Protection by rhynchophylline against MPTP/MPP+-induced neurotoxicity via regulating PI3K/Akt pathway

Meizhu Zheng, Minghui Chen, Wenli Wang, Mi Zhou, Chunming Liu, Yajun Fan, Dongfang Shi
PII: S0378-8741(20)33456-5
DOI: https://doi.org/10.1016/j.jep.2020.113568 Reference: JEP 113568

To appear in: Journal of Ethnopharmacology

Received Date: 9 October 2020
Revised Date: 3 November 2020
Accepted Date: 3 November 2020

Please cite this article as: Zheng, M., Chen, M., Wang, W., Zhou, M., Liu, C., Fan, Y., Shi, D., Protection by rhynchophylline against MPTP/MPP+-induced neurotoxicity via regulating PI3K/Akt pathway, Journal of Ethnopharmacology, https://doi.org/10.1016/j.jep.2020.113568.

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Protection by rhynchophylline against MPTP/MPP+-induced neurotoxicity via regulating PI3K/Akt pathway
Meizhu Zhenga,Minghui Chenb,Wenli Wangb, Mi Zhoub, Chunming Liua*,Yajun Fanb, Dongfang Shia
a The Central Laboratory, Changchun Normal University, Changchun, Jilin, PR China
b College of Life Science, Changchun Normal University, Changchun, Jilin, PR China

*Corresponding author:

The Central Laboratory, Changchun Normal University, Changchun 130032, China Tel: 86 431 86168777; Fax: 86 431 86168777
E-mail address: [email protected]

List of authors’ email addresses

Meizhu Zheng E-mail address:[email protected] Minghui Chen E-mail address:[email protected]
Wenli Wang E-mail address:[email protected] Mi Zhou E-mail address:[email protected]
Chunming Liu E-mail address: [email protected] Yajun Fan E-mail address:[email protected]
Dongfang Shi E-mail address:[email protected]

Abbreviations
PD, Parkinson’s disease; MPTP, 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine; MPP+, 1-Methyl-4- phenylpyridinium ; SNpc, substantia nigra pars compacta; MAO-B, monoamine oxidase B; ROS, reactive oxygen species; TLR, toll-like receptor; LPS, lipopolysaccharides; PI, propidium iodide; LDH, lactate dehydrogenase; Bax, Bcl-2 associated X protein ; Bcl-2, B-cell lymphoma 2; TBST, Tris-buffered saline containing 0.05% Tween-20; PAGE, polyacrylamide gel electrophoresis; TFA, trifluoracetic acid; Rhy, rhynchophylline; Cell Counting Kit-8, cck-8; TH, tyrosine hydroxylase; PI3K, phosphatidylinositol 3-Kinase; FBS, fetal bovine serum; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; HRP, horseradish peroxidase; DTT, dithiothreitol; SDS, sodium dodecyl sulfate; PVDF, polyvinylidene difluoride; i.g. intragastric; PBS, phosphate belanced solution; PBST, phosphate-buffered saline with Tween; DMEM, Dulbecco’s modification of Eagle’s medium Dulbecco.

Abstract

Ethnopharmacological relevance: Isolated from Uncaria rhynchophylla (U. rhynchophylla), rhynchophylline (Rhy) has been applied for treating diseases related to central nervous system such as Parkinson’s disease. Nevertheless, the molecular mechanism of the neuroprotective effect has not been well interpreted.
Aim of the study: To investigate the effects of Rhy on MPTP/MPP+-induced neurotoxicity in C57BL/6 mice or PC12 cells and study the mechanisms involved.
Materials and methods: The neuroprotective effect of Rhy on MPTP-induced neurotoxicity was evaluated by spontaneous motor activity test, as well as a test of rota-rod on a rat model of Parkinson’s disease. The numbers of TH-positive neurons in the substantia nigra pars compacta (SNpc) was assessed by immunohistological. CCK-8, lactate dehydrogenase (LDH), reactive oxygen species (ROS), the concentration of intracellular calcium ([Ca2+]i) and flow cytometry analysis were performed to evaluate the pharmacological property of Rhy on 1-methyl-4-phenylpyridinium (MPP+) induced neurotoxicity in PC12 cells. Besides, LY294002, a PI3K inhibitor was employed to determine the underlying molecular signaling pathway revealing the effect of Rhy by western-blot analysis.
Results: The results showed that Rhy exhibited a protective effect against the MPTP-induced decrease in tyrosine hydroxylase (TH)-positive fibers in the substantia nigra at 30mg/kg, demonstrated by the immunohistological and behavioral outcomes. Furthermore, it has been indicated that cell viability was improved and the MPP+-induced apoptosis was inhibited after the treatment of Rhy at 20 µM, which were severally analyzed by the CCK-8 and the Annexin V/propidium iodide staining method. In addition, Rhy treatment attenuated MPP+-induced up-regulation of LDH, ([Ca2+]i), and the levels of ROS. Besides, it can be revealed from the western blot assay that LY294002, as a selective Phosphatidylinositol 3-Kinase (PI3K) inhibitor, effectively inhibited the Akt phosphorylation caused by Rhy, which suggested that Rhy showed its protective property through the activated the PI3K/Akt signaling pathway. Moreover, the Rhy-induced decreases of Bax and caspase-3 as the proapoptotic markers and the increase of Bcl-2 as the antiapoptotic marker, were blocked by LY294002 in the MPP+-treated PC12 cells.
Conclusions: Rhy exerts a neuroprotective effect is partly mediated by activating the PI3K/Akt signaling pathway.

Key words: Parkinson’s disease; MPTP; Rhynchophylline; MPP+; Phosphoinositide 3-kinase/Akt signaling pathway

Graphical Abstract

1. Introduction

As the second most epidemic age-related neurodegenerative disorder, Parkinson’s disease (PD) has universally influenced millions of people’s lives (Qin et al., 2011). The selectively and progressively degenerated dopaminergic neurons in substantia nigra pars compacta and the subsequently decreased dopamine in striatum can be identified as the characteristics of PD (Braak et al., 2003; Lang and Lozano, 1998). Although further investigation was still needed for accurately interpreting the mechanism, the integration of oxidative stress and the dysfunction of mitochondria were considered as the inducements of the selective degenerated nigrostriatal dopaminergic neurons (Eberhardt and Schulz, 2003; Kou and Bloomquist, 2007). When mitochondrial complex I defects, the genetic factors clearly modulate susceptibility may result in oxidative stress and increase the susceptibility of neurons to excitotoxic death. In this way, mitochondrial dysfunction may result in neurodegeneration. (Sudhakar and Marie, 2013).
1-methyl-4-phenylpyridinium (MPP+), an bioactive derivative of 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP), has been broadly applied as the common neurotoxin for it can induce the loss of dopaminergic cells and the apoptosis of neurons, which was similar with the syndromes of PD (Lotharius and O’Malley, 2000). The neurotoxic derivative of MPTP, MPP+ is produced in brain by Monoamine Oxidase B (MAO-B), which is transferred into the dopaminergic neurons via the dopaminergic transporters, and subsequently suppresses the activities of electron transport chain in mitochondria (Nicklas et al., 1985). It is well-known that MPP+ plays a role in elevating the oxidative stress (Jung et al., 2007) and initiating the apoptosis via inducing the dysfunctions in mitochondria, which relates to the changes of proteins belonging to the Bcl-2 family, the production of cytochrome c, and the activation of caspases (Blum et al., 2001; O’Malley et al., 2003). Consequently, the apoptosis of neurons may be effectively inhibited by regulating the intracellular reactive oxygen species (ROS) and alleviating the dysfunction of mitochondria, which provides a new prospective in preventing and treating with PD.
So far, there are no effective drugs for PD in clinical. A variety of authorized drugs, like levodopa, have certain limitations and serious side effects when treating with PD (Rascol et al., 2003). A significant study shift from chemically synthesized drugs to naturally active compounds is being witnessed. Therefore, natural products become the major sources of chemical diversity for starting materials while driving pharmaceutical discovery, which represent a better therapeutic

effect and lower side effects, have important theoretical significance and application value (Houghton et al., 2005; Chen et al., 2007).
As a traditional Chinese herb, U. rhynchophylla (Miq.) Miq. ex Havil., also named as “Gou-Teng” has been widely applied in the intervention of neurodegenerative diseases for many years (Zhou and Zhou, 2010). Extracted from the hooked stem of the plant, Rhynchophylline (Rhy) is an oxindole alkaloid which can be easily absorbed and passed through the blood-brain barrier. Consistent with the previous study mentioned above, rhynchophylline block calcium release from intracellular stores, which is protective against ischemia, glutamate or dopamine-induced damage or death, inhibition of NMDA receptors, and suppression of 5-HT2A receptor function, among others (Zhou and Zhou, 2012, Huang et al., 2014). The characteristics of Rhy made it a promising drug for treating with the neurodegenerative disorders (Kang et al., 2002; Kang et al., 2004). Previous study has demonstrated that the Ca2+ influx can be suppressed by Rhy, which prevents the granule cells in cerebellum from the glutamate-induced toxicity (Xu et al., 2012). It was reported that Rhy played an anti-convulsive role in the rats suffered from acute seizures via the regulation of the Toll-like Receptor (TLR) and neurotrophin signaling pathways (Huang et al., 2014). Rhy has also been found to reduce dopamine treatment-induced cell death in a teratocarcinoma cell line of human and effectually inhibit the release of pro-inflammatory cytokines in the lipopolysaccharides (LPS)-induced microglia (Yuan et al., 2009). As a result, the hypothesis was that Rhy on the basis of the anti-inflammatory and anti-convulsive properties, might hold the competence of blocking the harmful behaviors relevant to neurotoxicity under the induction of MPTP or MPP+, which eventually stop the degeneration of oxidative stress-mediated in dopaminergic neurons. Under this background, the study aims to the effect of Rhy and the underlying mechanism for MPTP/MPP+-induced neurotoxicity.
Belonging to the adrenal gland pheochromocytoma cell line in rats, PC12 cells were characterized by its synthetic, metabolic, and transporting systems of dopamine (Tuler et al., 1989). Because of the characteristics of the dopaminergic neurons, PC12 cells have been widely applied as an ideal model for in vitro researches of PD (Jenkins and Barone, 2004; Qin et al., 2011). PC12 cells at different division cycle have been selected for evaluating the changes of morphology, biochemistry and molecular biology of neurons during the apoptosis (Li et al., 2017).
To sum up, it was suggested from the former studies that Rhy played a protective role in

neurons, which made it a promising medicine to deal with PD. However, it has not been reported that Rhy played the protective roles of on the apoptosis of neurons mediated by MPP+. In the present work, the protective roles of Rhy on the neurotoxicity mediated by MPP+ in the PC12 cells and MPTP-induced PD mouse model were investigated and the molecular signaling pathway underlying the effects was also identified to explore how Rhy performs neuroprotective effect in PD in vitro and in vivo models. This process would contribute to developing drugs from natural products and provide a new perspective for Rhy used as a neuroprotective agent clarifying their molecular mechanisms in clinical treatment.
2. Materials and methods

2.1 Ethical statement

All animal procedures were in compliance with the Guide for the Care and Use of Laboratory Animals and have been authorized by the Committee on the Ethics of Animal Experiments at Changchun Normal University.
2.2 Chemicals

The selective PI3K inhibitor LY294002, MPP+, the annexin V labeled by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide fluorescein isothiocyanate, and Propidium Iodide (PI) were bought from Sigma (St. Louis, MO, USA). Madopar was purchased from Shanghai Roche Led., (Shanghai, China) in a pharmacy. The Eagle’s medium modified by Dulbecco and Fetal Bovine Serum (FBS) were obtained from Invitrogen (Carlsbad, CA, USA). A kit for caspase-3 activity, Lactate Dehydrogenase (LDH), and Cell Counting Kit-8 were bought from Beyotime Institute of Biotechnology (Haimen, China). Fluo-3AM, as a florescent probe sensitive to Ca2+, was bought from Sigma-Aldrich (St. Louis, MO, USA). The enhanced chemi-luminescent reagent and bicinchoninic acid protein assay kit were purchased from Pierce (Rockford, IL, USA).
All the antibodies and goat anti-rabbit horse-radish peroxidase-conjugated IgG secondary antibody, were bought from Abcam (Cambridge, UK). The other reagents adopted in the work came from China with the analytical grade.
2.3 Plant material and preparation of Rhy extracted from U. rhynchophylla

The Uncaria rhynchophylla (miq.) jacks were purchased from Changchun, China, in 2018,

and they were confirmed by Professor Shu-Min Wang (Changchun University of Chinese Medicine). The freshly collected U. rhynchophylla was cut into sections and placed in a cool and ventilated place (about 25℃) to dry in the shade for approximately 7 days. Hooks with stems of U. rhynchophylla were used.
U. rhynchophylla, weighing 10 g, was finely minced, and a triple extraction was performed in a reflux condenser with 200 mL 60% ethylalcohol by sonication at a temperature of 25 ± 2℃. Each extraction lasted for 1 h. After complete evaporation, a re-dissolution was performed with 20
mL of heated water, and modulation with sulphoacid produced a solution with a pH of 3.0, followed by a filtering process. The macro-porous resinous column (D101, 10 × 80 cm, Naikai Chemical Co., China) was incorporated to separate the filtrate in an orderly fashion using 100 mL purified water and 100 mL 70% ethanol. The extracted URA was obtained by evaporating the aqueous ethanol fraction. A small amount of the URA was as a sample (PLP-20-0639) and preserved by the Central Laboratory of Changchun Normal University.
A 2695 HPLC system with a Waters 2998 DAD detector (Milford, MA, USA) was employed in the current research. A C18 column (250 × 4.6 mm id, 5 µm) was selected, and the column temperature was 25°C. The following parameters were used: mobile phase A: 0.5% acetic acid in water; mobile phase B: methanol; mobile phase gradient procedure: 0 – 2min, 13% B, 2 – 120min, 13% – 100% B; flow rate: 0.8 mL/min. The detection wavelength was set at 254 nm. Rhy appeared at 9.2 min (Fig.1 and Table.1).
2.4 Animals and treatments

8-week-old adult male C57BL/6 mice (about 25 g) were selected for the study. The mice were maintained according to the standard method, under a 12-h day-night cycle with 50-55% humidity, and water and food ad libitum. Mice were grouped to six sets with 12 mice in every group. (1) control mice; (2) MPTP exposed; (3) MPTP + Rhy 30 mg/kg, ig-treated group; and (4) MPTP + madopar 100 mg/kg, ig-treated group (Mu et al., 2009; Zheng et al., 2017). MPTP was administrated to mice for 8 consecutive days (30 mg/kg/d, intraperitoneal administration) while the control animals was treated with equivalent volume of normal saline. After the treatment of MPTP, the last four groups were processed with Rhy or madopar by i.g. administration for another 7 days, while the MPTP and control groups were treated with equal volume of normal saline. The behavioral tests were performed after the treatment of MPTP or saline.

2.5 Spontaneous motor activity test

A robotized locomotion monitoring system equipped with two infrared video web cameras was applied to measure spontaneous motor activity. Methods were performed as demonstrated in the former researches (Sundstrom et al., 1990; Mu et al., 2009; Zheng et al., 2017).
2.6 Rotarod test

The rotarod test was carried out to evaluate the sensorimotor coordination on a moving Lucite rod with a 2.5 cm diameter. Methods were performed based on the former researches (Sundstrom et al., 1990; Li et al., 2011; Zheng et al., 2017).
2.7 Immunohistochemistry

For the immunohistochemical study, four of the mice were perfusion-fixed with 4% paraformaldehyde following a heparinized saline flush 7 days after the behavioral assessment. The
mice brains were dissected and postfixed in paraformaldehyde overnight at 4℃, followed by their transfer into 30% sucrose in 0.1 M PB at 4℃ for 24 h. Using a cryostat, a series of 20-µm-thick coronal sections were obtained through the ventral mesencephalon. Nigral brain sections were
rinsed in PBS+Triton X-100 (PBST), quenched in 3% H2O2, and incubated in a blocking solution. After incubation with the anti-tyrosine hydroxylase (TH, monoclonal mouse, Abcam, 1:200) at 4℃ overnight, the sections were treated with a biotinylated secondary antibody for 1 h at 37℃,
followed by incubation with streptavidin-peroxidase for 1 h. Subsequently, the sections were incubated with 3, 4-diaminobenzidine. The results were analyzed by counting the number of positive cells at a 400× magnification using Nikon microscope (Eclipse Ci-L, Nikon Corp., Janpan). The region of interest was captured using a camera and analyzed using Image-Pro Plus
6.0 software. The average number of positive cells was used to represent cell density.

2.8 The cultivation of cells and drug treatments

PC12 cells were obtained from American Type Culture Collection (Rockville, MD, USA).They were cultivated in the DMEM medium containing penicillin and streptomycin (100 unit/mL, 100 µg/mL, respectively), 10% FBS at 37℃ under a humid condition with 5% CO2 atmosphere.
A variety of concentrations of Rhy were added to the cells and incubated for 24 h, followed by treating with MPP+ for another 48 h with the final concentration of 500 µM. When studying the

suppression of the phosphoinositide 3-kinase (PI3K)/Akt signaling pathway, 50 µM LY294002 was added 1 h before the treatment of Rhy and/or MPP+.
2.8.1 The assessment of cell viability

PC12 cells, with 1×104 cells in each well, were seeded in the 96-well plate. After incubating overnight, the cells were separated equally to five groups to receive the treatments as follows: (1) non-treated control; (2) MPP+ 500 µM; (3) MPP+ 500 µM + Rhy-5 (Rhy 5 µM); (4) MPP+ 500 µM
+ Rhy-10; (5) MPP+ 500 µM + Rhy-20. Or (1) non-treated control; (2) MPP+ 500 µM; (3) MPP+
500 µM + Rhy-5 (Rhy 20 µM); (4) MPP+ 500 µM + LY294002 50 µM; (5) MPP+ 500 µM +
Rhy-20 + LY294002 50 µM. For the MTT and LDH activity analyses, n was set as 6 in each group. After the cells were seeded for 48 h, the cells were rinsed with serum-free medium, followed by the treatments of different reagents. After further incubating for 48 hours, the experiments were conducted.
Cell viability was evaluated based on the CCK-8 method as described previously (Zheng et al., 2016). After incubating for 48 h, 10 µL CCK-8 reagent was added. After further maintaining for 2 h at 37℃, the absorbent peaks were identified at A450 on a microplate reader (SpectraMax M5, Molecular Devices, Sunnyvale, CA).Cell viability was expressed as percentage of the
non-treated control.

2.8.2 The release test for Lactate dehydrogenase (LDH)

Aliquots of 1.5×105 cells per well were transferred into the 24-well plate and maintained overnight. The supernatants were obtained and 200 µL/well was given to a black 96-well plate. The leakage of LDH was analyzed based on the LDH cytotoxicity assay kit (In Vitro Toxicology Assay Kit, Sigma) according to the instructions. The results were detected by the microplate reader at 490 nm. The release of LDH was defined as the percentage of total (released + unreleased).
2.8.3 The analysis of the concentration of intracellular Ca2+

As a fluorescent probe response to Ca2+, Fluo-3AM, was adopted to detect the changes in [Ca2+]i based on spectrofluorometry. Cultivated and collected as mentioned before, PC12 cells were further rinsed twice using Phosphate Buffered Saline (PBS). [Ca2+]i was defined based on

the previous work by Cui et al. [Ca2+]i was represented as the proportion of the non-treated control.
2.8.4 The analysis of intracellular ROS

Intracellular ROS was analyzed using 2’,7’-dichlorodihydrofluorescin diacetate (DCFH-DA, Beyotime, Shanghai, China), a fluorescent dye sensitive to oxidation, according to the previous study (Zheng et al., 2016). The de-esterification of DCFH-DA was occurred in cells, which was further oxidized to 2’, 7’-dichlorofluorescein as a highly fluorescent dye. The fluorescence was detected by the microplate reader, the excitation of which was set as 488 nm and the emission as 525 nm. The percentage of the control was adopted to express the intracellular ROS.
2.8.5 The flow cytometric apoptosis analysis

The death of cells was analyzed based on the Annexin V-FITC/PI double staining kit (Sigma-Aldrich, USA) (Cui et al., 2017). PC12 cells were collected, rinsed twice using cold PBS, and cultivated with the Annexin V/PI labeling reagent for 20 min at room temperature in the darkness. The cultures were detected based on flow cytometry (FACSCanto II Analyzer, BD, USA). The dead cells were calculated from the cellular populations of annexin V-FITC/PI.
2.8.6 The analysis of caspase-3 activity

The caspase-3 activity was measured by a caspase-3 assay kit (Beyotime, Shanghai, China) based on the instructions (Zheng et al., 2016). After the lysis, the buffer including the substrate peptide Ac-DEVD-pNA for caspase-3 interacted to pNA was added to the lysate. The level of pNA was measured based on the absorbance at 405 nm. Data were expressed as percentage of the non-treated control.
2.8.7 Western blot

Western blot was conducted as described before (Zheng et al., 2019). After the respective treatments, PC12 cells were collected and lysed for western blotting and immunoprecipitation assays (Beyotime) with 1 mM phenylmethylsulfonyl fluoride as the protease inhibitor. Equivalent proteins (20 µg/well) were separated by the 10% SDS-polyacrylamide gels and shifted to the nitrocellulose membranes, which were further blocked with 5% defatted milk in Tris-buffered

saline with 0.05% Tween-20 (TBST) for 1 h at room temperature, and conducted immunoblotting with the primary antibodies at 4℃ for all night. Triply rinsed with TBST, the membrane was probed with peroxidase-conjugated secondary antibody for 45 minutes and the protein bands were
measured based on enhanced chemi-luminescent analyzing system. The visible density of protein band was calculated by the Gel-Pro analyzer 4.0 software (Media Cybernetics, China).
2.9 Statistics

All data were represented as mean ± Standard Deviation (SD) and further analyzed by the SPSS 13.0 software (IBM, Chicago, IL, USA). One-way analysis of variance combined with the Tukey’s multiple comparison test was adopted to evaluate the statistical significance, which was assessed by p < 0.05.
3. Results

3.1 The protective properties of Rhy against MPTP-induced behavioral damage in mice

The results of the spontaneous motor activity are shown in Figure 2. Compared with the control group, there was a remarkable behavioral degeneration in the spontaneous motor activity (P < 0.01) after the treatment of MPTP. However, MPTP-induced behavioral impairments were markedly improved by the Rhy treatment (P < 0.01). The mice treated with Madopar also showed significantly reduced rotational behavior (P < 0.01, Fig.2A).
The MPTP-induced mice represented a marked degeneration in latency to fall off the rotarod test compared to the control group (P < 0.01). However, as shown in Figure 3, Rhy dramatically prevented this MPTP-induced behavioral impairment (P < 0.01). The mice treated with Madopar also showed significantly reduced rotational behavior (P < 0.01, Fig. 2B).
3.2 Effect of the Rhy on MPTP-induced reduction of tyrosine hydroxylase immunoreactivity in the substantia nigra
Tyrosine hydroxylase immunoreactivity is a marker of dopaminergic neurons. Representative microphotographs of the TH immunostaining in the substantia nigra are shown in Fig.3 A-D. Animals that received vehicle-only treatment with MPTP injection showed a marked loss of TH-immunopositive neurons (P < 0.05). In contrast, compared to the MPTP group, the loss of neurons was inhibited by Rhy at a dose of 30 mg/kg and madopar treatment significantly,

accompanied by a concomitant increase in TH-positive neurons (P < 0.05, Fig.3E).

3.3 Rhy prevents the apoptosis caused by MPP+

To exclude the possibility that Rhy itself affected the survival of PC12 cells, different concentrations of Rhy (0, 5, 10, 20, and 50 µM) were added to the cells for 48 h. Rhy-induced loss of cell viability in PC12 cells was negligible below a concentration of 20 µM while it decreased markedly at 50 µM (P < 0.05) (Fig. 4A).
Cell viability results showed that in MPP+ group was remarkable lower than that in the control group (56.2 ± 3.93%, p < 0.01). Rhy-treated (5, 10 or 20 µM) significantly improved the
viability comparison with the MPP+ group (68.6 ± 2.56%, 75.4 ± 3.67%, and 82.7 ± 5.43% respectively)(Fig. 4B). Furthermore, it was indicated by the post hoc analyses that there were remarkable differences among the Rhy-treated groups (p < 0.01). As a consequence, PC12 cells were treated with 50 µM LY294002, the PI3K inhibitor. It was revealed in Fig.4C that LY294002 showed the property of reducing the viability of the MPP+-induced PC12 cells, which also dramatically decreased the positive role of Rhy on cell viability significantly (p < 0.01).
3.4 Rhy suppresses the release of LDH, increase of Ca2+ overload and ROS levels in the MPP+-induced cells
In contrast to control group, 500 µM MPP+ treated PC12 for 48 h produced an obvious promotion in LDH release (196 ± 3.2%, p < 0.01) and Ca2+ concentration(167 ± 4.8%, p < 0.01) and a remarkable up-regulation of the levels of intracellular ROS (168 ± 4.2%, p < 0.01) in comparison with the control group. However, Rhy (20 µM) decreased the release of LDH, Ca2+ concentration and ROS levels significantly (132 ± 1.6%, p < 0.01;121 ± 4.8%, p < 0.01; 121 ± 4.8%, p < 0.01, respectively). In addition, PI3K inhibitor LY294002 (50 µM) attenuated the effects of Rhy (p < 0.01, p < 0.01, p < 0.01) (Fig.5).
3.5 Rhy reduces apoptosis in cells treated with MPP+
It can be demonstrated from the Annexin V/PI staining assay that Rhy may protect the PC12 cells from the cell death induced by MPP+. As revealed in Fig.6, 500 μM MPP+ increased both early and late death of PC12 cells and the rate of apoptosis was 20.13% in total. Nevertheless, treatment with Rhy (20 μM) decreased the apoptosis rate to 8.72%. To verify the effect of PI3K/Akt signaling pathway in the neuroprotective properties of Rhy, 50 µM LY294002 were

firstly added to PC12 cells for 1 h, followed by 30 min treatment with 20 µM Rhy and then 24 h incubation with 500 µM MPP+. It was shown that the effects of Rhy on MPP+-mediated cell death were alleviated by LY294002 (p < 0.01), which suggested that the PI3K/Akt signaling pathway played a part in MPP+-induced cell death.
3.6 Rhy suppresses the activation of caspase-3 in MPP+-treated PC12 cells

The caspase-3 activity was increased markedly in the MPP+-induced group in comparison with the control group (228 ± 3.8% of the control, p < 0.01), However, treatment of cells with Rhy attenuated the increase in caspase-3 activity (145 ± 4.8%, p < 0.01). In addition, after treating with 50 µM LY294002, there was a remarkable improvement of the caspase-3 activity (160 ± 3.6%, p < 0.01). It was demonstrated from the results that Rhy can suppress the activation caspase-3 caused by MPP+, however, the influence of Rhy on the activity of caspase-3 was alleviated by LY294002(Fig.7).
3.7 Rhy reduces the Bcl-2/ Bax ratio in the PC12 cells treated with MPP+
Since the activated PI3K/Akt signaling pathway is considered to elevate the level of Bcl-2 and phosphorylate Bax helped in interacting with other proteins and inhibit the cell death, the roles of Rhy on Bcl-2 and Bax were assessed. It was shown in Fig.8 that the MPP+ treatment down-regulated the Bcl-2/Bax ratio in comparison with the control group (52 ± 1.5% of the control, p < 0.01) and there was a remarkable elevated Bcl-2/Bax ratio (72 ± 1.8%, p < 0.01) in the 20 µM Rhy-treated groups induced by MPP+ compared to the MPP+ group. It can be revealed that the intervention of Rhy changed the balance between pro-/anti-apoptotic components and cell survival, however, the roles of Rhy on the ratio of Bcl-2/Bax was alleviated by LY294002.
3.8 Rhy attenuates caspase-3 activity through the PI3K/Akt signaling pathway

Caspase-3 is one of the major executioners of apoptosis. The direct and indirect activated PI3K/Akt triggers the phosphorylation of caspase-3, which helps in interacting with other proteins and preventing the apoptosis. As represented in Fig.9, the levels of caspase-3 was markedly elevated by dealing with MPP+, compared to the untreated controls (178 ± 3.2%, p < 0.01). Nevertheless, the results of western blot indicated that caspase-3 was reduced by Rhy in the cells dealing with MPP+. LY294002 alleviating the functionsof Rhy (p < 0.01), suggesting that Rhy suppressed the activity of caspase-3 via activating PI3K/Akt signaling pathway.

3.9 Roles of Rhy on the PI3K/Akt signaling pathway in MPP+-induced PC12 cells

To further identify the effects of PI3K/Ak signaling pathway in the protective properties of Rhy against the cell death induced by MPP+, the levels of phospho-Akt (Ser473) and Akt were analyzed by western blot. As represented in Figure 14, there are no changes in the levels of Akt after MPP+-induced neurotoxicity or Rhy treatment. Moreover, compared to the controls, the levels of phospho-Akt (Ser473) were significantly down-regulated in the MPP+-induced group (p
< 0.01). In contrast, treating with Rhy alleviated the decrease of phospho-Akt (Ser473) significantly compared with the MPP+ group, suggesting that Rhy activated the PI3K/Akt signaling pathway (Fig.10). The effects of Rhy intervention was markedly inhibited by LY294002, additionally proving that the PI3K/Akt signaling pathway played a role in exerting the protective properties of Rhy.
4. Discussion

Uncaria rhynchophylla has been broadly applied in Chinese and Japanese Kampo medicine to deal with vascular dementia, stroke, and other neurodegenerative diseases (Yuan et al., 2009). As an alkaloid, Rhy is derived from Uncaria, and exerts an outstanding neuroprotective function (Kang et al., 2002; Kang et al., 2004). According to previous studies, Rhy has been reported to display various pharmacological activities, including antiaggregating effect on β-amyloid proteins in the Alzheimer disease (Fujiwara et al., 2006), and ameliorating cognitive disorders caused by D-gal-actose in mice (Xian et al., 2011). Rhy was known to play a prodominent role in the anti-convulsion in kainic acid (KA)-treated rats through regulating TLR and neurotrophin signaling pathways, and then inhibiting the expressions of IL-1β and BDNF (Ho et al., 2014; Chen et al., 2020). Finally, Rhy showed its neuroprotective properties against MPPC-triggered neurotoxicity in PC12 cells by stimulating MEF2D through the activation of the PI3-K/Akt/GSK3b cascade (Wang et al., 2018). In the present study, we conducted the first investigation of anti-PD effects of Rhy extracted from Uncaria rhynchophylla, in mice exposed to MPTP, using a behavioral and neurohistological alteration model. We observed changes in the expression of TH in the SN of the ipsilateral hemisphere. Moreover, we investigated the roles of Rhy in cell viability, nuclear and mitochondrial apoptosis, and the intracellular levels of Ca2+ concentration in the PC12 cell model of PD induced by MPP+, and examined the mechanisms

underlying the effect of Rhy. To sum up, it was the first time to indicate the neuroprotective property of Rhy against MPP+-mediated cytotoxicity in cultured PC12 cells through the PI3K/Akt signaling pathway.
The MPTP mouse model has been broadly applied to evaluate the neuroprotective effects of drugs for it reproduced the main pathological and biochemical characteristics of PD, including the energy metabolism disorder in mitochondria, oxidative stress, neuronal excitotoxicity, and apoptosis (Fang et al., 2019; Zheng et al., 2017). Our results confirmed that continued MPTP exposure evoked the behavioral deficits in mice, such as the reduced spontaneous motor behavior and motor coordination in the rotarod test, which were in accordance with the former study (Sundstrom et al., 1990). It has been revealed in Fig. 2 that the behavioral deficits induced by MPTP were largely prevented by the administration of Rhy. Through the behavioral tests, it can be concluded that Rhy, acting as a neuroprotective agents, effectively improved the symptoms in MPTP-induced mice.
In this work, the viability of PC12 cells was reduced by MPP+, which was reversed by Rhy and the morphology was improved with the concentrations of 5 to 20 µM. It was demonstrated in the former research that Rhy played a protective role in alleviating the damage induced by LPS in N9 cells with the concentrations of 1 to 30 µM (Yuan et al., 2009). The higher efficiant concentrations in this work may be affacted by the type of cells and/or toxins.
LDH is a soluble cytosolic enzyme that is present in most cells, which release LDH upon injury. Increased release of LDH was found in the cultured neurons teated with neurotoxins (Cao et al., 2010) and brain injury (Zandbergen et al., 2001). Consistent with the previous study, it was indicated from the current data that the release of LDH was promoted by MPP+, which was alleviated by Rhy in a dose-dependent manner. Consequently, the inhibition of LDH leakage may play part in the protective properties of Rhy against neurotoxicity and brain injury.
Ca2+ influx was reported to have an important impact in the production of ROS in cells (Zheng et al., 2016; Cui et al., 2017). However, in variety of conditions, the increase in [Ca2+]i was caused by free radicals. Further Ca2+ influx was induced by the impairment of membrane caused by ROS, which in turn promoted the production of extra ROS (Cotman et al., 1992). The dysfunction in mitochondria was induced by Ca2+ influx and ROS, which led to apoptosis (Cui et al., 2017). It has been reported that the intracellular Ca2+ overload may relate to the MPP+-induced

apoptosis in PC12 cells (Xu et al., 2012). Likewise, in our work, it was shown that the levels of intracellular Ca2+ and ROS in PC12 cells was significantly enhanced by treating with MPP+. Treatment with Rhy significantly decreased the [Ca2+]i and ROS in the MPP+-induced PC12 cells, indicating that neuroprotective role of Rhy was associated with the suppression of Ca2+ overload and ROS excess.
The dysfunction and the activation of apoptosis in mitochondria can be induced by MPP+ (Boada et al., 2000). The apoptotic pathway in mitochondria was controlled by the proteins of Bcl-2 family, containing several homologous proteins, including the anti-apoptotic proteins like Bcl-2 and the pro-apoptotic proteins like Bax (Kosten et al., 2008). Thus, the balance of these regulatory proteins was crucial for viability or apoptosis (Li et al., 2008; Wang et al., 2008). The relative proportion of pro- and antiapoptotic proteins has been applied to define the cell survival or apoptosis.
Caspase-3, acting as a common executioner of the signals of apoptosis which catalyzed the cleavage of various regulatory proteins, actively regulated apoptosis (May and Madge, 2007; Cheung et al., 2008). We found that MPP+ intervention up-regulated the level of Bax and cleaved caspase-3, and down-regulated the level of Bcl-2. Moreover, it was shown that the Bcl-2/Bax ratio was elevated and the cleaved caspase-3 was reduced by Rhy. The results were in accordance with of the data described previously (Lee et al., 2005; Cao et al., 2010; Liu et al., 2020). Overall, it was suggested that the apoptoic pathway in mitochondria was involved in the protective property of Rhy against the death of PC12 cells induced by MPP+.
A variety of researches have indicated that PI3K signaling pathway has a remarkable impact in the protection against various apoptotic stimuli (D’Astous et al., 2006; Cao et al., 2017; Chen et al., 2019). As a prominent downstream effector, Akt protein kinase was required for the PI3K/Akt signaling transduction (Abeyrathna and Su, 2015; Chen et al., 2020). A variety of researches have reported that Akt was involved in a large number of brain diseases, such as PD (Zheng et al., 2017), ischemia (Brywe et al., 2005), autism (Sheikh et al., 2010), seizures (Cote et al., 2005). The cell survival was improved by Akt by blocking the pro-apoptotic proteins and related processes. Furthermore, the level of Bcl-2 can be up-regulated by Akt, which results in cell survival (Pugazhenthi et al., 2000). Additionally, Bax, belonging to the pro-apoptotic Bcl-2 family, can be phosphorylated by Akt, which in turn inhibited the pro-apoptotic effects of Bax (Datta et al., 1997;

Peso et al., 1997). Furthermore, it has been reported that the activated PI3K/Akt inhibited the expression of caspase-3 and the cleavage of DNA for various cell lines (Wu et al., 2000; Araki et al., 2002). Our findings clearly indicated that MPP+ intervention dramatically down-regulated the protein level of p-Akt in PC12 cells, and Rhy showed the oppisite effect. In addition, it was revealed that Rhy improved the viability and inhibited the neurotoxicity in MPP+-induced PC12 cells, which can be diminished by LY294002, effectively supporting the role of PI3K/Akt signaling pathway in Rhy-treated protection for neurons. Rhy treatment up-regulated the Bcl-2/Bax ratio and reduced the cleaved caspase-3 caused by MPP+, however, LY294002 inhibited the above properties of Rhy. The results suggested that Rhy prevented the PC12 cells from apoptosis caused by MPP+ through the PI3K/Akt signaling pathway.
5. Conclusions

In conclusion, our study provided the evidence that Rhy was efficient in inhibiting the cell death in PC12 cells induced by MPP+ and exhibited a prominent neuroprotective effects against brain lesions induced by MPTP, which may be realized via the activated PI3K/Akt signaling pathway. It has been proven that Rhy can be applied as a promising drug for preventing or treating of the neurodegenerative disorders including the Parkinson's disease. However, further researches were required to interpret the molecular mechanisms.
Authors’ contributions

The present work was conducted in collaboration with all authors. Authors LCM and ZMZ designed the study and wrote the methodology. The authors ZMZ and FYJ performed the experiments. ZMZ, CMH, WWL, ZM and SDF performed the literature search and statistical analysis, author ZMZ wrote the paper, and author LCM supervised the research. All authors have read and approved the final manuscript.
Formatting of funding sources

This project was supported by the National Natural Science Foundation of China (grant numbers 31170326), and the Natural Science Foundation of Jilin Province (20160101209JC), and Natural Science Foundation of Education Department of Jilin Province (JJKH20181168KJ).
Conflict of interest None to declare.

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Figure Captions

Fig.1. High-performance liquid chromatograms of Uncaria rhynchophylla (Miq.) Jacks. extract (a) HPLC chromatograms of Uncaria rhynchophylla (Miq.) Jacks. extract (b) TIC of MS of Uncaria rhynchophylla (Miq.) Jacks. extract (c) The Mass spectrum and chemical structure of of Rhynchophylline

HPL chromatography peaks were as follows: 1, Rhynchophylline; 2, Corynantheine; 3, Corynoxeine; 4, Isocorynoxeine; 5, Isorhynchophylline; 6, Dihydrocorynantheine; 7, Hirsutine; 8,
Hirsuteine; 9, Angustoline; 10, Angustidine

Fig. 2. Effect of Rhy on spontaneous motor activity and on rotarod performance in MPTP-treated mice. (A) Spontaneous motor activity. (B) Rotarod performance. Values are expressed as means ± standard errors of the mean (SEMs; n = 18). ##P < 0.01, compared with the control group;*P < 0.05, **P < 0.01, compared with the MPTP group；△P <0.05，△△P < 0.01, compared with the Madopar group.

Fig. 3. Effect of Rhy on MPTP-induced reduction of tyrosine hydroxylase (TH) immunoreactivity in the substantia nigra of mice. (A) Representative microphotographs showing control; (B) Model (MPTP);(C)\MPTP+Rhy (30 mg/kg);(D) Madopar; (E) Summary of the effects of Rhy on MPTP-induced TH-immunoreactive neurons in the substantia nigra. The TH-positive neuron immunoreactivities are expressed as mean ± S.E.M. (n = 3). *P < 0.05 compared with the control group, #P < 0.05 compared with the MPTP/vehicle treatment group. Scale bars: 100 µm.

Fig.4. Cytotoxic and protective properties of Rhy in the PC12 cells. (A) Impacts of Rhy on cell survival. Cells were incubated with different concentrations of Rhy for 48 h, and cell viability was analyzed by the CCK-8 kit. (B) Neuroprotective properties of Rhy against the cytotoxicity induced by MPP+. Cells were incubated with 500 µM MPP+ and Rhy (5, 10, and 20 µM) for 48 h, and cell viability was also analyzed. (C) Protective role of Rhy on MPP+-treated PC12 cells. After 1 h pretreatment with 50 µM LY294002, the cells were incubated with 20 µM Rhy for 24 h, and

further treated with MPP+ for another 48 h. The results are represented as the mean ± SD (n = 6).
##p < 0.01 as compared with control; *p < 0.05, **p < 0.01 as compared with MPP+ group. $$p <
0.01 compared to the group treated with MPP+ and LY294002 (50 µM).

Fig.5. Effects of Rhy on lactate dehydrogenase (LDH) leakage, intracellular calcium concentrations ([Ca2+]i), and intracellular reactive oxygen species (ROS) levels in MPP+-induced PC12 cells. ##p < 0.01 as compared with control; *p < 0.05, **p < 0.01 as compared with MPP+ group; $$p < 0.01 compared to the group treated with MPP+ and LY294002 (50 µM).

Fig.6. The effects of Rhy on the apoptotic rate based on flow cytometry. (A) PC12 controls. (B) 500 µM MPP+. (C) 20 µM Rhy + 500 µM MPP+. (D) LY294002 + 500 µM MPP+. (E) 20 µM Rhy
+ 500 µM MPP+ + LY294002. Data are expressed as the mean ± SD (n = 3). #p < 0.05 and ##p <
0.01 vs. control; *p < 0.05, **p < 0.01 vs. MPP+-induced group; $$p < 0.01 compared to the group treated with MPP+ and LY294002 (50 µM).

Fig. 7. The impacts of Rhy on activating the caspase-3 activities in MPP+-treated PC12 cells. ##p <
0.01 as compared with control; *p < 0.05, **p < 0.01 as compared with MPP+ group; $$p < 0.01 compared to the group treated with MPP+ and LY294002 (50 µM).

Fig.8. Effects of Rhy on Bcl-2 and Bax expression in MPP+-induced PC12 cells. (a) Representative immunoblots of Bcl-2 and Bax (b) Bcl-2/Bax ratio. ##p < 0.01 as compared with control; *p < 0.05, **p < 0.01 as compared with MPP+ group; $$p < 0.01 compared to the group treated with MPP+ and LY294002 (50 µM).

Fig.9. Rhy treatment reduced the cleavage of caspase-3 that had been upregulated by treating with MPP+ in PC12 cells. (a) Western blot assay of the cleaved caspase-3 after the various interventions. (b) The level of the cleaved caspase-3 was quantitated and relatively represented to the control. ##p < 0.01 as compared with control; *p < 0.05, **p < 0.01 as compared with MPP+ group; $$p < 0.01 compared to the group treated with MPP+ and LY294002 (50 µM).

Fig.10. The role of PI3K/Akt pathway in the protection of Rhy against apoptosis induced by MPP+. (a) Western blot analysis of Akt and p-AKT expression in PC12 cells after the different treatments. (b) Quantification of western blot data. ##p < 0.01 as compared with control; *p < 0.05,
**p < 0.01 as compared with MPP+ group; $$p < 0.01 compared to the group treated with MPP+ and LY294002 (50 µM).

Table 1 The MS-MS fragmentation patterns of the main alkaloids in the total alkaloids extracted from Uncaria rhynchophylla (Miq.) Jacks.

Protection by rhynchophylline against MPTP/MPP+-induced neurotoxicity via regulating PI3K/Akt pathway
Highlights

• Rhynchophylline has a protective against MPTP/MPP+-induced neurotoxicity in mouse model and PC12 cell.
• Phosphoinositide 3-kinase (PI3K) inhibitor, LY294002, completely abolished the protective effects of Rhy against MPP+-induced neuronal cell apoptosis.
• The PI3K/Akt signaling pathway have mediated the protection of Rhynchophylline against MPP+-induced apoptosis in PC12 cells. MPP+ iodide