Tianma Gouteng Yin, a Traditional Chinese Medicine decoction, exerts neuroprotective effects in animal and cellular models of Parkinson’s disease

Tianma Gouteng Yin (TGY) is a traditional Chinese medicine (TCM) decoction widely used to treat symptoms associated with typical Parkinson’s disease (PD). In this study, the neuroprotective effects of water extract of TGY were tested on rotenone-intoxicated and human α-synuclein transgenic Drosophila PD models. In addition, the neuroprotective effect of TGY was also evaluated in the human dopaminergic neuroblastoma SH-SY5Y cell line treated with rotenone and the rotenone intoxicated hemi-parkinsonian rats. In rotenone-induced PD models, TGY improved survival rate, alleviated impaired locomotor function of Drosophila, mitigated the loss of dopaminergic neurons in hemi-parkinsonian rats and alleviated apoptotic cell death in SH-SY5Y cells; in α-synuclein transgenic Drosophila, TGY reduced the level of α-synuclein and prevented degeneration of dopaminergic neurons. Conclusively, TGY is neuroprotective in PD models both in vivo and in vitro.

Scientific RepoRts | 5:16862 | DOI: 10.1038/srep16862 paralysis 1 . However, conclusive experimental evidence to support TGY's clinical application in the treatment of PD is lacking. In this study, rotenone-intoxicated and α -synuclein overexpressed Drosophila models and rotenone-induced hemiparkinsonian rats were employed as in vivo models to study the anti-PD activity of TGY. The underlying neuroprotective mechanism was also analyzed in vitro using rotenone-intoxicated SH-SY5Y cells.

Results
Preparation and LC/MS analysis of TGY. Similar to most TCM formulas, in clinical application, TGY is prepared in decoction. Controlled quality of TGY is pivotal to its pharmacological activity and repeatability of the experiments. In order to keep the consistent quality of TGY as it is used in clinical practice, TGY was extracted by water and prepared scrupulously. The preparation procedure is summarized in Fig. 1A. Quality analysis of TGY was performed with a method we developed (to be published elsewhere) using ultra high-performance liquid chromatography (UPLC) coupled with quadrupole-tandem time-of-flight mass spectrometry (Q-TOF-MS). The TGY powder used in this study, prepared with this method, was analyzed. As shown in Fig. 1B, the MS spectra reveals the presence of genipin-1-beta-gentiobioside, geniposide, baicalin, oroxylin-7-O-glucuronide and wogonoside, by comparison with reference compounds (Fig. 1C) and matrix solvent (Fig. 1D).
TGY antagonized rotenone toxicity in Drosophila. Rotenone has been reported as an environmental neurotoxin inducing PD-like phenotypes in D. melanogaster 7 . Based on the preliminary toxicity assay, male w1118 flies receiving 125 μ M rotenone died within 14 days ( Fig. 2A) and the median lethal dose (LD 50 ) of rotenone was calculated to be 42 μ M (Fig. 2B). To test whether TGY is protective against rotenone intoxication, we fed the flies with rotenone and different dosages of TGY concurrently. As shown in Fig. 2A, almost 80% of the flies died within 7 days and all died by the end of the experiment. However, the flies fed with TGY in addition to rotenone showed better survival rates. More than 80% of the flies treated with 100 mg of TGY were alive on the 7 th day and no less than 30% of flies survived at the end of the experiment. In addition, TGY significantly alleviated motor dysfunction caused by rotenone intoxication (Fig. 2C). In comparison with the normal flies, the flies intoxicated by the sublethal dosage of rotenone (42 μ M) for 14 days exerted distinct motor function loss: most of the flies were unable to climb and stayed at the bottom of the vials. This phenotype was quantified by climbing assay. The result (Fig. 2C) shows that, in the presence of rotenone, the flies commonly could not reach the finishing line within the time limit (50 s). In contrast, in the absence of rotenone, only 15 s were needed for the flies to reach the finishing line. The climbing defect induced by rotenone was partially rescued by TGY treatment. The climbing ability of the flies treated with 100 mg of TGY was significantly improved as indicated by the improvement in the climbing score (~20 s).

TGY reduced α-synuclein level and suppressed neurotoxicity in transgenic Drosophila.
Abnormal α -synuclein accumulation is one of the major culprits causing PD. Drosophila overexpressing α -synuclein recapitulates the key features of human PD 8,9 , such as loss of dopaminergic neurons, neuronal inclusion containing α -synuclein and locomotor defects [8][9][10] . These Drosophila PD models have been successfully utilized in testing the activities of small molecules and gene function 11,12 . In the current study, we tested TGY on flies overexpressing α -synuclein. Wild type human α -synuclein (WT-α -synuclein) was pan-neurally overexpressed by elav-GAL4. The 30-day-old α -synuclein transgenic flies exhibited locomotor dysfunction as indicated by the climbing test scores. The aged α -synuclein transgenic flies took more than 30 s to reach the finishing line while the normal control flies (elav-GAL4/+ ) spent less than 20 s (Fig. 3A). In addition to the behavioral results, the level of the marker protein of dopaminergic neurons, tyrosine hydroxylase (TH), was decreased strikingly in the presence of α -synuclein (Fig. 3B). As a result of α -synuclein toxicity, the flies lost 40% of TH in comparison with normal control flies (Fig. 3D). In contrast, TGY dose-dependently alleviated locomotor dysfunction (Fig. 3A). The flies treated with 100 mg of TGY spent less than 20 s to reach the finishing line, which was similar to the normal controls. As shown in the typical blots, TGY prominently reduced the α -synuclein protein levels ( Fig. 3B,C) in the brains of the flies. Meanwhile, the flies treated with TGY preserved more TH then than untreated α -synuclein overexpressed flies (Fig. 3B,D). The results indicate that TGY alleviated the neurotoxicity triggered by α -synuclein overexpression and subsequently protected the dopaminergic neurons in the brains of the flies.
TGY reduced α-synuclein induced dopaminergic neuron loss in transgenic Drosophila. To further verify whether TGY could specifically counteract dopaminergic neuron degeneration caused by overexpression of α -synuclein, Ddc-GAL4, a dopaminergic neuron-specific GAL4 driver, was employed to overexpress human WT-α -synuclein exclusively in the flies' dopaminergic neurons. Full spectrum analyses of the dopaminergic neuron systems of the flies' brains were performed. The flies were fed with TGY for 30 days and then decapitated. The heads were immunostained with TH through whole mount assay. TH-positive neurons in dorsomedial (DM) dopaminergic neuronal cluster of Drosophila brains were shown and counted (Fig. 4A,B). The number of TH positive neurons suggested that TGY prevented (B) The LD50 of rotenone on w 1118 was tested. (C) Climbing assay was performed to test the motor function of the flies. TGY alleviated the impairment of rotenone on motor function. *p < 0.05, **p < 0.01, and ***p < 0.001 compared with rotenone treated flies. Results were obtained from three independent replicates. dopaminergic neuron loss caused by WT-α -synuclein. The immunoblot data (Fig. 4C,D) showed that TGY maintained the TH level in transgenic PD flies, which was consistent with the immunohistochemistry analysis data. To further confirm these findings, dopamine levels in the flies' brains were determined using LC/MS analysis. The result (Fig. 4E) showed that TGY preserved dopamine levels in the brains of transgenic Drosophila. The data of dopamine analysis and the TH protein level data obtained in immunoblot analyses together prove that TGY prevents dopaminergic neuron loss resulting from α -synuclein toxicity. In the climbing assay, TGY exhibited a protective effect in comparison with the untreated model flies (Fig. 4F), which was also consistent with the previous findings.

TGY alleviated rotenone-induced apoptotic cell death in SH-SY5Y cells.
To further investigate the neuroprotective mechanisms of TGY, we tested the anti-apoptosis effect of TGY on human dopaminergic SH-SY5Y cells by adding rotenone to induce apoptotic cell death 13 . SH-SY5Y cells were treated with 20 μ M of rotenone and co-treated with different dosages of TGY for 24 hrs. The cell viability was tested by MTT assay. Result of the MTT assay indicates that TGY prevented cell death induced by rotenone (Fig. 5A). In immunoblot analysis, the pro-survival protein Bcl-2 and the apoptosis marker cleaved-caspase3 were detected. TGY preserved the level of Bcl-2 and reduced the cleavage of caspase3, which indicates TGY alleviated rotenone-induced apoptosis ( Fig. 5C-E).

TGY mitigated dopaminergic neurons loss in hemi-parkinsonism rats. The neuroprotective effect
of TGY was further examined in mammalian PD model. The rotenone-intoxicated hemi-parkinsonian rats were used in the study. Rats stereotaxically infused with rotenone to ventral tegmental area (VTA) and substantia nigra pars compacta (SNpc) increasingly displayed rotational behavior that provoked by apomorphine (APO) injection time dependently 14 . Four weeks after infused with rotenone, the rats displayed strong rotational behavior (~10 circles/min, Fig. 6A) after challenged with APO. However, the rats receiving TGY significantly reduced the APO-provoked rotations. The APO-provoked rotations in rotenone-intoxicated rats receiving 3 and 6 g/kg TGY were reduced by nearly 60% compared with those rotenone-intoxicated rats with vehicle treatment. The rotational behavior was further tested at the end of treatment (8 weeks). As shown in Fig. 6B, the rotenone intoxicated rats showed intensified APO-provoked rotations while TGY treatment notably prevented the deterioration. This data indicated TGY antagonize the damaged dopaminergic system function caused by rotenone infusion. To further verify whether TGY could prevent dopaminergic neuron loss, the ipsilateral part of the SNpc of the rats WT-α -synuclein was over expressed under the neuronal specific driver elav-GAL4. The flies were treated with TGY for 30 days. It was found that (A) TGY protects the flies from the impairment of motor function induced by WT-α -synuclein. (B-D) The level of WT-α -synuclein was decreased while the level of Tyrosine hydroxylase (TH), the marker protein of dopaminergic neuron, increased accordingly. *p < 0.05, **p < 0.01, and ***p < 0.001 compared with model flies without treatment. Results were obtained from three independent replicates.
were harvested and subjected to immunoblot analysis. The marker protein of dopaminergic neuron, tyrosine hydroxylase (TH), was probed. The typical blot was shown in Fig. 6C. As shown in the figure, rotenone infusion drastically reduced the level of TH, which indicated rotenone resulted in severe degeneration of dopaminergic neurons. TGY alleviated the degeneration dose dependently. As indicated by the densitometry analysis of the blots (Fig. 6D), almost ~80% of the dopaminergic neurons were loss 8 weeks after rotenone intoxication, while the rats treated with 6 g/kg TGY preserved about 60% of the dopaminergic neurons. The results were further confirmed by histological examination of brain tissues. The typical TH 3,3′-diaminobenzidine (DAB) staining photos of SNpc from each group were shown in Fig. 6E. The integrated intensity of TH immunostaining was examined by densitometry analysis. In SNpc, the results were quite consistent with the immunoblot result, the immunoreactivity of rotenone intoxicated rats showed ~80% loss TGY treated rats showed ~60% of immunoreactivity compared to that of the sham rats (Fig. 6F).

Discussion
In the present study the anti-PD activity of TGY with controlled quality was evaluated in in vivo and in vitro models of PD. Treatment with TGY significantly suppressed rotenone-induced death and locomotor dysfunction of Drosophila. Feeding with TGY notably reduced α -synuclein protein levels and comparatively prevented loss of dopaminergic neurons in the brains of Drosophila. The neuroprotective effect of TGY was further confirmed on hemi-parkinsonian rats. In stereotaxic rotenone intoxication rats, TGY significantly improved the behavior and mitigated dopaminergic neurons loss. Meanwhile, the anti-apoptosis activity of TGY was also verified in SH-SY5Y cells. These results provide experimental evidences supporting the application of TGY for the treatment of PD.
TGY is traditionally used in the form of decoction. Hence, in this study, we used water to extract TGY following a standardized preparation. In the TGY water extract, flavonoids and iridoids, such as baicalin and geniposide, are the most abundant small molecules (Fig. 1B). These compounds have been shown to have anti-inflammatory, anti-oxidative and anti-apoptotic activities [15][16][17][18] . All of the compounds in TGY constitute the chemical foundation of its pharmacological activity. How the components of TGY exert anti-PD activity deserves further investigation.
In this study, the insecticide rotenone was introduced to established in vivo and in vitro PD models. Rotenone was reported as an inhibitor of mitochondrial complex I and an inducer of dopaminergic lesion 19 . Through treating Drosophila with lethal and sublethal doses of rotenone, a PD model was established to evaluate the anti-PD activity of TGY (Fig. 2). In using this model, we found that special attention must be paid to the variability in susceptivity to rotenone of different Drosophila strains. In the present study, we performed the experiments all with the same w 1118 strain 20 . We also treated SH-SY5Y cells 21 with rotenone to induce apoptotic cell death. Mild anti-apoptotic cell death activity of TGY was observed (Fig. 5), indicating anti-apoptotic mechanism plays an important role in the neuroprotective activity of TGY. The neuroprotective effect of TGY in terms of preventing dopaminergic neuron loss caused by rotenone was further confirmed in hemi-parkinsonian rat (Fig. 6). In this model, direct infusion of rotenone into the SNpc and VTA resulted in loss of dopaminergic neurons in these two brain regions. We pretreated the rats prior to rotenone infusion to evaluate the preventive effect of TGY. The data obtained on this hemi-parkinsonian rat model confirmed our finding in Drosophila. In summary, TGY treatment could mitigate the toxicity of rotenone.
In PD, α -synuclein is the major pathogenic protein. The mutations in this protein accelerate its aggregation then contribute to the onset and progress of PD 22 . Thus, reducing the formation and increasing the clearance is a reasonable strategy for developing a therapy for PD. Drosophila is a widely used model organism in studying neurodegenerative diseases 23 . By using the GAL4/upstream activating sequence (UAS) system 24 , ectopic overexpression of human α -synuclein in the brain of Drosophila was established as a genetic PD model 11 . In the current study, we utilized elav-GAL4 25 to drive pan-neural expression of α -synuclein. Overexpressed α -synuclein impaired dopaminergic neurons as indicated by decreased TH level in immunoblot analysis and defective ability in the climbing assay. TGY treatment reduced α -synuclein and rescued the dopaminergic neurons (Fig. 3). In this result, the flies receiving 10 mg TGY did not reduce the level of α -synuclein but alleviated the decrease of TH. This result indicated TGY exhibited neuroprotectivity that prevented the loss of dopaminergic neurons without affecting the level of α -synuclein at this dosage. In other words, TGY protected against α -synuclein-induced neurotoxicity not only through degrading α -synuclein. Another issue is that elav-GAL4 drive the expression of the transgene in the whole neural system. Thus, all of the neurons were affected by α -synuclein. Hence we used a dopaminergic neuron-specific GAL4 driver, Ddc-GAL4, to exclusively express α -synuclein in the dopaminergic neurons of adult flies 26 . We implemented a broad spectrum of analyses focusing on dopaminergic neurons. The results demonstrate that TGY protected dopaminergic neurons from α -synuclein impairment. From these two Drosophila PD models, we provided sufficient evidence that TGY decreases α -synuclein in the neurons and thereby, prevents dopaminergic neuron loss consequently.
In conclusion, our data indicate that TGY is neuroprotective in PD models. This corroborates its ongoing use in traditional Chinese medicine to treat PD-like symptoms. Evidence that it works immediately initiates a flood of further inquiries as to how it works. For instance, how does TGY impact the misfolded and oligomerized α -synuclein that is thought to be more toxic than the monomer 27 ? How does TGY regulate abnormalities in lysosome or proteasome function that is important in clearance of α -synuclein 28 ? In addition, since PD is a disease affecting central nervous system, it would be very important to understand the brain permeability of TGY phytochemicals and their pharmacokinetics features. Further experiments are needed to answer all of these questions.

Preparation of TGY extract and fly food. Dry materials of the ingredients in proportions as listed
here were pulverized. Roots and rhizomes were cut into small pieces prior to being pulverized. One (human) dose of TGY is: Gastrodiae Rhizoma (Tianma) 9 g, Uncaria Ramulus Cum Uncis (Gouteng) 12 g, Haliotidis Concha (Shijueming) 18 g, Gardeniae Fructus (Zhizi) 9 g, Scutellariae Radix (Huangqin) 9 g, Cyathulae Radix (Chuanniuxi) 12 g, Eucommiae Cortex (Duzhong) 9 g, Leonuri Herba (Yimucao) 9 g, Taxilli Herba (Sangjisheng) 9 g, Polygoni Multiflori Caulis (Shouwuteng) 9 g, Poria (Fuling) 9 g. After pulverizing, the powder was steeped with threefold tap water (v/w) for 0.5 h, boiled for 1 h, and then filtered. The residue was extracted with tap water another two times. Climbing assay. Climbing assay was performed as described 29 with minor modification. Briefly, groups of ten flies were transferred into 18 cm transparent plastic tubes 1 hr prior to the assay for environmental acclimatization. A finishing line at 10 cm from the bottom of the tubes was marked. The tubes were tapped to send flies to the bottoms of the tubes. The climbing time was recorded when at least five flies had passed the finishing line. When flies took more than 50 s to climb above the finishing line, the climbing time was recorded as 50 s. Five trials were performed for each group. The experiment was repeated at least three times. The mean climbing times were calculated and plotted as climbing scores.
Stereotaxic rotenone intoxication hemi-parkinsonisom rat model and TGY treatment. Total 40 rats were used, the rats were randomly allocated to 5 groups: Sham group (Infused with 1 μ l Dimethyl sulfoxide). The rest of the rats were infused with rotenone and then assigned into the other 4 groups including Rotenone group (Model group, received vehicle treatment); TGY low dosage group (TGY-L, received 1.5 g/kg TGY extract); TGY middle dosage group (TGY-M, received 3 g/kg TGY extract) and TGY high dosage group (TGY-H, received 6 g/kg TGY extract). All TGY groups received two weeks pretreatment prior to rotenone intoxication and two months post treatment. The stereotaxic rotenone infusion was performed as previously described with minor modification 14 . Briefly, the rats were anesthetized with intraperitoneal injection of chloral hydrate (3.0% w/v in normal saline, 1 ml/100 g b.d.w.) and then placed and fastened on the stereotaxic frame (Stoelting, Wood Dale, USA). Rotenone was dissolved in DMSO with a final concentration of 3 μ g/μ l, 1 μ l of this solution was infused into the right VTA (AP: 5.3 mm; ML: 0.9 mm; DV: 8.0 mm) at a flow rate of 0.2 μ l/min. The needle was hold for extra five minutes for the thorough diffusion of rotenone. After that, the needle was withdrawn slowly. Then rotenone was infused into the right SNc (AP: 5.3 mm; ML: 2.0 mm; DV: 8.0 mm) at a flow rate of 0.2 μ l/ min, with additional five-minute needle retention. After operation, rats received 200 k Unit Penicillin by subcutaneous injection per day for total three days to prevent postsurgical infection.

Apomorphine induced rotation.
All groups of the rats were tested for APO-provoked rotations at the fourth and eighth week after drug administration. The rats were placed in an opaque plastic cylinder (diameter = 30 cm) for 10 min acclimation, and then challenged with APO (3 mg/kg) via intraperitoneal injection. The following rotational behavior was then recorded for 35 min. The rotations were counted from 6 to 35 min. The rats were placed back into their cages after rotational testing.
Immunohistochemical analysis. For Drosophila whole brain staining, protocol was followed as reported with minor modification 30 . Flies were anesthetized under CO 2 and then decapitated with forceps. Heads were transferred to 1.5 ml microcentrifuge tubes containing fixative (4% paraformaldehyde, 0.3% Triton X-100). Tubes stayed on ice for 3 hrs to make sure that fly heads were submerged in fixative. The brains were dissected under stereomicroscope, fixed at room temperature for 30 min and incubated with 0.3% PBST (0.3% Triton X-100, 1 × PBS) three times on a rotator, each time 20 min; then blocked for 1 hr at room temperature with blocking buffer (5% normal goat serum, 1 × PBS, 0.1% Triton X-100), then incubated with anti-TH (1:200 dilute in blocking buffer) for 36 hrs at 4 °C on a rotator. The primary antibody was removed and the brains were washed with 0.3% PBST three times on a rotator, each time 20 min, then incubated in goat anti-rabbit Alexa 488(1:200 dilute in blocking buffer). Next, brains were washed with 0.3% PBST three times on a rotator, each time 20 min, and finally mounted with FluorSave ™ Reagent (Merck). Mounted brain samples were analyzed under a confocal microscope (Leica, TCS SP5, Germany). For the rat samples, coronal sections (30 μ m thickness) were cut throughout the striatum (from 1.0 mm to 2.0 mm caudal to the bregma) by using Shandon Cryotome SME Cryostat (Ramsey, Minnesota, USA). The sections were placed on the slides, air-dried, and then treated with 1% H 2 O 2 for 10 min to quenched the endogenous peroxidase activity. After washed with 0.3% Triton-X100, the sections were blocked with diluted horse serum for 30 min at room temperature. After blocking, the sections were incubated with TH antibody (Millipore, AB152, 1:500) for 24 hrs at 4 °C. The sections were incubated with ABC ® Elite (VECTOR, Burlingame, USA) secondary biotinylated goat anti-rabbit IgG for 30 min at room temperature. Finally the sections were incubated with DAB for 5 min. The sections were then air-dried and mounted with Leica mounting buffer. The slides were then observed under motic SMZ-171 microscope (Hong Kong, China).

Immunoblot analysis.
For Drosophila samples, immunoblot analyses were carried out as previously described 9 with minor adjustments. Twenty fly heads were homogenized in lysis buffer (50 mM Tris, 1%NP-40, 0.35% sodium deoxycholate, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM PMSF, pH7.4) and lysed on ice for 20 min. The supernatants were collected after centrifugation (14,000 rpm, 10 min, 4 °C) and denatured with 5 × Laemmli sample buffer and then separated on 12% SDS-PAGE gels. The protein on the gels were then transferred to Polyvinylidene fluoride (PVDF) membranes (GE Healthcare, RPN303F) and processed for immunoblotting. Membranes were blocked with 5% nonfat milk and probed with primary and secondary antibodies. The bands were developed with an ECL kit (Pierce, 32106). The density of the bands was quantified by densitometry (ImageJ, NIH). For cell samples, lysis buffer was added and the cells were scraped off, transferred to microcentrifuge tubes and then lysed on ice for 30 min. The remaining steps were the same as those used for the fly samples. For the rat brain samples, the ipsilateral striatum was dissected and homogenized with phosphate buffered saline (PBS) containing 1 mM PMSF (1 mg brain added 10 μ l PBS). The striatum was homogenized by sonication (120 w, 60 Hz, 2 s per cycle, total 25 blasts, BRANSON, USA) performed on ice. Then the lysate was centrifuged at the speed of 14,000 rpm for 30 min at 4 °C. The remaining steps were the same as those used for the fly samples. Dopamine quantification. Drosophila brain dopamine levels were analyzed by HPLC/MS method 31 .
After treatment, 10 heads of flies from each group were homogenized in 100 μ L pre-chilled 0.1 M formic acid. The samples were centrifuged at 13,000 rpm, 4 °C for 10 min. The supernatants were collected and freeze-dried, and then reconstituted with 100 μ L ice-cold methanol; they were then centrifuged at 13,000 rpm, 4 °C for 10 min. The supernatants were collected and subjected to LC/MS analysis. Dopamine levels were measured with triple quadrupole LC/MS system (Agilent 6460) equipped with a ZIC pHILIC 150 × 2.1 mm, 5 μ m column (Merck).
Cell culture and cell viability assay. Human neuroblastoma cells SH-SY5Y were maintained in Dulbecco's Modified Eagle's medium (DMEM, Invitrogen, 12800017), supplemented with 15% Fetal bovine serum (FBS, Invitrogen, 10099141). MTT assay was employed to evaluate cell viability as previously described 32 . SH-SY5Y cells were seeded in 96-well plates (5,000 cells/well). After 24 hrs, different treatments were performed. In the assay, 20 μ M of rotenone was used to induce cytotoxicity 33 . After treatment, the media were removed and phenol red-free DMEM containing MTT (final concentration 0.5 mg/mL) was added, and cells were incubated for 4 hrs. The MTT solution was removed, and the cell crystals were dissolved using 100 μ L of 20% SDS in 50% DMF. The intensity was measured using a plate reader at 570 nm with the reference of 620 nm.

Statistics analysis.
Each experiment was repeated at least three times unless indicated. The data was expressed as means ± SD. One-way ANOVA followed by Dunnett's multiple comparison tests were conducted. All statistics analyses were performed in GraphPad Prism6 (GraphPad Software, Inc.).