Baicalin regulates neuronal fate decision in neural stem/progenitor cells and stimulates hippocampal neurogenesis in adult rats

Abstract

Background: Recent studies revealed that baicalin, a flavonoid compound derived from the root of Scutellaria baicalensis Georgi, could promote neuron differentiation of NSPCs after commencing the differentiation process in vitro. However, this may not be the most efficacious strategy to determinate cell fate. Here, we have investigated whether baicalin can influence early events of neuron generation and stimulate adult neurogenesis.

Results: Transient exposure of NSPCs to baicalin during proliferation could activate Mash1 to alter the differential fate and increase the proportion of cells expressing neuronal markers. Seven days after, rats were exposed to transient cerebral ischemia, they were treated for 3 weeks with baicalin, BrdU labeling study showed that exposure to baicalin increased the number of newly generated cells in hippocampus, BrdU/NeuN double staining analysis indicated that baicalin could promote new neuron production after cerebral ischemia. Additionally, Morris water maze test showed that delayed postischemic treatment with baicalin improved cognitive impairment.

Conclusions: These results identify the existence of a single molecule, baicalin, which can specify the neuronal fate of multipotent NSPCs and stimulate neurogenesis, making it a promising candidate for developing clinically relevant strategies to manipulate neuronal fate of NSPCs for brain repair.

Figure 1

Effect of baicalin on the cell viability of NSPCs. Dissociated NSC/NPCs were cultured on a 96‐well plate with the indicated baicalin (A) concentration (1, 1.875, 3.75, 7.5, 15, 30 μM) for 2 days. (B) Cell proliferation was measured using a MTS assay kit. Values were expressed as means ± SD of three individual experiments. n = 6, *P < 0.05, **P < 0.01 compared with control.

Figure 2

Effect of baicalin on the bHLH transcription factors expression and the neural fate of NSPCs. The mRNA or protein levels of Mash1 treated with designated concentrations (7.5, 15, 30 μM) of baicalin analyzed by real‐time RT‐PCR or Western blotting. (A) The mRNA levels quantified were calculated by the ΔΔCt method, normalized with GAPDH internal control. Mash1 expression were significantly upregulated in baicalin‐treated NSPCs compared with control (n = 6); (B) Semiquantified scores of Mash1 expression were significantly upregulated in baicalin‐treated NSPCs compared with control, normalized with Actin internal control (n = 6); (C) The mRNA levels of β‐tubulin III, GFAP treated with designated concentrations (7.5, 15, 30 μM) of baicalin for 3 days were analyzed by RT‐PCR. The mRNA levels were semiquantified by densitometric measurements, normalized with GAPDH internal control (n = 6), and expressed as means ± SD of three individual experiments. Values were expressed as means ± SD of three individual experiments. *P < 0.05, **P < 0.01 compared with control.

Figure 3

Baicalin primed toward a neuronal fate of NSPCs during their subsequent differentiation. (A) Fluorescence microscopic photos showing the neural differention of NSPCs treated with designated concentrations (7.5, 15, 30 μM) of baicalin for 3 days in proliferation medium. Red, MAP‐2; Green, GFAP; blue, DAPI. Scale bar, 100 μm. (B) Baicalin significantly increased the fraction of MAP‐2‐positive cells. (C) There was no significant difference in the fraction of GFAP‐positive cells. The data expressed as means ± SD of three individual experiments. n = 8, *P < 0.05, **P < 0.01 compared with control.

Figure 4

Baicalin increased the number of BrdU and BrdU/NeuN double staining‐positive cells in vivo. (A) Representative photomicrographs showing BrdU‐positive cells in the hippocampus of each group. BrdU‐labeled cells (Green) were indicated by arrows. (B) Quantification of BrdU‐positive cells in the hippocampus. (C) Representative photomicrographs showing BrdU/NeuN double staining‐positive cells (newly generated neurons) in the hippocampus of each group. New generated neurons were indicated by arrows. (D) Quantification of new generated neurons in the hippocampus. Scale bar: 200 μm. Each column represents the represent the mean ± SD (n = 3). *P < 0.05, **P < 0.01. Significant difference from the vehicle.

Figure 5

Effects of baicalin on spatial learning in Morris water maze. (A) The ordinate indicated escape latency to the hidden platform, and the abscissa represented the training time. There was a decrease in escape latencies with training in all three groups. Baicalin‐injected rats showed improved performance in the Morris water maze task compared with vehicle on day 5. Data are expressed as means ± SD of 10 rats in each group. *Significant difference from the Sham at P < 0.05, #Significant difference from the vehicle at P < 0.05. (B) Representative examples of search patterns in the pool of each group rats on day 6 after the platform removed.