Ningdong Granule Upregulates the Striatal DA Transporter and Attenuates Stereotyped Behavior of Tourette Syndrome in Rats

Abstract

This study aimed to evaluate the possible mechanism of Ningdong granule (NDG) for the treatment of Tourette syndrome (TS). The rats with stereotyped behavior were established by microinjection with TS patients' sera; then, the model rats were divided into NDG and haloperidol (Hal) group, and the nonmedication model rats were regarded as treatment control (TS group). The stereotyped behavior of the rats was recorded, the level of dopamine (DA) in striatum, and the content of homovanillic acid (HVA) in sera were tested, and dopamine transporter (DAT) expression was measured in the study. The experimental results showed that NDG effectively inhibited the stereotyped behavior (P < 0.01), decreased the levels of DA in the striatum (P < 0.05), increased the content of sera HVA (P < 0.01), and enhanced the protein and mRNA expression of DAT in the striatum (P < 0.01). Additionally, the results also revealed Hal could improve the stereotyped behavior as well but had no remarkable influence on DAT expression and DA metabolism. In conclusion, NDG attenuates stereotyped behavior, and its mechanism of action might be associated with the upregulation of DAT expression to regulate DA metabolism in the brain.

Figure 1

The behavior score was recorded at the start (week 0) and the last day of each week during the medication. Data were presented as mean ± SD (n = 12). There was no significant difference among the four groups at week 0. A week later, the scores of stereotyped behavior in both NDG and Hal groups were, respectively, significantly lower than the TS group, and score of the two groups continued to reduce until week 8. Repeated-measures ANOVA revealed that the NDG-treated rats had a lower score compared with nontreated TS rats (<0.05) and no significant differences with Hal group (P > 0.05). Score of TS group was significantly higher than the other three groups (NDG, Hal, and CTR) (P < 0.01).

Figure 2

DA expression level in striatal homogenate at week 8. Compared to NDG, the content of DA was significantly higher than that in control, Hal, and CTR (a). Compared to week 0, the content of HVA significantly increased after medication at week 8 in NDG, but no significant difference existed in the Hal, CTR, and TS (b). ^∗P < 0.05; ^∗∗P < 0.01.

Figure 3

DAT expression in different groups at week 8. Immunochemistry labeling for DAT in different groups was performed at week 8 during medication (n = 5/group). Insets in (A1-A4) are shown at high magnification in (B1-B4); the arrows point to the area with DAT positive expression (A1-B4). Scale bars = 50 μm in (A4) (applies to A1-A4); scale bars = 25 μm in (B4) (applies to B1-B4). Graphical representation shows more positive area of DAT in the NGD-treated group than the Hal and TS group (C) (n = 5). Representative western blots of DAT expression (n = 5/group), and GAPDH was a loading control (D). Semiquantitative measurements were obtained by normalizing GAPDH loading (E). ^∗∗P < 0.05^∗∗P < 0.01. CTR stands for the CTR group microinfused by normal sera; TS stands for the TS model control without medication; Hal stands for the Haloperidol treatment group; NDG stands for the NDG treatment group.

Figure 4

Relative mRNA expression of DAT was analyzed by the 2-Delta Delta C(T) method. Compared to the control group, significantly different from the NDG group (^∗∗P < 0.01), but no significant difference from the Hal group and the CTR group.

Figure 5

The relationship among the expression of DAT and HVA and score of stereotyped behavior in the NDG group. Scatterplot of correlative analysis between the increased HVA concentration in sera and the DAT protein expression at week 8 showing a positive correlation in the NDG group (r = 0.917, n = 12, P < 0.01) (a). Scatterplot of correlative analysis between the improved score of stereotyped behaviors and the DAT protein expression at week 8 showing a positive correlation in the NDG group (r = 0.895, n = 12, P < 0.01) (b). Correlative analysis scatterplot between the improved score of stereotyped behavior and the increased HVA concentration in sera showing a positive correlation upon an 8-week period of medication in the NDG group (r = 0.936, n = 12, P < 0.01) (c).