For animal studies, ICA (purity $>$98%) was purchased from Scidoor Hi-tech Biology (Xi’an, Shaanxi, China). ICA was diluted with normal saline and intragastrically administered to mice.

For the cell culture, ICA (purity $>$99%) was purchased from the National Institutes for Food and Drug Control (Beijing, China). ICA was added to the cell culture medium after dilution with phosphate-buffered saline (PBS).

A53T Tg mice (B6. Tg (PDGF-h-$\alpha$-synuclein A53T)-GC/ILAS, CSTR: 16397.09.0H01000945) were purchased from the Center for Experimental Animal Research, Chinese Academy of Medical Sciences (Beijing, China) [18]. Age-matched C57BL/6 (wild type, WT) mice were purchased from Beijing HFK Encapee (Beijing, China).

All mice were housed under a 12 h light/dark cycle with relative humidity of 55–60% and a temperature of 22 $\pm$ 2 °C and free access to water and food. Animal studies were approved by the Bioethics Committee of Xuanwu Hospital of the Capital Medical University (approval number: 20120912) and were performed in accordance with the National Institutes of Health Guide for Care and Use of Laboratory Animals.

A53T Tg and WT mice of two different ages (5 and 10 months old) were allocated to two experimental groups. (1) 5-month-old A53T mice received a daily dose of ICA (either 50 or 100 µmol/kg) or saline (as the model group) over a period of 3 months; coetaneous WT mice were treated with normal saline or 100 µmol/kg ICA; n = 8–10 per group. (2) 10-month-old A53T Tg mice received a daily dose of ICA (either 50 or 100 µmol/kg) or saline (as the model group) over a period of 3 months; WT mice of the same age were treated with normal saline or 100 µmol/kg ICA; n = 10–12 per group. According to the molecular weight of ICA (676.65), the dose of 100 µmol/kg was converted to 67.7 mg/kg and 50 µmol/kg was converted to 33.8 mg/kg at the time of administration to the mice. All treatments were intragastrically administered.

The rotarod test (YLS-4C, Yanyi Life Science, Jinan, Shandong, China) was applied to evaluate the motor coordination and balance of the mice [21]. Mice were trained three times in 5-min trials before the test at a speed of 10 rotations per minute (rpm). The mice were then individually placed on the rotarod with a fixed speed (30 rpm) and cut-off time (180 s). The test was performed five times at intervals of at least 30 min, and the mean of the results was then calculated.

The pole test (XPS-2, Chinese Academy of Medical Sciences and Peking Union Medical, Beijing, China) was conducted to evaluate the coordination function of the mice [22]. The mice were placed head-up on the top of a pole with high rough-surface (height, 50 cm; diameter, 2 cm). The time taken for the 8-month-old mice to climb down the pole was recorded. The test was then performed in three trials at intervals of at least 30 min. Trials were excluded if the mouse jumped off or slid down the pole. The behaviors of the 13-month-old mice were observed as they descended from the top to the bottom of the pole and evaluated using a scoring method. Each mouse was allowed to descend the pole three times and the average score was then calculated. The scoring criteria for the pole test were set as follows: 5 points, use of all limbs to climb down the pole smoothly; 4 points, step-by-step downward spiral crawling, dragging the hind limbs; 3 points, pausing several times during the climb down but holding tightly to the pole; 2 points, sliding on the pole and falling off; and 1 point, inability to grab the pole, directly dropping.

Four mice from each group were anesthetized using 2.5% Avertin (Sigma-Aldrich, St. Louis, MO, USA) and euthanized after behavioral testing. The brain was rapidly removed, and the striatum isolated and homogenized in lysis buffer containing 20 mM Tris-HCl (pH 7.5), 10% glycerol, 150 mM NaCl, 0.5 mM ethylene glycol tetraacetic acid, 1 mM ethylenediaminetetraacetic acid, and a protease inhibitor cocktail (Cat. No. 04693116001; Sigma-Aldrich, St. Louis, MO, USA) [17, 23]. Protein concentration was detected using an RC-DC Protein Assay Kit (Bio-Rad Laboratories, Hercules, CA, USA), and the protein samples were boiled for 5 min before storage.

Proteins were separated using sodium dodecyl sulfate (SDS)–Tris-glycine polyacrylamide gel and transferred onto polyvinylidene difluoride (PVDF) membranes. The following primary antibodies were used: mouse anti-$\alpha$-synuclein (Cat. No. ab1903; Abcam, Cambridge, UK), rabbit anti-p-$\alpha$-syn (Ser129) (Cat. No. ab51253, Ser129-phosphorylated $\alpha$-synuclein, Abcam), rabbit anti-parkin (Cat. No. P6248; Merck Millipore, Darmstadt, Germany), rabbit anti-PLK2 (snk, Cat. No. sc25421; Santa Cruz Biotechnoloy, Santa Cruz, CA, USA), and mouse anti-$\beta$-actin (Cat. No. A5316, Sigma-Aldrich). PVDF membranes were then incubated with horseradish peroxidase-conjugated anti-mouse or anti-rabbit IgG antibody (1:2000). The immune complex was then visualized using enhanced chemiluminescence detection reagents (Cat. No. WBLUF0500, Merck Millipore). Images were captured using the FluorChem E System (Bio-Techne, Minneapolis, MN, USA) and analyzed using AlphaView software (Bio-Techne).

The human dopaminergic cell line SH-SY5Y was purchased from the Cell Resource Center of Peking Union Medical College (Beijing, China). The cell line was previously authenticated by STR and tested for Mycoplasma infection by the Cell Resource Center of Peking Union Medical College. The results indicated that the cell line was derived from human and showed no mycoplasma contamination. The cells transfected with Green fluorescent protein (GFP)-tagged WT $\alpha$-synuclein or vector (gifted by Prof. Hong Ma, Beijing Institute of Technology, Beijing, China) were maintained in Dulbecco’s modified Eagle’s medium (DMEM) (Cat. No. A1896701, Thermo Fisher Scientific, Fair Lawn, NJ, USA) supplemented with 10% fetal bovine serum (FBS) and 0.3 g/L G418 (Cat. No. A1720, diluted in 0.1 M HEPES, Thermo Fisher Scientific, Fair Lawn, NJ, USA) at 37 °C in an incubator humidified with 5% CO${}_2$. All experiments were conducted in triplicate.

Cell viability was detected using the 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. Cells (1 $\times$ 10${}^4$ cells/mL) were seeded and incubated with different doses of ICA (1.56, 3.125, 6.25, 12.5, 25, 50, 100, and 200 µM) for 24 or 48 h. After incubation, MTT was mixed with the medium at a final concentration of 0.5 mg/mL. The MTT solution was removed after 4 h incubation at 37 °C, and 200 µL dimethyl sulfoxide (DMSO) was added to completely dissolve the formazan crystals. The absorbance of each well was measured at 570 nm using a microplate reader (Multiskan Spectrum, Thermo Fisher Scientific, Fair Lawn, NJ, USA). Cell viability was calculated as follows: absorbance (optical density, OD) of the drug-treated groups/absorbance of the vehicle-treated group.

For the western blot assay, 2 $\times$ 10${}^5$ cells were seeded in a flask and treated with ICA for 48 h. Cultured cells were harvested and lysed to obtain the protein.

Cells were seeded in 24-well plate and treated with 40 µM ICA for 48 h. After removing the culture medium, cultured cells were fixed in by 4% paraformaldehyde and 0.1% Triton X-100 for 30 min and then washed three times in 0.01 M PBS. After blocking with serum, the cultured cells were incubated with mouse anti-$\alpha$-synuclein (1:200, Cat. No. ab1903, Abcam, Cambridge, UK) at 4 °C overnight. After washing in PBS, the fixed cells were incubated with goat anti-mouse IgG (Alexa Fluor 594, Cat. No. A-11005, 1:200, Thermo Fisher Scientific) and Hoechst33342 (Cat. No. R37165, Thermo Fisher Scientific). The cells were covered with mounting medium before visualization using a Nikon 80i microscope (Nikon, Tokyo, Japan).

All data were analyzed using SPSS software (version 20.0, IBM Corp, Armonk, NY, USA). Data were analyzed using one-way analysis of variance (ANOVA) followed by Dunnett’s post-hoc comparisons to identify significant differences among groups. Numerical data are provided as the mean $\pm$ standard error of the mean (SEM). Statistical significance was set at p 0.05.

The rotarod test was applied to detect motor balance and coordination in A53T Tg mice after daily intragastric administration of ICA for 3 months. Both the 8- and 13-month-old A53T Tg mice fell off the rotarod at 30 rpm quicker than the WT control mice, with the 13-month-old A53T Tg group showing a statistically significant difference (p 0.05, Fig. 1A,B). ICA (50 and 100 µmol/kg) treatment increased the time to fall off the rotarod in both age groups of A53T Tg mice, with the ICA (100 µmol/kg) treatment in the 8-month-old A53T Tg group showing a statistically significant difference (p 0.05, Fig. 1A,B).

Fig. 1.

ICA attenuates behavior dysfunction in young and aged A53T $\alpha$-synuclein transgenic mice. The rotarod and pole tests were applied to assess the motor balance and coordination of the A53T Tg mice after intragastric administration of ICA for 3 months. (A) Time on the rotarod (latency to fall off the rotarod) of the 8-month-old mice in the rotarod test. (B) Time on the rotarod of the 13-month-old mice in the rotarod test. (C) Pole test; time spent climbing down the pole by the 8-month-old mice. (D) Pole test; performance score for climbing down the pole of mice at 13 months of age. Data are provided as the mean $\pm$ SEM, n = 4–9 per group (8-month-old mice), n = 9–12 per group (13-month-old mice). *p 0.05, **p 0.01, vs. model group. ICA, icariin; SEM, standard error of the mean; Tg (+), A53T $\alpha$-synuclein transgenic mice; WT, wild type.

The pole test assesses the coordination ability of mice. The 8-month-old A53T Tg mice climbed down the pole quicker than the WT control mice, whereas ICA-treated A53T Tg mice took longer to climb down the pole than the vehicle-treated A53T Tg mice, albeit without statistically significant differences (Fig. 1C). For 13-month-old mice, we evaluated the pole test performance score. A53T Tg mice had lower scores than the WT group (p 0.01), and ICA (50 and 100 µmol/kg) treatment significantly increased the scores of A53T Tg mice (p 0.05, p 0.01, Fig. 1D). These results indicate that ICA was able to alleviate impaired motor function and coordination in A53T Tg mice at 8 and 13 months of age.

We used western blotting to detect the expression levels of $\alpha$-synuclein in the striatum of 8-month-old and 13-month-old mice. The results showed that the levels of $\alpha$-synuclein monomers and polymers were increased in the striatum of A53T Tg mice compared with WT mice at 8 months of age (p 0.05, Fig. 2A,B). ICA treatment at doses of 50 and 100 µmol/kg significantly decreased the level of $\alpha$-synuclein monomers (p 0.01), with 100 µmol/kg ICA treatment significantly decreasing the level of $\alpha$-synuclein polymers in the striatum of A53T Tg mice at 13 months of age (p 0.05, Fig. 2C,D). These results indicate that ICA reduced the expression and aggregation of $\alpha$-synuclein in the striatum of A53T mice.

Fig. 2.

Effects of ICA on the expression level and aggregation of $\alpha$-synuclein ($\alpha$-syn) in the striatum of A53T Tg mice (western blotting). (A) Representative blot images of $\alpha$-synuclein in 8-month-old mice. (B) Quantitative analysis of the $\alpha$-synuclein monomer and polymer forms in 8-month-old mice. (C) Representative blot images of $\alpha$-synuclein in 13-month-old mice. (D) Quantitative analysis of the $\alpha$-synuclein monomer and polymer forms in 13-month-old mice. The ratio of $\alpha$-synuclein to $\beta$-actin was taken as 1. Data are provided as the mean $\pm$ SEM, n = 4. *p 0.05, **p 0.01, vs. Tg (+) model group. ICA, icariin; SEM, standard error of the mean; Tg (+), A53T $\alpha$-synuclein transgenic mice; WT, wild type.

Phosphorylation of $\alpha$-synuclein at Ser129 is an important marker of pathological forms of PD and related synucleinopathies. A53T Tg mice at 8 and 13 months of age showed elevated phosphorylation levels of $\alpha$-synuclein at Ser129 in the striatum compared with WT mice (p 0.01, Fig. 3). ICA treatment (100 µmol/kg) significantly decreased the phosphorylation level of $\alpha$-synuclein at Ser129 in the striatum of A53T Tg mice at 8 months old (p 0.01, Fig. 3A,B), but no significant difference was observed in A53T Tg mice at 13 months old (Fig. 3C,D). These results indicate that ICA decreased the phosphorylation of $\alpha$-synuclein, which might inhibit the formation of pathological $\alpha$-synuclein.

Fig. 3.

Effects of ICA on the phosphorylation of $\alpha$-synuclein at serine 129 in the striatum of the A53T Tg mice (western blotting). (A,B) Representative blots and quantitative analysis of p-$\alpha$-syn (Ser129) in the striatum of mice at 8 months of age. (C,D) Representative blots and quantitative analysis of p-$\alpha$-syn (Ser129) in the striatum of mice at 13 months of age. The ratio of p-$\alpha$-syn (Ser129) to $\beta$-actin was taken as 1. Data are provided as the mean $\pm$ SEM, n = 4 per group. **p 0.01, vs. Tg (+) model group. p-$\alpha$-syn (Ser129), $\alpha$-synuclein phosphorylated at serine 129.

Wild-type $\alpha$-synuclein-transfected SH-SY5Y cells were used to investigate the potential effects of ICA on $\alpha$-synuclein expression and phosphorylation in vitro. $\alpha$-Synuclein-transfected SH-SY5Y cells were treated with different dosages of ICA for 48 h. Cell viability after 24 h or 48 h of treatment with ICA was assessed using an MTT assay. The results indicated that ICA did not exhibit any toxic or inhibitory effects on SH-SY5Y cells at a dose range of 1.56–200 µM (Fig. 4A,B).

Fig. 4.

Effects of ICA on the expression and phosphorylation of $\alpha$-synuclein ($\alpha$-syn) in SH-SY5Y cells. (A) Cell viability after a 24-h treatment with ICA at different doses. (B) Cell viability after a 48-h treatment with ICA at different doses; cell viability = optical density (OD) in ICA-treated group/OD in control group. (C,D) Representative western blot images and quantitative analysis of $\alpha$-synuclein in SH-SY5Y cells. (E,F) Representative blots and quantitative analysis of Ser129-phosphorylated $\alpha$-synuclein in SH-SY5Y cells. The ratio of $\alpha$-syn and p-$\alpha$-syn (Ser129) to $\beta$-actin in the vehicle-treated $\alpha$-syn-transfected group was taken as 100%. (G) Representative images of immunocytochemistry staining for $\alpha$-synuclein ($\alpha$-syn, red), GFP (green), and nucleus (Hoechst33342, blue) as well as the merged images; scale bar = 50 µm. Data are provided as the mean $\pm$ SEM, n = 3. ${}^{\mathrm{\#}\mathrm{\#}}$p 0.01, vehicle-treated $\alpha$-syn-transfected group vs. vector control group; *p 0.05, **p 0.01, ICA-treated $\alpha$-syn-transfected group vs. vehicle-treated $\alpha$-syn-transfected group. GFP, green fluorescent protein; p-$\alpha$-syn (Ser129), $\alpha$-synuclein phosphorylated at serine 129.

The expression and phosphorylation levels of $\alpha$-synuclein were detected using western blotting. Compared with vehicle-treated cells transfected with GFP-tagged $\alpha$-synuclein, ICA decreased the elevated expression level of GFP-tagged $\alpha$-synuclein (p 0.05, p 0.01, Fig. 4C,D). Moreover, the level of Ser129-phosphorylated $\alpha$-synuclein was significantly increased in vehicle-treated SH-SY5Y cells transfected with $\alpha$-synuclein, and ICA treatment dose-dependently decreased the level of the Ser129-phosphorylated $\alpha$-synuclein (p 0.01, Fig. 4E,F).

In the western blot assay, ICA at a dose of 40 µM decreased the levels of Ser129-phosphorylated $\alpha$-synuclein without decreasing the elevated expression level of GFP-tagged $\alpha$-synuclein. Since Ser129-phosphorylated $\alpha$-synuclein tends to aggregate into its pathological form, we assessed $\alpha$-synuclein aggregation in SH-SY5Y cells using immunocytochemistry. Morphologically, GFP staining was similar in the two groups of cells, indicating comparable expression levels of transfected $\alpha$-synuclein (shown in green, Fig. 4G). However, detection with the $\alpha$-synuclein antibody revealed that ICA treatment (40 µM) of SH-SY5Y cells transfected with $\alpha$-synuclein reduced the deposition of aberrant $\alpha$-synuclein compared with the vehicle-treated cells (shown in red, Fig. 4G).

These results suggest that ICA may inhibit the overexpression and aggregation of $\alpha$-synuclein in SH-SY5Y cells.

To explain the possible mechanisms through which ICA affects protein expression and phosphorylation, we applied western blotting to detect the expression levels of parkin and PLK2. The expression of parkin was decreased in the brain of A53T Tg mice compared with WT mice at 13 months of age (p 0.05) and ICA treatment significantly increased the expression level of parkin (p 0.05, p 0.01, Fig. 5A,B). This result suggests that ICA may activate parkin-related pathways, including the UPS-related pathways.

Fig. 5.

Effects of ICA on the expression of Parkin and PLK2. (A,B) Representative blots and quantitative data of parkin in the brain of 13-month-old A53T Tg mice; n = 4; *p 0.05, **p 0.01, vs. Tg (+) model group. (C,D) Representative blots and quantitative data of PLK2 in the $\alpha$-synuclein-transfected SH-SY5Y cells; n = 3; ${}^{\mathrm{\#}}$p 0.05, vehicle-treated $\alpha$-syn-transfected group vs. vector control group; *p 0.05, **p 0.01, ICA-treated $\alpha$-syn-transfected group vs. vehicle-treated $\alpha$-syn-transfected group. The ratio of parkin and PLK2 to $\beta$-actin was taken as 1. Data are provided as the mean $\pm$ SEM. PLK2, polo-like kinase 2.

In SH-SY5Y cells transfected with $\alpha$-synuclein, the expression level of PLK2 was significantly increased (p 0.05), while ICA treatment decreased its expression (p 0.05, p 0.01, Fig. 5C,D). This result suggests that ICA inhibits the phosphorylation of $\alpha$-synuclein by regulating PLK2.