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

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 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

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

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

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

Reversal of Muscle Atrophy by Zhimu-Huangbai Herb-Pair via Akt/mTOR/FoxO3 Signal Pathway in Streptozotocin-Induced Diabetic Mice

<div><p>Skeletal muscle atrophy is one of the serious complications of diabetes. Zhimu-Huangbai herb-pair (ZB) is widely used in Chinese traditional medicine formulas for treating Xiaoke (known as diabetes) and its complications. However, the effect of ZB on reversal of muscle atrophy and the underlying mechanisms remain unknown. In this research, we investigated the effect and possible mechanisms of ZB on skeletal muscle atrophy in diabetic mice. Animal model of diabetic muscle atrophy was developed by high fat diet (HFD) feeding plus streptozotocin (STZ) injection. After oral adminstration of ZB for 6 weeks, the effects of ZB on reversal of muscle atrophy and the underlying mechanisms were evaluated by biochemical, histological and western blot methods. The skeletal muscle weight, strength, and cross-sectional area of diabetic mice were significantly increased by ZB treatment. Biochemical results showed that ZB treatment reduced the serum glucose level, and elevated the serum insulin-like growth factor 1 (IGF-1) and insulin levels significantly compared with untreated diabetic group. The western blot results showed that ZB activated the mTOR signal pathway, shown as increased phosphorylations (p-) of Akt, mTOR, Raptor, S6K1 and reduced Foxo3 expression compared with the model group. ZB could reverse muscle atrophy in diabetic mice. This may be through activation of mTOR signaling pathway that promotes protein synthesis, and inactivation foxo3 protein that inhibits protein degradation. These findings suggested that ZB may be considered as a potential candidate drug in treatment of diabetic muscle atrophy.</p></div

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 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

ZB increased muscle strength and coordination of diabetic mice.

<p>A: grip strength of the mice during treatment in each group. B: time to fall from an accelerating rotarod of the mice during treatment in each group. Data are expressed as the means±SD. *<i>P</i><0.05, **<i>P</i><0.01 vs. nondiabetic control. ^#<i>P</i><0.05, ^##<i>P</i><0.01 vs. Model group.</p