Ginseng-Angelica-Sansheng-Pulvis Boosts Neurogenesis Against Focal Cerebral Ischemia-Induced Neurological Deficiency

Abstract

Background: The traditional Chinese medicine Ginseng-Angelica-Shanseng-Pulvis (GASP) has been used to treat stroke for 300 years. This present study investigated if it can induce increases in neurogenesis following cerebral ischemic injury.

Methods: Rats following middle cerebral artery occlusion were orally treated with high, medium, and low doses of a standardized GASP extract.

Results: After 14 days, treatment with GASP improved regional blood flow and infarction volume by magnetic resonance imaging scanning, enhanced Ki67⁺ expression in the subventricular zone, increased brain-derived neurotrophic factor (BDNF) secretion, Nestin, and bone morphogenetic protein (BMP) 2/4 expressions in the hippocampus in a dose-dependent manner. Interestingly, low-dose treatment with GASP downregulated doublecortin and Notch1 expressions in the hippocampus, as well as upregulated glial fibrillary acidic protein expression in the subgranular zone and hairy and enhancer of split (Hes) 5 expression in the hippocampus, while treatment with middle and high doses of GASP reversed these results. Meanwhile, the consumed time was shortened in the basket test and the adhesive removal test and the spending time on exploring novel objects was prolonged by GASP treatment whose effects were more obvious at day 14 post-ischemia.

Conclusion: Our study demonstrates that treatment with GASP increases neurogenesis and ameliorates sensorimotor functions and recognition memory. We hypothesize that these effects are thought be mediated by an effect on the BMP2/4 pathway and Notch1/Hes5 signal. Due to its beneficial efficacy, GASP can be recognized as an alternative therapeutic agent for ischemic stroke.

Keywords: Ginseng-Angelica-Sansheng-Pulvis; ischemic stroke; neural differentiation; neurogenesis; neurological deficiency.

FIGURE 1

(A) The overall experimental process timeline. (B) The schematic of the novel object recognition test. (C) Rat in adhesive removal testing: the rat finds paper type sticking on its forelimb and tries to remove them using its tooth.

FIGURE 2

Assessments of basket test and novel object recognition test outcomes. (A) The basket test was performed before operation as well as 4, 7, and 14 days after operation in each group. (B) Ratio of time spent on novel object was counted at the same time. Data presented as mean ± standard deviation. ^∗p < 0.05, ^∗∗∗p < 0.001 vs. Sham group; ^$p < 0.05, ^$$$p < 0.001 vs. middle cerebral artery occlusion (MCAo) group; ^£££p < 0.001 vs. MCAo + Low-dose Ginseng-Angelica-Shanseng-Pulvis (GASP) (L) group.

FIGURE 3

MRI results of regional cerebral blood flow (CBF) deficits and infarction volume evaluation in T2W after 14 days post-MCAo. (A) Regional CBF deficit (red zones) images in each group. (B) Quantitative analysis of CBF deficits. (C) T2W images of infarction volume in different groups. (D) Quantitative analysis of cerebral infarction volume. Data presented as mean ± standard deviation. ^$$p < 0.01, ^$$$p < 0.001 vs. MCAo group; ^££p < 0.01, ^£££p < 0.001 vs. MCAo + Low-dose GASP (L) group; ^&&p < 0.01, ^&&&p < 0.001 vs. MCAo + Middle-dose GASP (M) group.

FIGURE 4

Ginseng-Angelica-Shanseng-Pulvis extraction advanced cell proliferation in the subventricular zone (SVZ) of the ipsilateral hemisphere. (A) Immunofluorescence staining with Ki67 (green) was presented (magnification ×200). (B) Relative expression of Ki67 in the SVZ was analyzed. Scale bar: 50 μm. Data presented as mean ± standard deviation. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001 vs. Sham group; ^$$$p < 0.001 vs. MCAo group; ^££p < 0.01, ^£££p < 0.001 vs. MCAo + Low-dose GASP (L) group; ^&&&p < 0.001 vs. MCAo + Middle-dose GASP (M) group.

FIGURE 5

Ginseng-Angelica-Shanseng-Pulvis extraction regulated the expressions of doublecortin, NeuN, glial fibrillary acidic protein, and Nestin in the subgranular zone (SGZ) of the ipsilateral hemisphere. (A) Immunofluorescence staining with DCX (green) and NeuN (red) are presented (magnification ×200). Relative expressions of DCX (B) and DCX and NeuN merging (C) were analyzed. (D) The image of immunofluorescence staining with GFAP (green) and Nestin (red) are presented (magnification ×200). Relative expressions of GFAP (E) and Nestin (F) were analyzed. Scale bar: 50 μm. Data presented as mean ± standard deviation. ^∗∗p < 0.01, ^∗∗∗p < 0.001 vs. Sham group; ^$p < 0.05, ^$$$p < 0.001 vs. MCAo group; ^£p < 0.05, ^£££p < 0.001 vs. MCAo + Low-dose GASP (L) group; ^&&p < 0.01, ^&&&p < 0.001 vs. MCAo + Middle-dose GASP (M) group.

FIGURE 6

Ginseng-Angelica-Shanseng-Pulvis extraction regulated hippocampal Nestin, DCX, and brain-derived neurotrophic factor (BDNF) expressions. (A) Representative Western blot results for Nestin, DCX, and BDNF in each group’s hippocampus and quantitative analysis for Nestin (B), DCX (C), and BDNF (D). Data presented as mean ± standard deviation.^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001 vs. Sham group; ^$$p < 0.01, ^$$$p < 0.001 vs. MCAo group; ^££p < 0.01, ^£££p < 0.001 vs. MCAo + Low-dose GASP (L) group; ^&&&p < 0.001 vs. MCAo + Middle-dose GASP (M) group.

FIGURE 7

Ginseng-Angelica-Shanseng-Pulvis extraction manipulated BMP2/4 and Notch1/Hes5 pathways in the hippocampus. (A) Representative Western blot belts for BMP2, BMP4, Notch1, and Hes5 in each group. (B–D) Quantitative analysis for the above proteins. Data presented as mean ± standard deviation. ^∗∗∗p < 0.001 vs. Sham group; ^$p < 0.05, ^$$$p < 0.001 vs. MCAo group; ^£££p < 0.001 vs. MCAo + Low-dose GASP (L) group; ^&&p < 0.01, ^&&&p < 0.001 vs. MCAo + Middle-dose GASP (M) group.

FIGURE 8

The schematic diagram of different effects of three dosages of GASP on neurogenesis in SGZ and SVZ after ischemic stroke.