Neuroprotective Efficacy of Astragalus mongholicus in Ischemic Stroke: Antioxidant and Anti-Inflammatory Mechanisms

Abstract

Stroke affects over 12 million people annually, leading to high mortality, long-term disability, and substantial healthcare costs. Although East Asian herbal medicines are widely used for stroke treatment, the pathways of operation they use remain poorly understood. Our study investigates the neuroprotective properties of Astragalus mongholicus (AM) in acute ischemic stroke using photothrombotic (PTB) and transient middle cerebral artery occlusion (tMCAO) mouse models, as well as an in vitro oxygen-glucose deprivation (OGD) model. Post-OGD treatment with AM improved cell viability in mouse neuroblastoma cells, likely by reducing reactive oxygen species (ROS). Mice received short-term (0-2 days) or long-term (0-27 days) AM treatment post-stroke. Infarct size was assessed using a 2,3,5-triphenyl tetrazolium chloride (TTC) staining procedure alongside magnetic resonance imaging (MRI). Neuroprotective metabolites including inositol (Ins), glycerophosphocholine+phosphocholine (GPc+ PCh), N-acetylaspartate+N-acetylaspartylglutamate (NAA+NAAG), creatine + phosphocreatine (Cr+PCr), and glutamine+glutamate (Glx) were analyzed via magnetic resonance spectroscopy (MRS). Gliosis was assessed using GFAP and Iba-1 immunohistochemical markers, while neurological deficits were quantified with modified neurological severity scores (mNSS). Motor and cognitive functions were assessed using cylinder, rotarod, and novel object recognition (NOR) tests. AM treatment significantly reduced ischemic damage and improved neurological outcomes in both acute and chronic stages of PTB and tMCAO models. Additionally, AM increased neuroprotective metabolites levels, reduced gliosis, and decreased oxidative stress, as evidenced by reduced inducible nitric oxide synthase (iNOS). These findings highlight the antioxidant properties of AM and its strong therapeutic potential for promoting recovery after ischemic stroke by alleviating neurological deficits, reducing gliosis, and mitigating oxidative stress.

Keywords: Astragalus mongholicus; brain injury; cognitive impairment; photothrombotic-induced mouse model; stroke; transient middle cerebral artery occlusion.

Figure 1

AM recovered the cytotoxicity caused by oxygen-glucose deprivation (OGD) conditions in vitro. (A) Experimental scheme. (B) Bright-field microscope image of NS-1 cells after 6 h of OGD. Arrows indicate morphological changes, including distorted cell bodies and prolonged neurites. Scale bar 100 μm. (C) Cell viability was detected in NS-1 cells after 10 h of OGD and after 24 h of reoxygenation with vehicle or AM administration. (D) ROS levels were assessed using the DCFDA assay in NS-1 cells after 24 h of OGD and 24 h of reoxygenation with vehicle or AM treatment. Value points are expressed as mean ± SEM. Statistical analysis was conducted with one-way ANOVA followed by Tukey’s multiple comparison test for cell viability and an unpaired t-test for ROS levels. ** p < 0.01, **** p < 0.0001.

Figure 2

AM administration in the PTB stroke mouse model significantly reduced infarct volume and improved neurological severity. (A) Experimental schematic. (B) mNSS score (PTB-V, n = 7, PTB-AM, n = 7) and (C) TTC staining images and quantification of the infarct ratio between the PTB-V and PTB-AM groups (PTB-V, n = 6, PTB-AM, n = 7). Statistical significance was evaluated using an unpaired t-test. Values are presented as mean ± SEM. Significance thresholds: * p < 0.05, and ** p < 0.01.

Figure 3

AM administration lowered cerebral I/R injury and increased the level of metabolites in tMCAO mouse brain. (A) Experimental schematic. (B) TTC staining and (C) quantification of infarct ratio between tMCAO-V and tMCAO-AM groups (tMCAO-V, n = 7, tMCAO-AM, n = 7). (D) Representative images of the penumbra region (red dotted lines) in tMCAO-V and tMCAO-AM groups, and (E) quantification of penumbra area in MRI T2 imaging (tMCAO-V, n = 8, tMCAO-AM, n = 5). (F) Magnetic resonance spectroscopy (MRS) signal intensities in tMCAO-V and tMCAO-AM groups. (G) The analysis of the levels of metabolites comparing tMCAO-V and tMCAO-AM groups (Ins, Contralateral. n = 14; tMCAO-V, n = 8; tMCAO-AM, n = 5; GPc+PCh, Contralateral. n = 13–14; tMCAO-V, n = 8; tMCAO-AM, n = 6; Cr+PCr, Contralateral. n = 14; tMCAO-V, n = 8; tMCAO-AM, n = 6; NAA+NAAG, Contralateral. n = 14; tMCAO-V, n = 8; tMCAO-AM, n = 6; Glx, Contralateral. n = 14; tMCAO-V, n = 8; tMCAO-AM, n = 6). Data are shown above as mean ± SEM. Statistical significance was assessed using an unpaired t-test for TTC staining and infarct ratio and a one-way ANOVA followed by Tukey’s multiple comparison test for MRS. Significance thresholds: * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Figure 4

AM alleviates gliosis and oxidative stress in tMCAO mouse brain. (A,B) Representative images and quantification of GFAP immunofluorescence in subventricular zone (SVZ) and hippocampus (HP). Scale bars, 100 μm. (sham, n = 6; tMCAO-V, n = 6; tMCAO-AM, n = 6). (C,D) Representative images and quantification of Iba-1 immunofluorescence in the SVZ and HP. (sham, n = 6; tMCAO-V, n = 6; tMCAO-AM, n = 6). (E,F) Representative Western blot images and quantification of TNF-α protein levels in the penumbra area of the tMCAO mouse brain. (sham, n = 7, tMCAO-V, n = 8; tMCAO-AM, n = 7). Representative Western blot images and quantification of iNOS protein levels in the penumbra region of the tMCAO mouse brain (sham, n = 7, tMCAO-V, n = 8; tMCAO-AM, n = 7). Data points are presented as mean ± SEM. Statistical analyses were conducted using the one-way ANOVA followed by Tukey’s multiple comparisons test for three group, and unpaired t-test for two-group comparisons. Significance thresholds: * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Figure 5

Experimental design and AM’s effects on memory function deficits in the tMCAO mouse model. (A) Experimental scheme. (B) Neurological score (mNSS) comparing tMCAO-V and tMCAO-AM groups at 1, 3, 7, 10, 14, and 21 days post-tMCAO (tMCAO-V, n = 5; tMCAO-AM, n = 10). (C) Survival probability comparison between tMCAO-V and tMCAO-AM groups (tMCAO-V, n = 16; tMCAO-AM, n = 14). (D) Quantification of body weight recovery (tMCAO-V, n = 16, tMCAO-AM, n = 14). (E) Rotarod test performance on day 14 (sham, n = 6, tMCAO-V, n = 5, tMCAO-AM, n = 10). (F) Novel object recognition (NOR) test results (sham, n = 6; tMCAO-V, n = 5; tMCAO-AM, n = 10). Values are presented as the mean ± SEM. Statistical analysis for comparisons of mNSS and body weight was conducted using an unpaired t test. Survival rate were analyzed with the log-rank (Mantel–Cox) test. One-way ANOVA followed by Tukey’s multiple comparisons test was used for Rotarod test data. Two-way ANOVA followed by Sidak’s multiple comparisons test was applied for NOR test analysis. Significance thresholds: * p < 0.05, ** p < 0.01, and *** p < 0.001.