Kaifei LIU, Yao HUANG, Guokai GUAN, Shimin WU, Xunyi LI, Linglin CHEN,2*, Yifei CHEN,2*

1. College of Pharmacy, Guilin Medical University, Guilin 541199, China; 2. Key Laboratory of Pharmacology, Guangxi Department of Education/College of Pharmacy, Guilin Medical University, Guilin 541199, China

Abstract [Objectives] To investigate the effect of rhynchophylline on the behavior of Alzheimer’s disease zebrafish induced by aluminum trichloride (AlCl3), to provide theoretical basis for the development and application of rhynchophylline in the field of Alzheimer’s disease. [Methods] The video recording was made on zebrafish before training, so as to judge whether zebrafish training was successful or not. After training zebrafish for 7 d, 60 zebrafish were randomly divided into 6 groups: blank group, model group, positive group, low-dose rhynchophylline group, medium-dose rhynchophylline group and high-dose rhynchophylline group. The blank group was video-recorded, while the model group, positive group, low-dose group, medium-dose group and high-dose group were exposed to AlCl3 for modelling. After the end, the model group was video-recorded, and the other groups were given drug intervention. Both the positive group and the rhynchophylline group were given drug for 6 d, and finally all the groups were video-recorded. After all the videos were finished, the behavioral analysis was carried out by using the behavioral record analysis software smart3.0, and the conclusion was drawn by analyzing the data. [Results] The data of length of stay of zebrafish in red short arm area of 6 groups were compared and analyzed: there was a significant difference between blank group and model group (P<0.05), and there was a significant difference between model group and positive group, medium-dose group or high-dose group (P<0.05). The percentage of time spent in the red short arm area of 6 groups of zebrafish was compared and analyzed: there was a significant difference between blank group and model group (P<0.05), and there was a significant difference between model group and positive group, medium-dose group or high-dose group (P<0.05). The data of swimming distance of 6 groups of zebrafish in red short arm area were compared and analyzed: there was no significant difference between the blank group and the model group (P>0.05), but there was only significant difference between the model group and the high-dose group (P<0.05). The percentage of swimming distance of zebrafish in the red short arm area of the six groups was compared and analyzed: there was a significant difference between the blank group and the model group (P<0.05), and there were significant differences between model group and positive group, medium-dose group or high-dose group (P<0.05). [Conclusions] Rhynchophylline can improve the behavior of Alzheimer’s disease zebrafish.

Key words Rhynchophylline, Alzheimer’s disease, Zebrafish, Behavioral analysis

1 Introduction

Alzheimer’s disease (AD) is a neurodegenerative disease, which often occurs in the elderly. It often shows decreased cognitive function, decreased daily activities and mental and behavioral symptoms. Cognitive dysfunction first shows impaired memory of recent events and impaired ability to learn new information, such as misplacing objects, repeating questions. In language barriers, grammatical errors, impairment of language logic and coherence and so on will be shown. In the executive dysfunction, there will be impaired reasoning ability, declining social communication ability and so on. On the visual structure space disorder, it shows getting lost in familiar places and not being able to identify common objects[1]. There is no effective treatment drug to improve the etiology of Alzheimer’s disease. At present, drug treatment is mainly to control symptoms[2], so basic research in related fields of Alzheimer’s disease is particularly important.

There are many causes of Alzheimer’s disease, and the accumulation of aluminum ions and other metals in the brain is one of the reasons for the occurrence and development of Alzheimer’s disease. Aluminum is one of the common metal elements in our life, which can react in the body and isolate Al3+, causing damage to the nervous system. Current studies have shown that the level of aluminum in the brain of patients with Alzheimer’s disease is significantly higher than that of normal people[3]. Excessive Al3+induces the polymerization of Aβ-peptide and phosphorylated tau protein in the brain, which leads to the occurrence and development of Alzheimer’s disease[4]. Therefore, in this study, zebrafish was exposed to AlCl3to induce AD-related symptoms.

In recent years, more and more traditional Chinese medicine has been found to have an intervention effect on Alzheimer’s disease. Rhynchophylline is derived from the alkaloids extracted from the branches ofUncariarhynchophylla(Miq.)Miq.ex Havil. The action ofUncariais in the liver and heart. It is sweet, bitter and slightly cold in nature, and has the effects of relieving wind and spasm. It is often used as a medicine for calming the liver to stop the wind.Uncariacontains a variety of chemical components, and rhynchophylline is one of the important effective components ofUncaria, accounting for a large proportion of chemical components inUncaria. However, rhynchophylline is very unstable[5], so the instability of rhynchophylline may affect the experiment to a certain extent.

It has been found that rhynchophylline can inhibit the excessive production of β-amyloid protein precursor, APP lyase and amyloid protein in the brain of dementia model mice[6]. However, there is no related research on rhynchophylline used in the model of Alzheimer’s disease zebrafish. Zebrafish was used as experimental animal in this experiment. Zebrafish has a spindle-shaped body, a short snout, a small and slightly pointed head. Compared with rodents, zebrafish has shorter growth cycle and faster embryo developmentinvitro, so it is suitable for high-throughput experiments, and zebrafish is economical and low-cost[7]. Because zebrafish is very sensitive to harmful substances, zebrafish is more likely to be exposed to AlCl3as an experimental animal.

The T-maze was designed according to the method of behavioral detection of T-maze in reference[9]. The green short arm area of T-maze (right area) was used as enriched chamber (EC) area. It was fed in EC area to make zebrafish produce memory and achieve training effect. Therefore, zebrafish should remember the EC area after training, and zebrafish should gather to the EC area after fasting and swim less to the red short arm area. After modeling, zebrafish wandered aimlessly in the T-maze because of memory impairment. At this time, zebrafish would appear in the red short arm area for a longer time than before modeling, so we could judge whether rhynchophylline had an effect on the behavior of Alzheimer’s disease zebrafish according to the swimming situation of zebrafish in the red short arm area.

The purpose of this experiment was to determine the behavioral effect of rhynchophylline on Alzheimer’s disease zebrafish through behavioral analysis.

2 Materials and methods

2.1 Materials

2.1.1Experimental animals. The adult zebrafish was about 2-4 cm long and weighed 0.5-1.0 g, and was provided by the zebrafish experiment center of Southern Medical University.

2.1.2Experimental drugs and reagents. Rhynchophylline (Beijing Solarbio Technology Co., Ltd.); Huperzine-A (Chenxin Pharmaceutical Co., Ltd.); pure AlCl3(China National Pharmaceutical Group).

2.1.3Experimental instruments. Precision balance (Mettler-Toledo Co., Ltd.); ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.); T-maze (self-made).

2.2 Experimental methods

2.2.1Animal grouping and training. First of all, put the zebrafish in the fish tank, control the temperature and density, and feed adaptively for 2 d. 2 d later, the zebrafish was put into the T-maze for training. The T-maze is divided into four areas (Fig.1), namely, the long arm area (start area), the middle area, the red short arm area (left area), and the green short arm area (EC area).

Fig.1 Schematic diagram of T-maze

At the beginning of the training, the zebrafish was put into the long arm area and fed in the EC area to make the zebrafish remember the EC area and achieve the training effect. The zebrafish was trained for 7 d, 1 h a day. Sixty qualified zebrafish were randomly divided into 6 groups: blank group, model group, positive group, low-dose rhynchophylline group, medium-dose group and high-dose group. The blank group was video-recorded for 5 min. During the experiment, zebrafish could swim freely, but no feed was given two days before each video recording, making zebrafish hungry and reducing the experimental error.

2.2.2Establishment of model of Alzheimer’s diseasezebrafish. The other 5 groups of zebrafish except the blank group were exposed to 100 μg/L AlCl3. 5 mg of AlCl3crystals were put into 1 L culture solution to prepare 5 mg/L mother liquid. After that, 200 ml AlCl3mother liquid was added to 9 800 mL culture solution to prepare 100 μg/L working liquid, and the PH of the solution was adjusted to 5.80 ±0.05. Five groups of zebrafish were exposed to poisonous liquid for 6 d. At the end of exposure, the model group was video-recorded for 5 min.

2.2.3Administration. Positive group: 50 μg/L mother liquid was made with Huperzine-A, and then 80 mL mother liquid was added to 920 mL culture solution to form 4 μg/L solution for administration. Rhynchophylline administration group: 33.6 mg of rhynchophylline was put into 525 mL culture solution to form 64 mg/L mother liquid, and then prepared into 8 mg/L low-dose group, 16 mg/L medium-dose group and 32 mg/L high-dose group. At the end of administration, 4 groups were video-recorded for 5 min.

2.2.4Behavioral analysis of zebrafish. The behavior trajectory of zebrafish was analyzed by the behavioral record analysis software smart 3.0, and the time and distance of swimming in each area and the percentage of time and distance were analyzed.

2.2.5Observation of experimental zebrafish. During the experiment, the activity, death and appearance of zebrafish were observed every day.

3 Results and analysis

3.1 Analysis of the time spent in the red short arm area for each group of zebrafishAs shown in Table 1, the average time for zebrafish to reach the red short arm in the blank group was significantly less than that in the model group, while the average time for zebrafish to reach the red short arm in the positive group, low-dose group, medium-dose group and high-dose group was more than that in the blank group but less than that in the model group.

Table 1 Average length of stay and percentage in the red short arm area for zebrafish

As shown in Fig.2, the length of stay in the red short arm area in the model group was compared with that in the blank group (P<0.05), and the length of stay in the red short arm area in the positive group and rhynchophylline group was compared with that in the model group. Except that there was no significant difference between the low-dose rhynchophylline group and the model group (P>0.05), the time spent in the red short arm area in the other groups decreased (P<0.05).

Fig.2 The path diagram of each group of zebrafish

As shown in Table 1, the time spent in the red short arm area for the blank group was the shortest, while the time spent in the red short arm area for the model group was the longest. The time spent in the red short arm area for the positive group was significantly shorter than that for the model group but longer than that for the blank group. The time spent in the red short arm area for the low-dose group, the medium-dose group and the high-dose group decreased successively, and the average time for the three groups was shorter than that for the model group and longer than that for the blank group.

As shown in Fig.3, there was a significant difference in the percentage of length of stay in the red short arm area between the model group and the blank group (P<0.05); there was a significant difference between model group and positive group, medium-dose group or high-dose group of rhynchophylline (P<0.05).

Note: Compared with the blank group, ###P<0.001; compared with the model group, *P<0.05, **P<0.01, ***P<0.001.

3.2 Analysis of swimming distance of zebrafish in the red short arm areaAs shown in Table 2, the average swimming distance of zebrafish in the red short arm area for each group showed irregular changes, there was no statistical difference between the blank group and the model group (P>0.05), and there was only significant difference between the model group and the high-dose group (P<0.05). However, Fig.4 shows a trend similar to the time spent by zebrafish in the red short arm area and the percentage of time spent by zebrafish in the red short arm area.

Table 2 The average swimming distance of zebrafish in the red short arm area and average percentage of swimming distance in the total distance n=10)

4 Discussion

According to the data in Table 1 and 2, the average time for the model group in the red short arm area was 63.53 s, accounting for 21.14%; the average time for the blank group was 4.82 s, accounting for 1.594%; the time for the model group was significantly longer than that for the blank group (P<0.05), which indicated that the model of Alzheimer’s diseasezebrafishwas established successfully. The average time spent in the red short arm area for the blank group was the least. It can be seen that after training, zebrafish will follow the training route to find feed and rarely stay in the red short-arm area where there is no feed. The model group developed Alzheimer’s disease after exposure, began to lose memory and got lost in familiar places, that is, they could not find the EC area in a state of hunger, and entered the red short arm area for feed many times.

Note: Compared with the blank group, ###P<0.001; compared with the model group, *P<0.05, **P<0.01, ***P<0.001.

After administration of Huperzine-A, the average time in the red short arm area for the positive group was 12.28 s, accounting for 4.107%, which was less than that for the model group, indicating that the positive group was given drug successfully. When Huperzine-A was used in the positive group, the time spent in the red short arm area decreased significantly compared with that before administration, so it can be seen that Huperzine-A has an improving effect on Alzheimer’s diseasezebrafish. Rhynchophylline was used in the treatment group.

The concentration of rhynchophylline was 8 mg/L in the low-dose group, 16 mg/L in the medium-dose group and 32 mg/L in the high-dose group. The average time in the red short arm area for the low-dose group was 33.02 s, accounting for 10.964%, which was less than that for the model group, but there was no statistical significance (P>0.05); the average time for the medium-dose group was 29.94 s, accounting for 9.965%, which was less than that for the model group; the average time for the high-dose group was 25.63 s, accounting for 8.487%, and the time for the high-dose group was less than that for the model group. The time spent in the red short arm area for the three groups was less than that for the model group, but more than that for the blank group, which indicated that rhynchophylline had a certain effect on Alzheimer’s disease in these three concentration ranges, but it was not as effective as Huperzine-A. In addition, the length of stay in the red short arm area for the three groups decreased successively from the low-dose group to the high-dose group, indicating that the higher the concentration of rhynchophylline, the better the improvement effect on Alzheimer’s disease zebrafish.

From Table 2 and Fig.4, it can be clearly seen that the swimming distance of zebrafish in the red short arm area for each group changed irregularly, and the average swimming distance of the high-dose group was much higher than that of other groups. The average swimming distance of the model group was 28.769 cm, and the swimming distance in the red short arm area was not the longest. In fact, swimming distance is not very convincing. Clinically, the clinical manifestations of Alzheimer’s disease include worse memory, having difficulty getting about, seeing things vaguely and so on, as well as emotional anxiety[10]. It may be because the zebrafish is extremely excited and swims very fast, so it will swim a long distance in a short time. Due to the different swimming speed of zebrafish, the swimming distance of each group of zebrafish is different. From a statistical point of view, the swimming distance of zebrafish in the red short arm area also showed irregular changes, and there was no significant difference between the model group and the blank group, positive group, low-dose group or medium-dose group (P>0.05).

The percentage of swimming distance in the red short arm area was 1.679% for the blank group and 18.457% for the model group. There was a significant difference between the two groups (P>0.05). The percentage for positive group, low-dose group, medium-dose group and high-dose group was 4.742%, 10.239%, 8.829% and 8.587%, respectively. There was a significant difference between model group and positive group, medium-dose group or high-dose group. This data is different from the trend of the swimming distance of zebrafish in the red short arm area, but it is the same as the trend of the time spent by zebrafish in the red short arm area, so it is difficult to analyze according to the swimming distance of zebrafish in the red short arm area. However, it can be analyzed from the percentage of swimming distance of zebrafish in the red short arm area.

Based on the above analysis, it can be concluded that rhynchophylline can improve the symptoms of memory loss, inability to identify common objects and getting lost in familiar places for Alzheimer’s disease zebrafish.