A method for establishing a parkinson's disease animal model

Abstract

The application discloses a Parkinson's disease model animal, a method for establishing the Parkinson's disease model animal, and application of the Parkinson's disease model animal. In the Parkinson's disease model animal, a mouse Citrobacter rodentium (C.R) and / or a dopaminergic neurotoxin 1-methyl-4-phenyl-1,2,3.6-tetrahydropyridine (MPTP) is given to the animal. The phenotype of the Parkinson's disease model animal is stable, and the Parkinson's disease model animal can be used for researching the pathology of Parkinson's disease and screening and developing a therapeutic drug for Parkinson's disease caused by intestinal infection.

Description

Technical Field

[0001] This invention relates to the fields of medicine and pharmaceutical technology. Specifically, this invention relates to a method for creating an animal model of Parkinson's disease, and the uses of this animal model. More specifically, this invention relates to utilizing the pathological functions of Citrobacterium murineis in vivo, and further discovers that it can be used to prepare an animal model of Parkinson's disease, and can be used for the screening and development of drugs for Parkinson's disease caused by intestinal infection. Background Technology

[0002] Parkinson's disease (PD) is a neurodegenerative disease caused by the degeneration and loss of dopaminergic neurons in the substantia nigra compacta (SNpc) of the midbrain. Pathological features include the presence of Lewy bodies containing α-synuclein and neuroinflammation. Approximately 1.7% of people over 65 years of age are affected. There are currently nearly 4 million PD patients in my country, and this number is expected to rise further with the aging population, placing a significant economic and psychological burden on society and families. The pathogenesis of the disease remains unclear, and clinical diagnosis and intervention methods are very limited. Furthermore, there is a lack of experimental animal models that accurately mimic the pathological characteristics and clinical manifestations of PD. Intestinal infection is associated with the pathogenesis of PD, but evidence in patients and animal models is still insufficient, and the mechanism is unclear. Establishing a new animal model of Parkinson's disease caused by intestinal infection is necessary, but there are currently no reported examples in the literature.

[0003] Citrobacter rodentium (CR) is a rodent-specific pathogenic bacterium that primarily accumulates in the colon, causing transient colitis (Luperchio SA et al (2000) Citrobacterrodentium, the causative agent of transmissible murine colonic hyperplasia, exhibits clonality: synonymy of C. rodentium and mouse-pathogenic Escherichiacoli. J Clin Microbiol 38:4343-4350; Mundy R et al (2005) Citrobacterrodentium of mice and man. Cell Microbiol 7:1697-1706.). MPTP is a neurotoxin of mitochondrial complex I that can cause motor deficits, dopaminergic neuron death, upregulation of α-synuclein expression, and glial activation in the substantia nigra-striatum, mimicking PD-like features in animal models (Jackson-Lewis, V.; Przedborski, S., Protocol for the MPTP mouse model of Parkinson's disease. Nat Protoc 2007, 2, (1), 141-51.).

[0004] This invention is based on CR and MPTP, aiming to establish a phenotypic stable animal model of Parkinson's disease. Summary of the Invention

[0005] The purpose of this invention is to provide a method for establishing an animal model of Parkinson's disease.

[0006] Another object of the present invention is to provide the use of the mouse model of Parkinson's disease.

[0007] In a first aspect of the invention, a method for producing a mammal with Parkinson's disease is provided, comprising the steps of:

[0008] (a) An inflammation model caused by intestinal flora infection was established using Citrobacter rodentium (CR): Of course, it is not limited to specific strains of Citrobacter rodentium, but only as an example, DBS100 and DBS770 were used. It was found that CR DBS770 infection affected the changes in monoamine neurotransmitters, and the dopamine neurotransmitter metabolic pathway was particularly significant.

[0009] (b) The Citrobacterium tumefaciens (DBS770) obtained in step (a) was administered to mammals, such as mice, at a dose of 3-5 x 10⁸ CFU / mouse, more preferably 4 x 10⁸ CFU / mouse, which caused intestinal flora dysbiosis and inflammation in mice.

[0010] (c) Identification of the mammal obtained in step (b) showed that Citrobacter infection affects the metabolism of dopamine neurotransmitters in the brain, which is a first discovery based on existing technology;

[0011] (d) For the mammals obtained in step (c), MPTP can be further administered. Intestinal flora dysbiosis can promote the pathogenesis of PD by aggravating neuroinflammation, thus completing the establishment of an animal model of Parkinson's disease.

[0012] In a second aspect of the invention, there is provided the use of mammals obtained by the method described above, which are used as models of Parkinson's disease or for screening drugs for the prevention and / or treatment of Parkinson's disease caused by intestinal infections.

[0013] In another preferred embodiment, the mammal is preferably a mouse or a rat.

[0014] Through in-depth and extensive research, the inventors have established a phenotypically stable animal model of Parkinson's disease, which is induced by a combination of intestinal infection and MPTP administration to mice or other mammals. This animal model of Parkinson's disease can be used for Parkinson's disease research and for screening drugs to prevent and / or treat PD caused by intestinal infection. This invention was completed based on this foundation.

[0015] Specifically, the inventors established intestinal flora infection-induced inflammation models using two types of Citrobacter rodentium (CR): DBS100 and DBS770. They found that CR DBS770 infection affected changes in monoamine neurotransmitters, particularly the dopamine neurotransmitter metabolic pathway. CR (4 x 10⁸ CFU / mouse) caused intestinal flora dysbiosis and inflammation in mice, representing a preferred dosage. Combined with the dopaminergic neurotoxin MPTP (40 mg / kg), the combination method and order of administration are not limited. If a preferred method is required, CR is the initial administration, followed by MPTP administration 5-7 days later, preferably 6 days. The number of injections is not particularly limited, but based on the above dosage, one injection is preferred. This method can establish a new animal model of Parkinson's disease.

[0016] The method of administration of MPTP is not particularly limited, as long as a certain blood drug concentration can be achieved. If a limitation is required, it can be intramuscular injection, subcutaneous injection, intraperitoneal injection, etc., with intraperitoneal injection being preferred.

[0017] Beneficial effects

[0018] DBS770 and MPTP have a significant synergistic effect, mainly manifested in the overactivation of microglia and astrocytes in the substantia nigra-striatal pathway affected by PD in the brain, the aggravation of the loss of dopaminergic neurons in the substantia nigra pars compacta and dopaminergic nerve endings in the striatum, the more obvious decrease in the level of dopamine neurotransmitters in the striatum, and the obvious motor coordination disorder in mice.

[0019] CR DBS770 infection can promote the metabolic transformation of dopamine neurotransmitters in the brain and synergistically exacerbate the pathogenesis of Parkinson's disease (PD) with MPTP. CR DBS770 infection or the combination of CR DBS770 and MPTP can establish a novel animal model of PD, which can be used to study the clinical phenomenon of the correlation between intestinal inflammation and PD, and to discover and optimize clinical medications for PD. Based on this, this invention has broad translational and application prospects, and can provide new models and ideas for PD research. Attached Figure Description

[0020] Figure 1 An infection model was successfully constructed using CR DBS770.

[0021] Figure 2 CR DBS770 infection causes intestinal flora imbalance.

[0022] Figure 3 CR DBS770 infection causes the accumulation of α-synuclein in the intestine.

[0023] Figure 4 CR DBS770 infection affects the metabolism of dopamine neurotransmitters in the brain.

[0024] Figure 5 CR infection exacerbates MPTP-induced dopaminergic neuronal death.

[0025] Figure 6 CR infection exacerbates the loss of dopaminergic nerve endings induced by MPTP.

[0026] Figure 7 Detection of neuroinflammatory factors.

[0027] Figure 8 Detection of motor dysfunction in mice. Detailed Implementation

[0028] The detection method used in this embodiment is a known technique in the prior art. Conventional methods are omitted here. If needed, please refer to the following literature:

[0029] RNA extraction, referencing the RNA extraction kit "TRNzol Universal Reagent"

[0030] "TRNzol Universal Total RNA Extraction Reagent", ELISA method, performed according to the instructions of "Mouse IL-1β ELISA MAX™ Deluxe"; "FastKing gDNA Dispelling RT SuperMix" can be used for genomic removal; "FastFire qPCR PreMix (SYBR Green)"

[0031] The "FastFire Premixed Reagent for Rapid Quantitative PCR" can be used for quantitative PCR steps.

[0032] Example 1: CR DBS770 infection induces intestinal inflammation.

[0033] Citrobacter (CR DBS770) was administered to mice (4 x 10⁸ CFU / mouse) for intestinal colonization, and the establishment of an animal model of intestinal inflammation was verified from multiple perspectives.

[0034] 1. Detection of inflammatory factors

[0035] An intestinal inflammation model was induced, and the degree of model establishment was reflected by detecting colonic inflammatory factors at mRNA levels. IL-1β, IL-6, IL-12, INOS, and TNF-α are common inflammatory factors in intestinal inflammation. Analysis of mRNA levels showed that, compared with the control group, all of the above factors, except TNF-α, showed statistically significant increases. IL-6 and INOS showed the most significant increases, followed by IL-1β and IL-12. TNF-α showed a relatively lower increase, but still a slight increase. (See attached results). Figure 1 A.

[0036] 2. Calprotectin Detection

[0037] Calprotectin is a calcium-containing protein derived from neutrophils and macrophages. Its expression is tissue- or cell-specific and can serve as a marker of acute inflammatory cell activation. By detecting calprotectin in feces using ELISA and comparing it with a control group, the experimental group showed a significant increase in calprotectin levels. (See results below.) Figure 1 B

[0038] 3. Intestinal flora detection

[0039] Abundance analysis of the gut microbiota at the phylum level revealed the following phyla: Dependentiae, Teneriicutes, Cyanobacteria, Deferribacteres, Patescibacteria, Actinobacteria, Epsilonbacteraeota, Verrucomicrobia, Proteobacteria, Bacteroidetes, and Firmicutes. The results (see [link to relevant documentation]) indicate that... Figure 2 (A) Analysis at the phylum level revealed significant changes in the proportion of different phyla in the gut microbiota after colonization by CR DBS770. Firmicutes, Bacteroidetes, Proteobacteria, and Verrucous microbes also showed significant changes compared to the control group. Therefore, the changes in gut microbiota homeostasis indicate that gut inflammation was successfully induced.

[0040] Example 2: Parkinson's Pathological Features Caused by CR DBS770 Infection

[0041] Citrobacter (CR DBS770) was administered to mice (4 x 10⁸ CFU / mouse) for intestinal colonization, and the establishment of a Parkinson's disease animal model was verified from multiple perspectives.

[0042] 1. Abnormal aggregation of α-synuclein in the gut

[0043] Citrobacter (CR DBS770) was administered to mice (4 x 10⁸ CFU / mouse) for intestinal colonization.

[0044] α-synuclein is a soluble protein expressed presynaptally and perinuclearly in the central nervous system. It is closely related to the pathogenesis and related functional impairments of Parkinson's disease, is a major component of Lewy bodies, and can be used to evaluate neurodegenerative disease models.

[0045] The model's construction level can be assessed by observing changes in the aggregation location of α-synuclein before and after CR DBS770 infection using an imaging system through immunofluorescence staining.

[0046] The experimental results show that α-synuclein exhibits abnormal aggregation optical properties after CR DBS770 infection, and statistical calculations of fluorescence intensity reveal a significant change in the fluorescence intensity of α-synuclein. (See results below) Figure 3 )

[0047] 2. Metabolic transformation of dopamine neurotransmitters in the brain

[0048] Dopamine (DA), homovanillic acid (HVA), and dihydroxyphenylacetic acid (DOPAC) are pathological changes in Parkinson's disease (PD). The most significant pathological change in PD is the degeneration and death of dopamine (DA) neurons in the substantia nigra of the midbrain. Changes in the levels of DOPAC, DA, and HVA in the striatum can reflect the pathological progression of PD. Detection of these three indicators showed a significant decrease in DA and a significant increase in DOPAC and HVA, indicating significant changes in the metabolic transformation of dopamine neurotransmitters in the brain. (Results included in...) Figure 4 )

[0049] Example 3: CR DBS770 synergistic MPTP enhancement can exacerbate the progression of Parkinson's disease.

[0050] To demonstrate the synergistic effect of CR and MPTP, an animal model was established by setting up groups and a drug control group. TH-positive cells in the substantia nigra were labeled using immunohistochemistry. Cell morphology was observed from the staining results, and cell number labeling results were compared. The results showed that CR DBS770 infection significantly exacerbated the loss of dopaminergic neurons in the substantia nigra induced by MPTP alone. (See...) Figure 5 Immunoblotting analysis of TH characteristic proteins showed that, compared with the control β-actin which maintained a consistent level, CR significantly enhanced the reduction of TH characteristic proteins when MPTP was used alone, and the difference was statistically significant (see [link to relevant documentation]). Figure 6 The transcriptional level was reflected by detecting the mRNA levels of inflammatory factors IL-6, IL-1β, iNOS, CD86, CD68, and COX2. CR DBS770 infection synergistically with MPTP significantly induced or exacerbated neuroinflammation, with statistically significant differences (see [link to relevant documentation]). Figure 7 The pole climbing behavior test, tumbling bar behavior test, and hanging behavior test can reflect the level of neurodegenerative diseases. Through testing, CR DBS770 infection combined with MPTP significantly induced motor dysfunction in mice in all three aspects (see...). Figure 8 ).

[0051] The results showed that DBS770 and MPTP had a significant synergistic effect, mainly manifested in the overactivation of microglia and astrocytes in the substantia nigra-striatal pathway affected by PD in the brain, the aggravation of the loss of dopaminergic neurons in the substantia nigra pars compacta and dopaminergic nerve endings in the striatum, the more obvious decrease in the level of dopamine neurotransmitters in the striatum, and the obvious motor coordination disorder in mice.

[0052] Group settings

[0053]

Claims

1. A method of establishing a mouse model of Parkinson's disease, characterized by, including colonization of mice with C. rodentium (C. RDBS770) at a dose of 3-5 x 10 8 CFU / mouse, 5-7 days post colonization, injection of 30-50 mg / kg 1-methyl-4-phenyl-1,2,3.6-tetrahydropyridine (MPTP). ​ 2. The method for establishing a mouse model of Parkinson's disease according to claim 1, further wherein the colonization is intestinal colonization.

3. The method for establishing a mouse model of Parkinson's disease according to claim 2, wherein the colonization method further comprises administering a suspension of Citrobacter murineis via gavage.

4. The method for establishing a mouse model of Parkinson's disease according to claim 3, wherein the mouse is colonized with C. ruddii (C. RDBS770) at a dose of 4 x 10 8 CFU / mouse.

5. The method for establishing a mouse model of Parkinson's disease according to claim 1, wherein the MPTP dosage is 40 mg / kg.

6. The method for establishing a mouse model of Parkinson's disease according to claim 5, wherein the MPTP is administered via intraperitoneal injection.

7. The method for establishing a mouse model of Parkinson's disease according to claim 5, further wherein the MPTP is performed 6 days after gavage administration of a suspension of Citrobacter murineis.

8. The method for establishing a mouse model of Parkinson's disease according to claim 3, wherein the mouse citrobacter suspension is prepared with physiological saline.

9. The method for establishing a mouse model of Parkinson's disease as described in claim 1, characterized in that, It also includes the step of identifying the obtained mammals.

10. Use of a mammal obtained by the method of any one of claims 1-9, characterized in that, Used as a model of Parkinson's disease; or used to screen drugs for the prevention and / or treatment of Parkinson's disease caused by intestinal infections.

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