Parkinson's disease animal model and construction method and application thereof

Abstract

The invention relates to a Parkinson's disease animal model and a construction method and application thereof. Relates to the technical field of animal model construction. The construction method comprises the steps that a heterocyclic arylamine compound is adopted for administration to animals, namely the animal model of the Parkinson's disease is constructed, and the heterocyclic arylamine compound is 2-amino-1-methyl-6-phenylimidazole [4, 5-b] pyridine. According to the construction method, 2-amino-1-methyl-6-phenylimidazole [4, 5-b] pyridine is creatively used for constructing the Parkinson's disease animal model, Parkinson's disease sample movement symptoms and nerve injury expressions can be effectively simulated in a short time, and the construction method is an efficient Parkinson's disease model construction method.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of animal model construction, and particularly relates to a Parkinson's disease animal model and a construction method and application thereof BACKGROUND

[0002] Parkinson's disease (PD) is a common neurodegenerative disease characterized by motor disorders, and its onset is related to the interaction of aging, environment and genetic factors. PD is mainly manifested in clinical practice as motor symptoms such as static tremor, motor retardation, muscle rigidity and gait abnormalities, and these symptoms tend to progress. Motor symptoms are the main basis for the current clinical diagnosis of PD. The main reason for the motor symptoms is the degeneration of the substantia nigra dopamine (DA) neurons in the midbrain, which leads to insufficient dopamine neurotransmitters in the striatum. In addition, LBs, which are mainly formed by the aggregation of α-Synuclein (α-Syn) in neurons, are a hallmark of PD and are the basis for the pathological diagnosis of PD.

[0003] Traditional PD animal models mainly use various neurotoxins, such as MPTP, rotenone, paraquat and 6-hydroxydopa, to cause selective damage to DA neurons. This type of animal model well simulates the damage to DA neurons in the substantia nigra and motor symptoms of PD, but lacks progressive LB-like pathological changes. In recent years, recombinant human α-Syn is incubated in phosphate buffer (PBS) to form preformed fibrils (PFF), which are then injected into the striatum or other neural sites of mice or other animals, such as the gastrointestinal tract. This model well simulates the progressive development of α-Syn or LB-like pathological changes, a certain degree of DA neuronal damage and changes in motor function, but the modeling period is relatively long, generally 3-6 months, and the success of the modeling is greatly related to the preparation of PFF.

[0004] Diet has been investigated as a cause of major neurodegenerative diseases such as Alzheimer's disease (AD) and PD. Importantly, the impact of dietary factors can be much greater than other environmental pollutants over the lifetime of most people. There are no effective treatments for most major neurodegenerative diseases. Therefore, identifying and preventing modifiable risk factors that cause the disease are key approaches to reduce the public health burden of neurodegenerative diseases. Importantly, epidemiological studies have identified meat consumption as a potential risk factor for AD and PD. The toxins formed during meat preparation as human risk factors for neurological diseases have so far received limited attention. However, an increasing number of laboratory data suggest that these toxins, especially heterocyclic aromatic amines (HAAs), are potential neurotoxins.

[0005] A large number of studies have shown that eating well-cooked roasted meat containing high levels of HAAs is a risk factor for colorectal cancer. 2-amino-1-methyl-6-phenylimidazol[4,5-b]pyridine (PhIP) has been extensively studied as a genotoxic and mutagenic agent, and is also the most abundant HAA isolated from cooked meat, with a content of about 15 μg / kg in uncooked meat (about 75% of genotoxic substances). PhIP exposure accounts for about two-thirds of total HAA consumption. However, there is no report on the establishment and evaluation of a PD model using the potential neurotoxic effects of PhIP. SUMMARY

[0006] To solve the above technical problems, the purpose of the present application is to provide a Parkinson's disease animal model and a construction method and application thereof, the present application innovatively uses 2-amino-1-methyl-6-phenylimidazol[4,5-b]pyridine (PhIP) to construct a Parkinson's disease animal model, which can effectively simulate PD-like motor symptoms and nerve injury performance in a relatively short time, is a high-efficiency PD model construction method, greatly shortens the time-consuming of PD course research, and effectively improves the research efficiency.

[0007] The technical scheme for solving the above technical problems of the present application is as follows: The first purpose of the present application is to provide a construction method of a Parkinson's disease animal model, which comprises administering a heterocyclic aromatic amine compound to an animal, i.e. constructing a Parkinson's disease animal model.

[0008] On the basis of the above technical scheme, the present application can also be improved as follows.

[0009] Further, the heterocyclic aromatic amine compound is 2-amino-1-methyl-6-phenylimidazol[4,5-b]pyridine.

[0010] The Parkinson's disease animal model is constructed by using 2-amino-1-methyl-6-phenylimidazopyridine (PhIP) innovatively, and the PD-like motor symptoms and nerve injury performance can be effectively simulated in a short time, so the method is a high-efficiency method for constructing the PD model.

[0011] Further, the administration is to give the animal 2-amino-1-methyl-6-phenylimidazopyridine by gavage for 10 days.

[0012] Further, the single administration amount is 20 mg / kg-40 mg / kg.

[0013] Further, the administration frequency is 20 mg / kg / d-40 mg / kg / d.

[0014] Further, the animal is a C57BL / 6N mouse.

[0015] Further, the administration is by gavage.

[0016] Further, the construction method further comprises verifying the constructed Parkinson's disease animal model.

[0017] The second object of the present application is to provide a Parkinson's animal model.

[0018] The Parkinson's animal model constructed by using PhIP for short-time modeling can better simulate the clinical symptoms and pathological performance of PD.

[0019] The third object of the present application is to provide an application of the Parkinson's disease animal model, and the Parkinson's animal model is used in the preparation of a drug for screening and / or treating Parkinson's disease. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1Figure 1 is a graph showing the PhIP-induced PD-like movement disorder in mice, wherein (A) is a comparison of the rotarod fall latency of PhIP-20 mg / kg / d group, PhIP-40 mg / kg / d group, and Control group mice; (B) is a typical gait sequence diagram and footprint record of the gait analysis experiment of PhIP-20 mg / kg / d group, PhIP-40 mg / kg / d group, and Control group mice; (C) is a comparison of the average movement speed of the gait analysis experiment of PhIP-20 mg / kg / d group, PhIP-40 mg / kg / d group, and Control group mice; (D) is a comparison of the normal gait sequence ratio of the gait analysis experiment of PhIP-20 mg / kg / d group, PhIP-40 mg / kg / d group, and Control group mice; (E) is a comparison of the three-foot support time ratio of the gait analysis experiment of PhIP-20 mg / kg / d group, PhIP-40 mg / kg / d group, and Control group mice (n=6, ***P<0.001, ****P<0.0001); Figure 2 Figure 2 is a graph showing the PhIP-induced abnormal dopamine metabolism in the nigrostriatal system of mice, wherein (A) is an electrophoresis diagram of the TH, DAT, and β-actin protein expression in the striatum of each group of mice; (B) is a statistical result of the relative expression level of tyrosine hydroxylase (TH) in the striatum of each group of mice; (C) is a statistical result of the relative expression level of DAT in the striatum of each group of mice; (D) is a statistical result of the dopamine (DA) level in the striatum of each group of mice (n=3, *P<0.05, **P<0.01); Figure 3 Figure 3 is a graph showing the PhIP-induced damage to dopaminergic neurons in the nigrostriatal system of mice, wherein (A) is a staining result of TH-positive dopaminergic neurons in the substantia nigra of each group of mice; (B) is a statistical result of the relative number of TH-positive dopaminergic neurons in the substantia nigra of each group of mice (n=3, *P<0.05, **P<0.01); Figure 4 Figure 4 is a graph showing the PhIP-induced damage to dopaminergic neurons MES23.5 cells and mitochondrial dysfunction, wherein (A) is a change in cell viability of dopaminergic neuron MES23.5 cells treated with different concentrations of PhIP; (B) is a Mito-Tracker Red CMXRos staining result of MES23.5 cells treated with 150 μM PhIP; (C) is a statistical result of the Mito-Tracker Red CMXRos staining of MES23.5 cells (n=3, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001). DETAILED DESCRIPTION

[0021] The principles and features of the present invention will be described below. The examples given are only for explaining the present invention and are not intended to limit the scope of the present invention.

[0022] Example 1: (1) Establishment of PD mouse model: 18 male C57BL / 6N mice at 8 weeks old were purchased from Cyagen (Gu'an) Biotechnology Co., Ltd. (Production License No.: SCXK(Hebei)2021-003), and were housed in the SPF-class animal room of Guilin Medical University, with free access to food and water, and a 12h / 12h day-night cycle. They were randomly divided into 3 groups: PhIP-20mg / kg / d group, PhIP-40mg / kg / d group, and Control group, with 6 mice in each group. The PhIP-20mg / kg / d group was given intragastric administration of PhIP (Product No.: P275746, CAS No.: 105650-23-5, Shanghai Aladdin Biochemical Technology Co., Ltd.) at a dose of 20mg / kg / day for 10 consecutive days; the PhIP-40mg / kg / d group was given intragastric administration of PhIP at a dose of 40mg / kg / day for 10 consecutive days; the Control group was given intragastric administration of an equal volume of normal saline for 10 consecutive days; (2) Behavioral detection After 10 days of drug administration, behavioral detection was carried out, and the detection method was as follows: A. Gait analysis: Before the formal experiment, let the mice walk or run freely on the runway to adapt to the runway and experimental equipment. Place the mice on one side of the gait analyzer runway (XR-FP101, Shanghai Xinruan Information Technology Co., Ltd.), and train the mice to walk from one end of the runway to the other end. Each mouse was trained 3 times. During the formal experiment, place the mice at one end of the runway, and use the camera and equipment footprint sensor of the gait analysis system (XR-FP101, Shanghai Xinruan Information Technology Co., Ltd.) to record the video and footprints of the mice walking from one end to the other end. By analyzing the step sequence, the proportion of three-foot support time, and the average moving speed in the same-frequency stride. To evaluate the gait and motor ability differences of mice in each group.

[0023] B. Rotarod test: The rotarod test was used to detect the coordinated motor ability of mice. The rotarod (XR1514, Shanghai Xinruan Information Technology Co., Ltd.) was used to train the mice in each group. They were trained 3 times at a speed of 4rpm / min for 2 minutes each time. Subsequently, the rotation speed of the rotarod was adjusted to accelerate from 4rpm / min to 40rpm / min within 5 minutes, and the time required for each mouse to fall off the rotarod was recorded, that is, the fall latency of the rotarod.

[0024] The detection results are as Figure 1 shown: (1) Figure 1In A, the results of the rotarod test showed that the coordination ability of PhIP gavage-treated mice was impaired, as evidenced by a significant shortening of the rotarod drop latency compared with the Control group. Specifically, the rotarod drop latency of the PhIP-20 mg / kg / d group was shortened from 300.00 s in the Control group to 147.50 s, and the rotarod drop latency of the PhIP-40 mg / kg / d group was shortened to 179.33 s, with a statistically significant difference. (2) The results of the gait analysis experiment showed that, as shown in Figure 1 B, Figure 1 C, PhIP gavage-treated mice exhibited obvious gait abnormalities. Specifically, the average movement speed of the PhIP-20 mg / kg / d group was reduced to 60.41 mm / s compared with 141.87 mm / s in the Control group, and the average movement speed of the PhIP-40 mg / kg / d group was reduced to 64.72 mm / s, with a statistically significant difference (B, C). Figure 1 B, C); (3) As shown in Figure 1 B, Figure 1 D, the proportion of normal step sequences in the PhIP-20 mg / kg / d group was reduced to 70.32% compared with 98.72% in the Control group, and the proportion of normal step sequences in the PhIP-40 mg / kg / d group was reduced to 71.52%, with a statistically significant difference. (4) As shown in Figure 1 B, Figure 1 E, the proportion of three-foot support time in the PhIP-20 mg / kg / d group was increased to 51.16% compared with 19.73% in the Control group, and the proportion of three-foot support time in the PhIP-40 mg / kg / d group was increased to 42.64%, with a statistically significant difference.

[0025] These results indicate that PhIP can induce obvious motor disorders in mice.

[0026] Example 2: (1) Western blotting Three mice from each group after behavioral test in Example 1 were euthanized by over-dose of 3% isoflurane and then the striatum brain region was isolated. The protein concentration was evaluated using BCA Protein Assay Kit (Thermo Fisher Scientific, Massachusetts, USA). Then 20 μg of protein from each group was separated by SDS-PAGE and transferred to NC membrane. The membrane was blocked with 5% skim milk and then incubated with mouse anti-TH antibody (1:10000, T2928, Sigma-Aldrich, MO, USA), rabbit anti-DAT antibody (1:1000, DF2243, affinity, Affinity, OH, USA) and mouse anti-β-actin (C1313, Applygen, Beijing, China) antibody overnight. The secondary antibody (1:10000, 926-68070, IRDye 680RD Goat anti-Mouse Secondary Antibody; 926-32211, IRDye 800CW Goat anti-Rabbit Secondary Antibody, LI-COR, Nebraska, USA) was combined with the primary antibody for 1 h at room temperature. After four washes, the membrane was scanned by ODYSSEY imaging system (LI-COR, Nebraska, USA) as shown in Figure 2

[0027] (2) Immunofluorescent staining a Sample preparation: Three mice from each group after behavioral test in Example 1 were anesthetized by 1% isoflurane and then fixed on the wax plate. The abdominal skin and muscle tissue were cut along the midline of the abdomen, and the ribs were cut along the lower edge of the ribs on both sides to expose the heart. The needle was inserted from the interventricular septum, the right auricle was cut, and the whole body blood was flushed with physiological saline. Then the whole body was fixed by perfusion with pre-cooled 4% paraformaldehyde. The mouse brain was peeled off and fixed in 4% paraformaldehyde for 48 h, and then dehydrated with 20%-30% sucrose gradient. The mouse brain tissue was sectioned at 20 μm on a CryoStar NX50 freezing microtome (Thermo Fisher Scientific, MA, USA).

[0028] ​b Detection: Subsequently, the sections of the substantia nigra region were permeated in 0.3% PBST for 1 h, blocked with 2% goat serum in PBST for 2 h at room temperature, and then incubated with anti-TH antibody (1:1000, T2928) overnight at 4°C. The next day, the sections were rinsed with 1 M PBS for 3 times, 5 min each time, and then incubated with Goat anti-Mouse IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 594 fluorescent secondary antibody for 1 h at room temperature. After rinsing the sections with 1 M PBS for 3 times, 5 min each time, the sections were mounted with DAPI anti-fluorescence quenching mounting medium. The sections were imaged under a fluorescence microscope, and the results are shown in Figure 3 .

[0029] As shown by Figure 2 , Figure 3 , (1) As shown in Figure 2 A and Figure 2 B, after the behavioral test, the tissues of the striatum and substantia nigra of the mice were subjected to pathological change detection. First, the protein expression of TH, a key rate-limiting enzyme of DA metabolism in the substantia nigra striatal system, was detected. The results of Western blotting detection showed that, compared with the detection result of 100.00% of the Control group, the TH protein expression in the striatum of the mice after PhIP treatment decreased significantly. Among them, the TH protein expression in the PhIP-20 mg / kg / d group decreased more obviously, to 57.30%, and the TH protein expression in the PhIP-40 mg / kg / d group also decreased significantly, to 49.14%. (2) As shown in Figure 2 A and Figure 2 C, the expression level of dopamine transporter DAT also decreased, and the DAT protein expression in the PhIP-20 mg / kg / d group decreased to 94.65%, and the DAT protein expression in the PhIP-40 mg / kg / d group decreased to 87.35%, indicating abnormal DA metabolism. As shown in Figure 2 D, the detection results of the DA level of the striatum showed that, compared with the detection result of 34.95 pg / mg protein of the Control group, the DA content in the striatum after PhIP treatment decreased significantly, the DA content in the PhIP-20 mg / kg / d group decreased to 23.82 pg / mg protein, and the DA content in the PhIP-40 mg / kg / d group decreased to 20.43 pg / mg protein, and the difference was statistically significant. These results further proved that PhIP treatment caused obvious DA metabolism abnormality in the substantia nigra striatal system.

[0030] (3) In addition, we performed immunohistochemical analysis on the substantia nigra brain region, as shown in Figure 3A、 Figure 3 The results of B show that compared with the results of the Control group 100.00%, there is a certain degree of reduction in TH-positive dopaminergic neurons in the substantia nigra after PhIP treatment, among which the PhIP-20 mg / kg / d group is reduced to 82.56%, and the PhIP-40 mg / kg / d group is reduced to 58.36%, and the difference is statistically significant, especially in the PhIP-40 mg / kg / d group. These results prove that PhIP can induce obvious PD-like pathological changes in mice.

[0031] Example 3: (1) Cell culture: The mouse dopaminergic neuron MES23.5 cell line (Professor Xun of Capital Medical University Xuanwu Hospital) was inoculated and cultured in DMEM / F12 medium (11320033, Thermo Fisher Scientific, MA, USA) containing 5% FBS (10099141C, Thermo Fisher Scientific, MA, USA) and 1% Sato's supplement (250mg / L Transferring, 2.43g / L Pyruvic acid, 250μg / L Na selenite, 315μg / L Progesterone (Sigma-Aldrich, MO, USA). After 48 h, the cells were digested with 0.25% trypsin (25200072, Thermo Fisher Scientific, MA, USA) and inoculated into a 96-well culture plate.

[0032] (2) Cell viability detection Different concentrations (0 μM, 25 μM, 50 μM, 100 μM, 150 μM) of PhIP were added to the culture medium of the mouse dopaminergic neuron MES23.5 cell line planted in the 96-well plate for 48 h. After 48 h, the old culture medium was discarded, 100 μL of fresh complete culture medium was added to each well, 10 μL of Cell count kit-8 reagent (Dojindo, Japan) was added, and the absorbance value was read at 450 nm wavelength on the microplate reader (Molecular Devices, USA) after incubation at 37°C for 1 h.

[0033] (3) Mitochondrial membrane potential detection Different concentrations (0 μΜ, 150 μΜ) of PhIP were added to the culture medium of mouse dopaminergic neuron MES23.5 cells planted in 96-well plates for 48 h. Mito-Tracker Red CMXRos staining was performed using a mitochondrial membrane potential and apoptosis detection kit (C1071M, Biyun Tian, Shanghai, China) to detect the mitochondrial membrane potential and apoptosis status of cells. The specific operation is briefly described as follows: After 48 h, the old culture medium was discarded, the cells were rinsed once with 0.1 M PBS, 94 μL of Annexin V-FITC binding solution was added to each well, and then 2 μL of Mito-Tracker Red CMXRos staining solution and 2.5 μL of Hoechst 33342 staining solution were added. After gentle mixing, the mixture was incubated at room temperature for 20 min in the dark, and then observed under a fluorescence microscope.

[0034] As shown in Figure 4 (1) Figure 4 A shows that after MES23.5 cells were treated with different concentrations of PhIP for 48 h, the cell viability was detected by CCK8 experiment, and the results showed that with the increase of PhIP concentration, the cell viability of MES23.5 cells showed a dose-dependent decrease, and the cell viability of the 150 μΜ PhIP treatment group decreased to 51.75%.

[0035] (2) The results of the mitochondrial membrane potential detection experiment are shown in Figure 4 B, Figure 4 C shows that compared with the Control group (100.00%), the relative fluorescence intensity of Mito-Tracker Red CMXRos staining of the PhIP treatment group was significantly weakened (43.00%), indicating that the mitochondrial membrane potential of the cells decreased, suggesting that PhIP caused obvious mitochondrial damage to the cells. These results prove that PhIP can induce damage and mitochondrial dysfunction of dopaminergic neuron MES23.5 cells.

[0036] Although the embodiments of the present application have been shown and described above, it should be understood that the above-described embodiments are exemplary and should not be construed as limiting the present application, and those skilled in the art can make changes, modifications, replacements and variations to the above-described embodiments within the scope of the present application.​

Claims

1. A method for constructing an animal model of Parkinson's disease, characterized in that, The construction method includes administering heterocyclic aromatic amine compounds to animals to construct an animal model of Parkinson's disease.

2. The method for constructing an animal model of Parkinson's disease according to claim 1, characterized in that, The heterocyclic aromatic amine compound is 2-amino-1-methyl-6-phenylimidazolium[4,5-b]pyridine.

3. The method for constructing an animal model of Parkinson's disease according to claim 2, characterized in that, The administration involved treating animals with 2-amino-1-methyl-6-phenylimidazole[4,5-b]pyridine for 10 days.

4. The method for constructing an animal model of Parkinson's disease according to claim 3, characterized in that, The single dose is 20 mg / kg to 40 mg / kg.

5. The method for constructing an animal model of Parkinson's disease according to claim 4, characterized in that, The dosing frequency is 20 mg / kg / day to 40 mg / kg / day.

6. The method for constructing an animal model of Parkinson's disease according to claim 3, characterized in that, The animal in question was a C57BL / 6N mouse.

7. A method for constructing an animal model of Parkinson's disease according to any one of claims 1 to 6, characterized in that, The medication was administered via gavage.

8. The method for constructing an animal model of Parkinson's disease according to claim 1, wherein the method further includes validating the constructed animal model of Parkinson's disease.

9. An animal model of Parkinson's disease, characterized in that, The Parkinson's animal model is constructed using the construction method described in any one of claims 1 to 8.

10. An application of an animal model of Parkinson's disease, characterized in that, The Parkinson's animal model of claim 9 is used in the preparation of drugs for screening and / or treating Parkinson's disease.

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