A Chinese Herbal Preparation, Xiao-Er-An-Shen Decoction, Exerts Neuron Protection by Modulation of Differentiation and Antioxidant Activity in Cultured PC12 Cells

Abstract

Xiao-Er-An-Shen Decoction (XEASD), a Chinese herbal formula, has been used in clinic for treating insomnia and mental excitement in children and adolescents. However, less of scientific data supports its effectiveness in clinic. Here, we aim to study the role of XEASD in regulating neuron differentiation and antioxidant activity. An HPLC-MS was used to chemically standardize herbal extract of XEASD. The standardized herbal extracts of XEASD (0.3-3.0 mg/mL) were applied onto cultured PC12 cells for 48 hours. The treatment with XEASD extract induced neurite outgrowth of PC12 cells in a dose-dependent manner, having the highest response by ~50% of differentiated cells. Application of XEASD extract dose dependently stimulated expressions of NF68, NF160, and NF200 in cultured PC12 cells. Furthermore, XEASD activated the phosphorylation of cAMP responsive element binding protein on PC12 cells, the effect of which was blocked by H89, a protein kinase A inhibitor. Moreover, XEASD showed free radical scavenging activity and stimulated the transcriptional activity of ARE. These results supported the neurobeneficial effects of XEASD in the induction of neurite outgrowth and protection against oxidative stress and could be useful for neurological diseases, in which neurotrophin deficiency and oxidation insult are involved.

Figure 1

The representative HPLC-MS chromatogram of XEASD. (a) Structures of chemical markers identified in XEASD extract, including liquiritin (1), calycosin 7-O-β-glucoside (2), ammonium glycyrrhizinate (3), naringin (4), 3,6′-disinapoylsucrose (5), hesperidin (6), neohesperidin (7), astragaloside IV (8), and puerarin (internal standard, ISTD). (b) The representative LC-MS chromatogram of XEASD extract. The LC condition was indicated in the Materials and Methods. The denotations from 1 to 8 in the chromatogram correspond to the chemical markers as shown in (a). The identification of the chemical markers was analyzed by a MS detector in a negative mode except astragaloside IV (8) in a positive mode. Representative chromatograms were shown, n = 3.

Figure 2

XEASD induces neurite outgrowth of cultured PC12 cells. (a) Cultured PC12 cells were treated with XEASD extract at various concentrations (0.3–3.0 mg/mL) for 48 hours. NGF (50 ng/mL) served as the positive control. After treatment, cells were fixed with ice-cold 4% paraformaldehyde, and then the neurite outgrowth was measured under microscope. Representative images were shown, n = 5. Bar = 20 μm. Arrowheads indicate cell neurite. (b) To quantify the differentiation effect, the % of differentiated cell numbers (upper panel) and length of neurite (lower panel) were calculated as indicated in the Materials and Methods. The cells were defined as differentiated if one or more neurites were longer than the diameter of cell body and also classified according to their neurite length in <15 μm, 15–30 μm, and >30 μm. Data are expressed as % of cells in 100 counted cells. Mean ± SEM; n = 4. Statistical comparison was made with the control; ^∗p < 0.05; ^∗∗p < 0.01.

Figure 3

XEASD stimulates neurofilament expression of cultured PC12 cells. (a) XEASD extracts (0.3–3.0 mg/mL) were applied onto cultured PC12 cells for 48 hours. The cell lysates were collected to determine the expressions of NF68, NF160, and NF200. NGF (50 ng/mL) served as the positive control. GAPDH served as a loading control. (b) Quantification plot was shown in histograms. Values are expressed as the fold of increase to basal reading (untreated culture, set as 1). Mean ± SEM; n = 4. Statistical comparison was made with the control; ^∗p < 0.05; ^∗∗p < 0.01; ^∗∗∗p < 0.001.

Figure 4

XEASD induces CREB phosphorylation on PC12 cells. (a) Cultured PC12 cells were serum starved over 5 hours before the treatment with XEASD extract (1 mg/mL) or NGF (50 ng/mL) for different time. Total CREB and phosphorylated CREB (both at ~ 43 kDa) were determined by using specific antibodies. (b) Cultured PC12 cells with serum starvation for over 5 hours were pretreated with or without H89 (5 μM; a PKA inhibitor) for 3 hours prior to the treatment with NGF (50 ng/mL) and XEASD (1.0 mg/mL) for 10 minutes. Total CREB and phosphorylated CREB were determined by using specific antibodies (upper panel). Quantification plot was indicated in histograms (lower panel). Values are expressed as the fold of change (x Basal) against the control (no treatment; set as 1). Mean ± SEM; n = 5. Statistical comparison was made with the H89-treated group; ^∗∗p < 0.01.

Figure 5

Antioxidation activities of XEASD. (a) An aliquot of 50 μL of extracts and vitamin C (Vit. C) was added to a 150 μL DPPH solution. After incubating for 30 min, the decrease in the absorbance of the mixture was determined at 495 nm. The DPPH radical scavenging effect was expressed as % scavenging relative to control (without samples). (b) A luciferase reporter containing four AREs and a downstream luciferase reporter gene, namely, pARE-Luc, was used as a study tool (upper panel). Cultured PC12 cells, transfected with pARE-Luc, were treated with XEASD extracts (0.3–3.0 mg/mL) for 24 hours. The cell lysates were subjected to luciferase assay. Values are expressed as the fold of increase to basal reading (untreated culture), and they are in mean ± SD, where n = 4, each with triplicate samples. Statistical comparison was made with the control; ^∗p < 0.05; ^∗∗p < 0.01.