinositide 3-Kinase / Protein Kinase B, PI3K/AKT）信号通路相关。

研究结论：

1.CCL7可能是大鼠心脏骤停复苏模型心脏和脑损伤的潜在共同关键基因。

2.PYR改善心脏骤停复苏大鼠的缺氧状态，减轻心肌损伤、炎症反应及氧化应激，改善左心室收缩功能。

3.PYR减轻复苏大鼠的大脑损伤和炎症反应，并部分恢复自主神经功能。

4.调节CCL2/CCL7信号转导减轻复苏后大鼠心脑损伤的炎症免疫反应是PYR改善PCAS的部分潜在机制。

Effects and Mechanisms of Pyridostigmine in Post-Resuscitation Cardiac and Brain Injury

Abstract

Background:

Post-cardiac arrest syndrome (PCAS) refers to a group of syndromes characterized by pathophysiological processes such as post-resuscitation myocardial dysfunction, brain injury, and systemic ischemia-reperfusion injury. PCAS is a significant clinical problem with high mortality and extremely poor prognosis. Cardiac arrest (CA) and cardiopulmonary resuscitation (CPR) are essentially severe systemic ischemia-reperfusion events, with the brain and heart as the primary target organs. Severe post-resuscitation myocardial dysfunction and brain injury are the main causes of early death in patients successfully resuscitated from initial cardiac arrest. Although the underlying pathological mechanisms of post-resuscitation cardiac and brain injury differ, they share common pathogenesis, such as ischemia-reperfusion injury, oxidative stress, inflammatory immune responses, and mitochondrial damage. Exploring the pathophysiological mechanisms of PCAS and identifying potential common targets and interventions for cardiac and brain injury are of great significance for the clinical treatment of PCAS and improving long-term outcomes.

CCL7 encodes monocyte chemoattractant protein 3 (MCP-3), a chemokine that recruits macrophages to secrete inflammatory factors during inflammatory responses. Studies have shown that CCL7 is a potential therapeutic target gene in myocardial ischemia-reperfusion injury, and intervention in the CCL2/CCL7 signaling pathway can improve cardiac remodeling after myocardial infarction. Meanwhile, early bioinformatics analysis suggests that CCL7 is a key immune molecular marker in cerebral ischemia-reperfusion injury after stroke. Recent studies also indicate that modulating CCL7 signal transduction can ameliorate microglial inflammatory responses and brain injury in a mouse cerebral ischemia model. In the first part of our study, we verified that Ccl7 may be a key gene in early cardiac and brain injury in a rat cardiac arrest resuscitation model, and that inflammatory immune responses may play an important role in early post-resuscitation brain injury. Therefore, CCL7 may simultaneously contribute to cardiac and brain injury after cardiac arrest resuscitation.

Pyridostigmine (PYR) is a cholinesterase inhibitor primarily used to treat myasthenia gravis and can also reverse the effects of non-depolarizing muscle relaxants or improve certain autonomic dysfunctions. Previous studies have shown that PYR can act on the CCL2/CCL7 signaling pathway via the non-neuronal cholinergic cardiac system to reduce myocardial damage and inflammation. Additionally, PYR can activate the parasympathetic nervous system to alleviate myocardial ischemia-reperfusion injury after myocardial infarction. Recent animal studies have demonstrated that vagus nerve stimulation can improve survival rates, cerebral blood flow, and neurological outcomes in resuscitated rats following an asphyxial cardiac arrest model. However, due to the complex pathophysiological features of PCAS, evidence for the use of PYR in cardiac arrest resuscitation models remains limited.

Objectives:

1.To explore common key genes and potential mechanisms of post-resuscitation cardiac and brain injury in a CA/CPR rat model.

2.To investigate the effects of low-dose PYR on early cardiac and brain injury in rats following cardiac arrest resuscitation.

3.To determine whether PYR ameliorates post-resuscitation cardiac and brain injury by modulating CCL7-related signaling pathways.

Methods:

Part 1:Male SD rats weighing 400–500 g were randomly divided into two groups: the Con group and the CA/CPR group. The Con group underwent sham surgery without right ventricular electrical stimulation, while the CA/CPR group received electrical stimulation to induce ventricular fibrillation (VF) and underwent CPR. Rats surviving for 24 hours had their left ventricular (LV) and hippocampal tissues collected for RNA sequencing (RNA-seq) and subsequent analyses. Whole heart and brain tissues were preserved for histopathological examination.

Part 2:Male SD rats weighing 400–500 g were randomly divided into four groups: Con, Con-PYR, CA, and CA-PYR. The Con and CA groups received a single intraperitoneal injection of saline (0.25 mg/kg) 30 minutes before surgery, while the Con-PYR and CA-PYR groups received a single intraperitoneal injection of PYR (0.25 mg/kg, Sigma) 30 minutes before surgery. Rats were euthanized 24 hours later for sample collection and further testing. The Con and Con-PYR groups underwent sham surgery without electrical stimulation, whereas the CA and CA-PYR groups received right ventricular electrical stimulation to induce VF and underwent CPR. Surviving rats were assessed 24 hours later via cardiac ultrasound, 15-minute electrocardiogram (ECG) for heart rate variability (HRV) analysis, invasive hemodynamic monitoring, arterial blood gas analysis, and collection of heart and brain tissues for histopathological and molecular biological examinations.

Results:

Part 1:RNA-seq analysis of hippocampal tissues 24 hours post-resuscitation revealed 159 upregulated and 58 downregulated genes in the CA/CPR group compared to the Con group. Gene Ontology (GO) enrichment analysis highlighted the positive regulation of tumor necrosis factor (TNF) production as the most significant biological process, potentially critical in early brain injury in VF-induced CA/CPR rats. Additionally, GO analysis of differentially expressed genes (DEGs) in brain tissues indicated their involvement in various immune-inflammatory pathways, including immune system processes, inflammatory responses, integrin-mediated signaling, cytokine responses, and phagocytosis. RNA-seq analysis of myocardial tissues identified 79 DEGs (32 downregulated and 47 upregulated) in the CA/CPR group compared to the Con group. GO enrichment suggested that the most altered biological process was amyloid-β clearance, which was also observed in hippocampal tissue analysis. A Venn diagram intersection of DEGs in LV and hippocampal tissues from the CA/CPR model identified 11 common genes. RT-PCR validation confirmed significant differences in the mRNA expression levels of Ccl7, Timp1, Apln, and Lgals3 in both LV and hippocampal tissues, consistent with transcriptomic trends.

Part 2: PYR pretreatment improved hypoxia, reduced serum levels of LDH, CK, and CK-MB, and decreased TUNEL-positive cardiomyocytes in resuscitated rats. It also increased LV ejection fraction (LVEF) and LV fractional shortening (LVFS), as well as LV systolic pressure (LVSP), LV dP/dt_max, and LV dP/dt_min in CA rats. PYR downregulated CCL7 protein expression, reduced CD68-positive cells, and lowered mRNA levels of CCL2, CCL7, and IL-6 in LV tissues. It also reversed elevated serum MDA and restored GSH and SOD levels while reducing ROS content in LV tissues. PYR improved mitochondrial dysfunction by increasing ATP content and modulating mitochondrial-related proteins DRP1 and MFN1. Additionally, PYR decreased m2AchR protein expression in LV tissues, enhanced HRV, and partially restored autonomic function. In the brain, PYR reduced hippocampal TUNEL-positive cells, CD68-positive cells, and CCL2 mRNA levels while suppressing serum NSE elevation. PYR also activated the PI3K/AKT signaling pathway in both LV and hippocampal tissues of resuscitated rats.

Conclusion:

1.CCL7 may be a potential common key gene in cardiac and brain injury in a rat cardiac arrest resuscitation model.

2.PYR improves hypoxia, mitigates myocardial injury, inflammation, and oxidative stress, and enhances LV systolic function in resuscitated rats.

3.PYR alleviates brain injury and inflammation while partially restoring autonomic nervous function.

4.Modulation of CCL2/CCL7 signaling to attenuate inflammatory immune responses is a partial mechanism by which PYR improves PCAS.