Li Dongli, Wu Qihui, Li Zunjiang, Chen Baijian, Sun Xing, Wu Qiqi, Ding Zhenzhu, Liu Linling, Fang Jiansong, Zeng Ruifeng, Gu Yong, Ding Banghan
The Second Affiliated Hospital of Guangzhou University of Chinese Medicine, Guangdong Provincial Hospital of Chinese Medicine, Guangzhou, China.
Guangdong Provincial Key Laboratory of Research on Emergency in TCM, Guangzhou, China.
CNS Neurosci Ther. 2025 Mar;31(3):e70297. doi: 10.1111/cns.70297.
Post-cardiopulmonary resuscitation brain injury (PBI) is essentially the cerebral ischemia reperfusion (CIR) injury, which is the main cause of death and long-term disability in patients with cardiac arrest. So far, there is no treatment for PBI; thus, it is urgent to develop new drugs or therapies for the prevention and treatment of brain injury after cardiopulmonary resuscitation. Although multiple constituent herbs or active ingredients of Xinbao Pill (XBP) have shown neuroprotective effects, whether XBP could play a therapeutic role on PBI is still unknown. This study aimed to illustrate the neuroprotective effect of XBP on PBI and probe the underlying mechanisms.
We first performed the cell and animal experiments to validate the protective effect of XBP on neurological function. We next identified the potential differential metabolites via metabolomics analysis. We further conducted a comprehensive network pharmacology analysis including overlap gene analysis, protein-protein interaction network, and gene-biological process-module function network to preliminarily investigate the specific mechanism of action (MOA) of XBP against PBI. Finally, PCR, MTT, ELISA assay, as well as Western blotting experiments were made to validate our proposed molecular mechanisms.
The in vitro experiment showed that XBP could increase cell viability and ameliorate cell morphological damage in PC12 cells exposed to oxygen-glucose deprivation and reoxygenation (OGD/RO) conditions. The in vivo experiment demonstrated that XBP improved the Neurologic Deficit Score (NDS), lowered the Neuron-Specific Enolase (NSE) level as well as reversed the typical neuropathological changes in PBI rats, indicating its neuroprotective effect on PBI. Further metabolomics analysis identified 94 differential metabolites after XBP treatment, and multiple metabolites were highly related to CIR. Moreover, network pharmacology results revealed that the therapeutic effect of XBP on PBI may be relevant to mitochondrial quality control (MQC). Mechanistically, XBP could not only promote the expressions of marker proteins including PGC1α, NRF1, TFAM, OPA1, MFN1 as well as MFN2 in mitochondrial biogenesis and mitochondrial fusion but also inhibit those proteins containing DRP1, MFF, FIS1, p62, PINK1, Parkin as well as LC3 in mitochondrial fission and mitophagy. Finally, AMP-activated protein kinase (AMPK) inhibitor was demonstrated to play a crucial role in regulating MQC.
Our study first determined that XBP might be an underlying anti-PBI formula, which also deciphered the potential MOAs of XBP against PBI by a network pharmacology approach combined with in vivo and in vitro experimental validation.
心肺复苏后脑损伤（PBI）本质上是脑缺血再灌注（CIR）损伤，是心脏骤停患者死亡和长期残疾的主要原因。迄今为止，尚无针对PBI的治疗方法；因此，迫切需要开发新的药物或疗法来预防和治疗心肺复苏后的脑损伤。尽管心宝丸（XBP）的多种组成草药或活性成分已显示出神经保护作用，但XBP是否能对PBI发挥治疗作用仍不清楚。本研究旨在阐明XBP对PBI的神经保护作用并探究其潜在机制。
我们首先进行细胞和动物实验以验证XBP对神经功能的保护作用。接下来，通过代谢组学分析鉴定潜在的差异代谢物。我们进一步进行了全面的网络药理学分析，包括重叠基因分析、蛋白质-蛋白质相互作用网络和基因-生物学过程-模块功能网络，以初步研究XBP抗PBI的具体作用机制（MOA）。最后，进行PCR、MTT、ELISA检测以及蛋白质印迹实验以验证我们提出的分子机制。
体外实验表明，XBP可提高暴露于氧糖剥夺和复氧（OGD/RO）条件下的PC12细胞的活力，并改善细胞形态损伤。体内实验表明，XBP改善了神经功能缺损评分（NDS），降低了神经元特异性烯醇化酶（NSE）水平，并逆转了PBI大鼠的典型神经病理变化，表明其对PBI具有神经保护作用。进一步的代谢组学分析鉴定出XBP处理后有94种差异代谢物，且多种代谢物与CIR高度相关。此外，网络药理学结果显示，XBP对PBI的治疗作用可能与线粒体质量控制（MQC）有关。机制上，XBP不仅可促进线粒体生物发生和线粒体融合中包括PGC1α、NRF1、TFAM、OPA1、MFN1以及MFN2等标记蛋白的表达，还可抑制线粒体分裂和线粒体自噬中包含DRP1、MFF、FIS1、p62、PINK1、Parkin以及LC3等的蛋白。最后，证明AMP激活的蛋白激酶（AMPK）抑制剂在调节MQC中起关键作用。
我们的研究首次确定XBP可能是一种潜在的抗PBI方剂，并且还通过网络药理学方法结合体内和体外实验验证，解读了XBP抗PBI的潜在作用机制。
