Method for constructing COPD (chronic obstructive pulmonary disease) combined depression rat model and application thereof

Abstract

The invention discloses a method for constructing a COPD (chronic obstructive pulmonary disease) combined depression rat model and application thereof, and belongs to the technical field of medical biological research. The method comprises the following steps: firstly, performing ovarian removal operation on 4-week-old female rats, then raising the rats to 6-8 weeks old, and performing tobacco smoke exposure, with the size of a tobacco smoke exposure box being 90 * 60 * 60 cm, lighting 9 cigarettes during each exposure, exposing for 4 times a day and 2 times in the morning and afternoon respectively, and exposing for at least 3 hours in the morning and afternoon; after the tobacco smoke is exposed for 2 months, the COPD combined depression rat model is successfully constructed. The built model rat not only has the phenomena of pathological changes related to chronic obstructive pulmonary disease and lung function decline, but also has depression-like behaviors. By using the method provided by the invention, the probability of animal modeling can be obviously increased, the modeling time is only 2 months, and the model construction period is greatly shortened.

Description

Technical Field

[0001] The invention belongs to the technical field of medical biological research, and in particular relates to a method for constructing a rat model of COPD combined with depression and an application thereof. Background Art

[0002] Chronic obstructive pulmonary disease (COPD) is one of the leading causes of morbidity and mortality worldwide, placing a heavy economic, social and healthcare burden on patients. Nearly 90% of COPD deaths under the age of 70 occur in low- and middle-income countries. In China, nearly 100 million people suffer from COPD. Depression, as a common mental disorder, also has a high prevalence. Globally, an estimated 5% of adults suffer from depression, and more women than men are affected by depression. More importantly, COPD patients often suffer from depression. The survey found that 10.79% of COPD patients suffer from anxiety, 13.65% suffer from depression, and 7.08% of patients have both anxiety and depression. When COPD patients have psychological comorbidities (anxiety, depression), COPD will worsen, manifested as more severe dyspnea and decreased lung function, and the prevalence and mortality of exertional dyspnea will also increase, and there is even a risk of suicide. Patients with COPD and depression are at higher risk of re-admission due to acute exacerbation of COPD. This has an adverse effect on the prognosis of COPD patients. Compared with the severity of COPD, the quality of life of COPD patients is more related to the presence of depressive symptoms. Patients believe that anxiety and depression are the main factors affecting their ability to cope with and self-manage their disease. Elderly COPD patients with anxiety or depression have a higher clinical burden and economic burden than patients with only COPD. Although depression caused by COPD has a significant impact on patients' daily life and medical expenses, it is still not treated or effectively treated. There is still a lack of methodological research to provide clear evidence for the antidepressant efficacy of antidepressants or psychotherapy in COPD patients. Therefore, it is of great significance to explore the pathogenesis of COPD combined with depression. In addition, through reviewing the literature, it was found that the prevalence of depression in female COPD patients was higher than that in male patients, and female gender was a risk factor for depressive symptoms in adult COPD patients. This may be closely related to the different levels of sex hormones in the body. Therefore, it is necessary to establish a female animal model of COPD combined with depression to explore whether estrogen levels affect the occurrence of COPD combined with depression. Summary of the invention

[0003] In order to solve the above technical problems, the present invention provides a method for constructing a rat model of COPD combined with depression and its application. The present invention first performs ovariectomy on female rats aged 4 weeks, and then raises them to 6-8 weeks of age for tobacco smoke exposure. The size of the tobacco smoke exposure box is 90*60*60cm. 9 cigarettes are lit for each exposure, and the exposure is 4 times a day, 2 times in the morning and afternoon, and the exposure time in the morning and afternoon is at least 3 hours apart. After 2 months of tobacco smoke exposure, a COPD combined with depression rat model is successfully constructed. The constructed model rats not only have COPD-related pathological changes and decreased lung function, but also have depressive-like behaviors. The use of the method in the present invention can not only significantly increase the probability of animal modeling, but also the modeling time is only 2 months, which greatly shortens the cycle of model construction.

[0004] In order to achieve the above object, the present invention adopts the following technical solutions:

[0005] One of the purposes of the present invention is to provide a method for constructing a rat model of COPD combined with depression, which method at least comprises a tobacco smoke exposure step: firstly, adaptive exposure for 6-8 days, the number of cigarettes gradually increases from 5-7 cigarettes / time, by 1 cigarette per day, until 8-10 cigarettes / time, the daily exposure time starts from 25-35 minutes in the morning and afternoon, and increases by 15-30 minutes per day according to the state of the rat, until a total of 220-260 minutes of exposure per day are carried out; at this time, formal exposure is carried out, the frequency is 5-7 days / week, the morning and afternoon exposure is 110-130 minutes respectively, the rest is 15-25 minutes in between, the morning and afternoon interval is at least 3 hours, and the formal exposure time is 7-10 weeks; after the formal exposure is completed, the COPD combined with depression rat model is constructed.

[0006] Furthermore, the tobacco smoke exposure step is carried out in a tobacco smoke exposure box, and the specifications of the tobacco smoke exposure box are (50-70)×(50-70)×(80-100) cm.

[0007] Furthermore, the specifications of the tobacco smoke exposure box are 60×60×90 cm.

[0008] Furthermore, the adaptive exposure time is 7 days, the number of cigarettes is gradually increased from 6 / time, by 1 cigarette per day, until 9 / time, the daily exposure time starts from 30 minutes in the morning and afternoon, and increases by 15-30 minutes per day according to the condition of the rat, until a total exposure of 4 hours per day.

[0009] Furthermore, the frequency of formal exposure is 6 days / week, with 2 hours of exposure in the morning and afternoon respectively, a 20-minute break in between, and a gap of at least 3 hours between the morning and afternoon, and the formal exposure time is 2 months.

[0010] Furthermore, the method also includes the step of ovarian removal.

[0011] Furthermore, the ovariectomy step is performed when the rats are 3-5 weeks old.

[0012] Furthermore, the ovariectomy step is performed when the rats are 4 weeks old.

[0013] Furthermore, the tobacco smoke exposure step is performed when the rats are 6-8 weeks old.

[0014] The second object of the present invention is to provide an application of any of the methods described in screening drugs for the prevention and treatment of COPD combined with depression.

[0015] The animal model constructed in the present invention can be used in the study of diseases related to COPD combined with depression, or can be used in the screening of preventive and therapeutic drugs for COPD combined with depression.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] (1) There are literature reports that tobacco smoke exposure can induce a COPD-related depression model in mice, but the experimental period is as long as 6 months. The present invention has established a method for tobacco smoke-induced COPD in rats, with a period of only 2 months. The model rats also have not only COPD-related pathological changes and decreased lung function, but also depressive-like behaviors.

[0018] (2) There are many methods for establishing animal models of depression, and ovariectomy (OVX) is one of them. However, in other studies, adult rats or mice are usually directly induced to have depressive-like behaviors through OVX. In the present invention, 4-week-old rats are first subjected to OVX, and then raised to 8 weeks of age for tobacco smoke exposure. The present invention also successfully established a COPD-related depression model, and this method not only increases the probability of animal modeling, but also shortens the model construction cycle. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The effects of each treatment on the lung function of rats in each treatment group of Example 1 of the present invention, wherein A: functional residual capacity (FRC); B: deep inspiratory capacity (IC); C: vital capacity (VC); D: dynamic lung compliance (Cdyn); E: total lung resistance (RI); F: 50 millisecond rate (FEV50 / FVC); Note: data are expressed as mean ± SEM (n = 5-8 per group). *P < 0.05, **P < 0.01, ***P < 0.001.

[0020] Figure 2The effects of each treatment on rat behavior in each treatment group of Example 1 of the present invention, where A: rat movement trajectory in the open field; B: sucrose preference rate; C: total horizontal movement distance; D: total number of entries into the central area; E: central area retention time; F: number of upright positions; Note: Data are expressed as mean ± SEM (n = 5-8 per group). *P < 0.05, **P < 0.01, ***P < 0.001. DETAILED DESCRIPTION

[0021] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Without departing from the spirit and essence of the present invention, modifications or substitutions made to the methods, steps or conditions of the present invention are within the scope of the present invention. The products, equipment, etc. used in the following examples are commercially available unless otherwise specified, and the methods used are consistent with conventional methods unless otherwise specified.

[0022] The technical solution of the present invention is further described in detail below in conjunction with embodiments.

[0023] Example 1

[0024] 1. Animal grouping

[0025] Rats aged 6-8 weeks (purchased from Hunan Slake Jingda Experimental Animal Co., Ltd., license number: SCXK (Xiang) 2021-0002, housed in the SPF animal room of Guangdong Huawei Testing Co., Ltd., ethics number: 202305004) were divided into a blank control group (CTL), an ovariectomy group (OVX), a tobacco smoke exposure group (CS), and an ovariectomy followed by tobacco smoke exposure group (CS OVX), with n = 8 rats in each group.

[0026] 2. Rat Ovariectomy

[0027] 1) Rats underwent OVX surgery at 4 weeks of age. Rats were fasted for 12 h before surgery. An appropriate amount of isoflurane was poured into the small animal inhalation gas anesthesia machine, and the small animal anesthesia machine was turned on. The rats were placed in the anesthesia induction box. After the rats were successfully anesthetized, they were taken out and fixed in the supine position, and anesthesia was maintained by continuous mask inhalation of isoflurane.

[0028] 2) Prepare the lower abdomen, disinfect thoroughly with iodine, and deiodinate twice with 75% ethanol.

[0029] 3) Make a 1-2 cm longitudinal incision in the middle of the lower abdomen, cut the skin, fascia, and muscle layers in turn, open the abdominal cavity, use toothless forceps to find the "Y"-shaped uterus deep in the abdominal cavity, find the green bean-sized pink cauliflower-shaped ovary along one side of the uterus, tie it near the uterus at the junction of the ovary and uterus, and then tie it at the fat tissue at the upper end of the ovary, completely cut off the ovary and remove it, find the other ovary along the uterus and remove it, and perform the same operation as before.

[0030] 4) Put the rat's uterus, adipose tissue, etc. back into the abdominal cavity, suture the peritoneum and muscle layer together, check if there is no bleeding, then suture the skin, and disinfect the area with iodine.

[0031] 5) The same volume of fat tissue was removed from the rats in the control group, and the rest of the operations were the same as before.

[0032] 6) Rats were housed in individual cages after surgery to prevent them from biting each other's wounds. They were given autoclaved melon seeds to supplement their nutrition and were observed for 3-5 days before being put back into the cages after their wounds had healed.

[0033] 3. Establishment of COPD rat model

[0034] Rats in the CTL and OVX groups were exposed to indoor air, and rats in the CS and CS OVX groups were placed in a homemade tobacco smoke exposure box (60×60×90cm) for exposure. The frequency of tobacco smoke exposure was 6 days per week, with 2 hours of exposure in the morning and afternoon, 20 minutes of rest in between, and at least 3 hours between the morning and afternoon. At the beginning of the experiment, the rats were exposed for 7 days. The number of cigarettes (Hongmei brand cigarettes, tar content 11mg, smoke nicotine content 0.9mg) was gradually increased from 6 cigarettes / time, and 1 cigarette was added every day until 9 cigarettes were added each time. The daily exposure time started with 30 minutes in the morning and afternoon, and increased by 15-30 minutes every day according to the animal's condition, until a total of 4 hours of exposure per day, and 6 days per week. The formal tobacco smoke exposure time was 2 months. During the experiment, the rats were weighed on a fixed day every week, and the rats were observed every day after exposure. The exposure time was reduced according to the specific situation and the experimental records were kept.

[0035] 4. Detection of COPD model combined with depression in rats

[0036] Behavioral tests were performed 24 hours after the COPD modeling period, and sucrose preference test and open field test were used to detect depressive-like behavioral manifestations.

[0037] (1) Sucrose preference test: used to evaluate motivation, depression (and anhedonia) and related emotional states in rodent models. Impaired reward behavior will lead to a decrease in preference for sucrose solution, which is manifested as a decrease in intake.

[0038] 1) Rats were housed in individual cages and trained to drink sucrose for 48 hours. Two bottles of 1% sucrose water were given to the rats in the first 24 hours, and one bottle of 1% sucrose water and the other bottle of pure drinking water were given in the last 24 hours (the positions of the two water bottles were swapped during the training).

[0039] 2) After the sucrose drinking training, the subjects were deprived of food and water for 16 hours.

[0040] 3) Prepare a bottle of 1% sucrose water and pure water for each rat, weigh and record, make sure the water bottles are leak-proof, and then gently place the two water bottles into the cage and start timing. No one is allowed to enter the room during the test, and avoid making noise, light, and major environmental changes during the test. End the test after 1 hour, weigh the water bottles again and record, and calculate the rat's intake of sucrose water and pure water within 1 hour.

[0041] 4) Calculate the sucrose preference index, formula:

[0042] (2) Open field test: Detect the spontaneous activity and exploratory behavior of rats, and evaluate the autonomous behavior, exploratory behavior and tension of experimental animals in a new environment.

[0043] 1) Bring the rats into the laboratory 3 hours in advance to reduce their anxiety about the new environment.

[0044] 2) Prepare a homemade plastic box of 100cm×100cm×40cm, spray it with alcohol, wipe it dry to remove odor, and set the camera position.

[0045] 3) Set the parameters in the EthoVision XT 14 behavioral software, define the central area, place a test rat into the open field, and start the experiment after the software can recognize the rat at all positions in the open field.

[0046] 4) The experimental rat was gently placed at the edge of the open field with its back facing the center area. The software automatically recorded the rat's activities in the open field (total horizontal movement distance, total number of entries into the central area, and time spent in the central area). The number of times the rat stood upright was manually recorded for 5 minutes.

[0047] 5) After the experiment, clean the urine and feces of the rats in the open field, spray with alcohol to remove the odor, wipe it dry with a paper towel before testing the next rat.

[0048] 5. Pulmonary function test

[0049] Pulmonary function tests were performed on the second day after the behavioral experiment was completed. The small animal PFT pulmonary function instrument (DSI, USA) was used to detect the lung function of rats.

[0050] 1) Install and connect the spirometry instrument for rats and calibrate the instrument according to the instructions.

[0051] 2) Rats were anesthetized by intraperitoneal injection of 3% sodium pentobarbital (3 mL / kg).

[0052] 3) After the rat showed no response in the toe clip test, the rat was fixed on the experimental platform and the teeth were fixed with surgical sutures to keep the neck straight.

[0053] 4) Disinfect the neck skin with 75% ethanol, cut the skin with scissors, and use forceps to bluntly separate to fully expose the airway.

[0054] 5) Use ophthalmic scissors to cut 2 mm horizontally at the airway. Insert the rat endotracheal tube into the trachea with the oblique end rotated downward. Insert slowly and gently to avoid damaging the airway.

[0055] 6) Place the rats in the lung function test chamber, input the rat group, number, and weight, and test the lung function of the rats using the DSIPFT system.

[0056] 7) During the test, the airtightness of the cavity is maintained to ensure that the baseline of the respiratory curve is around 0. If the rat's breathing does not cooperate with the ventilator, additional anesthetics are required.

[0057] 8) After the lung function data are collected and saved, the rats are taken out and the endotracheal tube is removed.

[0058] Experimental Results

[0059] 1. Decreased lung function after CS exposure

[0060] Lung function decline is one of the main indicators for judging the success of COPD modeling. Figure 1 A, P<0.05), deep inspiration volume ( Figure 1 B, P<0.001), vital capacity ( Figure 1 C, P<0.001), dynamic lung compliance ( Figure 1 D, P<0.01) were significantly higher than those in the CTL group. The total lung resistance in the CS group was higher than that in the CTL group ( Figure 1 E, P<0.01), 50 ms rate decreased ( Figure 1 F, P<0.01). This indicates that after 2 months of CS exposure, rats developed ventilatory dysfunction, increased airway resistance, and other signs of decreased lung function ( Figure 1 FI, P<0.05)

[0061] 2. CS exposure and ovariectomy lead to depressive-like behavior

[0062] In this field, behavioral tests are mainly used to observe animal depressive behaviors. In the sucrose preference experiment, compared with the CTL group and the OVX group, the sucrose preference rate of rats in the CS group and the CS OVX group decreased significantly after 2 months of CS exposure ( Figure 2 B, P<0.001), and the sucrose preference rate of rats in the CS OVX group was lower than that in the CS group (P<0.05). In the open field test, compared with the CTL group, the total horizontal movement distance of each group was reduced ( Figure 2C, P<0.05, the total horizontal movement distance of the CS OVX group was shorter than that of the CS group (P<0.05). The total number of central area entry, central area retention time, and upright times of the OVX group, CS group, and CS OVX group were all lower than those of the CTL group ( Figure 2 DF, P<0.05), and the rats in the CS OVX group did not enter the central area. The above behavioral test results showed that rats showed depressive-like behavior after CS exposure, and ovariectomy aggravated the manifestation of depressive behavior.

[0063] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for constructing a rat model of COPD combined with depression, characterized in that: At least the tobacco smoke exposure step is included: first, the adaptive exposure is carried out for 6-8 days, the number of cigarettes is gradually increased from 5-7 cigarettes / time, and one cigarette is added every day until it reaches 8-10 cigarettes / time, and the daily exposure time starts from 25-35 minutes in the morning and afternoon, and increases by 15-30 minutes every day according to the condition of the rat, until the total exposure time is 220-260 minutes per day; at this time, formal exposure is carried out, the frequency is 5-7 days / week, and the morning and afternoon exposure time is 110-130 minutes each day, with a rest time of 15-25 minutes in between, and the interval between the morning and afternoon is at least 3 hours, and the formal exposure time is 7-10 weeks; After the formal exposure is completed, the COPD combined with depression rat model is constructed.

2. The method according to claim 1, characterized in that The tobacco smoke exposure step is carried out in a tobacco smoke exposure box, and the specifications of the tobacco smoke exposure box are (50-70)×(50-70)×(80-100) cm.

3. The method according to claim 2, characterized in that The specifications of the tobacco smoke exposure box are 60×60×90 cm.

4. The method according to claim 3, characterized in that: The adaptive exposure time is 7 days, the number of cigarettes is gradually increased from 6 / time, by 1 cigarette per day, until 9 / time, the daily exposure time starts from 30 minutes in the morning and afternoon, and increases by 15-30 minutes per day according to the condition of the rat, until a total exposure of 4 hours per day.

5. The method according to claim 4, characterized in that The frequency of formal exposure is 6 days / week, with 2 hours of exposure in the morning and afternoon respectively, 20 minutes of rest in between, and at least 3 hours of interval between the morning and afternoon. The formal exposure time is 2 months.

6. The method according to claim 5, characterized in that The method further comprises the step of ovarian removal.

7. The method according to claim 6, characterized in that The ovariectomy procedure was performed on rats at 3-5 weeks of age.

8. The method according to claim 7, characterized in that The ovariectomy procedure was performed on rats at 4 weeks of age.

9. The method according to any one of claims 1 to 8, characterized in that: The tobacco smoke exposure procedure was performed on rats when they were 6-8 weeks old.

10. Use of the method according to any one of claims 1 to 9 in screening drugs for the prevention and treatment of COPD combined with depression.

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