Department of Neurology, Guangdong Provincial Hospital of Traditional Chinese Medicine, Guangzhou, China; Department of Second Institute of Clinical Medicine, Guangzhou University of Traditional Chinese Medicine, Guangzhou, China.
Department of Neurology, Guangdong Provincial Hospital of Traditional Chinese Medicine, Guangzhou, China; State Key Laboratory of Dampness Syndrome of Chinese Medicine, The Second Affiliated Hospital of Guangzhou University of Chinese Medicine, Guangzhou, China; Department of Second Institute of Clinical Medicine, Guangzhou University of Traditional Chinese Medicine, Guangzhou, China; Guangdong Provincial Key Laboratory of Research on Emergency in TCM, Guangzhou, China.
Phytomedicine. 2022 Aug;103:154214. doi: 10.1016/j.phymed.2022.154214. Epub 2022 May 29.
Oxidative stress plays an important role in the pathology of ischemic stroke. Studies have confirmedthat scutellarin has antioxidant effects against ischemic injury, and we also reported that the involvement of Aldose reductase (AR) in oxidative stress and cerebral ischemic injury, in this study we furtherly explicit whether the antioxidant effect of scutellarin on cerebral ischemia injury is related to AR gene regulation and its specific mechanism.
C57BL/6N mice (Wild-type, WT) and AR knockout (AR) mice suffered from transient middle cerebral artery occlusion (tMCAO) injury (1 h occlusion followed by 3 days reperfusion), and scutellarin was administered from 2 h before surgery to 3 days after surgery. Subsequently, neurological function was assessed by the modified Longa score method, the histopathological morphology observed with 2,3,5-triphenyltetrazolium chloride (TTC) and hematoxylin-eosin (HE) staining. Enzyme-linked immunosorbent assay (Elisa) was used to detect the levels of ROS, 4-hydroxynonenal (4-HNE), 8-hydroxydeoxyguanosine (8-OHDG), Neurotrophin-3 (NT-3), poly ADP-ribose polymerase-1 (PARP1) and 3-nitrotyrosine (3-NT) in the ischemic penumbra regions. Quantitative proteomics profiling using quantitative nano-HPLC-MS/MS were performed to compare the protein expression difference between AR and WT mice with or without tMCAO injury. The expression of AR, nicotinamide adenine dinucleotide phosphate oxidases (NOX1, NOX2 and NOX4) in the ipsilateral side of ischemic brain were detected by qRT-PCR, Western blot and immunofluorescence co-staining with NeuN.
Scutellarin treatment alleviated brain damage in tMCAO stroke model such as improved neurological function deficit, brain infarct area and neuronal injury and reduced the expression of oxidation-related products, moreover, also down-regulated tMCAO induced AR mRNA and protein expression. In addition, the therapeutic effect of scutellarin on the reduction of cerebral infarction area and neurological function deficits abolished in AR mice under ischemia cerebral injury, which indicated that the effect of scutellarin treatment on tMCAO injury is through regulating AR gene. Proteomic analysis of AR and WT mice indicated AR knockout would affect oxidation reaction even as NADPH related process and activity in mice under cerebral ischemia conditions. Moreover, NOX isoforms (NOX1, NOX2 and NOX4) mRNA and protein expression were significant decreased in neurons of penumbra region in AR mice compared with that in WT mice at 3d after tMCAO injury, which indicated that AR should be the upstream protein regulating NOX after cerebral ischemia.
We first reported that AR directly regulates NOX subtypes (not only NOX2 but also NOX1 and NOX4) after cerebral ischaemic injury. Scutellarin specifically targets the AR-NOX axis and has antioxidant effects in mice with cerebral ischaemic injury, providing a theoretical basis and accurate molecular targets for the clinical application of scutellarin.
氧化应激在缺血性中风的病理中起着重要作用。研究已经证实，野黄芩苷具有对抗缺血性损伤的抗氧化作用，我们还报告了醛糖还原酶（AR）在氧化应激和脑缺血损伤中的参与，在这项研究中，我们进一步明确了野黄芩苷对脑缺血损伤的抗氧化作用是否与 AR 基因调节及其具体机制有关。
C57BL/6N 小鼠（野生型，WT）和 AR 敲除（AR）小鼠遭受短暂性大脑中动脉闭塞（tMCAO）损伤（1 小时闭塞，随后 3 天再灌注），从手术前 2 小时开始至手术后 3 天给予野黄芩苷。随后，通过改良的 Longa 评分法评估神经功能，用 2,3,5-三苯基氯化四唑（TTC）和苏木精-伊红（HE）染色观察组织病理学形态。酶联免疫吸附测定（ELISA）用于检测缺血半影区中 ROS、4-羟壬烯醛（4-HNE）、8-羟基脱氧鸟苷（8-OHDG）、神经营养因子-3（NT-3）、聚 ADP-核糖聚合酶-1（PARP1）和 3-硝基酪氨酸（3-NT）的水平。使用定量纳米 HPLC-MS/MS 进行定量蛋白质组学分析，比较 AR 和 WT 小鼠在有或没有 tMCAO 损伤时的蛋白质表达差异。通过 qRT-PCR、Western blot 和 NeuN 免疫荧光共染色检测缺血侧大脑中 AR、烟酰胺腺嘌呤二核苷酸磷酸氧化酶（NOX1、NOX2 和 NOX4）的表达。
野黄芩苷治疗减轻了 tMCAO 中风模型中的脑损伤，如改善神经功能缺损、脑梗死面积和神经元损伤，并降低了氧化相关产物的表达，此外，还下调了 tMCAO 诱导的 AR mRNA 和蛋白质表达。此外，在缺血性脑损伤中，AR 敲除小鼠的脑梗死面积和神经功能缺损的治疗效果在 AR 小鼠中被取消，这表明野黄芩苷治疗 tMCAO 损伤是通过调节 AR 基因。AR 和 WT 小鼠的蛋白质组学分析表明，AR 敲除会影响氧化反应，甚至影响 NADPH 相关过程和活性在缺血条件下的小鼠。此外，与 WT 小鼠相比，tMCAO 损伤后 3 天，AR 小鼠的半影区神经元中 NOX 同工型（NOX1、NOX2 和 NOX4）mRNA 和蛋白质表达显著降低，这表明 AR 应该是缺血后调节 NOX 的上游蛋白。
我们首次报道 AR 直接调节缺血性脑损伤后的 NOX 亚型（不仅是 NOX2，还有 NOX1 和 NOX4）。野黄芩苷特异性靶向 AR-NOX 轴，对脑缺血损伤小鼠具有抗氧化作用，为野黄芩苷的临床应用提供了理论依据和准确的分子靶点。
