A Chinese Herbal Formulation, Xiao-Er-An-Shen Decoction, Attenuates Tourette Syndrome, Possibly by Reversing Abnormal Changes in Neurotransmitter Levels and Enhancing Antioxidant Status in Mouse Brain

Abstract

Xiao-Er-An-Shen Decoction (XEASD) has been used clinically for the treatment of Tourette syndrome (TS) in children for more than 20 years in mainland China. The biochemical mechanism underlying the therapeutic action produced by XEASD treatment against TS remains unknown. However, a previous study has shown that pre-incubation of PC12 neuronal cells with XEASD can induce neurite outgrowth and protect against oxidative stress. In the present study, using a mouse model of TS induced by 3,3'-iminodipropionitrile (IDPN), stereotypy scoring, and locomotor activity were assessed. Levels of neurotransmitters including glutamate, aspartate, and gamma-aminobutyric acid (GABA) in brain tissue as well as plasma cyclic adenosine monophosphate (cAMP) were measured using assay kits. The ratio of reduced glutathione (GSH)/oxidized glutathione (GSSG) and Mn-superoxide dismutase (MnSOD) activity in brain mitochondrial fractions as well as mitochondrial glutathione reductase and cytosolic γ-glutamylcysteine activities were also examined. The phosphorylation of cAMP-responsive element binding protein (CREB) in brain tissue was measured by Western blot analysis. XEASD treatment was found to significantly ameliorate the severity of behavioral symptoms in affected mice, as evidenced by decreases in the stereotypy score and locomotor activity. The beneficial effect of XEASD was accompanied by the reversal of abnormal levels of GABA, glutamate, and aspartate, in brain tissue of IDPN-challenged mice. In addition, XEASD treatment increased plasma cyclic adenosine monophosphate (cAMP) levels and activated the phosphorylation of CREB in brain tissue of TS mice. Furthermore, XEASD treatment was found to enhance the antioxidant status of brain tissue in affected mice, as evidenced by increases in the GSH/GSSG ratio and the activity of MnSOD in brain mitochondrial fractions. Taken together, these experimental results will hopefully provide insight into the pharmacological basis for the beneficial effects of XEASD in children suffering from TS.

Keywords: Tourette sydrome; Xiao-Er-An-Shen Decoction; antioxidant status; brain; neurotrasmitters.

Figure 1

HPLC-MS chromatogram of XEASD extract. A representative LC-MS chromatogram from three batches of XEASD extract is shown. Eight chemical markers were identified in the XEASD extract. The denotation peaks 1–13 were caffeic acid (1), morroniside (2), loganin (3), liquiritin (4), tenuifolin (5), calycosin-7-O-β-glucoside (6), ammonium glycyrrhizate (7), naringin (8), 3,6’-disinapoyl sucrose (9), hesperidin (10), neohesperidin (11), astragaloside IV (12), and isoimperatorin (13).

Figure 2

Effects of XEASD treatment on locomotor activity of IDPN-induced TS mice. Locomotor activity was measured as described in Materials and Methods. Control 1 represents normal control mice; control 2 represents IDPN-challenged control mice. Each bar represents mean ± SEM, with n ≥ 23. *P < 0.05, significantly different from the control 1; ^# P < 0.05, significantly different from IDPN control 2. Locomotor activity of control 1 (mean ± S.E.M). = 247 ± 14.1.

Figure 3

Effects of XEASD treatment on glutamate and aspartate levels in brain tissue of IDPN-induced TS mice. (A) Biochemical assay of glutamate levels was performed as described in Materials and Methods. (B) Levels of aspartate in brain tissues were measured as described in Materials and Methods. Control 1 represents normal control mice; control 2 represents IDPN-challenged control mice. Data are expressed as the percentage of non-challenged control 1 values. *P < 0.05, significantly different from the control 1; ^# P < 0.05, significantly different from the IDPN control 2. The value of the glutamate levels of control 1 (mean ± S.E.M.) and aspartate level of control 1 (mean ± S.E.M.) were 2953 ± 440 (nmol/mg protein) and 0.73 ± 0.06 (nmol/mg protein), respectively.

Figure 4

Effects of XEASD treatment on gamma-aminobutyric acid (GABA) levels in brain striatum of IDPN-induced TS mice. Biochemical assay of striatal GABA levels was performed as described in Materials and Methods. Control 1 represents normal control mice; control 2 represents IDPN-challenged control mice. Data are expressed as the percentage of non-challenged control 1 values. *P < 0.05, significantly different from the control 1; ^# P < 0.05, significantly different from the IDPN control 2. The value of GABA level of control 1 (mean ± S.E.M.) = 2.03 ± 0.15 (pg/mg protein).

Figure 5

Effects of XEASD treatment on plasma cAMP levels in IDPN-induced TS mice. Biochemical assay of plasma cAMP levels was performed as described in Materials and Methods. Control 1 represents normal control mice; control 2 represents IDPN-challenged control mice. Data are expressed as the percentage of non-challenged control 1 values. *P < 0.05, significantly different from the control 1; ^# P < 0.05, significantly different from the IDPN control 2. The value of cAMP level of control 1 (mean ± S.E.M.) = 0.50 ± 0.05 (pmol/mg protein).

Figure 6

Effects of XEASD treatment on the phosphorylation of CREB in brain tissue of IDPN-induced TS mice. Phosphorylated CREB and total CREB were measured by western blot analysis. Control 1 represents normal control mice; control 2 represents IDPN-challenged control mice. The values (arbitrary units) for phosphorylated CREB were normalized with reference to total CREB levels (arbitrary units) in the samples and expressed in % control 1. *P < 0.05, significantly different from the control 1; ^# P < 0.05, significantly different from the IDPN control 2.

Figure 7

Effects of XEASD treatment on brain mitochondrial antioxidant status in IDPN-induced TS mice. (A) Biochemical assay of MnSOD activity was performed as described in Materials and Methods. (B) Mitochondrial GR activity was measured as described in Materials and Methods. (C) Mitochondrial GSSH/GSSG ratio was measured as described in Materials and Methods. Control 1 represents normal control mice; control 2 represents IDPN-challenged control mice. Data are expressed as the percentage of non-challenged control 1 values. *P < 0.05, significantly different from the control 1; ^# P < 0.05, significantly different from the IDPN control 2. The value of GSH/GSSG ratio of control 1 (mean ± S.E.M.) was 31.3 ± 2.68. The values of GR activity of control 1 (mean ± S.E.M.) and SOD activity of control 1 (mean ± S.E.M.) were 21.1 ± 1.42 (mU/mg protein) and 5.69 ± 0.66 (U/mg protein), respectively.

Figure 8

Effects of XEASD treatment on brain cytosolic GCL activity in IDPN-induced TS mice. Biochemical assay of cytosolic GCL activity was performed as described in Materials and Methods. Control 1 represents normal control mice; control 2 represents IDPN-challenged control mice. Data are expressed as the percentage of non-challenged control 1 values. *P < 0.05, significantly different from the control 1; ^# P < 0.05, significantly different from the IDPN control 2. The value of GCL activity of control 1 (mean ± S.E.M). was 73.4 ± 5.29 (mU/mg protein).