Direct Stimulation of Adult Neural Stem/Progenitor Cells In Vitro and Neurogenesis In Vivo by Salvianolic Acid B

Background: Small molecules have been shown to modulate the neurogenesis processes. In search for new therapeutic drugs, the herbs used in traditional medicines for neurogenesis are promising candidates. Methodology and Principal Findings: We selected a total of 45 natural compounds from Traditional Chinese herbal medicines which are extensively used in China to treat stroke clinically, and tested their proliferation-inducing activities on neural stem/progenitor cells (NSPCs). The screening results showed that salvianolic acid B (Sal B) displayed marked effects on the induction of proliferation of NSPCs. We further demonstrated that Sal B promoted NSPCs proliferation in dose- and time-dependent manners. To explore the molecular mechanism, PI3K/Akt, MEK/ERK and Notch signaling pathways were investigated. Cell proliferation assay demonstrated that Ly294002 (PI3K/Akt inhibitor), but neither U0126 (ERK inhibitor) nor DAPT (Notch inhibitor) inhibited the Sal B-induced proliferation of cells. Western Blotting results showed that stimulation of NSPCs with Sal B enhanced the phosphorylation of Akt, and Ly294002 abolished this effect, confirming the role of Akt in Sal B mediated proliferation of NSPCs. Rats exposed to transient cerebral ischemia were treated for 4 weeks with Sal B from the 7th day after stroke. BrdU incorporation assay results showed that exposure Sal B could maintain the proliferation of NSPCs after cerebral ischemia. Morris water maze test showed that delayed post-ischemic treatment with Sal B improved cognitive impairment after stroke in rats

Transcriptomic Analyses Shed Light on Critical Genes Associated with Bibenzyl Biosynthesis in Dendrobium officinale

The Dendrobium plants (members of the Orchidaceae family) are used as traditional Chinese medicinal herbs. Bibenzyl, one of the active compounds in Dendrobium officinale, occurs in low amounts among different tissues. However, market demands require a higher content of thes compounds to meet the threshold for drug production. There is, therefore, an immediate need to dissect the physiological and molecular mechanisms underlying how bibenzyl compounds are biosynthesized in D. officinale tissues. In this study, the accumulation of erianin and gigantol in tissues were studied as representative compounds of bibenzyl. Exogenous application of Methyl-Jasmonate (MeJA) promotes the biosynthesis of bibenzyl compounds; therefore, transcriptomic analyses were conducted between D. officinale-treated root tissues and a control. Our results show that the root tissues contained the highest content of bibenzyl (erianin and gigantol). We identified 1342 differentially expressed genes (DEGs) with 912 up-regulated and 430 down-regulated genes in our transcriptome dataset. Most of the identified DEGs are functionally involved in the JA signaling pathway and the biosynthesis of secondary metabolites. We also identified two candidate cytochrome P450 genes and nine other enzymatic genes functionally involved in bibenzyl biosynthesis. Our study provides insights on the identification of critical genes associated with bibenzyl biosynthesis and accumulation in Dendrobium plants, paving the way for future research on dissecting the physiological and molecular mechanisms of bibenzyl synthesis in plants as well as guide genetic engineering for the improvement of Dendrobium varieties through increasing bibenzyl content for drug production and industrialization

The effect of Salvianolic acid B on BrdU-incorporation in cultured NSPCs.

<p>Dissociated NSPCs were cultured on poly-L-lysine-coated 12-well chamber slides with or without Sal B for 12 h and were subsequently incubated with BrdU (10 µg/ml) for other 12 h. After treatment, cells were fixed, immunostained with anti BrdU antibody. (A) Visualization of BrdU-positive cells by the Immunofluorescence staining assay on NSPCs. Scale bar: 100 µm. (B) BrdU-positive cells were counted in 10 randomly selected fields from three different chambers. Data represent the mean ± S.D. from three independent experiments. *Significant difference from the control group at <i>P</i><0.05. **Significant difference from the control group at <i>P</i><0.01.</p

Salvianolic acid B up-regulated the self-renew genes of NSPCs.

<p>(A) The mRNA levels of NSPCs self-renew markers were analyzed by RT-PCR. (B) The expression levels were semi-quantified by densitometric measurements, normalized with GAPDH internal control, compared with control, and expressed as means ± S.D from three independent experiments. *Significant difference from the control group at <i>P</i><0.05. **Significant difference from the control group at <i>P</i><0.01.</p

The promotive effect of salvianolic acid B on NSPCs proliferation.

<p>(A) Sal B dose-dependently promoted NSPCs proliferation. (B) Sal B time-dependently promoted NSPCs proliferation. Data represent mean ± S.D. from three independent experiments. **Significant difference from the control group at <i>P</i><0.01. (C–D) Representative microphotographs of formed neurospheres in the absence (C) or presence (D) of Sal B (20 µM). Sal B increased the size of formed neurospheres. Scale bars: 200 µm.</p

Delayed post-ischemic treatment with salvianolic acid B improved cognitive impairment in Morris water maze task.

<p>(A) Place learning with multiple trials. There was a decrease in escape latencies with training in all three groups. (B) In the transfer task, the escape latencies (mean ± S.D.) are compared among sham-operated, untreated ischemic, and Sal B treated groups (n = 8). And the animals from the Sal B-treated group spent more time in the quadrant that contained the escape platform during the place learning than untreated group. *<i>P</i><0.05 as compared with sham group, #<i>P</i><0.05 as compared with model group.</p

Salvianolic acid B increased the number of BrdU-positive cells <i>in vivo</i>.

<p>(A) Representative photomicrographs showing BrdU-positive cells in the hippocampus of sham, ischemia and ischemia+Sal B (50 mg/kg)-treated rats. BrdU-labeled cells were indicated by arrows. Scale bar: 200 µm. (B) Quantification of BrdU-positive cells in the hippocampus. Each column represents the represent the mean ± S.D. (n = 10). **Significant difference from the sham group at <i>P</i><0.01.</p

Salvianolic acid B promoted NSPCs proliferation in a PI3K/Akt -independent pathway.

<p>NSPCs were cultured in the proliferation medium containing Sal B (20 µM) in the presence and absence of the PI3K inhibitor Ly294002 (20 µM), MEK inhibitor U0126 (10 µM) or Notch inhibitor DAPT (10 µM) for 2 days. Cell survival was assessed by MTS assay. Data represent the mean ± S.D. from three independent experiments. **<i>P</i><0.01 as compared with control,##<i>P</i><0.01 as compared with Sal B-treated cells.</p

Screening for NSPCs proliferation-inducing natural materials.

<p>The proliferation-inducing activities on NSPCs of a total of 45 natural compounds, which were from medicinal materials extensively used in China to treat stroke clinically, were tested using a MTS assay, and the results are expressed in fold change relative to the corresponding controls. The proliferation-inducing effect of the most potent compound, Sal B (A) and berberine (B), were indicated by the arrow and its structure is shown in the inset. Data represent the mean ± S.D. from three independent experiments. **Significant difference from the control group at <i>P</i><0.01.</p

Salvianolic acid B activated PI3K/Akt in NSPCs.

<p>Cell lysates from NSPCs treated or untreated with Sal B (20 µM) were subjected to Western blot analysis with antibodies against both total and phosphorylated forms of ERK1/2, and Akt. Actin was used as loading control. (A) Sal B increased the phosphorylation of Akt. Akt phosphorylation in Sal B-treated runners (15 min, 30 min, 60 min, 120 min) were significantly greater than control runners. Histograms represent the change in the phosphorylation of Akt normalized to anti-actin antibodies (n = 4). **Significant difference from the control group at <i>P</i><0.01. (B) PI3K/Akt inhibitors Ly294002 regulated the Sal B-mediated phosphorylation of Akt. Ly294002 abolished the phosphorylation of Akt induced by Sal B. Histograms represent the change in the phosphorylation of Akt normalized to anti-actin antibodies (n = 4). *<i>P</i><0.05 as compared with control, **<i>P</i><0.01 as compared with control, ##<i>P</i><0.01 as compared with Sal B-treated cells. No change in total Akt was observed.</p