Department of Pharmacology, Guangxi Institute of Chinese Medicine and Pharmaceutical Science, Nanning, 530022, China.
Guangxi Key Laboratory of Traditional Chinese Medicine Quality Standards, Nanning, 530022, China.
Phytomedicine. 2023 Dec;121:155104. doi: 10.1016/j.phymed.2023.155104. Epub 2023 Sep 18.
Damaged mitophagy and impaired angiogenesis involve in the pathogenic development of ischemic stroke. Active fraction of Polyrhachis vicina (Roger) (AFPR) showed great potential on neurological disease with it's remarkable anti-inflammatory and anti-oxidative effects.
This study designed to clarify the correlation between Pink1/Parkin-mediated mitophagy and angiogenesis after stroke, and to elucidate the role of SIRT3 in regulating mitophagy and angiogenesis, and to address the mechanism of AFPR on promoting mitophagy and angiogenesis in microvessels endothelium of ischemic brain.
A cerebral ischemia/reperfusion (CIR) rat model was developed by middle cerebral artery occlusion procedure. bEnd.3 cells were exposed to oxygen-glucose deprivation/reoxygenation (OGD/R) to mimic CIR process. Neurological function, mitophagy and angiogenesis related indicators were measured. SIRT3 siRNA and 3-MA were used to verify the interaction between SIRT3-mediated mitophagy and angiogenesis.
CIR rats were orally treated with AFPR (8 and 4 g raw drug /kg) and Nimodipine (10.8 mg/kg) for 12 days to mimic the recovery phase post-stroke. The neurological function assessment, TTC staining, HE staining, TUNEL staining and Nissl staining were performed to assess neuroprotective effects of AFPR against CIR. Then CD31-labeled microvessel density in brain was visualized and quantified by immunofluorescence staining. Mitochondrial ultrastructure was assessed by transmission electron microscope scanning. Expressions of relative proteins,e.g. SIRT3, Pink1, Parkin, LC3-II, p62, VEGFA, involving in mitophagy and angiogenesis, were detected by Western blotting analysis. In vitro, bEnd.3 cells were cultured with AFPR or in combination of autophagy inhibitor 3-MA during the reoxygenation. Then cell viability, and LDH releasing were measured. Angiogenic indicators,such as migration and tube formation activity, VEGFA level were determined. To assess effects of AFPR on mitophagy, mitophagy-related proteins were detected, as well as the autophagosome engulfment and lysosome degradation of mitochondria. To address the role of SIRT3, deacetylation activity of SIRT3 was validated by detecting acetylated FOXO3A level with co-immunoprecipitation (Co-IP) assay. Pre-treatment of siRNA or combination use of 3-MA were used to verify the detailed mechanism.
AFPR remarkably reduced neurological scores and infarct size, alleviated neuron apoptosis in cortex, and increased Nissl density in hippocampus of CIR rats. In addition, AFPR significantly promoted angiogenesis by increasing microvessels density and VEGFA expressions, increased SIRT3 expression, and activated Pink1/Parkin mediated mitophagy. In bEnd.3 cells, the combination use of 3-MA and AFPR further demonstrated that AFPR might promote angiogenesis after OGD/R injury through activating Pink1/Parkin mediated mitophagy. Co-IP assay suggested AFPR reduced acetylated FOXO3A level. This might be correlated with an elevation of SIRT3 expression and it's deacetylation activity. SIRT3 siRNA pretreatment significantly abolished the activation of mitophagy through Pink1/Parkin axis, eventually inhibited angiogenesis.
AFPR promoted angiogenesis through activating mitophagy after cerebral ischemia reperfusion, which might partially involved in the amelioration of SIRT3-mediated regulation on Pink1/Parkin axis. Our study will shed new light on the role of SIRT3 in ischemic brain, especially in regulating mitophagy and angiogenesis after stroke.
受损的线粒体自噬和受损的血管生成参与了缺血性中风的发病机制。中华蜜蜂工蜂（Polyrhachis vicina（Roger））的有效部位（AFPR）因其显著的抗炎和抗氧化作用，在神经疾病方面显示出巨大的潜力。
本研究旨在阐明 Pink1/Parkin 介导的自噬与中风后血管生成之间的关系，阐明 SIRT3 在调节自噬和血管生成中的作用，并探讨 AFPR 促进缺血性脑微血管内皮细胞自噬和血管生成的机制。
通过大脑中动脉闭塞术建立大脑缺血再灌注（CIR）大鼠模型。用氧葡萄糖剥夺/复氧（OGD/R）模拟 CIR 过程，使 bEnd.3 细胞暴露于其中。测量神经功能、自噬和血管生成相关指标。使用 SIRT3 siRNA 和 3-MA 来验证 SIRT3 介导的自噬和血管生成之间的相互作用。
用 AFPR（8 和 4 g 生药/kg）和尼莫地平（10.8 mg/kg）对 CIR 大鼠进行口服治疗 12 天，以模拟中风后的恢复阶段。通过神经功能评估、TTC 染色、HE 染色、TUNEL 染色和尼氏染色来评估 AFPR 对 CIR 的神经保护作用。然后通过免疫荧光染色观察和定量 CD31 标记的脑微血管密度。用透射电子显微镜扫描评估线粒体超微结构。通过 Western blot 分析检测与自噬和血管生成相关的相对蛋白，如 SIRT3、Pink1、Parkin、LC3-II、p62、VEGFA 的表达。在体外，用 AFPR 或结合自噬抑制剂 3-MA 培养 bEnd.3 细胞，在复氧期间。然后测量细胞活力和 LDH 释放。测定血管生成指标，如迁移和管形成活性、VEGFA 水平。为了评估 AFPR 对自噬的影响，检测了自噬相关蛋白，以及线粒体自噬体吞噬和溶酶体降解。为了探讨 SIRT3 的作用，通过共免疫沉淀（Co-IP）检测乙酰化 FOXO3A 水平来验证 SIRT3 的去乙酰化活性。用 siRNA 预处理或联合使用 3-MA 来验证详细的机制。
AFPR 显著降低了 CIR 大鼠的神经评分和梗死面积，减轻了皮质中的神经元凋亡，并增加了海马中的尼氏密度。此外，AFPR 通过增加微血管密度和 VEGFA 表达，增加 SIRT3 表达，激活 Pink1/Parkin 介导的自噬，显著促进了血管生成。在 bEnd.3 细胞中，3-MA 和 AFPR 的联合使用进一步表明，AFPR 可能通过激活 Pink1/Parkin 介导的自噬来促进 OGD/R 损伤后的血管生成。Co-IP 检测表明，AFPR 降低了乙酰化 FOXO3A 水平。这可能与 SIRT3 表达和去乙酰化活性的提高有关。SIRT3 siRNA 预处理显著抑制了通过 Pink1/Parkin 轴的自噬激活，最终抑制了血管生成。
AFPR 通过激活缺血再灌注后的自噬来促进血管生成，这可能部分涉及 SIRT3 对 Pink1/Parkin 轴的调节改善。我们的研究将为 SIRT3 在缺血性脑，特别是中风后调节自噬和血管生成方面的作用提供新的视角。
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