CN111165428B - Construction method and application of animal model addicted to chewing areca nuts - Google Patents
Construction method and application of animal model addicted to chewing areca nuts Download PDFInfo
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Abstract
The invention provides a construction method and application of an animal model addicted to chewing areca nuts, wherein the construction method comprises the following steps: quantitatively coating the betel nut chewing mass extracting solution on the surface of a limited amount of feed, feeding C57BL/6 mice in limited feeding for ingestion, and establishing an animal model addicted to chewing betel nuts. The experimental result shows that the method provided by the invention can establish an animal model for chewing areca addiction and has better reproducibility; the animal model for chewing areca addiction constructed by the invention can be used for evaluating chewing areca addiction of experimental animals and is also suitable for further exploration of neurobiological mechanisms. The construction method provided by the invention is simple, the operation is easy, and other medicines do not need to be combined.
Description
Technical Field
The invention belongs to the technical field of animal model construction, and particularly relates to a construction method and application of an animal model addicted to chewing areca nuts.
Background
Betel nut, which is a daily hobby, is the fourth most common psychoactive substance in the world after tobacco, alcohol and caffeine-containing beverages, and about 6 billion people worldwide have a habit of chewing betel nut at present. The betel nut can cause slight excitability and euphoria when being chewed, and simultaneously relieve mental stress to achieve the effect of refreshing, so that a certain addiction can be caused after long-term chewing. Although the preliminary findings at home and abroad show that long-term chewing of betel nuts can cause the symptoms of tolerance, craving and withdrawal of the chewers, the current research is not deep, is mostly based on small-sample investigation or experimental research, and does not form a conclusive view. Because human beings repeatedly chew the betel nuts and absorb pharmacological components of the betel nuts through oral mucosa, an ideal animal model is lacked at present, and the neurobiological mechanism exploration of betel nut addiction is greatly limited. Therefore, creating an experimental animal chewing betel nut addiction model simulating the behavior of human chewing betel nuts is highly desirable.
Disclosure of Invention
The invention aims to overcome the defects in the prior art and provides a construction method and application of an animal model addicted to chewing areca nuts.
In order to achieve the above objects, in a basic embodiment, the present invention provides a method for constructing an animal model addicted to chewing betel nut, comprising the steps of: quantitatively coating the betel nut chewing mass extracting solution on the surface of a limited amount of feed, feeding C57BL/6 mice in limited feeding for ingestion, and establishing an animal model addicted to chewing betel nuts.
In a preferred embodiment, the extract of betel nut chewing gum is administered in a dose of 100 mg/kg.
In a preferred embodiment, the betel nut chewing gum extract is administered in a volume of 10 ml/kg.
In a preferred embodiment, the application comprises: quantitatively applying with a pipette.
In a preferred embodiment, the limited feeding regime for mice comprises: carrying out single-cage feed-limiting feeding; the daily food intake (g) of the mice is the experimental feed amount (g) + the supplementary feed amount (g).
In a preferred embodiment, the experimental feed amount (g) <20 g-the pre-experimental body weight (g) of the mice was entered and the mice were guaranteed to eat all experimental feed within 2 h; the animals were fed with the supplementary feed 1h after the end of the experiment every day, and the supplementary feed amount (g) was 20 g-the body weight (g) of the mice 1h after the end of the experiment.
Another objective of the present invention is to provide the application of the animal model constructed by the above method in evaluating betel nut chewing addiction of experimental animals.
The invention also aims to provide a method for evaluating addiction of chewing areca nuts by experimental animals, which comprises the following steps: 1) constructing a mouse model addicted to chewing areca nuts by adopting the method; 2) applying a betel nut chewing gum extract to the mouse model; 3) and evaluating the chewing betel nut addiction of the experimental animal by adopting the mouse model.
Through the technical scheme, the areca chewing block extracting solution is taken as an inducer for chewing areca addiction, and is given to C57BL/6 mice to establish an animal model for chewing areca addiction, so that the animal model is used for deeply researching areca addiction and possible neurobiological mechanisms thereof. The experimental result shows that the method provided by the invention can establish an animal model for chewing areca addiction and has better reproducibility; the animal model for chewing areca addiction constructed by the invention can be used for evaluating chewing areca addiction of experimental animals and is also suitable for further exploration of neurobiological mechanisms. In addition, the construction method provided by the invention is simple, the operation is easy, and other medicines do not need to be used in combination.
Drawings
FIG. 1 is a graph showing the total distance of autonomic activity of 1d, 7d and 13d of each group of mice.
FIG. 2 is a schematic diagram of activity time of the CPP in the medicine accompanying box before and after training of each group of mice.
Detailed Description
In order to better understand the technical solutions, the technical solutions of the present application are described in detail with specific embodiments below, and it should be understood that the specific features in the embodiments and examples of the present application are detailed descriptions of the technical solutions of the present application, but not limitations of the technical solutions of the present application, and the technical features in the embodiments and examples of the present application may be combined with each other without conflict. It should be understood that the term "and/or" as used herein includes any and all combinations of one or more of the associated listed items.
The endpoints of the ranges and any values disclosed herein are not limited to the precise range or value, and such ranges or values should be understood to encompass values close to those ranges or values. For ranges of values, between the endpoints of each of the ranges and the individual points, and between the individual points may be combined with each other to give one or more new ranges of values, and these ranges of values should be considered as specifically disclosed herein.
The embodiment of the invention provides a construction method and application of an animal model addicted to chewing areca nuts, and the obtained animal model addicted to chewing areca nuts can be used for evaluating the addiction to chewing areca nuts of experimental animals and is also suitable for further exploration of a neurobiological mechanism. The Conditioned Place Preference test (CPP) and the behavior Sensitization test (Behavioral Sensitization) are two Behavioral test methods for evaluating the psychic dependence of the medicament, and the animal model for chewing areca addiction constructed by the embodiment of the invention is evaluated by the two test methods.
The embodiment of the invention mainly comprises the following steps: a method for constructing animal model addicted to chewing areca nut comprises the following steps: quantitatively coating the betel nut chewing mass extracting solution on the surface of a limited amount of feed, feeding C57BL/6 mice in limited feeding for ingestion, and establishing an animal model addicted to chewing betel nuts.
Unless otherwise specifically stated, various raw materials, reagents, instruments, equipment and the like used in the examples of the present invention are commercially available or can be prepared by an existing method. The following describes in detail the construction method of animal model for chewing areca addiction and evaluation experiment thereof.
1) Preparation of betel nut chewing gum extract
The instrument comprises the following steps: a condensing reflux device; a round bottom flask; a constant temperature water bath (HH. S21-4-S, Shanghai New Miao medical device manufacturing Co., Ltd.); rotary evaporator (RE2000A, shanghai yanglong biochemical instruments factory); a freeze dryer (FD-1A-50, Shanghai Yuming Instrument Co., Ltd.); electronic balance (BSA223S, beijing sidoris scientific instruments ltd); high-speed multifunctional disintegrator (PJ-10B, Shanghai Help trade Co., Ltd.).
The extraction method comprises the following steps: pulverizing semen Arecae chewing pieces sold in market, and sieving with 40-60 mesh sieve; extracting with 70% ethanol at ratio w (kg)/v (L) of 1:2 at 55 deg.C for 2 times (each for 2 hr), and mixing filtrates; filtering, concentrating, and freeze drying to obtain powder. The powder was diluted with distilled water and the following experiment would use three doses: 1mg/kg, 10mg/kg and 100 mg/kg.
2) Feeding and administration method for mice
Experimental animals: male C57BL/6 mice, weighing 18-20g, purchased from Beijing Wintolite laboratory animal technology Co., Ltd, production license number: SCXK (Jing) 2016-: no. 1100111911051800.
Feeding mode: all mice were fed a single cage of restricted feed, in short, daily food intake of mice was restricted in order to maintain the weight of the mice, i.e. daily food intake (g) was the experimental feed amount (g) + the supplemental feed amount (g). The experimental feed amount (g) <20 g-the weight (g) of the mice before entering the experiment and ensuring that the mice eat all experimental feeds within 2 h. The animals were fed with the supplementary feed 1h after the end of the experiment every day, and the supplementary feed amount (g) was 20 g-the body weight (g) of the mice 1h after the end of the experiment.
The administration mode comprises the following steps: the drug administration group evenly smears 10ml/kg of areca chewing mass extracting solution on the limited feed surface by using a liquid transfer gun, and the control group smears 10ml/kg of distilled water to be respectively gnawed by mice.
3) Influence of betel nut chewing mass extracting solution on behavior sensitization of mice
The instrument comprises the following steps: small animal autonomic activity tester (YHLA, Ningbo' an scientific instruments Co., Ltd.). After the test animal enters the device, the camera system at the top of the device monitors the behavior and activity of the test animal, and the behavior and activity are recorded and stored by a computer.
The experimental method comprises the following steps: 40 mice, pre-adapted to the experimental environment for 3d, were randomly divided into 4 groups, each: control group, low dose group (1mg/kg), medium dose group (10mg/kg), high dose group (100 mg/kg). The whole experiment is divided into 3 stages of a formation stage, a withdrawal stage and an expression stage. The method comprises the following specific steps: and (3) formation period: 1-7d, mice in the daily administration group eat feeds coated with 10ml/kg of areca chewing block extracting solution with different doses, mice in the control group eat feeds coated with 10ml/kg of distilled water, the mice are placed into a small animal autonomous activity tester after 2h, the test is continuously carried out for 60min, and the total distance of autonomous activity of the mice is recorded. A withdrawal period: 8-12d, all mice were fed restricted feed only, and fed feed without any treatment. And (3) expression period: and 13d, respectively gnawing the feed coated with 10ml/kg of volume of betel nut chewing block extracting solution with different doses by using mice in the administration group, gnawing the feed coated with 10ml/kg of volume of distilled water by using the control group, respectively putting the mice into a small animal autonomous activity tester after 2h, continuously measuring 60m in, and recording the total autonomous activity distance of the mice.
Data processing: data are presented as Mean ± SEM, statistical data analysis and plotting using GraphPadPrism5 software; meanwhile, the total distance of autonomous activities of each group of mice in a single administration, a formation period and an expression period is analyzed and compared by adopting single-factor variance analysis, and the significant difference that P is less than 0.05 is adopted.
The experimental results are as follows: the weight of the mice was maintained at 18-20g by gavage, and the average weight of each group of mice before daily entry into the experiment is shown in Table 1. The total distance of the independent activities of each group of mice in the single administration, the formation period and the expression period is statistically analyzed and compared respectively, and the experimental results are shown in figure 1. In fig. 1, P <0.05 compared to the control group on the same day is indicated. And represents P <0.05 compared to group 1 d. # denotes P <0.05 compared to group 7 d.
TABLE 1 average body weight of each group of mice before daily entry into the experiment
As can be seen from Table 1, the weight of the mice was maintained well, indicating that the daily food intake was better controlled during the experiment. As shown in FIG. 1, the total distance of autonomous activities in both the formation phase and the expression phase of the control group tended to be stable. The total distance of the independent movement of the mice in the formation period 100mg/kg dose group at the 1d and the 7d is obviously higher than that of the control group, and the total distance of the independent movement of the mice in the 100mg/kg dose group at the 7d is also obviously higher than that of the dose group at the 1d, which indicates that at the dose, the single administration can enhance the activity of the mice, and the continuous 7d administration can form the behavior sensitization of the mice. The total distance of the independent activities of the mice in the 100mg/kg dose group in the expression period at 13d is obviously higher than that of the control group, and the total distance of the independent activities of the mice in the 100mg/kg dose group at 13d is also obviously higher than that of the dose group at 7d, which indicates that the behavior sensitization of the mice is expressed at the dose. Indicating that the model molding is successful at a dose of 100 mg/kg.
4) Influence of betel nut chewing mass extracting solution on mouse conditional position preference
The instrument comprises the following steps: conditional Positional Preference (CPP) testing apparatus (XR-XT401, Shanghai Xin Soft information technology Co., Ltd.) which divides a box body into three parts, namely a white box, a black box and a middle area, by two middle partition plates. After the test animal enters the device, the camera system at the top of the device monitors the behavior and activity of the test animal, and the behavior and activity are recorded and stored by a computer.
The experimental method comprises the following steps: the whole experiment is divided into 3 stages of pretreatment, training and testing. The method comprises the following specific steps: a pretreatment stage: before the experiment, the mouse is placed in the CPP box with the drawn baffle plate to move freely, and after the mouse is adapted to 2d, the mouse position preference test is carried out at the 3 rd time. The baffles were removed before testing, the mice were placed in the middle area, and the time of movement of the mice in the black and white box was recorded within 15min, and the results were used as baseline values. As a result, it was found that > 90% of the mice in the natural state favored the black box, and therefore the black box was selected as the non-concomitant box and the white box was selected as the concomitant box. Mice with > 65% total time of activity on either side, or > 45% total time in the middle zone, were excluded. A training stage: taking 40 qualified mice, randomly dividing the mice into 4 groups, wherein the groups are respectively as follows: control group, low dose group (1mg/kg), medium dose group (10mg/kg), high dose group (100 mg/kg). And 4-13d, closing the partition plate, wherein, in the 4d, mice in the administration group gnaw the feed coated with 10ml/kg of the areca chewing block extract solution with different dosages respectively, in the control group gnaw the feed coated with 10ml/kg of distilled water, and after 2h, the mice are placed in a white box for 40min respectively. On the next day (5 d), all mice gnaw the feed smeared with 10ml/kg volume of distilled water, and after 2h, they were placed in black boxes for 40min, respectively. Repeat the first two days for 4 times. And (3) a testing stage: at 14d, the baffles were withdrawn before testing, all mice were placed in the middle area without any treatment, and the time of the mice's activity in the black and white box within 15min was recorded as the test value.
Data processing: data are presented as Mean ± SEM, statistical data analysis and plotting using GraphPadPrism5 software; meanwhile, analyzing and comparing the activity time of each group of mice in the companion medicine box by adopting single-factor variance analysis; calculating the amplification percentage of the activity time of the mouse in the medicine accompanying box before and after training, wherein the calculation formula is as follows: x 100% when P <0.05 is significant difference [ (time of activity of mouse in drug-accompanying box after training-time of activity of mouse in drug-accompanying box before training)/time of activity of mouse in drug-accompanying box before training ].
The experimental results are as follows: the weight of the mice was maintained at 18-20g by gavage, and the average weight of the mice before daily entry into the experiment is shown in Table 2. The 3 rd and 14 th mice in each group are statistically analyzed and compared with the activity time of the concomitant drug box and the amplification percentage of the activity time of the concomitant drug box, and the experimental results are shown in the table 3 and the figure 2.
TABLE 2 average body weight of each group of mice before daily enrollment into the experiment
TABLE 3 percentage increase and duration of activity in the concomitant kit before and after CPP training (Mean + -SEM) for each group of mice
Note: denotes P <0.05 compared to control group.
As can be seen from Table 2, the weight of the mice was maintained well, indicating that the daily food intake was better controlled during the experiment. As can be seen from Table 3 and FIG. 2, the CPP activity time before and after the control mice training tended to be stable. The time for the CPP to be activated after training of the mice in the 100mg/kg dose administration group is obviously longer than that in the control group, and the amplification percentage is also obviously longer than that in the control group. Indicating that at 100mg/kg dosing, mice develop CPP. Indicating that the model was successfully molded at this dose. P <0.05 compared to the control group is indicated in figure 2.
The preferred embodiments of the present invention have been described above in detail, but the present invention is not limited thereto. Within the scope of the technical idea of the invention, many simple modifications can be made to the technical solution of the invention, including combinations of various technical features in any other suitable way, and these simple modifications and combinations should also be regarded as the disclosure of the invention, and all fall within the scope of the invention.
Claims (5)
1. A method for constructing an animal model addicted to chewing areca nuts is characterized by comprising the following steps: the method comprises the following steps: quantitatively coating the betel nut chewing mass extracting solution on the surface of a limited amount of feed, feeding C57BL/6 mice in limited feeding for ingestion, and establishing an animal model addicted to chewing betel nuts;
the dosage of the betel nut chewing gum extracting solution is 100 mg/kg;
the limited feeding mode of the mice comprises the following steps: carrying out single-cage feed-limiting feeding; the daily feed intake of the mice = experimental feed amount + supplementary feed amount; the experimental feed amount is less than 20g, the weight of the mouse before the experiment is carried out, and the mouse is ensured to eat all experimental feeds within 2 h; feeding the mice 1h after the end of the experiment every day, wherein the feed supplement amount =20 g-the weight of the mice 1h after the end of the experiment;
wherein the daily food intake, experimental feed intake and supplementary feed intake of the mice, the weight of the mice before the experiment and the weight of the mice 1h after the experiment are in g.
2. The method of claim 1, wherein the animal model of addiction to chewing betel nut comprises: the administration volume of the betel nut chewing gum extracting solution is 10 ml/kg.
3. The method of claim 1, wherein the animal model of addiction to chewing betel nut comprises: the smearing comprises the following steps: quantitatively applying with a pipette.
4. Use of an animal model constructed according to the method of any one of claims 1-3 for evaluating betel nut chewing addiction in a test animal.
5. A method for evaluating addiction of experimental animals to chewing areca nuts is characterized in that: the method comprises the following steps: 1) constructing a mouse model of addiction to chewing areca using the method of any one of claims 1-3; 2) applying a betel nut chewing gum extract to the mouse model; 3) and evaluating the chewing betel nut addiction of the experimental animal by adopting the mouse model.
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| CN111826436B (en) * | 2020-07-21 | 2022-10-04 | 国家烟草质量监督检验中心 | Group high-throughput determination method for gene and corresponding protein expression change in hippocampal tissues in nicotine addiction, withdrawal and addiction reconstruction stages |
| CN112586444B (en) * | 2020-12-14 | 2023-03-31 | 江汉大学 | Construction method and application of conditional position preference animal model |
| CN112715474B (en) * | 2020-12-25 | 2022-10-04 | 江汉大学 | Method for forming animal model with conditional site preference by induction of arecoline and menthol and application |
| CN114766424B (en) * | 2021-01-22 | 2023-06-27 | 北京化工大学 | Preparation method of rhesus monkey addiction model |
| CN114903006A (en) * | 2021-02-09 | 2022-08-16 | 中国科学院脑科学与智能技术卓越创新中心 | Construction method and application of non-human primate substance addiction model |
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| WO2015021358A2 (en) * | 2013-08-09 | 2015-02-12 | Dominique Charmot | Compounds and methods for inhibiting phosphate transport |
| CN103656645A (en) * | 2013-10-27 | 2014-03-26 | 北京大学 | Novel molecular target for treating opiate addiction and application thereof in drug development |
| CN106511345A (en) * | 2016-09-21 | 2017-03-22 | 海南省人民医院 | Construction method of oral submucous fibrosis mouse model |
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| 槟榔碱对小鼠吗啡行为敏化的影响;韩容 等;《中国药物依赖性杂志》;20050630(第3期);第197页右栏倒数第5行-第198页右栏第4行 * |
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