Targeting autophagy as a therapeutic strategy for identification of liganans from Peristrophe japonica in Parkinson’s disease

Dear Editor, PD is characterized by the loss of dopaminergic neurons in substantia nigra, and the loss of dopamine resulting in motor deficit. Its main pathological hallmarks include the genetic mutations of gene such as α-synuclein. Increasing study showed that the dopaminergic neurons in midbrain are sensitive and damaged by the PD toxins. 6-Hydroxydopamine (6-OHDA) is widely used to induce lesion of nigrostriatal dopaminergic system in the PD model systems, including nerve cells, Caenorhabditis elegans (C. elegans), and rats. Autophagy (mitophagy) is a cellular self-digestive process that engulfs the damaged organelles, including injured mitochondria and misfolded proteins such as α-synuclein. Therefore, enhanced autophagy plays an important role in neuroprotection via cellular degradation of damaged mitochondria or mutant proteins in neurodegenerative diseases such as PD. Peristrophe japonica (PJ), a traditional Chinese medicine, is reported to exhibit anti-inflammatory, anti-bacterial, and anti-oxidative effects. However, the neuroprotective components of PJ and the mechanism remain un-elucidated. In this study, we found that the total ethanol extract of PJ (PJTEE) could protect against 6-OHDA-induced damage in PC-12 cells (Supplementary Fig. 1). Meanwhile, PJ-TEE exhibited potent autophagy effect by dose-dependently increasing the number of GFP-LC3 puncta formation in stable RFP-GFP-LC3 U87 cells (Supplementary Fig. 2a). Based on the autophagy activity-guided chemical separation, PJ-TEE was extracted using petroleum ether, ethyl acetate, and n-butanol reagents, and the components in these fractions were analyzed by UHPLC-DAD-TOF/MS (Supplementary Fig. 2b). In stable RFP-GFP-LC3 U87 and PC-12 cells, ethyl acetate fraction (EF) was demonstrated to induce the strongest autophagy effect (Supplementary Fig. 2c–e). After subfractionation of EF to 34 fractions, F11 to F34 were found to induce autophagy in stable RFP-GFP-LC3 U87 and PC-12 cells (Supplementary Fig. 3). Finally, 3 liganans, including justicidin A (JA), justicidin B (JB), and justicidin C (JC) (Fig. 1a) in F11, were isolated and identified by UHPLC-DAD-TOF/MS and nuclear magnetic resonance (NMR) instruments (Supplementary Fig. 4). To further confirm the autophagy effect of F11 was attributed to JA, JB, and JC, stable RFP-GFP-LC3 U87, PC-12, and SHSY5Y cells were adopted. The results showed that JA, JB, and JC dosedependently increased the number of GFP-LC3 puncta formation in stable RFP-GFP-LC3 U87 cells (Fig. 1b), and LC3-II protein expression in both PC-12 and SHSY5Y cells (Fig. 1c and Supplementary Fig. 5 and 7). Among them, JA time-dependently increased the ratio of RFP-LC3/GFP-LC3 puncta in stable RFP-GFPLC3 U87 cells (Supplementary Fig. 6 and Video 1). Meanwhile, JA, JB, and JC increased the ratio of RFP-LC3/GFP-LC3 puncta and the number of autophagosome in PC-12 cells (Supplementary Fig. 8a, b). In addition, LY294002 (LY) and Bafilomycin A1 (Baf) inhibited the autophagic sequestration and autophagosome-lysosome fusion induced by JA, JB, and JC (Fig. 1e and Supplementary Fig. 8c, d). All these evidences suggested that the liganans induced autophagic flux in neurons. Then, the mechanistic study demonstrated that JA, JB, and JC activated autophagy via the AMPK/ ULK1, Raf/MEK/ERK, but mTOR-independent signaling pathways (Fig. 1d and Supplementary Fig. 9, 10). In addition, compound C (CC, an AMPK inhibitor) and SCH772984 (SCH, an ERK inhibitor) decreased the number of GFP-LC3 puncta formation in stable RFP-GFP-LC3 U87 cells and LC3-II protein expression in PC-12 cells (Fig. 1e and Supplementary Fig. 11). Furthermore, JA, JB, and JC increased the number of GFP-LC3 puncta formation and LC3-II protein expression in MEF Atg7 but not in MEF Atg7 cells (Fig. 1f and Supplementary Fig. 12, 13), suggesting that JA, JB, and JC induced autophagy via Atg7. Moreover, Parkin/PINK1-mediated mitophagy was also activated by JA, JB, and JC as revealed by the colocalization of MitoTracker with GFP-LC3 puncta and the decreased GFP/RFP ratio illuminated by mCherry-GFP-FIS1101-152 (mito-QC) (Supplementary Fig. 14, 15 and Video 2). Next, the neuroprotective effect of JA, JB, and JC was examined in 6-OHDA-induced PC-12 and SHSY5Y cells. Our results showed that JA, JB, and JC dose-dependently increased the cell viability (Fig. 1g), improved cell morphology, and inhibited cell apoptosis (Supplementary Fig. 16a–d). Consistently, LY and CC could attenuate the effect of JA, JB, and JC on the improvement of cell viability (Supplementary Fig. 16e–h). The flow cytometric result demonstrated that JA, JB, and JC significantly decreased ROS production in 6-OHDA-induced PC-12 and SHSY5Y cells (Fig. 1h and Supplementary Fig. 16i, j). In addition, JA, JB, and JC could protect against oxidative damage in H2O2-induced PC-12 and SHSY5Y cells (Supplementary Fig. 17). Furthermore, JA, JB, and JC inhibited the oligomerization of α-synuclein in PC-12 cells and decreased the levels of WT-, A53T-, A30P-, and E46K-α-synuclein in MEF cells via Atg7 (Supplementary Fig. 18). However, JA, JB, and JC neither inhibited MAO activity nor activated muscarinic (M) receptor (Supplementary Fig. 19). Taken together, JA, JB, and JC exerted neuroprotective effect in 6-OHDA-, H2O2-, and αsynuclein-induced cell models of PD. To validate the autophagy and neuroprotective effect of JA, JB, and JC in vivo, both C. elegans and rat models of PD were employed. BC12921 strain expressing GFP-p62 fusion protein and DA2123 strain expressing GFP-LGG-1 fusion protein were used to detect the autophagic activity in C. elegans. The results showed that JA, JB, and JC significantly decreased the expression of p62 and increased GFP-LGG-1 punctate formation (Fig. 1i). Then, the neuroprotective effect of JA, JB, and JC was evaluated in 6-OHDA-induced BZ555 strain expressing GFP in its dopaminergic neurons, and NL5901 strain expressing human α-synuclein protein tagged with YFP in the body wall muscle. The results showed that JA, JB, and JC significantly decreased the ROS levels (Supplementary Fig. 20a), inhibited the degeneration of dopaminergic neurons (Fig. 1j), and improved the food sensing ability (Supplementary Fig. 20b) in 6-OHDA-induced BZ555 strain, as

The full-length Western blotting images for Fig. 1d. Supplementary Fig. 30 The full-length Western blotting images for Supplementary  Fig. 10. Supplementary Fig. 31 The full-length Western blotting images for Supplementary  Fig. 11 Supplementary Fig. 32 The full-length Western blotting images for Fig. 1f. Supplementary Fig. 33 The full-length Western blotting images for Supplementary  Fig. 15. Supplementary Fig. 34 The full-length Western blotting images for Supplementary  Fig. 19. Supplementary Fig. 35 The full-length Western blotting images for Fig. 1l. Supplementary Fig. 36 The full-length Western blotting images for Supplementary The test drugs used in this study were dissolved with DMSO and stored at -20 °C until further use. The viability of the cells treated as the indicated drugs was measured by MTT assay using the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (Sigma-Aldrich, MO, USA) reagent as previously described. Briefly, cells seeded in 96-well microplates were treated the drugs as the indicated concentrations for 24-48 h 1 . After treatments, the medium was removed and 100 μL of fresh medium with 10 µL of MTT solution (5 mg/mL) was added into each well, followed by a further incubation for 4 h. The formed formazan representing the viability of cells was then dissolved by 100 µL DMSO. The solution was then subjected to the spectrophotometer and the colorimetric reading was determined at OD 570 nm. Apart from MTT assay, the viability of cells was also measured by crystal violet staining 2 . In brief, cells seeded in 6-well plate were treated the drugs as the indicated concentrations for 24 h. After treatment, the medium was removed, then the cells were incubated with crystal violet solution for 20 min. The excessive crystal violet solution adhered on the cells was wash away by PBS and air-dried at room temperature. The representing images of stained cells were captured by CCD digital camera Spot RT3™ under the Nikon ECLIPSE 80i microscope. While the cell viability was quantified by dissolving the crystal violet in 10% acetic acid, and the colorimetric reading of the solution was determined by the spectrophotometer at OD 570 nm. The percentage of cell viability by these two methods was calculated using the following formula: Cell viability (%) = Cells number treated/Cells number DMSO control × 100. Data were obtained from three independent experiments.

Quantification of the number of GFP-LC3 puncta
Quantification of the number of GFP-LC3 puncta in GFP-LC3 transiently transfected MEF cells or stable RFP-GFP-LC3 U87 cells were conducted as previously described 3 . In brief, stable RFP-GFP-LC3 U87 cells or MEF cells were seeded on coverslips in 6-well plates. MEF cells were transfected with pEGFP-LC3 by the Exfect® Transfection Reagent (Vazyme Biotech Co., Ltd., Nanjing, China) according to the manufacture's instruction. Cells were then treated the drugs as the indicated concentrations for 24 h. After treatment, the cells were fixed by 4% paraformaldehyde (PFA) for 20 min at room temperature, followed by the wash with PBS for 2 times. The slides were then taken out for air-drying and mounted by FluorSave™ mounting media (Calbiochem, San Diego, CA, USA). Representative images of cells were captured by Nikon ECLIPSE 80i fluorescence microscope equipped with a CCD digital camera Spot RT3™ (Diagnostic Instruments, Inc., Melville, NY, USA). The number of GFP-LC3 puncta per cells using ImageJ software. Briefly, the green channel of RGB images was extracted and converted to the grayscale images by auto-thresholding, and GFP-LC3 granules were analyzed for the number. The criteria for the analysis are as follow: all particle sizes larger than background pixelation but smaller than the average nuclear were selected for the analysis, while the cells containing apoptotic nuclei or necrotic cell bodies were excluded from the analysis.

Determination of Autophagy Flux in Stable RFP-GFP-LC3 U87 Cells
The autophagy flux was determined as described previously by calculating the RFP/GFP ratio in stable RFP-GFP-LC3 cells 2 . In brief, stable RFP-GFP-LC3 U87 cells

mRFP-GFP Tandem Fluorescent-Tagged LC3 (tfLC3) Detection
The autophagosome was determined as described previously in transfected tfLC3 plasmid PC-12 cells 5 . In brief, tfLC3 transiently transfected PC-12 cells were seeded on coverslips in 6-well plates. After treatments with JA, JB, JC or rapamycin at the indicated concentrations for 24 h, cells were fixed with 4% PFA for 20 min at room temperature and mounted by FluorSave™ mounting media for the examination of GFP/RFP-LC3 in cells by a Leica SP8 confocal microscope with a LAS X 3D Visualization (Leica Microsystems Inc., Wetzlar, Germany). The images of cells with GFP-LC3 and RFP-LC3 at the same field were merged to determine the mean number of LC3B puncta per cell and the percentage of autophagosome per cell using ImageJ software (ImageJ 1.46r; National Institutes of Health, Bethesda, MD, USA).

Transmission electron microscopy
JA-, JB-, or JC-induced autophagosome morphology was examined by transmission electron microscopy according to the reported method 2 . Briefly, PC-12 cells were seeded in 100 mm dishes and treated with JA (0.13 μM), JB (0.13 μM), JC (4 μM), or rapamycin (0.5 μM) for 24 h. After treatment, PC-12 cells were collected and centrifugation at 1000 r/min for 5 min. The supernatant was discarded and the cells were resuspended by 0.5% glutaraldehyde fixative solution and stood still for 10 min at 4 °C. Then, cell suspension was centrifugated at a high speed of 12000 r/min for 10 min. The cell pellet was fixed by adding 3% glutaraldehyde fixative overnight, followed by a wash with PBS. Then, the cells were subjected to a post-fixation with 1% OsO4 and an embedment using Araldite 502. The obtained ultrathin sections were double stained with 3% uranyl acetate and lead citrate, then analyzed by a transmission electron microscope (JEM-1400plus, JEOL, Tokyo, Japan) at a voltage of 80 kV.

Colocalization of GFP-LC3 and Mito-Tracker Red CMXRos
The mitophagy was determined by visualizing the colocalization of GFP-LC3 and Mito-Tracker Red CMXRos in PC-12 cells 6 . In brief, PC-12 cells seeded on 6-well plates were transiently transfected with GFP-LC3 plasmid for 24 h, and followed by the treatment of JA, JB, JC or AICAR for 24 h. Then, cells were incubated with 200 nM Mito-Tracker Red CMXRos solution for 30 min at 37 °C. After incubation, the Mito-Tracker Red CMXRos solution was exchanged by the fresh cell culture medium.
The colocalization of GFP-LC3 and Mito-Tracker-Red was visualized and captured using the fluorescence microscope with 100× magnification. The overlap of green and red dot represents the mitochondrial-containing autolysosome.

Detection of mitophagy using mito-QC plasmid
Mito-QC is made up of a tandem-fusion protein (mCherry-GFP) that is targeted to the outer mitochondrial membrane (OMM) via the mitochondrial targeting sequence of the OMM protein, FIS1. Mito-QC is a pH-sensitive mitochondrial fluorescent probe. The GFP will be quenched when mitophagy is induced because mitochondria are delivered to the lysosome 7 . In this study, we also monitor the mitophagy using mito-QC plasmid.
In brief, PC-12 cells seeded on 6-well plates were transiently transfected with mito-QC plasmid for 24 h, and followed by the treatment of JA, JB, JC or AICAR for 24 h. Then, cells were examined and captured by a fluorescence microscope with 10× magnification. The images of cells with GFP and RFP at the same field were merged to determine the mitophagy by calculating the GFP/RFP fluorescence ratio using ImageJ software (ImageJ 1.46r; National Institutes of Health, Bethesda, MD, USA).
The intensities of GFP and RFP signals were obtained from 3 randomly selected fields.

Western blot
After drug treatments, cells were lysed by 1× RIPA buffer from Cell Signaling Technologies Inc. (Beverly, MA, USA). The proteins were harvested and the protein concentrations were measured by 1× Bradford reagent (Bio-Rad, Hercules, CA, USA).
Equal amount of lysate of each sample was resolved by SDS-PAGE for electrophoresis, then the proteins on the SDS-PAGE gel were transferred to the polyvinylidene difluoride (PVDF) membrane. After that, the membrane was blocked with 5% non-fat milk for 1 h at room temperature, then was incubated with primary antibodies overnight at 4 °C. After incubation, the membrane was washed using TBST for 3 times and further incubated with the HRP-conjugated secondary antibodies for 1 h at room temperature.
Then, the membrane was washed using TBST for 3 times, and the band of the targeted proteins was revealed using the ECL Western Blotting Detection Reagents (4A Biotech Co., Ltd, Beijing, China). In this study, the relative quantitation of target protein expression was carried out in the WB images of unsaturated blots and obtained by calculating the normalized band intensities to β-actin using the ImageJ software. All the data were from for three independent experiments.

Measurement of ROS levels
In this study, ROS level in PC-12 or SHSY5Y cells was determined by using flow cytometer as our previously described method 2 . In brief, cells seeded in 6-well plates were treated the indicated drug for 24 h. After treatments, the cells were collected and

Measurement of cell apoptosis
In this study, cell viability and apoptosis of PC-12 or SHSY5Y cells were measured by flow cytometry using the Annexin V-FITC/PI Apoptosis detection Kit from 4A Biotech Co., Ltd. (Beijing, China) 8 . In brief, PC-12 cells seeded in 6-well plates were treated with the indicated drugs for 24 h. After treatments, cells were collected to centrifugate at 2000 rpm for 5 min. Then, the cell pellet was resuspended by the 1× annexin V solution containing FITC and PI reagents, followed an incubation in dark for 15 min.
After that, the cells were subjected to the flow cytometric analysis by a FACSVerse flow cytometer according to the manufacturer's instructions. The rate of cell apoptosis or viability was calculated using the Flowjo software. Data were obtained from three independent experiments.

Spectrophotometric monoamine oxidase activity assay
The spectrophotometric monoamine oxidase (MAO) activity assay was measured based on the conversion of benzylamine to benzaldehyde in the role of MAO. Benzaldehyde is generated into aldehydephenylhydrazone which is measured by a spectrophotometer at OD 470 nm. After treatment of JA, JB, JC, or selegiline, PC-12 cells were trypsinzed, collected and washed by isotonic PBS for 1-2 times, followed by a homogenization with an ultrasonic disintegrator in an ice bath. After centrifugation at a speed of 1000 rpm for 10 min, the supernatant was collected for the determination of the protein concentration using BCA protein concentration determination kit. Then, the MAO activity of the samples was detected by the MAO detection kit (R22030-100T, Shanghai yuanye Bio-Technology Co., Ltd., Shanghai, China) according to the manufacturer's instructions. The MAO activity was calculated using the following formula: MAO activity (U/mg) = benzaldehyde (nmol) × Vt × N/(t×Vs×C). Vt, N, Vs, t, and C represents the total volume (mL) of samples to be detected, the dilution factor (N) of samples, the volume (mL) of samples being detected, the reaction time (min), and the protein concentration of samples, respectively.

Determination of the activation of muscarinic receptor
The effect of JA, one of the liganans, on the activation of muscarinic (M) receptor was performed in ex vivo rabbit intestinal muscle according to reported literature 9 . In brief, after 24 h of fasting, a male New Zealand rabbit was humanely euthanized by means of a blow to the head, 1.5-2 cm of jejunum was removed, washed, freed from mesenteric attachment. The segment of jejunum suspended in the direction of longitudinal and circular smooth muscle fibers in a thermostatically controlled (37 °C) organ bath (10 mL capacity) containing Krebs solution and was continuously gassed with air. The segment of jejunum was connected to an isometric force transducer. Then, the signal output of the mechanical activity was amplified and recorded by the RM6240 series Multi-channel physiological signal acquisition and analysis system (Chengdu Taimeng Technology Co., Ltd. Chengdu, China). Prior to each testing, the segment of jejunum was allowed to equilibrate in Krebs solution. At first, the activation and antagonism of M receptor was validated by 0.01% acetylcholine (Ach) chloride and 0.1% atropine (Atr) sulfate. After wash and equilibrium, 10 mg/mL of JA was added into the Krebs solution, which was followed by the addition of 0.2 mL of 0.01% acetylcholine (Ach) chloride. The signal was recorded by the RM6240 series Multi-channel physiological signal acquisition and analysis system.

Determination of autophagy in C. elegans
In this study, BC12921 expressing the SQST-1-GFP fusion protein, C. elegans homolog of mammal p62 and DA2123 expressing the C. elegans microtubule-associated protein

Analysis of food-sensing behavior
The function of C. elegans dopaminergic neurons was also assayed by evaluating the food-sensing behavior 13 . In brief, the L3 larvae of BZ555 strain were treated with 6-OHDA or co-treated with 6-OHDA and JA, JB, JC or L-Dopa for 72 h at 20 °C. The worms were then released to the center of NGM agar plate spotted with or without E. coli OP50 lawn. After 5 min, the body bends of each worm were counted for 20 s in the presence or absence of food. The slowing rate was calculated according to the following formula: Slowing rate = (N without food -N with food)/N without food where, N represents total number of body bends in the presence or absence of bacterial.

Analysis of movement in C. elegans
The motor impairment and dysfunction are the main symptom of PD patients. The strain NL5901 [Punc-54::α-synuclein::YFP; unc-119(+)] expressing human α-synuclein protein tagged with YFP in the body wall muscle displays the progressive decline in body movement 14 . In this study, the improvement of JA, JB and JC in the motor function was evaluated in NL5901 strain. Briefly, age-synchronized NL5901 animals were grown onto NGM plates containing JA, JB, JC or levodopa to reach L4 developmental stage. At the same time, FUDR (50 g/ml) was added to NGM plates to prevent the growth of progeny. When animals grew to L4+5 days, they were placed independently in a drop of M9 buffer, and allowed to recover for 120 s to avoid observing behavior associated with stress. Then the number of body bends was counted in 20 s. Meantime, the representative video of swimming worms in 20 s was captured under a Leica M205FA stereomicroscope (Leica, Wetzlar, Germany) equipped the Bandicam software (Bandicam Company, Seoul, Korea). 20 animals were used to analyze in each experiment, and all experiments were carried out in triplicate.

Quantitative assay of α-synuclein protein aggregation in C. elegans
As is known to us, the accumulation of α-synuclein aggregates results in the degeneration and loss of dopaminergic neurons. In this study, the analysis of αsynuclein accumulation were performed in the NL5901 strain 14 . In brief, synchronized L1 larvae were transferred onto the NGM plates containing JA, JB, JC or L-Dopa and incubated for 5 days at 20 ℃. Then the animals were collected and the representative images were captured for the analysis of α-synuclein aggregation under the fluorescence microscope. The expression of α-synuclein was measured by quantifying the fluorescence intensity using ImageJ software.

RNAi treatments in C. elegans
To examine whether the neuroprotective effect of liganans is associated with the autophagy induction in vivo. 6-OHDA-induced BZ555 or NL5901 strain were respectively transferred onto NGM plates which were seeded with the control bacteria HT115 or the RNAi bacteria expressing double-stranded RNA of two key autophagy-related genes including unc-51 and vps-34. At the same time, L-Dopa (2 mM) or JA (100 μM) was also added into the NGM plates with bacteria HT115 or RNAi bacteria.
After treatment, the fluorescence intensity indicating the live dopaminergic neurons in BZ555 worms, and the fluorescence intensity representing the α-synuclein protein aggregation and the movement of NL5901 worms were examined as described above. University (Luzhou, China). All efforts were made to minimize the animal suffering. 6-OHDA was dissolved in normal saline solution with 0.02% vitamin C to a concentration of 2 μg/μL. After anesthesia, the rats were fixed on a stereotaxic apparatus (DW-2000, Taimeng, Chengdou). Fur on the head was shaved by a pet clipper and 10% povidoneiodine solution was used to sterilize the incision site. The skin was slit and bluntly separated with tissue forceps, and the periosteum was burned with hydrogen peroxide to expose the bregma. According to the Rat Brain Atlas in Stereotaxic Coordinates Paxinos, the exposed skulls were trepanned by a dental drill (3 mm) at the appropriate position. 5 μL of 6-OHDA solution or saline was injected into the right midbrain Syringe was lowered into the brain at a rate of 1 mm/min, then the syringe was left in the place for 5 min and the reagents were injected at a rate of 0.2 μL/min. Afterward, the syringe was left in the place for 5 min again before drawing back at a rate of 1 mm/min. The incision was cleaned by povidone-iodine solution and closed using three sutures. 10 IU of penicillin solution was injected intraperitoneally to rats for 1 week so as to prevent infections. The postoperative behavioral changes of rats including bradykinesia, less movement, tremor, vertical hair, and olfactory abnormality were observed daily. On the 7 th , 14 th , and 21 st day after surgery, apomorphine was subcutaneously injected to induce rotational behavior, and the rats with more than 210 rotations per 30 min or 7 rotations per 1 min in 0.5 h were selected for the following behavioral training and tests.

Groups and administrations
All the rats were randomly divided into 5 groups (n=8 rats/group), including sham, model, 5 mg/kg JA, 10 mg/kg JA, and 10 mg/kg L-Dopa groups. In brief, rats in the sham group were intraperitoneally injected with equal volume of 0.9% normal saline.
6-OHDA-induced rats were intraperitoneally injected with equal volume of 0.9% normal saline, 5 mg/kg JA, 10 mg/kg JA, and L-Dopa. Before 1week of test, all rats were subjected to the following behavioral trainings and tests. All behavioral tests were performed in a double-blinded manner between 10 a.m. and 3 p.m.

Apomorphine-induced rotational test
The apomorphine-induced rotational test was performed as previously described 15 .
Briefly, after a subcutaneous injection of apomorphine hydrochloride dissolved in normal saline (0.5 mg/kg; GLPBIO, USA), the full 360-degree rotations of rats within 1 h were manually counted in a cylindrical container (75 cm long, 50 cm wide and 35 cm high). Finally, the net number of rotations was calculated as the positive scores (contralateral rotations) minus the negative scores (ipsilateral rotations).

Swimming test
The swimming test was used to evaluate the limb coordination ability and motility 16 .
In brief, rats were placed in a cylindrical swimming pool with a size of 100 cm × 100 cm × 70 cm. The pool was filled with water for 50 cm of depth and the temperature was maintained at 22-25 °C. Swimming test was performed and the score was recorded as previously described 17 . The scoring criteria were as follows: The test time of each rat was 10 min, and the time interval of score was 1 min. Three training were performed for each mouse before the test.

Forelimb hanging test
The Forelimb hanging test was used to evaluate the muscular strength and motor neuron integrity 18 . In brief, rats utilized their forelimbs to grab the wire with a diameter of 3 mm and suspend their body on it. The wire was stretched for 30 cm between two posts and height for 70 cm above a soft pad placed on the ground to prevent the rats from being injury. During the test, the time from the rats beginning to grab wire to fall down is recorded. The average forelimb hanging score was recorded according to the hanging time of rats on the wire. In general, the scoring criteria were as follows: 0-5 s: 0 point,