背景

脑卒中发病人群中大约有87％属于缺血性脑卒中。目前，溶栓和血栓切除术是治疗急性缺血性脑卒中的有效方法。但是，由于严格的时间窗（4.5 h以内）限制以及增加出血风险等不良反应，该种方法只能应用于少数患者。神经保护剂仍然是急性缺血性脑卒中治疗的有效方法，即抑制缺血半暗带神经元等细胞的死亡。到目前为止，单纯采取神经保护剂治疗的策略在实际临床治疗中疗效并不显著，采取有效方法修复神经血管单元的所有组分是促进神经修复的关键策略。三七皂苷制剂广泛用于缺血性脑卒中，三七皂苷R1（notoginsenoside R1，NGR1）是三七皂苷中分离出的特有皂苷成分。前期课题组研究发现，NGR1预防给药对缺血性脑卒中急性期的损伤具有明显的神经保护作用，但NGR1治疗给药能否对缺血性脑卒中神经修复期起到显著作用尚缺乏报道。

目的

本课题从血管新生（angiogenesis）和神经发生（neurogenesis）两个关键环节，通过深入挖掘NGR1促进缺血性脑卒中神经修复的作用途径和关键靶点，系统阐明NGR1促进缺血性脑卒中神经修复的作用机制，为进一步研究三七皂苷类成分治疗缺血性脑卒中的作用机制提供实验依据，并为三七的临床应用奠定理论基础。

方法

建立脑中动脉缺血/再灌（middle cerebral artery occlusion/reperfusion，  MCAO/R）大鼠脑缺血模型与氧糖剥夺（oxygen glucose deprivation/reperfusion，OGD/R）诱导的细胞模型。NGR1于MCAO手术后立即腹腔注射，连续给药28天。①采用TTC染色法检测NGR1不同给药剂量对大鼠脑梗死体积的影响；②采用免疫学手段（尼氏染色法）评价NGR1不同给药剂量对大鼠皮层和海马神经元形态和活力的影响；③通过多普勒成像、免疫荧光双标、双光子成像、透射电镜、划痕实验、EdU增殖实验和成管实验考察NGR1对血管新生的促进作用；④通过ELISA、基质辅助激光解吸附/电离质谱成像技术（matrix-assisted laser desorption/ionization mass spectrometry imaging, MALDI-MSI）、划痕实验、EdU增殖实验、western blot法分析NGR1促进血管新生的作用机制，并采用烟酰胺磷酸核糖转移酶（nicotinamide phosphoribosyltransferase，NAMPT）抑制剂FK866和SIRT1抑制剂Ex-527进一步验证NGR1对缺血性脑卒中血管新生的分子机制。

⑤通过行为学实验、免疫荧光双标、western blot、ELISA、MALDI-MSI、划痕实验和EdU增殖实验考察NGR1对修复神经功能和神经发生的促进作用。⑥通过免疫荧光双标、划痕实验、EdU增殖实验、western blot法分析NGR1促进神经发生的作用机制，并采用TrkB抑制剂ANA-12和PI3K 抑制剂LY294002进一步验证NGR1对缺血性脑卒中神经发生的分子机制。

结果

与模型组相比，高剂量（40 mg·kg^-1）和中剂量（20 mg·kg^-1）NGR1治疗给药7天能显著改善大鼠局部脑梗死体积；改善大鼠皮层和海马CA1区尼氏小体丢失情况、提升神经元活力。另外，NGR1中剂量（20 mg·kg^-1）对海马CA1区尼氏小体丢失的改善及神经元活力的提升作用更加明显。

NGR1（20 mg·kg^-1）治疗给药28天能够显著恢复脑缺血大鼠的脑血流量、促进新生神经元CD31⁺/EdU⁺的增殖、增加血管密度及血管分叉数、增长总血管长度、改善脑微血管内皮细胞结构；显著提高血管生成因子VEGF、TGF-β、b FGF2和PDGF在皮层组织及血清中的表达；显著改善能量代谢因子NAD⁺、ATP、ADP、AMP和GMP表达水平。体外实验结果表明，12.5~50 μM的NGR1孵育处理可显著提高HBMEC脑微血管内皮细胞的迁移、增殖与成管，而NGR1对HBMEC脑微血管内皮细胞迁移和增殖的作用被NAMPT抑制剂FK866和SIRT1抑制剂Ex-527抑制。通过体内外实验进一步发现，NGR1处理可以显著抑制由温度和蛋白酶引起的NAMPT降解，提高NAMPT的表达水平，上调SIRT1表达，SIRT1的上调能够使Notch细胞结构内域NICD脱乙酰化，从而抑制DLL4-Notch信号传导，引起内皮祖细胞中VEGFR-2的上调，从而增强了EPC血管生成功能，而NGR1促血管生成功能被FK866和Ex-527抑制。另一方面，NGR1（20 mg·kg^-1）治疗给药7天处理能够显著提高NAMPT的表达水平，上调NAD⁺，提高SIRT1/2/3的表达，增加线粒体保护作用，改善能量代谢，而NGR1改善能量代谢作用被FK866抑制。

NGR1（20 mg·kg^-1）治疗给药28天能够显著改善脑缺血大鼠各项行为学指标；促进新生神经元DCX⁺/EdU⁺、Nestin⁺/EdU⁺和NeuN⁺/EdU⁺的增殖；促进少突胶质细胞APC⁺/EdU⁺的增殖；提高神经营养因子BDNF、NGF和NT-4在皮层组织及血清中的表达；增加缺血再灌注损伤后突触蛋白SYN、PSD95、MAP-2和Tau-1的表达水平；促进神经递质谷氨酸、N-乙酰天冬氨酸和K⁺的释放。体外实验结果表明，12.5~100 μM的NGR1孵育处理可显著提高PC12神经元细胞的迁移与增殖，而NGR1对PC12神经元细胞迁移和增殖的作用被TrkB抑制剂ANA-12和PI3K抑制剂LY294002抑制。通过体内外实验进一步发现，NGR1处理能够显著提高BDNF的表达水平，特异性激活其受体TrkB的表达，上调BDNF信号传导下游效应因子CREB和Akt的表达, 而NGR1对BDNF/Akt/CREB信号通路的激活被ANA-12和LY294002抑制。

结论

NGR1（20 mg·kg^-1）治疗给药能够显著促进缺血性脑卒中大鼠神经修复，促进血管新生，其作用机制与调控NAMPT/Notch/VEGFR-2信号通路相关。另外，NGR1治疗给药能够显著改善缺血引起的神经功能损伤，促进神经发生，其作用机制与激活BDNF/Akt/CREB信号通路相关。以上发现为三七促进缺血性脑卒中的神经功能修复提供了新的科学依据。

Background

About 87% of patients with stroke are ischemic stroke. Currently, thrombolysis and thrombectomy are effective methods for the treatment of acute ischemic stroke. However, due to strict time window (within 4.5 h) restrictions and adverse effects such as increased bleeding risk, this method can only be applied to a small number of patients. Neuroprotective agents are still an effective method for the treatment of acute ischemic stroke, that is, inhibit the death of cells such as neurons in the ischemic penumbra. So far, this strategy of simply taking neuroprotective agents has not been effective in actual clinical treatment, we must try our best to promote the repair ability of the central nervous system. Notoginsenoside R1（NGR1）is a unique saponins isolated from panax notoginosides. The previous research of the research group found that the preventive administration of NGR1 has obvious neuroprotective effect on the acute stage of ischemic stroke injury, but whether the therapeutic administration of NGR1 can repair neurological function by promoting angiogenesis and neurogenesis has not yet been studied.

Objectives

To explores the targets of NGR1 promoting neurorestoration in ischemic stroke from the two-key links of angiogenesis and neurogenesis, and systematically clarifies the role of NGR1 in promoting neurorestoration in ischemic stroke by digging deeply into the pathways and key targets of NGR1. The mechanism of action provides experimental basis for further research on the treatment of ischemic stroke with administration of Panax notoginseng, and lays a theoretical foundation for the clinical application of Panax notoginseng.

Methods

Establish middle cerebral artery occlusion/reperfusion (MCAO/R)-induced rat cerebral ischemia model and oxygen glucose deprivation/reperfusion (OGD/R)-induced cells model. NGR1 was injected intraperitoneally immediately after MCAO surgery for 28 consecutive days. ①Using TTC staining method to detect the effect of different doses of NGR1 on cerebral infarction volume in rats. ②Using immunological methods (Nissl staining methods) to evaluate the effects of different doses of NGR1 on the morphology and activity of rat cortex and hippocampal neurons. ③Using Doppler imaging, immunofluorescence double labeling, two-photon imaging, transmission electron microscope, scratch experiment, EdU proliferation and tube experiments investigate the promoting effect of NGR1 on angiogenesis. ④Using ELISA, matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI), scratch experiment, EdU proliferation experiment, western blot method to analyze the mechanism of NGR1 promoting angiogenesis, and use the nicotinamide phosphoribosyltransferase (NAMPT) inhibitor FK866 and SIRT1 inhibitor Ex-527 for further verify the molecular mechanism of NGR1 on angiogenesis after ischemic stroke. ⑤Using behavioral experiment, immunofluorescence double labeling, western blot, ELISA, MALDI-MSI, scratch experiment and EdU proliferation experiment. ⑥Using immunofluorescence double labeling, scratch experiment, EdU proliferation experiment, western blot method to analyze the mechanism of NGR1 promoting neurogenesis, and use TrkB inhibitor ANA-12 and PI3K inhibitor LY294002 to further verify the molecular mechanism of NGR1 on neurogenesis after ischemic stroke.

Results

The results showed that the therapeutic administration of NGR1 for 7 days can significantly improve the volume of local cerebral infarction in rats, and the high dose (40 mg·kg^-1) and middle dose (20 mg·kg^-1) are more obvious. Nissl staining results showed that compared with the model group, the loss of Nissl corpuscles in the cortex and hippocampus CA1 area of rats was significantly improved in the NGR1 high dose (40 mg·kg^-1) and middle dose (20 mg·kg^-1) group, the activity of neurons was significantly improved, and no obvious vacuole structure was found. In addition, the middle dose of NGR1 (20 mg·kg^-1) improved the loss of Nissl corpuscles in the CA1 area of the hippocampus and increased neuronal activity more significantly.

The results showed that NGR1（20 mg·kg^-1）treatment for 28 days can significantly restore cerebral blood flow in cerebral ischemic rats, promote the proliferation of new neurons CD31⁺/EdU⁺, increase blood vessel density and the number of vascular bifurcations, increase total blood vessel length, and improve cerebral microvascular endothelial cells structure. NGR1（20 mg·kg^-1）treatment for 28 days can significantly increase the expression of angiogenic factors VEGF, TGF-β, b FGF2 and PGNF in cortical tissues and serum. NGR1（20 mg·kg^-1） treatment for 7 days can significantly improve the expression levels of energy metabolism factors NAD, ATP, ATPase, NADPH, ADP, AMP and GMP. In vitro, 12.5~50 μM NGR1 incubation treatment can significantly improve the migration, proliferation and tube formation of HBMEC brain microvascular endothelial cells. The effect of NGR1 on the migration and proliferation of HBMEC brain microvascular endothelial cells was inhibited by the NAMPT inhibitor FK866 and SIRT1 inhibitor Ex-527. Through in vivo and in vitro experiments, it was further found that NGR1 treatment can significantly inhibit the degradation of NAMPT caused by temperature and protease, increase the expression level of NAMPT, and up-regulate the expression of SIRT1. The up-regulation of SIRT1 can deacetylate the internal domain of Notch. This inhibits DLL4-Notch signal transduction, leading to the up-regulation of VEGFR-2 in endothelial progenitor cells, thereby enhancing EPC angiogenesis. The angiogenesis function of NGR1 is blocked by FK866 and Ex-527. On the other hand, NGR1（20 mg·kg^-1）treatment can significantly increase the expression level of NAMPT, up-regulate NAD⁺, increase the expression of SIRT1/2/3, increase mitochondrial protection, and improve energy metabolism, while the effect of NGR1 on improving energy metabolism is blocked by FK866.

The results showed that NGR1（20 mg·kg^-1）treatment for 28 days can significantly improve the behavioral indicators of cerebral ischemia rats, promote the proliferation of neonatal neurons DCX⁺/EdU⁺, Nestin⁺/EdU⁺, NeuN⁺/EdU⁺. NGR1（20 mg·kg^-1）treatment for 28 days promote the proliferation of oligodendrocyte APC⁺/EdU⁺. The increases the expression of neurotrophic factors BDNF, NGF and NT-4 in cortical tissue and serum, increases the expression levels of synaptic proteins SYN, PSD95, MAP-2 and Tau-1 after ischemia/reperfusion injury, and promotes release of the neurotransmitters, such as glutamate, N-acetylaspartate and K⁺. In vitro, 12.5~100 μM NGR1 incubation treatment can significantly increase the migration and proliferation of PC12 neuronal cells. The effect of NGR1 on PC12 neuronal cell migration and proliferation was inhibited by TrkB inhibitor ANA-12 and PI3K inhibitor LY294002. Through in vivo and in vitro experiments, it was further found that NGR1 treatment can significantly increase the expression level of BDNF, specifically activate the expression of its receptor TrkB, and up-regulate the expression of CREB and Akt, the downstream effectors of BDNF signaling. The activation of BDNF/Akt/CREB signal pathway by NGR1 was inhibited by ANA-12 and LY294002.

Conclusions

NGR1（20 mg·kg^-1）treatment for 28 days can significantly promote neurorestoration and angiogenesis in rats with ischemic stroke. Its mechanism of action is related to the regulation of NAMPT/Notch/VEGFR-2 signaling pathway. In addition, therapeutic administration of NGR1 can significantly improve neurological damage caused by ischemia, and promote neurogenesis. Its mechanism of action is related to the activation of the BDNF/Akt/CREB signaling pathway.