Abstract：
BACKGROUND: Ischemic stroke is a leading cause of death and long-term disability worldwide. Studies have suggested that cerebral ischemia induces massive mitochondrial damage. Valerianic acid A (VaA) is the main active ingredient of valerianic acid with neuroprotective activity.
OBJECTIVE: This study aimed to investigate the neuroprotective effects of VaA with ischemic stroke and explore the underlying mechanisms.
METHODS: In this study, we established the oxygen-glucose deprivation and reperfusion (OGD/R) cell model and the middle cerebral artery occlusion and reperfusion (MCAO/R) animal model in vitro and in vivo. Neurological behavior score, 2, 3, 5-triphenyl tetrazolium chloride (TTC) staining and Hematoxylin and Eosin (HE) Staining were used to detect the neuroprotection of VaA in MCAO/R rats. Also, the levels of ROS, mitochondrial membrane potential (MMP), and activities of NAD+ were detected to reflect mitochondrial function. Mechanistically, gene knockout experiments, transfection experiments, immunofluorescence, DARTS, and molecular dynamics simulation experiments showed that VaA bound to IDO1 regulated the kynurenine pathway of tryptophan metabolism and prevented Stat3 dephosphorylation, promoting Stat3 activation and subsequent transcription of the mitochondrial fusion-related gene Opa1.
RESULTS: We showed that VaA decreased the infarct volume in a dose-dependent manner and exerted neuroprotective effects against reperfusion injury. Furthermore, VaA promoted Opa1-related mitochondrial fusion and reversed neuronal mitochondrial damage and loss after reperfusion injury. In SH-SY5Y cells, VaA (5, 10, 20 μM) exerted similar protective effects against OGD/R-induced injury. We then examined the expression of significant enzymes regulating the kynurenine (Kyn) pathway of the ipsilateral brain tissue of the ischemic stroke rat model, and these enzymes may play essential roles in ischemic stroke. Furthermore, we found that VaA can bind to the initial rate-limiting enzyme IDO1 in the Kyn pathway and prevent Stat3 phosphorylation, promoting Stat3 activation and subsequent transcription of the mitochondrial fusion-related gene Opa1. Using in vivo IDO1 knockdown and in vitro IDO1 overexpressing models, we demonstrated that the promoted mitochondrial fusion and neuroprotective effects of VaA were IDO1-dependent.
CONCLUSIONS: VaA administration improved neurological function by promoting mitochondrial fusion through the IDO1-mediated Stat3-Opa1 pathway, indicating its potential as a therapeutic drug for ischemic stroke.
OBJECTIVE: This study aimed to investigate the neuroprotective effects of VaA with ischemic stroke and explore the underlying mechanisms.
METHODS: In this study, we established the oxygen-glucose deprivation and reperfusion (OGD/R) cell model and the middle cerebral artery occlusion and reperfusion (MCAO/R) animal model in vitro and in vivo. Neurological behavior score, 2, 3, 5-triphenyl tetrazolium chloride (TTC) staining and Hematoxylin and Eosin (HE) Staining were used to detect the neuroprotection of VaA in MCAO/R rats. Also, the levels of ROS, mitochondrial membrane potential (MMP), and activities of NAD+ were detected to reflect mitochondrial function. Mechanistically, gene knockout experiments, transfection experiments, immunofluorescence, DARTS, and molecular dynamics simulation experiments showed that VaA bound to IDO1 regulated the kynurenine pathway of tryptophan metabolism and prevented Stat3 dephosphorylation, promoting Stat3 activation and subsequent transcription of the mitochondrial fusion-related gene Opa1.
RESULTS: We showed that VaA decreased the infarct volume in a dose-dependent manner and exerted neuroprotective effects against reperfusion injury. Furthermore, VaA promoted Opa1-related mitochondrial fusion and reversed neuronal mitochondrial damage and loss after reperfusion injury. In SH-SY5Y cells, VaA (5, 10, 20 μM) exerted similar protective effects against OGD/R-induced injury. We then examined the expression of significant enzymes regulating the kynurenine (Kyn) pathway of the ipsilateral brain tissue of the ischemic stroke rat model, and these enzymes may play essential roles in ischemic stroke. Furthermore, we found that VaA can bind to the initial rate-limiting enzyme IDO1 in the Kyn pathway and prevent Stat3 phosphorylation, promoting Stat3 activation and subsequent transcription of the mitochondrial fusion-related gene Opa1. Using in vivo IDO1 knockdown and in vitro IDO1 overexpressing models, we demonstrated that the promoted mitochondrial fusion and neuroprotective effects of VaA were IDO1-dependent.
CONCLUSIONS: VaA administration improved neurological function by promoting mitochondrial fusion through the IDO1-mediated Stat3-Opa1 pathway, indicating its potential as a therapeutic drug for ischemic stroke.
摘要：
背景：缺血性卒中是全球范围内死亡和长期残疾的主要原因。研究表明，脑缺血可引起大量线粒体损伤。戊酸A（VaA）是戊酸的主要活性成分，具有神经保护活性。
目的：本研究旨在探讨VaA对缺血性卒中的神经保护作用及其机制。
方法：在本研究中，我们在体外和体内建立了氧糖剥夺和再灌注（OGD/R）细胞模型和大脑中动脉阻塞和再灌注（MCAO/R）动物模型。神经行为评分,采用2，3，5-三苯基氯化四唑（TTC）染色和苏木精和伊红（HE）染色检测VaA对MCAO/R大鼠的神经保护作用。此外，ROS的水平，线粒体膜电位（MMP），检测NAD+的活性以反映线粒体功能。机械上,基因敲除实验,转染实验，免疫荧光，达人,分子动力学模拟实验表明，与IDO1结合的VaA调节色氨酸代谢的犬尿氨酸通路，阻止Stat3去磷酸化，促进线粒体融合相关基因Opa1的Stat3激活和随后的转录。
结果：我们显示VaA以剂量依赖性方式降低梗死体积，并对再灌注损伤发挥神经保护作用。此外，VaA促进Opa1相关线粒体融合，逆转再灌注损伤后神经元线粒体损伤和丢失。在SH-SY5Y细胞中，VaA（5、10、20μM）对OGD/R诱导的损伤具有类似的保护作用。然后，我们检查了调节缺血性中风大鼠模型同侧脑组织犬尿氨酸（Kyn）途径的重要酶的表达，这些酶可能在缺血性卒中中发挥重要作用。此外，我们发现VaA可以与Kyn通路中的初始限速酶IDO1结合并阻止Stat3磷酸化，促进线粒体融合相关基因Opa1的Stat3激活和随后的转录。使用体内IDO1敲低和体外IDO1过表达模型，我们证明了VaA促进的线粒体融合和神经保护作用是IDO1依赖性的。
结论：VaA通过IDO1介导的Stat3-Opa1通路促进线粒体融合，从而改善神经功能，表明其作为缺血性中风治疗药物的潜力。
目的：本研究旨在探讨VaA对缺血性卒中的神经保护作用及其机制。
方法：在本研究中，我们在体外和体内建立了氧糖剥夺和再灌注（OGD/R）细胞模型和大脑中动脉阻塞和再灌注（MCAO/R）动物模型。神经行为评分,采用2，3，5-三苯基氯化四唑（TTC）染色和苏木精和伊红（HE）染色检测VaA对MCAO/R大鼠的神经保护作用。此外，ROS的水平，线粒体膜电位（MMP），检测NAD+的活性以反映线粒体功能。机械上,基因敲除实验,转染实验，免疫荧光，达人,分子动力学模拟实验表明，与IDO1结合的VaA调节色氨酸代谢的犬尿氨酸通路，阻止Stat3去磷酸化，促进线粒体融合相关基因Opa1的Stat3激活和随后的转录。
结果：我们显示VaA以剂量依赖性方式降低梗死体积，并对再灌注损伤发挥神经保护作用。此外，VaA促进Opa1相关线粒体融合，逆转再灌注损伤后神经元线粒体损伤和丢失。在SH-SY5Y细胞中，VaA（5、10、20μM）对OGD/R诱导的损伤具有类似的保护作用。然后，我们检查了调节缺血性中风大鼠模型同侧脑组织犬尿氨酸（Kyn）途径的重要酶的表达，这些酶可能在缺血性卒中中发挥重要作用。此外，我们发现VaA可以与Kyn通路中的初始限速酶IDO1结合并阻止Stat3磷酸化，促进线粒体融合相关基因Opa1的Stat3激活和随后的转录。使用体内IDO1敲低和体外IDO1过表达模型，我们证明了VaA促进的线粒体融合和神经保护作用是IDO1依赖性的。
结论：VaA通过IDO1介导的Stat3-Opa1通路促进线粒体融合，从而改善神经功能，表明其作为缺血性中风治疗药物的潜力。
