Notoginsenoside R1 Improves Cerebral Ischemia/Reperfusion Injury by Promoting Neurogenesis via the BDNF/Akt/CREB Pathway

Abstract

Notoginsenoside R1 (R1), a major component isolated from P. notoginseng, is a phytoestrogen that exerts many neuroprotective effects in a rat model of ischemic stroke. However, its long-term effects on neurogenesis and neurological restoration after ischemic stroke have not been investigated. The aim of this study was to evaluate the effects of R1 on neurogenesis and long-term functional recovery after ischemic stroke. We used male Sprague-Dawley rats subjected to middle cerebral artery occlusion/reperfusion (MCAO/R). R1 was administered by intraperitoneal (i.p.) injection immediately postischemia. We showed that R1 significantly decreased infarct volume and neuronal loss, restored neurological function, and stimulated neurogenesis and oligodendrogenesis in rats subjected to MCAO/R. More importantly, R1 promoted neuronal proliferation in PC12 cells in vitro. The proneurogenic effects of R1 were associated with the activation of Akt/cAMP responsive element-binding protein, as shown by the R1-induced increase in brain-derived neurotrophic factor (BDNF) expression, and with the activation of neurological function, which was partially eliminated by selective inhibitors of BDNF and PI3K. We demonstrated that R1 is a promising compound that exerts neuroprotective and proneurogenic effects, possibly via the activation of BDNF/Akt/CREB signaling. These findings offer insight into exploring new mechanisms in long-term functional recovery after R1 treatment of ischemic stroke.

Keywords: ischemic stroke; neurogenesis; neurological recovery; notoginsenoside R1; oligodendrogenesis.

FIGURE 1

Schematic graphic of the rat experimental design. On the left is the group information, and on the right is the timeline of the experimental process.

FIGURE 2

R1 reduces the infarction volume and neuronal loss in rats subjected to MCAO. R1 and edaravone were both intraperitoneally injected immediately after MCAO surgery (A) Effects of R1 on infarction volumes (n = 5) (B) Quantitative analysis of cerebral infarct volumes (C) H&E staining and Nissl staining of the cortex and hippocampal CA1 regions of each group (n = 5) (D) Relative density (% of sham) of the H&E and Nissl staining in the cortex and hippocampal CA1 regions in all the groups. Scale bar = 200 μm. Data are expressed as the mean ± SD and were analyzed by ANOVA. ^# p< 0.05 and ^## p< 0.01 vs. Sham group; *p< 0.05 and **p< 0.01 vs. MCAO/R group.

FIGURE 3

R1 restores long-term neurological function in rats subjected to MCAO. The behavioral tests were conducted on days 1, 7, 14, 21 and 28 after MCAO surgery (A) Zea-Longa score (B) cylinder tests (C) novel object recognition tests on day 28 after MCAO surgery (D) The body weight of the rats in each treatment group over 28 days. Data are expressed as the mean ± SD and were analyzed by ANOVA. ^# p< 0.05 and ^## p< 0.01 vs. Sham group; *p< 0.05 and **p< 0.01 vs. MCAO/R group.

FIGURE 4

R1 enhances neural reconstruction by stimulating neurogenesis after ischemic stroke. Representative images of the infarction area costained with antibodies against (A) DCX (green, marker of migrating and immature neurons) and EdU (red, marker of proliferating cells) on day 7 after R1 treatment, yellow arrows indicate migrating neuroblasts (EdU⁺/DCX⁺ cells) (B) Nestin (green, marker of proliferating NPCs) and EdU (red) on day 7 after R1 treatment, yellow arrows indicate proliferating NPCs (EdU⁺/Nestin⁺ cells) (C) NeuN (green, marker of mature neurons) and EdU (red) on day 28 after R1 treatment, yellow arrows indicate newly formed mature neurons (EdU⁺/NeuN⁺ cells). DAPI (blue) indicates the nucleus, Scale bar = 50 μm (D-F) Quantitative analysis of (A-C) in the striatum region respectively after R1 treatment. n = 3 brains per group. Data are expressed as the mean ± SD and were analyzed by ANOVA. ^# p< 0.05, ^## p< 0.01 vs. Sham group; ^* p< 0.05, **p< 0.01 vs. MCAO/R group.

FIGURE 5

R1 facilitates oligodendrogenesis and preserves myelin after ischemic stroke (A) Representative images of the infarction area costained with antibodies against APC⁺ (green, marker of oligodendrocytes) and EdU (red, marker of proliferating cells) on day 28 after R1 treatment, yellow arrows indicate proliferated oligodendrocytes (EdU⁺/APC⁺ cells), DAPI (blue) indicates the nucleus, Scale bar = 50 μm (B) Quantitative analysis of proliferating oligodendrocytes (EdU⁺/APC⁺ cells) in the cortex region after R1 treatment. n = 3 brains per group. Representative images of immunoblotting (C) and quantification of cnpase (cyclicnucleotide 30-phosphohydrolase, D), MBP (myelin basic protein, E), and vimentin (F) in the infarct cortex region of the Sham, MCAO/R, R1, and MCAO/R + R1 groups on day 28 after MCAO surgery. n = 3 in each group. Data are expressed as the mean ± SD and were analyzed by ANOVA. ^# p< 0.05, ^## p< 0.01 vs. Sham group; ^* p< 0.05, **p< 0.01 vs. MCAO/R group.

FIGURE 6

R1 increases neurotrophic factor expression and restores disrupted neural synaptic function after ischemic stroke. The serum and cortex tissue levels of neurotrophic factors, such as BDNF (A, B), NGF (C, D), and NT-4 (E, F) were detected by ELISA. (G) Representative images of the infarction area costained with antibodies against BDNF (red) and NeuN (green) on day 28 after R1 treatment, yellow arrows indicate NeuN-positive neurons expressed BDNF, DAPI (blue) indicates the nucleus, Scale bar = 50 μm. Representative images of immunoblotting (H) and quantification of the relative protein levels of SYN (I), PSD95 (J), MAP-1 (K) and Tau-1 (L) in the infarct cortex region of the Sham, MCAO/R, R1, and MCAO/R + R1 groups on day 28 after MCAO surgery. n = 3 in each group. Data are expressed as the mean ± SD and were analyzed by ANOVA. ^# p< 0.05, ^## p< 0.01 vs. Sham group; ^* p< 0.05, **p< 0.01 vs. MCAO/R group.

FIGURE 7

R1 activates the BDNF/Akt/CREB signaling pathway to promote the recovery of neurological function. Representative images of immunoblotting (A) and quantification of the relative protein levels of BDNF (B), p-TrkB/TrkB (C), p-CREB/CREB (D) and (E) p-Akt/Akt in the infarct cortex region of the Sham, MCAO/R, R1, and MCAO/R + R1 groups on days 7 and 28 after MCAO surgery. n = 3 in each group. In situ MALDI MSI of glutamate (F), N-acetylaspatate (G) and K⁺ (H) (I-K) Quantitative measurement of the corresponding indicators in the ischemic cortex. Scale bar = 2 cm, n = 5 in each group. Data are expressed as the mean ± SD and were analyzed by ANOVA. ^# p< 0.05, ^## p< 0.01 vs. Sham group; ^* p< 0.05, **p< 0.01 vs. MCAO/R group.

FIGURE 8

R1 promotes neuronal proliferation though the BDNF and PI3K signaling pathways in vitro (A) R1 promotes neuronal proliferation. PC-12 cells were treated with or without R1 (drug concentration reached up to 100 µM) for 12 h. Images of proliferating neurons detected by EdU staining. Nuclei were visualized by DAPI staining (blue). Scale bar = 200 μm (B) PC-12 cells were pretreated with or without R1 (25 µM) in the presence or absence of ANA-12 (10 μM) or LY294002 (10 μM) for 12 h. Images of proliferating neurons detected by EdU staining. Nuclei were visualized by DAPI staining (blue). Scale bar = 200 μm (C, D) Proliferation rate of EdU⁺ cells was analyzed. Representative images of immunoblotting (E) and quantification of the relative protein levels of BDNF (F), p-TrkB/TrkB (G), p-CREB/CREB (H) and p-Akt/Akt (I) in PC-12 cells. n = 3 in each group. Data are expressed as the mean ± SD and were analyzed by ANOVA. ^# p< 0.05, ^## p< 0.01 vs. Cont group; ^* p< 0.05, **p< 0.01 vs. OGD/R group; and p< 0.05, and p< 0.01 vs. R1 group.

FIGURE 9

Schematic diagram of the mechanisms underlying the R1-induced promotion of neurological recovery after ischemia-reperfusion injury. Overall, R1 enhances neurogenesis and oligodendrogenesis by promoting NPC proliferation, migration and the survival of newborn neurons. Therefore, R1 stimulates the expression of neural synaptic factors and facilitates the release of neurotransmitters. R1 activates the BDNF/Akt/CREB signaling pathway, thereby accelerating neurogenesis to promote neurological recovery.