Long-term administration of salvianolic acid A promotes endogenous neurogenesis in ischemic stroke rats through activating Wnt3a/GSK3β/β-catenin signaling pathway

Abstract

Stroke is the major cause of death and disability worldwide. Most stroke patients who survive in the acute phase of ischemia display various extents of neurological deficits. In order to improve the prognosis of ischemic stroke, promoting endogenous neurogenesis has attracted great attention. Salvianolic acid A (SAA) has shown neuroprotective effects against ischemic diseases. In the present study, we investigated the neurogenesis effects of SAA in ischemic stroke rats, and explored the underlying mechanisms. An autologous thrombus stroke model was established by electrocoagulation. The rats were administered SAA (10 mg/kg, ig) or a positive drug edaravone (5 mg/kg, iv) once a day for 14 days. We showed that SAA administration significantly decreased infarction volume and vascular embolism, and ameliorated pathological injury in the hippocampus and striatum as well as the neurological deficits as compared with the model rats. Furthermore, we found that SAA administration significantly promoted neural stem/progenitor cells (NSPCs) proliferation, migration and differentiation into neurons, enhanced axonal regeneration and diminished neuronal apoptosis around the ipsilateral subventricular zone (SVZ), resulting in restored neural density and reconstructed neural circuits in the ischemic striatum. Moreover, we revealed that SAA-induced neurogenesis was associated to activating Wnt3a/GSK3β/β-catenin signaling pathway and downstream target genes in the hippocampus and striatum. Edaravone exerted equivalent inhibition on neuronal apoptosis in the SVZ, as SAA, but edaravone-induced neurogenesis was weaker than that of SAA. Taken together, our results demonstrate that long-term administration of SAA improves neurological function through enhancing endogenous neurogenesis and inhibiting neuronal apoptosis in ischemic stroke rats via activating Wnt3a/GSK3β/β-catenin signaling pathway. SAA may be a potential therapeutic drug to promote neurogenesis after stroke.

Keywords: Wnt3a/GSK3β/β-catenin signaling; hippocampus; ischemic stroke; neurogenesis; salvianolic acid A; striatum.

Fig. 1. Chemical structure of SAA and the schematic diagram of the experimental protocols.

a Chemical structure of SAA. b The schematic diagram of the experimental protocols.

Fig. 2. The effects of SAA on neurological function and body weight in ischemic stroke rats.

a Survival curves after stroke, n = 33–47. b Body weight. c The mNSS score. d Longa behavior test. e Prehensile ability test. f The movement routes. g Path length and (h) average velocity of rats in a box within 5 min. The data are presented as the mean ± SEM, n = 7–14; ^#P < 0.05, ^##P < 0.01, and ^###P < 0.001 vs the sham group, ^*P < 0.05, ^**P < 0.01, and ^***P < 0.001 vs the model group.

Fig. 3. The effects of SAA on infarct volume, vascular thrombosis and histopathology after stroke.

a Representative images of DWI_T2 of brain. b Bar graph of infarct volume. c Representative images of TOF_3D of brain. d H&E staining of the hippocampal and striatum. The data are presented as the mean ± SEM, n = 6. ^###P < 0.001 vs the sham group, ^*P < 0.05 and ^**P < 0.01 vs the model group. The images are at ×200 magnification.

Fig. 4. The effect of SAA on the proliferation of NSPCs around the SVZ at 14 d after stroke.

a Representative images of double-labeling immunostaining of Ki67/BrdU⁺ (Ki67-red and BrdU-green) in or near the SVZ. b The cumulative optical density of Ki67 in or near the SVZ. c The number of Ki67-BrdU double (+) cells in or near the SVZ. d The ratio of Ki67-BrdU double (+) cells among BrdU positive cells in or near the SVZ. e Representative images of double-labeling immunostaining of Nestin/BrdU⁺ (Nestin-red and BrdU-green) in or near the SVZ. f The cumulative optical density of Nestin in or near the SVZ. g The number of Nestin-BrdU double (+) cells in or near the SVZ. h The ratio of Nestin-BrdU double (+) cells among BrdU positive cells in or near the SVZ. The data are presented as the mean ± SEM, n = 6; ^#P < 0.05 and ^##P < 0.01 vs the sham group, ^*P < 0.05 and ^**P < 0.01 vs the model group. The images are at ×200 magnification.

Fig. 5. The effects of SAA on neuronal density and neurogenesis at 14 d after stroke.

a Representative images of double-labeling immunostaining of MAP2/BrdU⁺ (MAP2-red and BrdU-green) in the striatum. b The cumulative optical density of MAP2 in the striatum. c The number of MAP2-BrdU double (+) cells in the striatum. d The ratio of MAP2-BrdU double (+) cells among BrdU positive cells in the striatum. e Representative images of double-labeling immunostaining of DCX/BrdU⁺ (DCX-red and BrdU-green) in or near the SVZ. f The cumulative optical density of DCX in or near the SVZ. g The number of DCX-BrdU double (+) cells in or near the SVZ. h The ratio of DCX-BrdU double (+) cells among BrdU positive cells in or near the SVZ. i The longest distance between the location of DCX ( + ) cells and the SVZ. j–m Representative images of MAP2 and GAP43 expression in the striatum and hippocampus. n Representative images of immunohistochemical staining and the pixel area of GAP43 in the striatum. o Bar graph of optical stain intensity from all groups. The data are presented as the mean ± SEM, n = 6; ^#P < 0.05, ^##P < 0.01, and ^####P < 0.0001 vs the sham group, ^*P < 0.05, ^**P < 0.01 and ^***P < 0.001 vs the model group. The images are at ×100 or 200 magnification.

Fig. 6. The effect of SAA on Wnt3a/GSK3β/β-catenin signaling pathway at 14 d after stroke.

a Representative images of expression of Wnt3a and β-catenin in the SVZ. b and c The cumulative optical density of Wnt3a and β-catenin in the SVZ. d–l Representative images of Wnt3a, p-GSK3β, GSK3β, β-catenin, TCF-4, CyclinD1, NeuroD1and BDNF expression in the striatum and hippocampus. The data are presented as the mean ± SEM, n = 3 or 6. ^#P < 0.05, ^##P < 0.01, and ^###P < 0.001 vs the sham group, ^*P < 0.05, ^**P < 0.01 and ^***P < 0.001 vs the model group.

Fig. 7. The effect of SAA on neuronal apoptosis at 14 d after stroke.

a Representative images of TUNEL staining in the SVZ of stroke rats. b The number of TUNEL immunoreactive cells in the SVZ. c The ratio of apoptosis cells relative to total cells in the SVZ. d–i Representative images of p-CREB, CREB, Bcl-2, Bax, cleaved caspase-3 and cleaved caspase-9 expression in the striatum and hippocampus. The data are presented as the mean ± SEM, n = 6. ^#P < 0.05, ^##P < 0.01 and ^###P < 0.001 vs the sham group, ^*P < 0.05, ^**P < 0.01 and ^***P < 0.001 vs the model group. The images are at ×100 magnification.

Fig. 8. Schematic diagram of the enhancement of SAA on endogenous neurogenesis via activating of Wnt3a/GSK3β/β-catenin signaling pathway during the recovery after ischemic stroke.

After stroke, the Wnt3a/GSK3β/β-catenin signaling pathway is inhibited, leading to neuronal apoptosis and neurological deficits. Treatment with SAA promotes the expression of Wnt3a ligand, leading to the Wnt3a-dependent inhibition of GSK3β. β-catenin accumulates in the cytoplasm and translocates to the nucleus, interacts with TCF/LEF and stimulates target genes expression, including CREB, BDNF, CyclinD1 and NeuroD1, facilitating the proliferation, migration and differentiation of NSPCs. In addition, SAA increases the ratio of Bcl-2/Bax and inhibits the expression of caspase-3 and casepase-9 through the enhancement of CREB, which promotes the survival of immature and mature neurons. SAA improves the neurological function through the enhancement of neurogenesis and inhibition of neuronal apoptosis via Wnt3a/GSK3β/β-catenin signaling pathway after ischemic stroke.