Construction method and application of a carfentanil mouse withdrawal model

Abstract

The application discloses a construction method and application of a carfentanyl mouse withdrawal model, and belongs to the field of biological medicine technology and animal model construction; carfentanyl is injected into the mouse, and the injection period is 6 days; 2 injections are injected every day in the first 5 days, and the interval between the injections is 12 hours; the injection dose of each injection on the nth day is n times of the injection dose of each injection on the first day; one injection is injected on the sixth day, and the injection dose is the same as the injection dose of each injection on the fifth day; the withdrawal is carried out after the injection is completed to obtain the carfentanyl mouse withdrawal model; the model of the application has fewer drug administration days, fewer drug administration times and uses less medicine, compared with the model constructed in the foregoing research; the model of the application has fewer drug administration days, fewer drug administration times and uses less medicine, and provides more economical, more efficient and more comprehensive reference evidence for the addiction and harmfulness of carfentanyl; the model can be widely applied to related researches such as pharmacological action evaluation or drug treatment of carfentanyl and similar new psychoactive substances.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and relates to a construction method of a carfentanil mouse withdrawal model and application thereof. BACKGROUND

[0002] New psychoactive substances (NPS), also known as “designer drugs”, “synthetic drugs” or “laboratory drugs”, fentanyl is one of the NPSs. According to the “2023 World Drug Report”, the number of new psychoactive substance users in the global market increased further in 2021, and opioid drugs are still the main cause of drug overdose deaths, which is due to the emergence of synthetic opioid drugs, especially fentanyl (synthetic opioid receptor agonist 50-100 times more potent than morphine). Carfentanil is 10,000 times more potent than morphine and is considered an extremely dangerous synthetic opioid drug. Like other fentanyl substances, carfentanil acts on the respiratory and central nervous systems and produces typical opioid effects in rodents, including analgesia, rigidity, and respiratory depression. In human studies, carfentanil was found to have similar effects, including euphoria, relaxation, analgesia, muscle weakness, drowsiness, sedation, bradycardia, hypotension, hypothermia, loss of consciousness, and respiratory depression.

[0003] Studies have found that carfentanil has a very high affinity for opioid receptors, and the binding affinity of carfentanil to μ opioid receptors is 20 times that of fentanyl. Plasma proteins can bind carfentanil tightly, allowing it to be widely distributed, and carfentanil is also a highly lipophilic compound that easily crosses the blood-brain barrier, making it very rapid in onset after administration. Therefore, it is a potent analgesic drug, and its analgesic dose is extremely low. Literature reports that the therapeutic index of carfentanil for analgesia is 10,600, that of fentanyl is 300, and that of morphine is 70, indicating that carfentanil is much safer than fentanyl, which makes people more complacent and ignore its dangers, leading to drug overdose poisoning and even death. Studies have found that in the mouse hot plate test, the analgesic ED50 of carfentanil (subcutaneous injection) is only 0.41 μg / kg, which is 50 times more potent than 3-methyl fentanyl, and the LD50 after tail vein administration has no significant difference from that of fentanyl (LD50 of carfentanil and fentanyl is 3.4 mg / kg and 3.1 mg / kg, respectively). This all indicates that it has pharmacological activity at low doses and has high addiction and dependence, thus increasing the risk of overdose and even death.

[0004] To further study its pharmacological activity, researchers have evaluated the abuse potential of carfentanil through drug discrimination experiments in rats, and found that the ED50(carfentanil) is only 0.6 μg / kg, and if pretreated with naloxone, the effect is reduced by about four times. Recent studies have found that subcutaneous injection of 1 μg / kg of carfentanil can induce the formation of conditioned place preference (CPP) in mice, while 100 μg / kg of fentanyl and 1000 μg / kg of heroin are required. In addition, intravenous injection of 0.05 μg / kg and 0.1 μg / kg of carfentanil can significantly maintain self-administration in rats, and if subcutaneously administered in a stepwise manner and induced by naloxone, the mice will have the same stereotyped jumping action as morphine.

[0005] The addiction, withdrawal symptoms and harm of carfentanil need to be further studied. The aforementioned recent study uses subcutaneous injection to construct the CPP and withdrawal model. Compared with intraperitoneal injection, subcutaneous injection has low drug utilization and low operation efficiency, and cannot well simulate the pharmacological activity of intravenous drug injection in real-life addicts. In addition, the study only analyzes the number of jumps and the degree of weight loss in the somatic symptoms after carfentanil withdrawal, and lacks research on other somatic and mental symptoms. Finally, the drug administration mode of this study is: taking 3 μg / kg as the initial dose, subcutaneously injecting drugs 3 times a day for 7 consecutive days, with an interval of 6 hours between each injection, and the daily dose is twice the previous day. On the 7th day of the model, only one injection is made, and naloxone (2 mg / kg) is injected intraperitoneally 2 hours after withdrawal to observe the stereotyped jumping. This drug administration model is long in time, high in frequency, and consumes a lot of drugs. According to the average body weight of 25 g per mouse (n = 12), the model uses about 190.5 μg of drugs. Carfentanil is expensive, so exploring a more efficient model not only explains its strong pharmacological activity, high addiction and harm, but also facilitates further research and screening of therapeutic drugs for carfentanil. SUMMARY

[0006] The purpose of the present application is to provide a construction method of a carfentanil mouse withdrawal model and its application, to solve the technical problems of the prior art that the mouse withdrawal model has a large number of drug administrations, a long time, and an incomplete evaluation.

[0007] To achieve the above-mentioned purpose, the technical scheme adopted by the present application is as follows:

[0008] In a first aspect, the present application provides a construction method of a carfentanil mouse withdrawal model, comprising the following steps:

[0009] The mouse is injected with carfentanil, and the injection cycle is 6 days; 2 injections are injected every day in the first 5 days, the injection dose of each injection on the nth day is n times of the injection dose of each injection on the first day; 1 injection is injected on the 6th day, and the injection dose is the same as the injection dose of each injection on the 5th day; after the injection is completed, withdrawal is carried out to obtain a carfentanil mouse withdrawal model.

[0010] Further, the carfentanil is injected in a way of intraperitoneal injection.

[0011] Further, the injection dose of each injection on the first day is 4 μg / kg.

[0012] Further, in the step of injecting 2 injections every day in the first 5 days, the interval between each injection is 12 h.

[0013] Further, the withdrawal time is 7 days.

[0014] Further, the mouse is an 8-week-old male C57BL / 6J mouse.

[0015] Further, the method further comprises: judging whether the model is successfully constructed by performing somatic symptom evaluation and mental symptom evaluation on the obtained mouse withdrawal model.

[0016] Further, the mental symptom evaluation comprises anxiety symptom evaluation and depression symptom evaluation, the anxiety symptom evaluation comprises an open field and an elevated plus maze, and the depression symptom evaluation comprises sucrose preference and tail suspension.

[0017] In the second aspect, the application provides an application of the mouse withdrawal model obtained by the method for constructing the carfentanil mouse withdrawal model in screening of new psychoactive drug addiction and withdrawal symptom treatment drugs.

[0018] Compared with the prior art, the application has the following beneficial effects:

[0019] The application discloses a construction method and application of a carfentanyl mouse withdrawal model, and the carfentanyl is injected into the mouse for 6 days, wherein a doubled dose is given on the basis of an initial dose every day for the first five days, 2 injections are given per day with an interval of 12 hours, and only 1 injection is given on the sixth day; the withdrawal is carried out after the injection is completed to obtain the carfentanyl mouse withdrawal model. Compared with the prior art, the carfentanyl mouse withdrawal model has the advantages of fewer days of administration, fewer times of administration, and less use of drugs (about 42 mu g of drugs are used when n=12 mice per group and the average weight of the mice is 25 g per mouse, which is less than 1 / 4 of the drugs used in the foregoing research), and the economic cost and time cost are optimized. The initial dose of the withdrawal model is selected according to the sensitization model and the open field results, and compared with the initial dose of the prior art research which is provided according to the ED50 of the hot plate experiment, the application is more closely connected with the addiction, is more rigorous on the evidence chain, and has the advantages of being more closely connected with the addiction and being more rigorous on the evidence chain. After the construction of the withdrawal model is completed, the mouse is evaluated in terms of physical symptoms and mental symptoms, and it is found that, compared with the saline group mouse, the carfentanyl mouse has obvious differences in physical symptoms and mental symptoms (including anxiety and depression symptoms), and the effect is equal to or even higher than that of the positive control morphine group. This not only proves that the model construction of the application is successful, but also further evaluates the strong pharmacological activity, addiction and harmfulness of carfentanyl, and can be widely applied to the evaluation of carfentanyl abuse and other related researches.

[0020] Further, the intraperitoneal injection method adopted by the application has higher drug availability than the subcutaneous injection used in the foregoing research, and the drug availability is embodied in that the CPP cannot be formed after the foregoing research is trained by subcutaneous injection of 0.3 mu g / kg carfentanyl, while the CPP can be formed by low-dose 0.5 mu g / kg carfentanyl in the application, and there is no difference in the CPP score between the 2 mu g / kg group and the 4 mu g / kg group. BRIEF DESCRIPTION OF DRAWINGS

[0021] In order to more clearly illustrate the technical solutions of the embodiments of the application, the following will briefly introduce the drawings needed to be used in the embodiments. It should be understood that the following drawings only show some of the embodiments of the application, and therefore should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can also be obtained without creative labor on the basis of these drawings.

[0022] Figure 1 The flow chart of the gradient carfentanyl-induced mouse conditioned place preference model of the embodiments of the application;

[0023] Figure 2 The result graph of the gradient carfentanyl-induced mouse conditioned place preference score of the embodiments of the application;

[0024] Figure 3A flow chart of a model of behavioral sensitization induced by gradient caphentanil in mice according to an embodiment of the present application;

[0025] Figure 4 A result graph of total distance of movement within 1 hour of day 3 and day 13 of behavioral sensitization induced by gradient caphentanil in mice according to an embodiment of the present application;

[0026] Figure 5 A result graph of 10 min open field of expression of sensitization induced by gradient caphentanil in mice according to an embodiment of the present application, Figure 5 a is a result graph of center time, Figure 5 b is a result graph of center distance;

[0027] Figure 6 A flow chart of administration of caphentanil withdrawal model according to an embodiment of the present application;

[0028] Figure 7 A flow chart of symptom evaluation of caphentanil withdrawal model according to an embodiment of the present application;

[0029] Figure 8 A result graph of somatic symptoms induced by naloxone after 2 h of withdrawal according to an embodiment of the present application;

[0030] Figure 9 A result graph of 10 min open field after 7 days of withdrawal according to an embodiment of the present application;

[0031] Figure 10 A result graph of open arm time of elevated plus maze after 7 days of withdrawal according to an embodiment of the present application;

[0032] Figure 11 A result graph of depressive symptoms after 7 days of withdrawal according to an embodiment of the present application. DETAILED DESCRIPTION

[0033] To enable persons skilled in the art to understand the features and effects of the present application, the following is a general description and definition of the terms and phrases mentioned in the specification and claims. Unless otherwise specified, all technical and scientific words used in the text are of the usual meaning understood by those skilled in the art of the present application, and in the event of conflict, the definition in the specification shall prevail.

[0034] Theories or mechanisms described and disclosed herein, whether correct or not, should not be regarded as limiting the scope of the present application, i.e., the present application can be practiced without regard to any particular theory or mechanism.

[0035] Herein, all features defined in the form of numerical ranges or percentage ranges, such as numerical values, quantities, contents and concentrations, are for the sake of brevity and convenience. Accordingly, the description of numerical ranges or percentage ranges should be considered to have encompassed and specifically disclosed all possible sub-ranges and individual numerical values within the range (including integers and fractions).

[0036] Herein, "comprising", "including", "containing", "having" or like terms are inclusive and meant to cover the terms "consisting of" and "consisting essentially of", e.g., "A comprising a" means "A comprising a and other" and "A consisting of a".

[0037] Herein, all possible combinations of the technical features in the various embodiments or examples are not described in order to make the description concise. Therefore, as long as there is no contradiction in the combination of the technical features, the technical features in the various embodiments or examples can be combined arbitrarily, and all possible combinations should be considered as the scope of the present specification.

[0038] The application will be further described in detail below with reference to the accompanying drawings:

[0039] The embodiment of the application discloses a method for constructing a carfentanil mouse withdrawal model, comprising the following steps: injecting carfentanil into a mouse, and the injection period is 6 days; 2 injections are injected into the mouse every day in the first 5 days, and the interval between the two injections is 12 hours, the injection dose of each injection on the nth day is n times of the injection dose of each injection on the first day; 1 injection is injected on the sixth day, and the injection dose is the same as the injection dose of each injection on the fifth day; and withdrawal is performed after the injection is completed to obtain the carfentanil mouse withdrawal model.

[0040] In an available embodiment of the application, the carfentanil is injected intraperitoneally, and the withdrawal time is 7 days. It should be noted that the intraperitoneal injection method is based on the results of conditional place preference induced by gradient carfentanil.

[0041] In an available embodiment of the application, the injection dose of each injection on the first day is 4 μg / kg, and the injection dose of each injection on each day is specifically shown in Table 1. Figure 6 The administration dosage refers to the administration dose, the saline represents a saline group, the morphine represents a morphine group, and the carfentanil represents a carfentanil group. It should be noted that the initial dose is based on the results of behavioral sensitization and open field results induced by gradient carfentanil.

[0042] In an available embodiment of the application, the method further comprises: judging whether the model is constructed successfully by performing somatic symptom evaluation and mental symptom evaluation on the obtained mouse withdrawal model.

[0043] The embodiment of the application discloses a method for evaluating a carfentanil mouse withdrawal model, comprising performing somatic symptom evaluation and mental symptom evaluation on a mouse.

[0044] The step of the somatic symptom evaluation comprises:

[0045] 2h after the last dose on day 6; then the mice were individually placed in a transparent plastic observation cylinder for 20 min, and the number of times of wet dog shakes, ear scratching, sniffing and jumping behaviors within 20 min were counted and analyzed; meanwhile, the occurrence of body tremors within every 5 min was observed, and if the body tremors occurred, 1 point was recorded, otherwise 0 point was recorded, and the scores of 4 segments were recorded in total; the total withdrawal symptom score of each mouse was calculated by weight, and the calculation method was as follows: total withdrawal symptom score = 0.5 x counted symptoms + 1 x scored withdrawal symptoms total score; the mice were weighed before and after withdrawal, and the weight loss rate was calculated, and the calculation method was as follows: weight loss rate = (after withdrawal-before withdrawal) / before withdrawal.

[0046] The mental symptom evaluation includes anxiety symptom evaluation and depression symptom evaluation after 7 days of withdrawal;

[0047] The method of anxiety symptom evaluation includes open field test and elevated plus maze test. The method of depression symptom evaluation includes sugar water preference test and tail suspension test. For details, refer to Figure 7 Wherein, SPT is Sugar Preference Test (sugar water preference test), OFT is Open Field Test (open field test), EPM is Elevated Plus Maze (elevated plus maze), TST is Tail Suspension Test (tail suspension test), withdrawal represents withdrawal, and a represents an initial dose.

[0048] The application further discloses application of the mouse withdrawal model obtained by the construction method of the capheterol mouse withdrawal model in screening of new psychoactive drug addiction and withdrawal symptom treatment drugs.

[0049] The application will be further described in conjunction with specific examples. It should be understood that the examples are only used to illustrate the application but not to limit the scope of the application. Furthermore, it should be understood that those skilled in the art can make various modifications or changes to the application after reading the content of the application, and these equivalent forms also fall within the scope defined by the appended claims of the application.

[0050] In the following examples, the instruments and equipment in the art are used. In the following examples, the experimental methods not specified in the specific conditions are usually carried out according to the conventional conditions or the conditions recommended by the manufacturers. In the following examples, various raw materials are used, unless otherwise specified, and the conventional commercially available products are used, and the specifications are conventional specifications in the art. In the specification of the application and the following examples, unless otherwise specified, “%” represents the weight percentage, “parts” represent the weight parts, and the ratio represents the weight ratio.

[0051] Examples:

[0052] Experimental animals: C57BL / 6J mice, SPF level, male, weight 18-25 g, 8 weeks old, provided by Beijing Vital River Laboratory Animal Technology Co., Ltd. Raising in SPF level animal room, adaptive feeding for 1 week. The animal room maintains constant temperature (20-24℃), humidity (40-60%), and lights off from 7:00 pm to 7:00 am. Before the start of the behavior experiment, touch the experimenter for 3-5 minutes every day.

[0053] Drugs:

[0054] Cafentanyl hydrochloride standard, provided by Xi'an Public Security Bureau of Shaanxi Province, dissolved in 0.9% saline, intraperitoneal injection for administration.

[0055] Morphine hydrochloride standard, provided by China Institute for Drug Control, dissolved in 0.9% saline, intraperitoneal injection for administration.

[0056] I. Gradient concentration administration to construct cafentanyl addiction model, determine the pharmacological activity intensity of intraperitoneal injection and clarify the initial dose of withdrawal model.

[0057] Literature research shows that the ED50 of rat drug discrimination experiment of cafentanyl is 0.41 μg / kg, and conditioned place preference (CPP) of subcutaneous administration in mice can be formed at 1, 3 μg / kg, while 0.3 μg / kg cannot form. We choose intraperitoneal injection, which has higher drug availability and efficiency, and select doses of 0.5, 2, and 4 μg / kg, while morphine (10 mg / kg) as a positive control, to construct gradient CPP and sensitization induced by cafentanyl in C57BL / 6J mice.

[0058] (1) Conditioned place preference model

[0059] ① Pre-test (Pre-test, Day 1): 30 min before the test, the experimental mice were moved from the animal feeding room to the behavior laboratory for adaptation. The arched door hole of the middle pull-out plate in the experimental box was placed at the lower end, and the mice without any treatment were gently placed in the experimental box, allowing them to freely move in the two boxes and record for 15 min. The test results were analyzed and mice with abnormal activity were excluded: a. Low motor activity (total movement distance too small, less than 20 times of shuttle); b. Significant natural preference (staying time on either side more than 600 seconds).

[0060] ②Condition training (Day 2-9): 30 min before the start of training, the mice were placed in the behavioral laboratory for adaptation. The horizontal stripes and grid floor were selected as the companion drug box; the vertical stripes and fence floor were selected as the non-companion drug box; during the training, the closed side of the middle operant partition was placed at the lower end to limit the movement of the mice in one side of the box. On the second day, the experimental group mice were divided into three dose groups, and were given carfentanil by intraperitoneal injection and placed in the companion drug side for 45 min of training; at the same time, the saline group was given normal saline by the same treatment and placed in the companion drug side for 45 min of training. On the third day, the experimental group and the saline group were given an appropriate amount of normal saline by intraperitoneal injection and placed in the non-companion drug side for 45 min of training. This was repeated for 4 cycles, and the mice were limited to move in the designated box each time.

[0061] ③Post-test (Day 10): 30 min before the start of the test, the mice were placed in the behavioral laboratory for adaptation, and the middle sliding partition in the experimental box was adjusted to place the arched door hole side at the lower end. All mice were not given any treatment and were gently placed in the experimental box to freely move in the left and right experimental boxes and the time spent in each box was recorded for 15 min. The total distance traveled, average activity speed and shuttle times of the mice in the left and right boxes were analyzed.

[0062] The specific experimental procedure is shown in Figure 1 .

[0063] According to the experimental results (see Figure 2 ), wherein Sal and S represent normal saline, Mor and M represent morphine, Carf and C represent carfentanil, Pre-test represents pre-test, Condition training represents formation training, and Post-test represents post-test.

[0064] Low dose (0.5 μg / kg), medium dose (2.0 μg / kg) and high dose (4.0 μg / kg) of carfentanil can induce the formation of CPP in mice (P<0.0001 before and after the test; P<0.0001 compared with the saline group), and there is no difference between groups, and the low dose of carfentanil exposure can achieve the effect of the positive control (morphine 10 mg / kg) (P<0.0001 before and after the test; P<0.0001 compared with the saline group), which indicates that carfentanil has a strong addictive property. In addition, compared with the previous study that failed to form CPP after subcutaneous injection of 0.3 μg / kg carfentanil, the experimental results suggest that intraperitoneal injection has better efficiency and blood absorption rate and drug utilization, and can better simulate the current situation of intravenous injection of addicted patients.

[0065] (2) Behavioral sensitization model

[0066] ①Pre-test period (Day 1-2): Before the experiment, the mice were moved from the animal housing room to the behavior test room and test box for 30 min, and each mouse was injected intraperitoneally with saline and then placed in the test box for 60 min. The data was analyzed and the animals with abnormal data were excluded after two consecutive pre-tests.

[0067] ②Development period (Day 3-7): Before the experiment, the mice were moved from the animal housing room to the behavior test room and test box for 30 min, and the saline group of mice was given saline every day, and the drug group of mice was given three doses of carfentanyl every day, all by intraperitoneal injection, and then placed in the test box for 60 min. The operation was repeated for 5 consecutive days.

[0068] ③Withdrawal period (Day 8-12): All experimental animals were not treated and were raised in the animal housing room for 5 days (equivalent to 5 days of withdrawal).

[0069] ④Expression period (Day 13): Before the experiment, the mice were moved from the animal housing room to the behavior test room and test box for 30 min, and no drug treatment was given during the test box adaptation period. The first 10 min of the open field test was performed on the mice, and the center time and center distance of the mice were recorded. After the adaptation period, the saline group of mice was given saline, and the drug group of mice was given the same dose of carfentanyl as in the development period, all by intraperitoneal injection, and then placed in the test box for 60 min. The activity of the mice in the behavior box was recorded.

[0070] The specific experimental procedure is shown in Figure 3 , wherein Sal represents saline, Mor represents morphine, Carf represents carfentanyl, Pre represents pre-test period, Development represents development period, Withdraw represents withdrawal period, and Post represents expression period.

[0071] This sensitization is the first time to construct a behavior sensitization model since the appearance of carfentanyl. According to the experimental results, the movement distance of mice at 4 μg / kg in the expression period (Day 13) was 4.5 times that of the first day of the development period (Day 3) (see Figure 4, Traveled Distance represents the distance of movement, SA represents the saline group, Mor represents morphine, 0.5Carf represents the 0.5 μg / kg carfentanil group, 2Carf represents the 2.0 μg / kg carfentanil group, and 4Carf represents the 4.0 μg / kg carfentanil group. *P < 0.05 represents the comparison between Day 3 of the formation period and Day 13 of the expression period, and the independent sample t test was used. The data are expressed as mean ± standard error, and n = 7. There was a significant difference (P < 0.05), and morphine had no statistical difference. In addition, the mice were recorded for 10 min in the open field during the expression period (see Figure 5 a and Figure 5 b, Central Time represents the proportion of central time, SA represents the saline group, Mor represents morphine, 0.5Carf represents the 0.5 μg / kg carfentanil group, 2Carf represents the 2.0 μg / kg carfentanil group, and 4Carf represents the 4.0 μg / kg carfentanil group. **P < 0.01 represents the comparison between groups of central time, and *P < 0.05 represents the comparison between groups of central distance. The one-way ANOVA was used. The data are expressed as mean ± standard error, and n = 7. It was found that the central time (P < 0.01) and the central distance (P < 0.05) of the mice of 4 μg / kg were significantly reduced compared with the saline group, and morphine also had no difference, indicating that the mice of 4 μg / kg had anxiety symptoms. Based on the experimental results, the present application selects 4 μg / kg carfentanil as the initial dose. In addition, as found in existing research, opioid drugs are prone to tolerance and are difficult to sensitize, but 4 μg / kg carfentanil can induce mice to form sensitization, further indicating the strong pharmacological activity thereof.

[0072] II. Carfentanil withdrawal symptoms.

[0073] Since carfentanil has a very high analgesic therapeutic index and can be active in the mind at a small dose, it is extremely harmful. In order to simulate the drug use and withdrawal of addicted patients in reality and provide more metabolic materials, we constructed a carfentanil withdrawal model. According to the previous addiction data, 4 μg / kg carfentanil was selected as the initial dose, and the dose was doubled every day on the basis of the initial dose, 2 injections per day, with an interval of 12 h, 6 days of administration, and only 1 injection on the last day. The mice in the somatic symptom group were given 2 mg / kg naloxone 2 h after the last injection, and the mice in the mental symptom group were subjected to the corresponding experiment 7 days after withdrawal. The specific experimental contents are as follows:

[0074] (1) Withdrawal syndrome (somatic symptoms)

[0075] Naloxone was injected immediately and the mice were placed individually in a transparent plastic observation cylinder for 20 min. According to the actual symptoms observed, five withdrawal symptom parameters were selected and statistically analyzed. Among them, the number of times of wet dog shake, scratching ears and cheeks, sniffing and jumping behavior within 20 min was counted and analyzed. At the same time, whether the body tremor appeared in every 5 min was observed, and if it appeared, it was scored as 1 point, and if it did not appear, it was scored as 0 point. The total score of 4 segments was recorded. Finally, the total withdrawal symptom score of each mouse was calculated by weight, and the higher the score, the more serious the withdrawal symptoms. Total score = 0.5 x count symptoms + 1 x total score of withdrawal symptoms. In addition, the mice were weighed before and after withdrawal for 2 h, and the weight loss rate = (after withdrawal-before withdrawal) / before withdrawal.

[0076] The results show (see Figure 8 a, using one-way ANOVA, data are expressed as mean ± standard error, n = 7) that under this model, the mice showed obvious somatic symptoms after caphentane withdrawal (P < 0.001 compared with the saline group), and the withdrawal symptom score was significantly higher than that of the morphine group (P < 0.01). In addition, compared with the mice of the saline group, the mice of the caphentane group (P < 0.05) and the mice of the morphine group (P < 0.0001) showed significant weight loss after 2 h of withdrawal (see Figure 8 b, using one-way ANOVA, data are expressed as mean ± standard error, n = 7).

[0077] (2) Anxiety symptoms

[0078] a. Open field

[0079] Before the experiment, the mice were moved from the animal housing room to the behavior test room for 30 min of adaptation, and then the mice were placed in the test box for 15 min. The spontaneous activity trajectory of the mice was recorded by smart 3.0 behavior analysis system, and the total distance of activity in the test area and the activity time in the central area were evaluated. Mice with anxiety-like behavior will stay in the central area for a shorter time than normal mice. This experiment can evaluate whether the withdrawal mice have anxiety symptoms, and the effect of different withdrawal times on anxiety symptoms.

[0080] b. Elevated plus maze

[0081] The mouse elevated plus maze device includes two open arms, two closed arms, and a central open area of the maze, and the distance from the ground is 50 cm high. A video acquisition system is provided above the device, and the residence time and entry times of the mice in the open arms and closed arms can be obtained by smart 3.0 behavior analysis system.

[0082] Before the experiment, the mice were moved from the animal housing room to the behavior test room for 30 min of adaptation, and then the mice were placed in the test box for 15 min. The spontaneous activity trajectory of the mice was recorded by smart 3.0 behavior analysis system, and the total distance of activity in the test area and the activity time in the central area were evaluated. Mice with anxiety-like behavior will stay in the central area for a shorter time than normal mice. This experiment can evaluate whether the withdrawal mice have anxiety symptoms, and the effect of different withdrawal times on anxiety symptoms.

[0083] After the experiment, four indicators need to be counted, including the percentage of open arm residence time in total arm entry time; the percentage of open arm entry times in total arm entry times; the percentage of closed arm residence time in total arm entry time; and the percentage of closed arm entry times in total arm entry times. Compared with non-anxious mice, anxious mice will significantly increase the residence time or number of times in the closed arm. This experiment can explore whether the withdrawal mice have anxiety symptoms, and the effect of different withdrawal times on anxiety symptoms.

[0084] The results show that the model mice after carfentanil withdrawal have obvious anxiety symptoms, which are shown in the open field (see Figure 9 a and Figure 9 b, Saline represents the saline group, Morphine represents morphine, and Carfentanil represents the carfentanil group. *P<0.05, **P<0.01 for comparison of center time between groups, using one-way ANOVA, data expressed as mean ± standard error, n=9) The center time (P<0.05) and center activity distance (P<0.05) decreased significantly, and the elevated plus maze (see Figure 10 , Saline represents the saline group, Morphine represents morphine, and Carfentanil represents the carfentanil group. *P<0.05, **P<0.01 for comparison of open arm time between groups, using one-way ANOVA, data expressed as mean ± standard error, n=9) The residence time decreased significantly (P<0.05); and the severity of anxiety symptoms and the positive control morphine group were close.

[0085] (3) Depression symptoms

[0086] a. Sugar preference

[0087] The experimental device is a double-bottle experiment with 1% sugar water concentration, and the breeding cage is equipped with double water bottles.

[0088] ① Training period (training, Day 1-4)

[0089] On the first day, the experimental mice need to be single-caged in the behavior room for 24 hours to adapt to the environment, and the mice need to be given 2 bottles of 1% sugar water for free drinking. On the second day, the mice were given 1 bottle of 1% sugar water and 1 bottle of drinking water for free drinking for 48 hours, and the positions of sugar water and drinking water were exchanged after 24 hours; On the third day, the mice were deprived of water and food for 24 hours.

[0090] ② Test period (test, Day 5)

[0091] The weight of sugar water and drinking water was measured before the test, and then the test was conducted for 2 h, during which the positions of sugar water and drinking water were exchanged at the end of 1 h. The remaining sugar water and drinking water were measured after 2 h. The sugar water preference rate was calculated, i.e. sugar water preference rate = sugar water consumption / (sugar water consumption + drinking water consumption) x 100%. Taking advantage of the natural preference of mice for sweets, this experiment was used to evaluate the motivation, depression and lack of pleasure of mice, and the related emotional state, i.e. to explore whether the withdrawal mice have depressive symptoms, and the effect of different withdrawal times on depressive symptoms.

[0092] This model avoids the influence of long training period on the state of mice when testing sugar water preference, so a round of training and testing is completed before the model is built to determine the sugar water preference baseline. After building the model, only 24 h of water and food deprivation is required, followed by a 2 h test.

[0093] b. Tail suspension

[0094] The tail suspension device includes 3 compartments, with a hook fixed at the top, the hook being 30 cm from the floor, the floor being black, and the side and back panels being white. Before the experiment, the mice were moved from the animal housing room to the behavior test room for 30 min, and the middle 1 / 3 of the mouse tail was stuck to one side of the pressure-sensitive tape, and the other side was fixed to the hook. The fixed time is 6 min. A video acquisition system is provided in front of the device, and the immobility time of the mice can be obtained through the smart 3.0 behavior analysis system. The depression state of the mice is reflected by recording the immobility time of the mice. This experiment can explore whether the withdrawal mice have depressive symptoms, and the effect of different withdrawal times on depressive symptoms.

[0095] The results show that the mice in the carfentanil withdrawal model have obvious depressive symptoms (see Figure 11 a and Figure 11 b, Saline represents the saline group, Morphine represents morphine, and Carfentanil represents the carfentanil group. *P<0.05, **P<0.01, ****P<0.0001 for inter-group comparison, using one-way ANOVA, data are expressed as mean ± standard error, n = 9), which is manifested as a decrease in sugar water preference rate (P<0.05) and an increase in tail suspension immobility time (P<0.0001); and the severity of depressive symptoms is slightly higher than that of the positive control morphine group.

[0096] III. Statistical analysis

[0097] The experimental results are expressed as mean ± standard error (mean ± SEM), the mean values of multiple groups are compared using one-way ANOVA, and then Bonferroni post-hoc test is performed, the comparison between two groups is performed using independent t-test, and the significant test standard is two-sided P<0.05. The statistical analysis and graphical presentation of all experimental data are performed using GraphPad Prism 9.

[0098] The results of the present application show that the present model is relatively efficient and comprehensive in improving and optimizing the prior art model, not only in terms of drug dosage, time consumption and efficiency, but also in terms of success in model construction, obvious withdrawal symptoms, comprehensive evaluation of somatic symptoms and mental symptoms, and wide application in caphentanil mouse addiction and harm and other related experiments.

[0099] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. Various modifications and changes can be made by those skilled in the art based on the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A method for constructing a mouse model of carfentanil withdrawal, comprising: The method comprises the following steps: injecting carfentanil to mice, the mice being 8-week-old male C57BL / 6J mice, the injection cycle being 6 days, 2 injections being performed per day in the first 5 days, the injection interval being 12 hours, the injection dose of each injection on the nth day being n times of the injection dose of each injection on the first day, the injection dose of each injection on the first day being 4 μg / kg, 1 injection being performed on the 6th day, the injection dose being the same as that of each injection on the 5th day, and withdrawal being performed after the injection to obtain a carfentanil mouse withdrawal model, the carfentanil being injected intraperitoneally, and the withdrawal time being 7 days.

2. The method of claim 1, wherein the method is for constructing a mouse model of carfentanil withdrawal. The method further comprises judging whether the model is successfully constructed by performing somatic symptom evaluation and mental symptom evaluation on the obtained mouse withdrawal model.

3. The method of claim 2, wherein the method is for constructing a mouse model of carfentanil withdrawal. The mental symptom evaluation comprises anxiety symptom evaluation and depression symptom evaluation, the anxiety symptom evaluation comprising an open field and an elevated plus maze, and the depression symptom evaluation comprising sucrose preference and tail suspension.

4. Application of a mouse withdrawal model obtained by the method for constructing the carfentanil mouse withdrawal model according to any one of claims 1 to 3 in screening of new psychoactive drugs addictive to opioids and treatment drugs for withdrawal symptoms.

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