Abstract：
BACKGROUND: Ischemic stroke (IS) is a major cause of mortality and disability worldwide. Inflammatory response is crucial in the pathogenesis of tissue injury in cerebral infarction. Arctium lappa leaves are traditionally used to treat IS.
OBJECTIVE: To investigate the neuroprotective effects and molecular mechanisms of the ethanolic extract of A. lappa leaves (ALLEE) on cerebral ischemia-reperfusion (CIR).
METHODS: Middle cerebral artery obstruction reperfusion (MCAO/R) rats and an oxygen-glucose deprivation/reoxygenation (OGD/R) cell model were used to evaluate ALLEE pharmacodynamics. Various methods, including neurological function, 2,3,5-triphenyltetrazolium chloride, hematoxylin and eosin, and Nissl, enzyme-linked immunosorbent, and TdT-mediated dUTP nick-end labeling assays, were used to analyze the neuroprotective effects of ALLEE in vitro and in vivo. The major chemical components and potential target genes of ALLEE were screened using network pharmacology. Molecular docking, western blotting, and immunofluorescence analyses were performed to confirm the effectiveness of the targets in related pathways.
RESULTS: ALLEE exerted potent effects on the MCAO/R model by decreasing the neurological scores, infarct volumes, and pathological features (p < 0.01). Furthermore, network pharmacology results revealed that the treatment of IS with ALLEE involved the regulation of various inflammatory pathways, such as the tumor necrosis factor (TNF) and chemokine signaling pathways. ALLEE also played key roles in targeting key molecules, including nuclear factor (NF)-κBIA, NF-κB1, interleukin (IL)-6, TNF-α and IL1β, and regulating the histone deacetylase (HDAC)-9-mediated signaling pathway. In vivo and in vitro analyses revealed that ALLEE significantly regulated the NF-κB pathway, promoted the phosphorylation activation of NF-κB P65, IκB and IKK (p < 0.01 or p < 0.05), and decreased the expression levels of the inflammatory factors, IL-1β, IL-6 and TNF-α (p < 0.01). Moreover, ALLEE significantly decreased the expression of HDAC9 (p < 0.01) that is associated with inflammatory responses. However, HDAC9 overexpression partially reversed the neuroprotective effects of ALLEE and its suppressive effects on inflammation and phosphorylation of NF-κB (p < 0.01).
CONCLUSIONS: In conclusion, our results revealed that ALLEE ameliorates MCAO/R-induced experimental CIR by modulating inflammatory responses via the inhibition of HDAC9-mediated NF-κB pathway.
OBJECTIVE: To investigate the neuroprotective effects and molecular mechanisms of the ethanolic extract of A. lappa leaves (ALLEE) on cerebral ischemia-reperfusion (CIR).
METHODS: Middle cerebral artery obstruction reperfusion (MCAO/R) rats and an oxygen-glucose deprivation/reoxygenation (OGD/R) cell model were used to evaluate ALLEE pharmacodynamics. Various methods, including neurological function, 2,3,5-triphenyltetrazolium chloride, hematoxylin and eosin, and Nissl, enzyme-linked immunosorbent, and TdT-mediated dUTP nick-end labeling assays, were used to analyze the neuroprotective effects of ALLEE in vitro and in vivo. The major chemical components and potential target genes of ALLEE were screened using network pharmacology. Molecular docking, western blotting, and immunofluorescence analyses were performed to confirm the effectiveness of the targets in related pathways.
RESULTS: ALLEE exerted potent effects on the MCAO/R model by decreasing the neurological scores, infarct volumes, and pathological features (p < 0.01). Furthermore, network pharmacology results revealed that the treatment of IS with ALLEE involved the regulation of various inflammatory pathways, such as the tumor necrosis factor (TNF) and chemokine signaling pathways. ALLEE also played key roles in targeting key molecules, including nuclear factor (NF)-κBIA, NF-κB1, interleukin (IL)-6, TNF-α and IL1β, and regulating the histone deacetylase (HDAC)-9-mediated signaling pathway. In vivo and in vitro analyses revealed that ALLEE significantly regulated the NF-κB pathway, promoted the phosphorylation activation of NF-κB P65, IκB and IKK (p < 0.01 or p < 0.05), and decreased the expression levels of the inflammatory factors, IL-1β, IL-6 and TNF-α (p < 0.01). Moreover, ALLEE significantly decreased the expression of HDAC9 (p < 0.01) that is associated with inflammatory responses. However, HDAC9 overexpression partially reversed the neuroprotective effects of ALLEE and its suppressive effects on inflammation and phosphorylation of NF-κB (p < 0.01).
CONCLUSIONS: In conclusion, our results revealed that ALLEE ameliorates MCAO/R-induced experimental CIR by modulating inflammatory responses via the inhibition of HDAC9-mediated NF-κB pathway.
摘要：
背景：缺血性卒中（IS）是全球范围内死亡和残疾的主要原因。炎症反应在脑梗死组织损伤的发病机制中至关重要。Arctiumlappa叶传统上用于治疗IS。
目的：研究拉帕叶乙醇提取物(ALLEE)对脑缺血再灌注(CIR)的神经保护作用及其分子机制。
方法：采用大脑中动脉阻塞再灌注(MCAO/R)大鼠和氧糖剥夺/复氧(OGD/R)细胞模型评价ALLEE药效学。各种方法,包括神经功能,氯化2,3,5-三苯基四唑，苏木精和伊红,还有Nissl,酶联免疫吸附，和TdT介导的dUTP缺口末端标记测定，用于分析ALLEE的体外和体内神经保护作用。利用网络药理学方法筛选了ALLEE的主要化学成分和潜在的靶基因。分子对接,西方印迹,我们进行了免疫荧光分析,以确认靶标在相关途径中的有效性.
结果：ALLEE通过降低神经评分对MCAO/R模型产生了有效的影响，梗死体积,病理特征(p<0.01)。此外，网络药理学结果表明，用ALLEE治疗IS涉及各种炎症途径的调节，如肿瘤坏死因子(TNF)和趋化因子信号通路。ALLEE还在靶向关键分子中发挥了关键作用，包括核因子（NF）-κBIA，NF-κB1、白细胞介素(IL)-6、TNF-α和IL1β,并调节组蛋白去乙酰化酶（HDAC）-9介导的信号通路。体内和体外分析显示，ALLEE显著调节NF-κB通路，促进NF-κBP65，IκB和IKK的磷酸化激活（p<0.01或p<0.05），并降低炎症因子的表达水平，IL-1β,IL-6和TNF-α(p<0.01)。此外，ALLEE显著降低与炎症反应相关的HDAC9的表达(p<0.01)。然而,HDAC9过表达部分逆转了ALLEE的神经保护作用及其对炎症和NF-κB磷酸化的抑制作用(p<0.01)。
结论：结论：我们的结果表明,ALLEE通过抑制HDAC9介导的NF-κB通路调节炎症反应,从而改善MCAO/R诱导的实验性CIR.
目的：研究拉帕叶乙醇提取物(ALLEE)对脑缺血再灌注(CIR)的神经保护作用及其分子机制。
方法：采用大脑中动脉阻塞再灌注(MCAO/R)大鼠和氧糖剥夺/复氧(OGD/R)细胞模型评价ALLEE药效学。各种方法,包括神经功能,氯化2,3,5-三苯基四唑，苏木精和伊红,还有Nissl,酶联免疫吸附，和TdT介导的dUTP缺口末端标记测定，用于分析ALLEE的体外和体内神经保护作用。利用网络药理学方法筛选了ALLEE的主要化学成分和潜在的靶基因。分子对接,西方印迹,我们进行了免疫荧光分析,以确认靶标在相关途径中的有效性.
结果：ALLEE通过降低神经评分对MCAO/R模型产生了有效的影响，梗死体积,病理特征(p<0.01)。此外，网络药理学结果表明，用ALLEE治疗IS涉及各种炎症途径的调节，如肿瘤坏死因子(TNF)和趋化因子信号通路。ALLEE还在靶向关键分子中发挥了关键作用，包括核因子（NF）-κBIA，NF-κB1、白细胞介素(IL)-6、TNF-α和IL1β,并调节组蛋白去乙酰化酶（HDAC）-9介导的信号通路。体内和体外分析显示，ALLEE显著调节NF-κB通路，促进NF-κBP65，IκB和IKK的磷酸化激活（p<0.01或p<0.05），并降低炎症因子的表达水平，IL-1β,IL-6和TNF-α(p<0.01)。此外，ALLEE显著降低与炎症反应相关的HDAC9的表达(p<0.01)。然而,HDAC9过表达部分逆转了ALLEE的神经保护作用及其对炎症和NF-κB磷酸化的抑制作用(p<0.01)。
结论：结论：我们的结果表明,ALLEE通过抑制HDAC9介导的NF-κB通路调节炎症反应,从而改善MCAO/R诱导的实验性CIR.
