Inhibiting effects of rhynchophylline on methamphetamine-dependent zebrafish are related with the expression of tyrosine hydroxylase (TH)

    Inhibiting effects of rhynchophylline on methamphetamine-dependent zebrafish are related with the expression of tyrosine hydroxylase (TH) Chen Zhu, Wei Liu, Chaohua Luo, Yi Liu, Chan Li, Miao Fang, Yingbo Lin, Jinying Ou, Minting Chen, Daoqi Zhu, Ken Kin-Lam Yung, Zhixian Mo PII: DOI: Reference:

S0367-326X(16)30870-X doi:10.1016/j.fitote.2017.01.001 FITOTE 3550

To appear in:

Fitoterapia

Received date: Revised date: Accepted date:

28 November 2016 2 January 2017 3 January 2017

Please cite this article as: Chen Zhu, Wei Liu, Chaohua Luo, Yi Liu, Chan Li, Miao Fang, Yingbo Lin, Jinying Ou, Minting Chen, Daoqi Zhu, Ken Kin-Lam Yung, Zhixian Mo, Inhibiting effects of rhynchophylline on methamphetamine-dependent zebrafish are related with the expression of tyrosine hydroxylase (TH), Fitoterapia (2017), doi:10.1016/j.fitote.2017.01.001

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Inhibiting effects of rhynchophylline on

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methamphetamine-dependent zebrafish are

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related with the expression of tyrosine hydroxylase (TH)

Yingbo Lin a, Jinying Ou a, Minting Chen a, Daoqi Zhu a, Ken Kin-Lam

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a,

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Chen Zhu a,1, Wei Liu a,1 , Chaohua Luo a, Yi Liua,Chan Li a, Miao Fang

Yung b,**, Zhixian Mo a,*

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a School of Traditional Chinese Medicine, Southern Medical University, Guangdong Guangzhou, 510515, China

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b Department of Biology, Hong Kong Baptist University, Kowloon Tong, Hong Kong

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*Corresponding author. School of Traditional Chinese Medicine, Southern Medical University, 1063 Shatai Road, Guangzhou, 510515, China. Tel.: +86 20 61648261, fax: +86 20 61648244.

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Email addresses: [email protected] (Z. Mo) **Corresponding author. Department of Biology, Hong Kong Baptist University, Kowloon Tong, Hong Kong. Tel.: + 852 90482038, fax: + 852 34117060. 1

These authors contributed equally to this work

ACCEPTED MANUSCRIPT Abstract

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In this study, to study the effect of rhynchophylline on TH in midbrain of methamphetamine-induced conditioned place preference (CPP) adult zebrafish, place preference adult zebrafish models were established by methamphetamine (40μg/g) and the expression of TH was observed by immunohistochemistry technique and Western blot. Ketamine (150μg/g), high dose of rhynchophylline (100μg/g) group can significantly reduce the place preference; immunohistochemistry results showed that the number of TH-positive neurons in midbrain was increased in the methamphetamine model group, whereas less TH-positive neurons were found in the ketamine group and high dosage rhynchophylline group. Western blot results showed that the expression of TH protein was significantly increased in the model group, whereas less expression was found in the ketamine group, high dosage rhynchophylline group. Our data pointed out that TH plays an important role in the formation of methamphetamine-induced place preference in adult zebrafish. Rhynchophylline reversed the expression of TH in the midbrain demonstrates the potential effect of mediates methamphetamine induced rewarding effect.

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Chemical compounds studied in this article Methamphetamine hydrochloride (PubChem CID:10836); Rhynchophylline (PubChem CID: 5281408); Ketamine hydrochloride (PubChem CID:15851)

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Keywords: Methamphetamine dependence; Rhynchophylline; Tyrosine hydroxylase (TH);

ACCEPTED MANUSCRIPT Introduction [1-3]

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The zebrafish, Danio rerio, is widely used in different areas of biological research

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including neuroscience[4]. Our previous report has strongly suggested that transgenic zebrafish are

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a good live biosensorfor persistent organic pollutants[5]. Zebrafish have a functional nervous system after several days of embryonic development, and thus it’s an excellent model for studying

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vertebrate brain development.

The basic structure of the central nervous system in zebrafish has all of the major domains

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that are found in the mammalian brain. Neurotransmitters such as dopamine and other catecholamines are also found in both interneuron systems and in long neuronal pathways [6,7].

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Tyrosine hydroxylase (TH) is necessary for catecholamine biosynthesis. It is commonly used as a marker furcate cholaminergic neurons[8,9]. The distributions of catecholaminergic neuron

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sinamniotes of mouse and rat have been extensively described[10,11]. The largest difference in the zebrafish catecholaminergic system compared with the mammalian system is the lack of midbrain

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dopaminergic neuron populations. A projection of dopaminergic neurons in the posterior tuber culum in the zebrafish is found to be equivalent to the mammalian ascending dopaminergic system[12,13], which is the major focus of the present study. Methamphetamine is a highly addictive stimulant drug that activates certain systems in the brain, including Central nervous system. Although it belongs to amphetamine family whose members are generally much more potent, longer lasting, and more harmful to the central nervous system

[14]

, methamphetamine may also affect the expression of functional proteins in multiple

regions in the brain, including hippocampus, prefrontal cortex, ventral tegmental area, striatum, nucleus accumbens and amygdale[15]. The accurate mechanism responsible for the addiction is

ACCEPTED MANUSCRIPT complicated and uncovered. As drug addiction has become a worldwide problem and numerous

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compound which can stop people from drug abusing is highly expected.

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studies are focusing on the risk, prevention, and intervention for addictions, an effective

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In the central nervous system, rhynchophylline could be beneficial for memory impairment induced by the dysfunction of cholinergic systems in brain [16,17]. Rhynchophylline is also effective in sedation, anti-convulsion, anti-epilepsy[18-21], thus rhynchophylline has been widely used in

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traditional Chinese medicines prescribed to treat ailments in central nervous system and

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cardiovascular systems, such as lightheadedness, convulsions, numbness, and hypertension. Rhynchophylla is one of the main components of traditional Chinese medicine and the main

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stem is extracted from Uncaria alkaloid. In recent years, rhynchophylline has been confirmed with

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all kinds of pharmacological activities, such as anti-arrhythmic, neuroprotective and

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anti-inflammatory effects, indicating rhynchophylline drug dependence treatment may have a certain effect. Early experiments show rhynchophylline amphetamine dependence caused by the

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elimination of the position preference effect, and in itself does not cause drug dependence[22-24]. Our study aimed to investigate the effect of methamphetamine dependence place preference in zebrafish. This experiment replicated the zebrafish model derived position preference zebrafish activity routes, using immunohistochemistry and western blot to detect TH expression in zebrafish, to explore rhynchophylline detoxification mechanisms and further research to provide the scientific rhynchophylline in accordance with.

ACCEPTED MANUSCRIPT Materials and methods

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Animals

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Wild-type adult male zebrafish (AB strain, age: 3-6-month-old; weight: 0.5-1g) were provided by Fish Facilities of the Key Laboratory of Zebrafish Modeling and Drug Screening for Human Diseases of Guangdong Higher Education Institutes (Guangzhou, China). All animals

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were maintained in a multi-tank system (temperature: 28°C; 14-h light: 10-h dark cycle) to

flake fish food and live brine shrimps.

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simulate their environmental condition. Adult zebrafish were fed twice a day with a mixture of

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All experimental protocols and animal handling procedures were performed in accordance

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with the National Institutes of Health (NIH, USA) Guide for the Care and Use of Laboratory

University.

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Animals and ere approved by the Experimental Animal Ethics Committee of Southern Medical

Drugs and reagents Methamphetamine hydrochloride was purchased from the the National Narcotics Laboratories (NO.1212-9802). Rhynchophylline was bought from Matsuura Yakugyo Co., Ltd., Japan(No. H1I2, purity 99.7%). Ketamine hydrochloride (Jiangsu Hengrui Medicine Co., Ltd.). Anti-TH(AB152，Millipore). Dual Mark(161-0374,BIO-RAD). MagicMark(LC5602, Invitrogen). Laemmli Sample Buffer(161-0737,BIO-RAD). SDS(L5750, sigma).TRIS Ultrapure(75825 ， USB).TEMED(T9281，sigma). Protein Assay Reagent A(500-0113，BIO-RAD). Protein Assay

ACCEPTED MANUSCRIPT Reagent B(500-0114，BIO-RAD). Extraction Reagent (71009-3, Novagen).

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Apparatus

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Aquarium conditioned place preference (CPP): cylinder volume of about 16 cm × 9cm × 9 cm. Fish tank in the middle of a 9cm high movable partition, the tank can be divided into two equal volume boxes, taken out after the partition, the fish can swim freely between the

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two cabinet. Brown side of the box, and the other side is transparent, the transparent bottom

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side with two black dots having a diameter of 2cm. Noldus animal behavior analysis system (Ethovision XT 8.5).

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Methamphetamine and ketamine treatment

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Methamphetamine behavioral, physiological and neuroregulatory effects were examined in different assays. A dose of methamphetamine (40μg/g) hydrogen tartrate salt (Sigma) or ketamine hydrochloride was employed on zebrafish. The methamphetamine solution was

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prepared by dissolved with methamphetamine hydrogen tartrate salt powder, while the ketamine-containing media was adjusted by diluting the ketamine hydrochloride solution in aquarium water. After preparation, zebrafish were directly exposed to methamphetamine or ketamine in solution during treatment.

Experimental design According to previous study and our pre-test[25; 26], zebrafish have an innate preference for brown environment. Therefore we identified the white compartment as the METH-paired compartment. Before the experiment, position preference test was performed on each of the

ACCEPTED MANUSCRIPT zebrafish and excluded the animal which did not meet the natural preference. Before the test, the middle partition of the box was taken out. When the zebrafish were between the two boxes, started

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the timer and observed for 15 minutes. When the hand of zebrafish arrived at the white box,

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recorded the residence time of the zebrafish. The observation process is carried out under the condition of sound insulation. 50 zebrafish were selected according to the experimental conditions and divided into 5 groups equally and randomly included blank control group, methamphetamine

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model group, rhynchophylline low dose (50mg/kg) group, rhynchophylline high dose (100 mg/kg)

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group, the ketamine (150mg/kg) group.

CPP test consisted of 3 phases and was preceded within 9 consecutive days. For the

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pre-conditioning phase, the zebrafish is placed in a separate CPP box for training. The water level

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of each CPP box is not less than 5cm to ensure enough water pressure. After two days of adaptive

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feeding, on day 3, the amount of time spent in the methamphetamine-paired compartment in 15 minutes was calculated and used Noldus Ethovision XT software to track their roadmap within 5

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minutes.

During the conditioning phase (on days 4-8), the door was closed so that the two compartments were separated. On days 4, 6 and 8, the zebrafish were anesthetized in 200mg/L tricaine methanesulfonate and injected methamphetamine (40mg/kg) by microinjection in methamphetamine-paired groups or the same volume of fish physiological saline in the blank control group and then confined to the white compartment for 45 min. After an interval of 12h, the zebrafish were anesthetized and injected corresponding drug (rhynchophylline low dose group:50ug/g; rhynchophylline high dose group :100ug/g; the ketamine group: 150ug/g ) and then moved to a large blue aquarium environment while blank and control group and model group

ACCEPTED MANUSCRIPT zebrafish were injected with the same volume of fish physiological saline. It was agreed with the administration of other treatments. On the same time of days 5 and 7, all fish were anesthetized in

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tricaine and injected intraperitoneally the same volume of fish physiological saline in each group

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and then were immediately confined to the brown compartment for 45 min.

24h after the last methamphetamine-paired conditioning trial ( on day 9), a 5-min trial was recorded to observe the time that the zebrafish spent on the drug-paired side (the white

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compartment) and the activities of the roadmap and compare them with kit before and after the

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residence time of the difference.

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Immunohistochemistry TH expression

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After the test of CPP, adult zebrafish were anesthetized in tricaine and killed in iced water.

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Following removal of the skull and exposure of the brains, zebrafish were decapitated and the brains were fixed with 4% paraformaldehyde in 0.1 mol/L phosphate buffer saline (PBS, pH 7.4)

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at 4 °C for 24 h. Immunohistochemistry was performed as described previously, with minor modifications. Brain tissues were sent for paraffin wax-embedding and processing. The brains were sectioned with vibratome at 5μm and the sections were deparaffinized with xylene, then dehydrated in decreasing concentrations of alcohol. After blocking endogenous peroxidase activity in 3% H2O2in PBS for 30 min, the sections were boiled for 10 min under pressure in citrate buffer for antigen retrieval. Nonspecific binding was blocked with 5% bovine serum albumin in PBS for 15 min and the tissues were incubated with primary antibodies for anti-TH (1:200 dilution; Millipore, Billerica, MA, USA) in PBS containing 5% bovine serum albumin overnight at 4 °C. The sections were then rinsed 3 times with PBS and incubated with secondary antibodies for 60

ACCEPTED MANUSCRIPT min at room temperature. After rinsing, the tissue sections were immersed in a freshly prepared DAB/H2O2for a reasonable time, stained with hematoxylin for 80 s and then sealed with neutral

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gum. 3 clear sections were selected randomly from each group and then 3 horizons were selected

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in the light microscope (40×) randomly to observe the distribution of positive particles in the cell at 400× magnification. Positive expression of TH in the posterior tuberculum and pretectal area of zebrafish brain was defined as appearance of brown particles in the cell nuclei. Image-Pro plus 6.0

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image analysis software was used to measure positive cells integrated optical density and the mean

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value of the group is regarded as the relative content of TH.

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Western blot for TH expression

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The sample of membrane was prepared according to the instruction of membrane protein extraction kit. After determinating the concentration of the protein by BCA method, the sample

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was preserved at 20 degrees below zero degree. Protein samples were added to 200% sample buffer, and 12% sodium dodecyl sulfate–polyacrylamide gel (SDS–PAGE), electrophoresed at 100V for 120min and transferred to PVDF membranes. The membranes were incubated in PBS with 3% nonfat dry milk. Primary antibodies (1:1000 anti-TH, Millipore) were diluted in PBS with 3% milk, and then added to the member overnight with agitation at 4°C. The membrane was washed with PBS, incubated with a horseradish peroxidase-conjugated secondary antibody(goat anti-rabbit IgG) at a 1:3000 dilution for 1 h, and then washed with PBST (0.05% Tween 20 in PBS) and developed by using a Super Enhanced chemiluminescence detection kit and recorded on X-ray film (Eastman Kodark, Rochester NY, USA). Relative intensities of all bands

ACCEPTED MANUSCRIPT were quantified using image analysis software (Gel Media System, China).

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Statistical analysis

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Values are expressed as mean ± SD. All data were analyzed using one-way analysis of variance (ANOVA), followed by the least significant difference (LSD) post hoc test (two-tailed).

considered to be statistically significant.

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Results

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All statistical analyses were performed using SPSS software (version17.0). P < 0.05 was

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methamphetamine

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Rhynchophylline blocked the behavioral alteration induced by

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The effect of rhynchophylline on methamphetamine-induce CPP zebrafish was shown in Table 1. Difference of time spend of methamphetamine model group before and after training in

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the methamphetamine-paired compartment was increased significantly compared with blank control group (P<0.05). Time difference of rhynchophylline high does group and ketamine group was dramatically reduced compared with model group and the value is similar to that in control group while comparing with the model group rhynchophylline low does group had no significant difference.

Adult zebrafish activities routes in CPP As is shown in Fig 1, compared with the blank control group, there was a significant difference in the active route of the methamphetamine model group. Compared with

ACCEPTED MANUSCRIPT methamphetamine model group, the road map of zebrafish in rhynchophylline high does group and ketamine group was significantly different and rhynchophylline low does group was

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Immunohistochemistry for TH expression

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insignificantly different.

As is shown in Fig 2 and Table 2, adult zebrafish after CPP training, TH-positive cells in the

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midbrain are significantly different. Compared with the zebrafish in control group, the number of

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TH positive cells in the hippocampus region in methamphetamine-treated zebrafish was increased significantly. Both the low dosage, the high dosage of rhynchophylline and ketamine group

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reduced the number of positive TH cells, but the high-dosage of rhynchophylline group and the

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ketamine group reduced significantly for TH positive cells (P< 0.01).

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Western blot for the expression of TH Compared with the blank control group, the TH expression in the brain of zebrafish in

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methamphetamine model group were significantly increased (P<0.05). Compared with the methamphetamine model group, the TH expression in the brain of zebrafish in rhynchophylline high does group and ketamine group was significantly different (P<0.05) while rhynchophylline low does group had insignificant difference, as shown in Table3 and Fig 3.

Discussion Conditioned place preference test is to detect drug psychological dependence classic experiments. The results showed that a new coordinator to determine gene regulatory networks, influence, or accompanied by amphetamine-induced conditioned place preference in zebrafish

ACCEPTED MANUSCRIPT behavior, could easily lead to drug dependence

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. Therefore, the model of adult zebrafish

conditioned place preference can be induced by methamphetamine [28].

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Tyrosine hydroxylase is the rate-limiting enzyme of dopamine synthesis, which controls the

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movement and behavior associated with the rewarding effects [29]. Tyrosine hydroxylase is the rate-limiting enzyme of catecholamine biosynthesis, which uses tetrahydrobiopterin and molecular oxygen on the tyrosine into dopa. Tyrosine hydroxylase is a key enzyme in catecholamine

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synthesis. The main role of cytoplasmic enzyme is to catalyze the conversion of tyrosine into

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L-L-dopa[30].

Tyrosine hydroxylase has a close relationship with drug addiction in humans and animals.

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Tyrosine Hydroxylase as a key enzyme in the synthesis of dopamine formation of drug

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dependence also plays an important role.

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Our previous research indicates that the neurobiological basis of drug addiction is addiction to drugs can induce reward center nucleus accumbens(NAc) dopamine concentration. Tyrosine

[31,32]

.

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hydroxylase was a catecholamine biosynthesis key enzyme dopamine neurons marker enzyme

This experimental study showed that adult zebrafish brain methamphetamine drug dependency model TH-positive cells were significantly increased intervention by rhynchophylline had improvement. Tips TH zebrafish model CPP effects are closely related, and is one of the neurobiological mechanisms of animal behavior change. Compared with control group, a significant increased activity time in the side of the transparent with drug; rhynchophylline high dose group, ketamine group and model group was statistically significant. To some extent, it shows rhynchophylline can shorten the duration of adult

ACCEPTED MANUSCRIPT zebrafish staying in the drug-cylinder. Immunohistochemistry results showed that model group zebrafish after CPP training

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increases the number of TH-positive cells in the midbrain regions. Compared with the model

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group, ketamine group and high dosage of rhynchophylline group TH-positive cells had reduced with statistically significant in zebrafish brain areas. It implies that rhynchophylline could reduce the number of TH-positive cells.

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Western blot results showed that TH expression in zebrafish midbrain of the model group

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was statistically significant. Compared to the model group, the TH expression in zebrafish midbrain of high-dose rhynchophylline group and ketamine group was statistically significant.

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What’s more, the TH expression in zebrafish brain of high-dose rhynchophylline had an increasing

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trend.

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In summary, methamphetamine-induced CPP adult zebrafish may be associated with the increasement of TH expression in the brain of zebrafish. Because Chinese medicine detoxification

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mechanism is very complex, rhynchophylline still need further study the drug dependent mechanism.

Author contributions Designed the study: Z.C. W.L. Z.X.M. Coordinated the study and finalized the manuscript: Z.C. W.L. Read and approved the manuscript: C.L. Performed the experiments: M.T.C. O.J.Y. Y.B.L. Y.L. M.F. D.Q.Z. Analyzed the data: C.Z. W.L. Wrote the paper: Z.C.

Competing financial interests The authors declare no competing financial interests.

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This work was supported by Fund Projects: the National Natural Science Foundation of

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China (No. 81229003, 81673628, 81403063); the Guangzhou Major Science and Technology

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Project (No. 201300000050); Project of Natural Science Foundation of Guangdong Province

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(No. 2014A030310251).

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Fig.1. Road maps of zebrafish in CPP compartment: A: Blank control group before modeling; B: Blank control group after modeling; C: Methamphetamine model group before modeling; D: Methamphetamine model group after modeling; E: Rhynchophylline low does group before modeling; F: Rhynchophylline low does group after modeling; G: Rhynchophylline high does group before modeling; H: Rhynchophylline high does group after modeling; I: Ketamine group before modeling; J: Ketamine group after modeling.

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Figure 2. Micrographs of TH positive cells in the midbrain A:black control group; B:model group of methamphetamine; C:low dose of rhynchophylline group; D:high dose of rhynchophylline group; E:ketamine group.

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β-actin

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Fig 3. The expression of TH by Western blotting. A: Ketamine group; B: high dose of rhynchophylline; C:l ow dose of rhynchophylline; D:control group; E: methamphetamine model group

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zebrafish activity time in non-preferred compartment(s)

Control Methamphetamine model Methamphetamine+ rhynchophylline Methamphetamine+ rhynchophylline Methamphetamine+ ketamine

-40 50 100 150

226.10 ±37.37 432 .20±60.88** 392.10 ±80.84 188.00±74.42▲▲ 200.60 ±45.08▲▲

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Group

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*P<0.05，**P<0.01 vs control group；▲P< 0.05，▲▲P<0.01 vs methamphetamine model group。

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Table 2 The IOD of TH positive cells in area of midbrain of mice in each group（n=10， x  s ） Group (TH)IOD Dose（μg/g） -16972.94±5078.71 Control 40 59297.94±22066.62** Methamphetamine model 50 64921.96±17833.67 Methamphetamine+ rhynchophylline 100 17477.72±9242.88▲▲ Methamphetamine+ rhynchophylline 150 19640.50±5115.86▲▲ Methamphetamine+ ketamine

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*P<0.05，**P<0.01 vs control group；▲P< 0.05，▲▲P<0.01 vs methamphetamine model group。

ACCEPTED MANUSCRIPT Table 3 The OD of TH expression in zebrafish brain（n=10， x  s ） Dose（μg/g）

(TH)OD

Control Methamphetamine model Methamphetamine+ rhynchophylline Methamphetamine+ rhynchophylline Methamphetamine+ ketamine

-40 50 100 150

3580.5±602.61 5123.3±1257.21* 4395.5±1152.75 3452.1±885.68▲ 3854.5±1006.87▲

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*P<0.05，**P<0.01 vs control group；▲P< 0.05，▲▲P<0.01 vs methamphetamine model group。

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Graphical Abstract

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As shown in the figure above，it is road maps of zebrafish in CPP compartment: A: Blank control group before modeling; B: Blank control group after modeling; C: Methamphetamine model group before modeling; D: Methamphetamine model group after modeling; E: Rhynchophylline low does group before modeling; F: Rhynchophylline low does group after modeling; G: Rhynchophylline high does group before modeling; H: Rhynchophylline high does group after modeling; I: Ketamine group before modeling; J: Ketamine group after modeling. Compared with the blank control group, there was a significant difference in the active route of the methamphetamine model group. Compared with methamphetamine model group, the road map of zebrafish in rhynchophylline high does group and ketamine group was significantly different and rhynchophylline low does group was insignificantly different.