CN114557317B - Construction method and application of heart-spleen deficiency type depression mouse model - Google Patents
Construction method and application of heart-spleen deficiency type depression mouse model Download PDFInfo
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Abstract
The invention discloses a method for constructing a heart-spleen deficiency type depression mouse model and application thereof, and relates to the field of medical biology. After the modeling is finished, the successful establishment of the mouse depression model based on continuous pressure stimulation is confirmed through a syrup preference experiment, an open field experiment, a tail suspension experiment, a forced swimming experiment and neurotransmitter content measurement and verification of drugs, the mouse is dialectical after the establishment of the depression disease model is confirmed, carotid artery ultrasonic measurement, laser speckle cerebral blood flow measurement and corresponding type Chinese medicine prescription syndrome verification are carried out, so that the success of the establishment of the heart-spleen deficiency depression mouse model is judged. The method is simple and stable, has high success rate and good repeatability, can provide support for prescription medication of the depression, is beneficial to precisely preventing and treating the depression, and has important value for research on subsequent mechanisms.
Description
Technical Field
The invention relates to the field of medical biology, in particular to a method for constructing a heart-spleen deficiency type depression mouse model and application thereof.
Background
Depression is a clinically common mental disorder, and according to world health organization reports, about 3.4 million depressed patients worldwide are likely to become the world second-most disease to heart disease. Depression is mainly manifested by depressed mood, impaired interest, pessimistic mind, slow thinking, lack of initiative, etc. With the aggravation of the illness, the patient can develop from stuffy to sad to absolute, and the more serious patient can have mental symptoms such as fantasy, mania and the like. Often, patients are afflicted with depression and go to the extreme of suicide. The model of depression mainly uses a chronic unpredictable mild stimulation model, the model has long modeling time, large workload and higher cost, has more requirements on manpower and material resources and time, and simultaneously, the pathogenesis of clinical depression is various, and the model of the model only depends on the chronic unpredictable mild stimulation model is difficult to meet the requirements of researching the pathogenesis of depression and researching and developing new antidepressant drugs.
Thus, stress generated by people who are in a fear environment for a long time also persists. In addition, with the development of social economy, many people are under the stress of work, life and academic for a long time, and scientific researches prove that the incidence rate of depression in such high-pressure environment for a long time is greatly improved.
At present, the traditional Chinese medicine depression symptoms are combined with animal models, are mostly molded by adopting composite factors, and have great difference with clinical syndrome formation.
Disclosure of Invention
The invention aims to at least solve one of the technical problems in the prior art and provides a method for constructing a heart-spleen deficiency type depression mouse model and application thereof.
The technical scheme of the invention is as follows:
a depression mouse model construction method adopts a plantar electric shock mode construction.
Preferably, the construction is carried out by a single-factor stimulation mode.
Preferably, the plantar electric shock mode specifically comprises: the electric shock current of the sole is 0.1-1000mA, the molding duration is 1-1000 days, the stimulation frequency is 1-5000 times/day, and each time is 1-5000s.
Preferably, the mice are housed in a single cage at the time of construction.
Preferably, the mice used in the construction are male mice of SPF class C57BL/6J strain.
Preferably, the behavioral assessment of apparent efficacy, neurotransmitter differential assay assessment of structural efficacy and validation drug assessment are employed for mouse depression model assessment.
Preferably, the behavioral assessment mode comprises open field experiments, cross maze experiments, tail suspension experiments and forced swimming experiments; neurotransmitters are measured by UPLC-MS/MS, the materials are blood and brain, and the content of serotonin in the neurotransmitters is detected.
The invention also discloses application of the depression mouse model construction method in screening of the anti-anxiety and depression drugs for treating heart-spleen deficiency.
Preferably, the medicament for treating anxiety and depression is used as a positive medicament to verify whether the model construction of the depression mice is successful, and after dialectical treatment of carotid ultrasound and laser speckle cerebral blood flow indexes, the classical prescription for treating heart-spleen deficiency is used to verify that the depression is depression heart-spleen deficiency.
Preferably, the positive medicine adopts paroxetine, and the classic prescription for heart-spleen deficiency syndrome adopts Guipi decoction.
The beneficial effects of the invention are as follows: according to the depression type mouse model, the mouse depression model caused by continuous panic pressure is manufactured by adopting a plantar electric shock mode, so that the variety of depression etiology animal models is enriched, and the syndrome type formed by the mouse is judged and verified after modeling is successful, so that the model is more in line with the rule of manufacturing the disease combined animal model. Simultaneously, a novel modeling mode is provided for researching an animal model of the disease caused by panic pressure; also provides a new animal model for screening depression drugs; the research foundation is also provided for the development of a pathogenesis and prevention and treatment method of depression; provides a research basis for the action mechanism of depression drugs; provides reference and reference for establishing animal models of symptoms combined with traditional Chinese medicine symptoms.
Drawings
FIG. 1 is a diagram of the experimental results of open field;
FIG. 2 is a graph of the results of tail suspension experiments;
FIG. 3 is a diagram of the results of forced swimming experiments;
FIG. 4 shows changes in serotonin levels in blood;
FIG. 5 shows changes in serotonin levels in the brain;
FIG. 6 mean change in cerebral blood flow index for each group;
FIG. 7 is a carotid artery systolic caliber;
FIG. 8 is a carotid artery diastolic vessel diameter;
FIG. 9 is a graph showing carotid blood flow peak;
FIG. 10 is a graph of the experimental trajectory of the control mice in the open field;
FIG. 11 is a graph of the experimental trajectories of model group mice in open field;
FIG. 12 is a graph of the open field experimental trajectories of Paroxetine group mice;
FIG. 13 is a graph showing the experimental trajectories of the mice of the spleen-invigorating decoction verification group in the open field;
in the figure, control-blank, model-model, paroxetine-test, guili decotion-spleen return soup-test.
In the figure, P is less than or equal to 0.05, P is less than or equal to 0.01, P is less than or equal to 0.001, and P is less than or equal to 0.05, which has statistical significance.
Detailed Description
The following detailed description of the invention is provided for a better understanding of the invention, but the scope of the invention is not limited thereto:
the feeding conditions, model detection modes, methods and results described in the embodiments of the present invention are applicable only to the present invention.
It should be noted that: UPLC-MS/MS is ultra-high performance liquid chromatography-tandem mass spectrometry.
Example 1
1. Materials and instruments
1. Experimental animal
30 mice (weight 20-24 g) of male SPF-grade C57BL/6J strain are provided by animal experiment science center of university of Chinese medicine in Jiangxi, and the feeding conditions are as follows: the environment temperature is 24-26 ℃, the humidity is 40-60%, standard pellet feed is fed, free diet and water intake are carried out, and after one week of adaptive feeding, experiments are carried out.
2. Instrument and medicament
Instrument: one ten thousandth electronic balance (model: BS224S type, sartorius Corporation), precision pipette (Eppendorf Corp. Germany), disposable syringe (2.5 ml x 100 Jiangxi Sanxin medical science Co., ltd.), 5810R cryocentrifuge (Eppendorf, germany), ice maker (Severe snow Electrical Co., ltd.), tissue grinder (Tianjin Jiling instruments Co., ltd.), forced swimming glass bucket (TorSus domestication), open field laboratory box (self-made), overhead maze (self-made), behavioural analysis software smart version3.0.06 (Dongle Natural Gene life sciences Co., ltd.), ultra-high resolution small animal ultrasound imaging system vevo2100 (Yoonto, canada), couplant (Siqing district Temple) laser speckle blood flow imager (Shengzhen Rui Warewrites Co., ltd.).
Reagent: methanol, acetonitrile (chromatographic purity, fisher Scientific, fairlaw, NJ, USA), formic acid (chromatographic purity, sigma-Aldrich Co.Ltd, stLouis, MO, USA), distilled water (Chen's) and the rest of the reagents are analytically pure, paroxetine (Jiuzhou Tongmedical Co., hubei) and the dosage of the spleen-returning decoction is referred to "prescriptions", and the used materials are purchased from Xiamen materia medica true source biological medicine science and technology Co. Specifically, each medicine is prepared from 18g of bighead atractylodes rhizome, 9g of ginseng, 18g of astragalus root, 18g of poria with hostwood, 3g of Chinese angelica, 6g of honey-fried licorice root, 3g of polygala tenuifolia, 18g of wild jujube seed, 9g of costustoot, 18g of longan pulp, one jujube and five ginger sheets according to the raw materials: water 1:10 (g: ml) adding water, adding 5 pairs of the original prescription, soaking for 30 minutes, decocting for 2 hours, and concentrating to obtain 175ml of extract.
2. Method of
1. Animal grouping and model establishment:
(1) Blank (control): and (5) normal feeding.
(2) Model group (model): and (5) performing model making by adopting a plantar electric shock mode. The plantar electric shock current is 6mA, the molding duration is 28 days, the stimulation frequency is 600 times/day, and 30s each time.
(3) Paroxetine validation group (paroxetine): and (3) performing model making by adopting a plantar electric shock mode, and after finishing model making, performing intraperitoneal injection on a mouse to administer paroxetine, wherein the dosage is 8mg/d/kg, and verifying whether the model is successful.
(4) Spleen-invigorating soup validation set (guili device): after the model is successfully manufactured by adopting a plantar electric shock mode, each mouse is perfused with the extract according to the volume of 0.15ml/d, so as to examine and verify whether the established syndrome model is successful.
The above 4 groups of 6 mice were fed in a single cage, and diet was normally given during the experiment. After the molding is finished, each group is subjected to a behavioural test; the blank group and the model group were dissected and obtained after the end of the behavioural test, and the positive drug verification group was given paroxetine at 8mg/d/kg for two weeks. After one week of molding, the spleen-invigorating decoction is administered with the decoction for two weeks. And after the administration is finished, performing a behavioral test, dissecting, sampling, performing carotid artery ultrasonic measurement, laser speckle measurement on cerebral blood flow and neurotransmitter measurement, and verifying the effectiveness of the model.
1. Open field experiments:
the operation process comprises the following steps: mice were placed in 45 x 45 open field test boxes, artificial lighting was maintained, and at the beginning of the experiment, mice were placed rapidly in open field test boxes, recording and timing was started. The total duration of the experiment is 6min, after the experiment is finished, the activity condition of the mice in the open field test box for 4min after the experiment is analyzed by using behavioral analysis software, and the total time of the depression group model group and the drug verification group entering the open field central area is counted.
The experimental results are shown in fig. 1 and fig. 10-13, and the open field experimental results prove that the time for the model mice to enter the open field central region is remarkably reduced (P < 0.05), and the time for the paroxetine and the spleen-invigorating decoction to enter the open field central region is remarkably different from the time for the model mice to enter the open field central region (P < 0.05), so that the paroxetine and the spleen-invigorating decoction are proved to have remarkable improvement effect on the depression behaviors of the model mice.
2. Tail suspension experiment:
the operation process comprises the following steps: fixing the tail end part of the mouse on a tail suspension test frame by using an adhesive tape, so that the head of the mouse is suspended downwards, the head of the mouse is consistent with the horizontal plane, the sight between adjacent mice is separated, timing and video recording are started at the beginning of the experiment, the total duration of the experiment is 6min, after the experiment is finished, the activity condition of the mice 4min after the experiment is analyzed by using behavioural analysis software, and the immobility time is used as a tail suspension test index.
The experimental results are shown in fig. 2, and the tail suspension experimental results prove that the immobility time of the mice in the model group is obviously improved (P < 0.05) compared with that of the mice in the control group, the immobility time of the mice in the Paroxetine and spleen-invigorating decoction verification group is obviously different from that of the mice in the model group (P < 0.05), and the Paroxetine and spleen-invigorating decoction are proved to have obvious improvement effect on the depression behaviors of the mice.
3. Forced swimming experiment:
the operation process comprises the following steps: putting the mice into a glass barrel with a height of 50cm and a diameter of 25cm, and placing warm water with a temperature of 24-25 ℃ into the barrel, wherein the water depth is 25cm, so that the mice cannot touch the bottom of the barrel. At the beginning of the experiment, timing and video recording was performed. The total duration of the experiment is 6min, and after the experiment is finished, the behavior analysis software is used for analyzing the activity condition of the mice in water for 4min after the experiment, and the immobility time of the mice in water is used as the index of the forced swimming test.
The experimental results are shown in fig. 3, and the forced swimming experimental results prove that the immobility time of the mice in the model group is obviously improved (P < 0.05) compared with that of the mice in the control group, the immobility time of the mice in the Paroxetine and spleen-invigorating soup verification group is obviously different from that of the mice in the model group (P < 0.05), and the Paroxetine and spleen-invigorating soup are proved to have obvious improvement effect on the depression behaviors of the mice.
4. Carotid artery ultrasound measurement
The operation process comprises the following steps: after the mice were anesthetized, the neck was dehaired, the carotid artery was sonicated, and carotid artery systolic, diastolic vessel diameter, and menstrual blood flow were measured.
The experimental results are shown in fig. 7-9, and it can be seen from the figures that the carotid artery systolic phase, the diastolic phase, the caliber and the blood flow of the mice in the model group have significant differences (P < 0.05) compared with the mice in the control group, and the positive medicament verification group and the Chinese medicament prescription verification group have significant differences (P < 0.05) compared with the mice in the model group, so that paroxetine and the spleen-invigorating soup have an improving effect on the carotid artery systolic phase, the diastolic phase, the caliber and the blood flow of the mice in the depression model group.
5. Laser speckle measurement of cerebral blood flow
The operation process comprises the following steps: after the mice are anesthetized, the heads of the mice are dehaired, scalp surface layers are cut off after dehairing, and cerebral blood flow measurement is carried out by using laser speckles.
The experimental results are shown in fig. 6 (BFI is blood flow index, and refers to mean value of cerebral blood flow index), and the graph shows that the cerebral blood flow of mice in the model group has significant difference (P < 0.05) compared with mice in the control group, and the positive drug verification group and the traditional Chinese medicine prescription verification group have significant difference (P < 0.05) compared with mice in the model group, so that paroxetine and spleen-invigorating soup have significant improvement effect on the cerebral blood flow of mice in the depression model group.
6. Neurotransmitter determination using UPLC-MS/MS:
the operation process comprises the following steps: after the abdominal cavity of the mouse is anesthetized, the mouse is dissected after the eyeball is picked up for blood taking, the taken blood sample is centrifuged, and the supernatant is taken and stored in a refrigerator at the temperature of minus 80 ℃. When the orbit blood collection is completed, the mice are dissected, a proper amount of PBS buffer solution is poured into the heart tips of the mice, then the brain tissues of the mice are rapidly collected, and the mice are stored in a refrigerator at the temperature of minus 80 ℃. At the time of the experiment, blood samples and brain tissue samples were treated separately.
Preparation of brain tissue samples:
taking out brain tissue, drying water by using filter paper, weighing whole brain, cutting with forceps, placing into an EP tube, adding physiological saline with the volume consistent with the weight, homogenizing for 2 minutes on a homogenizer, taking 0.1ml of homogenate, adding 2000 mu L of acetonitrile, centrifuging, setting the temperature of a centrifuge to 4 ℃, and centrifuging at a high speed of 15000r/min for 15 minutes; the supernatant was aspirated and placed into a sample vial.
Preparation of blood samples:
taking 100 mu L of a supernatant sample of the blood sample, adding 380 mu L of acetonitrile and 20 mu L of an internal standard into an EP centrifuge tube with the volume of 1.5mL, carrying out centrifugation after vortexing for 30s, setting the temperature of the centrifuge to be 4 ℃, and carrying out high-speed centrifugation at 15000r/min for 15min; the supernatant was aspirated and placed into a sample vial.
Chromatographic conditions:
controls and samples were analyzed on the Shimadzu Controlle CBM Alite system of AB Sciex Instruments, which was combined with the UPLC system of Shimadzu. The control and sample were separated in SIL-30AC at 40℃and the mobile phase consisted of 0.01% formic acid-water (A) and acetonitrile (B), and the linear gradient elution procedure was performed as follows: 2% B0-0.001 min; 2% B for 0.001-1 min; 2% -30% B for 1-4 min; 30% -90% B for 4-10 min; 90% -100% B for 10-10.1 min; 100% B for 10.1-13 min; 100% -5% B13-13.1 min; STOP for 15 minutes. The total flow rate is 0.2000ml/min, and the injection amount is 2.00ul each time.
Mass spectrometry conditions:
mass spectrometry was performed in positive ion mode using Triple Quad 5500. The ionization parameters were as follows: air curtain gas (CUR) 35.00psi; ion source gas 1 (GS 1) 45.00; ion source gas 2 (GS 2) 40.00; ion source Temperature (TEM) 550.00 ℃; the Entry Potential (EP) is 10.00V; collision cell outlet voltage (CXP) 14V; collision induced cracking voltage (CAD) 7psi. Mass spectrometer and UPLC system analysis 1.6.2 software control.
The experimental result shows that 4-5 shows that compared with the blank group, the blood and brain tissue contents of the mice in the model group are obviously reduced, the blood and brain tissue contents of the mice in the Paroxetine and spleen-invigorating decoction verification group are obviously different from those of the blood and brain tissue contents of the mice in the model group (P is less than 0.05), and the Paroxetine and spleen-invigorating decoction is proved to have obvious improvement effect on the blood and brain tissue contents of the mice in the depression model group.
7. Data analysis:
statistical analysis was performed using statistical software GraphPad prism8.0.2, and single factor analysis of variance was used between groups. P <0.05 is statistically different and P >0.05 is not statistically different.
3. Conclusion:
after the modeling of the mice is finished, apparent efficiency, structural efficiency and effectiveness of the model are verified through open field experiments, tail suspension experiments, forced swimming experiments, carotid artery ultrasonic measurement, laser speckle measurement, neurotransmitter content measurement, positive verification drugs and traditional Chinese medicine formulas. The results of the open field experiments prove that the time for the model mice to enter the open field central region is remarkably reduced (P < 0.05), and the time for the group mice to enter the open field central region is remarkably different from that of the model group mice (P < 0.05), so that the effect of paroxetine and the spleen-invigorating soup on remarkably improving the depression behaviors of the model group mice is proved. The tail suspension experiment result proves that the immobility time of the mice in the model group is obviously improved compared with that of the mice in the control group (P < 0.05), and the time of the immobility of the mice in the model group is obviously different from that of the mice in the model group (P < 0.05), so that the paroxetine and the spleen-invigorating soup have obvious improvement effect on the depression behavior of the mice. The forced swimming experiment result proves that the immobility time of the mice in the model group is obviously improved (P < 0.05) compared with that of the mice in the control group, and the time of the immobility of the mice in the model group is obviously different (P < 0.05), so that paroxetine and the spleen-invigorating soup are proved to have obvious improvement effect on the depression behavior of the mice. The carotid ultrasonic experimental result shows that the carotid artery systolic phase, diastolic phase caliber and blood flow of the model group mice have significant differences (P < 0.05) compared with the control group mice, and the positive drug verification group and the traditional Chinese medicine prescription verification group have significant differences (P < 0.05) compared with the model group mice, so that paroxetine and the spleen-invigorating soup have significant improvement effects on carotid artery systolic phase, diastolic phase caliber and blood flow of the depression model group mice. The results of laser speckle experiments show that the cerebral blood flow of mice in the model group is significantly different (P < 0.05) from that of mice in the control group, and the positive drug verification group and the traditional Chinese medicine prescription verification group are significantly different (P < 0.05) from those of mice in the model group, so that the paroxetine and the spleen-invigorating soup have significant improvement effect on the cerebral blood flow of mice in the depression model group. Through neurotransmitter detection, the content of the serotonin in the blood and brain tissues of the mice in the model group is obviously reduced compared with that in the blank group, the significant difference (P < 0.05) between the serotonin content in the blood and brain tissues of the mice in the model group and the serotonin content in the blood and brain tissues of the mice in the model group is verified, and the paroxetine and the spleen-invigorating soup are proved to have a significant improving effect on the serotonin content in the blood and brain tissues of the mice in the depression model group.
The experimental results show that the depression model accords with the apparent effectiveness index and the structural effectiveness index of depression evaluation as well as the test result of neurotransmitters, and the positive drug verification proves that the mouse depression model based on continuous fear pressure stimulation is successfully established. After the establishment of the depression disease model is confirmed to be successful, carotid artery ultrasonic measurement, laser speckle cerebral blood flow measurement and corresponding type traditional Chinese medicine prescription syndrome verification are carried out on the mice to judge that the establishment of the heart-spleen deficiency depression mouse model is successful. The method is simple and stable, has high success rate and good repeatability, can provide support for prescription medication of the depression, is beneficial to precisely preventing and treating the depression, and has important value for research on subsequent mechanisms.
The above additional technical features can be freely combined and superimposed by a person skilled in the art without conflict.
The foregoing is only a preferred embodiment of the present invention, and all technical solutions for achieving the object of the present invention by substantially the same means are included in the scope of the present invention.
Claims (4)
1. A method for constructing a heart-spleen deficiency type depression mouse model is characterized in that a plantar electric shock mode is adopted for construction;
the electric shock current of the sole is 6mA, the molding duration is 28 days, the stimulation frequency is 600 times/day, and each time is 30s;
the evaluation of the heart-spleen deficiency type depression mouse model adopts behavioural evaluation apparent efficiency and neurotransmitter difference measurement evaluation structural efficiency and verification drug evaluation;
the behavioral assessment mode comprises open field experiments, cross maze experiments, tail suspension experiments and forced swimming experiments; neurotransmitters are measured by UPLC-MS/MS, the materials are blood and brain, and the content of serotonin in the neurotransmitters is detected;
an application of a heart-spleen deficiency type depression mouse model construction method in screening and treating heart-spleen deficiency type anxiolytic and depression medicaments;
the preparation method comprises the steps of adopting a medicament for treating anxiety and depression as a positive medicament, verifying whether a model is successfully constructed for a depression mouse, and verifying that the model is heart-spleen deficiency type depression by using a classical prescription for heart-spleen deficiency type depression after dialectical treatment of carotid ultrasound and laser speckle cerebral blood flow indexes;
paroxetine is adopted as the positive medicine, and the spleen-invigorating soup is adopted as the classical prescription for treating heart-spleen deficiency.
2. The method for constructing a heart-spleen deficiency type depression mouse model according to claim 1, wherein the method is constructed by adopting a single factor stimulation mode.
3. The method for constructing a heart-spleen deficiency type depression mouse model according to claim 1, wherein the mice are fed in a single cage during construction.
4. The method for constructing a heart-spleen deficiency type depression mouse model according to claim 1, wherein the mouse is a male mouse of SPF class C57BL/6J strain.
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