CN115777625A - Method for establishing animal model of Parkinson's disease - Google Patents

Abstract

The invention discloses a Parkinson disease model animal, a method for establishing the Parkinson disease model animal and application of the Parkinson disease model animal. In the animal model of Parkinson's disease of the invention, animals are administered Citrobacter (C.R) and/or dopaminergic neurotoxin 1-methyl-4-phenyl-1,2,3.6-tetrahydropyridine (MPTP) to mice. The Parkinson disease animal model has stable phenotype, can be used for researching the pathology of the Parkinson disease and screening and developing treatment medicines for the Parkinson disease caused by intestinal infection.

Description

Method for establishing animal model of Parkinson's disease

Technical Field

The invention relates to the technical field of medicine and pharmacy. In particular, the invention relates to a method of a Parkinson's disease animal model and application of the Parkinson's disease animal model. More particularly, the invention relates to the utilization of the pathological function of the Citrobacter murinum in vivo, and further finds that the Citrobacter murinum can be used for preparing animal models of Parkinson's disease and can be used for screening and developing medicaments for Parkinson's disease caused by intestinal infection.

Background

Parkinson's Disease (PD) is a neurodegenerative disease caused by degeneration and loss of dopaminergic neurons in the substantia nigra compacta (SNpc) of the middle brain, and the pathological features comprise existence of lewy bodies containing alpha-synuclein and neuritis, about 1.7 percent of patients in people over 65 years old, nearly 400 million PD patients in China can be further increased along with the aggravation of the aging of the population in China, and huge economic and mental pressure is brought to the society and families. The pathogenesis of the disease is not clear, the clinical diagnosis and treatment and intervention means are very limited, and in addition, an experimental animal model which accurately simulates the pathological characteristics and clinical manifestations of PD is lacked. Intestinal infection is related to the pathogenesis of PD, but the evidence in patients and animal models is still insufficient, the mechanism is unclear, and the establishment of a new animal model of Parkinson's disease caused by intestinal infection is necessary and has no literature report.

Citrobacter murinum (C.R) is a specific rodent pathogenic bacterium that mainly accumulates in the colon and causes transient colitis (Luerchio S A et al (2000) Citrobacter rodentium, the functional agent of transmissible microbial community, inhibition: synthesis of C.rodentium and methyl-pathogenic Escherichia coli. J. Clin Microbiol 38. MPTP is a mitochondrial complex I neurotoxin that can cause motor deficits, dopaminergic neuronal death, upregulation of alpha-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 Protocol 2007,2, (1), 141-51.).

The invention aims to establish the Parkinson disease animal model with stable phenotype based on C.R and MPTP.

Disclosure of Invention

The invention aims to provide a method for establishing an animal model of Parkinson's disease.

Another purpose of the invention is to provide application of the mouse model for Parkinson's disease.

In a first aspect of the invention, there is provided a method of producing a mammal having parkinson's disease, comprising the steps of:

(a) The use of Citrobacter rodentium (C.R) in mice to establish a model of inflammation caused by intestinal flora infection: certainly not limited to a specific strain of citrobacter murinus, but by way of example only, DBS100 and DBS770, found: the C.R DBS770 infection affects the change of monoamine transmitters, and the metabolic pathway of the dopamine transmitters is particularly obvious;

(b) Administering the selected Citrobacter from step (a) (DBS 770) to a mammal, such as a mouse, at a dose of 3-5x108 CFU/mouse, more preferably 4x108 CFU/mouse, which causes disturbances in the intestinal flora and inflammation in the mouse;

(c) Identifying the mammal obtained in step (b) that Citrobacter infection affects dopamine transmitter metabolism in the brain for the first time based on the prior art;

(d) MPTP can be further given to the mammal obtained in the step (c), and the disturbance of intestinal flora can promote the pathogenesis process of PD by intensifying neuroinflammation, thereby completing the establishment of the animal model of Parkinson disease.

In a second aspect of the invention, there is provided the use of the mammals obtained by the above-described method of the invention as models of parkinson's disease or for screening drugs for the prevention and/or treatment of parkinson's disease caused by intestinal infections.

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

The present inventors have conducted extensive and intensive studies to establish a phenotypically stable animal model of Parkinson's disease, which is a mouse or other mammal resulting from intestinal infection in combination with MPTP administration. The model animal can be used for studying Parkinson's disease and can be used for screening medicines for preventing and/or treating PD caused by intestinal infection. The present invention has been completed on the basis of this finding.

Specifically, the inventors utilized two murine Citrobacter rodentium (c.r): the DBS100 and the DBS770 respectively establish an inflammation model caused by intestinal flora infection, and the C.R DBS770 infection influences the change of monoamine transmitters, so that the dopamine transmitter metabolic pathway is particularly obvious; r (4x108 CFU/mouse) can cause disturbance and inflammation of intestinal flora of mice, and belongs to a more preferable dosage; in combination with the dopaminergic neurotoxin MPTP (40 mg/kg), the combination mode is not limited, the administration sequence is not limited, if a preferred mode is needed, C.R is initial administration, MPTP is used for administration after 5-7 days, and 6 days are preferred; the number of injections to be administered is not particularly limited, but one injection may be preferable based on the above dose, and a novel animal model of Parkinson's disease can be established in the above manner.

The administration mode of MPTP is not particularly limited, and it is sufficient that a certain blood concentration can be achieved, and if necessary, it may be intramuscular injection, subcutaneous injection, intraperitoneal injection, etc., preferably intraperitoneal injection.

Advantageous effects

The DBS770 and MPTP have obvious synergistic effect when used together, and are mainly reflected in the excessive activation of microglia and astrocytes in substantia nigra-striatum of a PD affected pathway in brain, the loss of dopaminergic neurons in substantia nigra pars compacta and dopaminergic nerve endings in striatum is aggravated, the reduction of dopamine transmitter level in striatum is more obvious, and the obvious motor coordination obstacle appears in mice.

R DBS770 infection can promote metabolic conversion of dopamine transmitter in the brain and exacerbate the progression of PD pathogenesis in cooperation with MPTP. The C.R DBS770 infection or the combination of the C.R DBS770 and MPTP can establish a new PD experimental animal model, can be used for researching the clinical phenomenon of the correlation between the intestinal inflammation and the PD, and finds and optimizes the clinical medication aiming at the PD. Based on the method, the method has wide conversion and application prospects, and can provide a new model and thought for the research of PD.

Drawings

Fig. 1 successfully constructs an infection model by using c.r DBS770, and the infection of c.r DBS770 induces the detection of intestinal inflammatory factors: (A) analysis of inflammatory factor mRNA levels in colon tissue; n =3-5; (B) ELISA detects the content of Calprotectin (Calprotectin) which is an inflammation marker protein in the excrement; n =7; statistical methods used unpaired T test, p <0.05, p <0.01, p <0.001, p <0.0001.

Figure 2c.r dbs770 infection caused steady state detection of intestinal dysbacteriosis: (a) phylum-level intestinal flora abundance; (B-E) abundance levels of firmicutes, bacteroidetes, proteobacteria and verrucomicrobia, respectively; n =8-10; statistical methods used unpaired T test, p <0.05, p <0.01, p <0.0001.

Figure 3c.r dbs770 infection caused aggregation of α -synuclein in the gut: (A) Representative alpha-synuclein (alpha-Syn) immunofluorescence maps in colon tissue; scale bar: 50 μm; (B) A statistical plot of the fluorescence intensity of alpha-Syn expressed in the mucosal layer of the intestinal tract; n =5, statistical methods used unpaired T test, p <0.05.

Fig. 4c.r dbs770 infection affects metabolite detection of dopamine transmitter in brain: (A-C) percentage changes of DA, DOPAC and HVA, respectively, relative to wild type mice; (D-E) metabolic conversion rates for DOPAC and HVA, respectively; n =5-6, statistical method used unpaired T test, p <0.05.

Figure 5c.r infection exacerbation MPTP-induced dopaminergic neuron death loss assay: (A) Marking TH positive cells in substantia nigra by adopting an immune tissue chemistry method, wherein the proportion is as follows: 50 μm. (B) Stereoscopic counting of TH-positive cells in substantia nigra

Figure 6c.r infection aggravated MPTP-induced dopaminergic nerve terminal loss: dopaminergic nerve terminal loss assay, (a) TH representative immunoblots; (B) TH immunoblot statistical plot, n =5-7, statistical analysis using One-way ANOVA, two-by-two comparisons using LSD test, × p <0.0001.

Figure 7 neuroinflammatory factor assay: (A-F) analysis of the transcription levels of IL-6, IL-1 β, iNOS, CD86, CD68 and COX2, (G) ELISA detection of the IL-6 content in serum; n =4-5, statistical analysis was performed using One-way ANOVA, and two comparisons were performed using LSD test, p <0.05, p <0.01, p <0.001.

Figure 8 mouse motor dysfunction detection map: (A) climbing behavior test; (B) testing the rotating rod behaviours; (C) suspension behavioural testing; n =7-12, statistical analysis using One-way ANOVA, two-by-two comparisons using LSD test, p <0.05, p <0.0001.

Detailed Description

The detection method employed in the present embodiment is a technical means known in the prior art, and is omitted here for conventional means, and the following documents may be referred to if necessary:

RNA extraction, reference RNA extraction kit "TRNzol Universal Reagent

TRNzol Universal total RNA extraction reagent, ELISA method, adopting Mouse IL-1 beta ELISA MAX ^TM Deluxe is carried out by the method described in the specification; "FastKing gDNA dispering RT SuperMix" can be used to remove the genome; "FastFire qPCR PreMix (SYBR Green)

The FastFire rapid fluorogenic quantitative PCR premixed reagent "can perform the fluorogenic quantitative PCR step.

Example a c.r DBS770 infection induces intestinal inflammation,

the citrobacter (c.r DBS 770) was given to mice (4 × 108 CFU/mouse) for intestinal colonization and the establishment of animal models of intestinal inflammation was verified from multiple angles.

1 detection of inflammatory factors

Inducing an intestinal inflammation model, and detecting the constructed degree of the colon inflammation factor embodiment model through mRNA level detection. IL-1 beta, IL-6, IL-12, INOS and TNF-alpha are common inflammatory factors of intestinal inflammation, and the results show that the factors except TNF-alpha can generate statistically different significant improvement by comparing with a control group, wherein the improvement of IL-6 and INOS is most significant, and the relative improvement degree of TNF-alpha is lower but still has small improvement on IL-1 beta and IL-12. The results are shown in FIG. 1A.

2 calprotectin assay

Calprotectin (Calprotectin) is a calcium-containing protein derived from neutrophils and macrophages, the expression of which has tissue or cell specificity and can be used as a marker for acute inflammatory cell activation. The experimental group was able to produce significant elevations of calprotectin by detection of calprotectin in feces, by ELISA and comparison with the control group. The results are shown in FIG. 1B

3 detection of intestinal flora

Through the abundance detection of the phylum level of the intestinal flora, the phylum which is detected together comprises Dependentiae, tenericucules, cyanobacteria, deuterobacteria, patescibacteria, actinob bacteria, epsilonbacterium aeota, verrucomicrobia, proteobacteria, bacteroides and Firmic bacteria, and the results show that the phylum level abundance is analyzed, and the proportion of the phylum of the intestinal flora after the C.R DBS770 is planted is obviously changed. The phylum firmicutes, bacteroidetes, proteobacteria and verrucomicrobia also produced significant changes relative to the control group, respectively. Therefore, intestinal inflammation can be successfully induced from steady state changes of intestinal strains.

Example di c.r DBS770 infection causes parkinsonian physical features

The mice (4 x108 CFU/mouse) were given the citrobacter (c.r DBS 770) for intestinal colonization, and the establishment of parkinson animal model was verified from multiple angles.

Abnormal aggregation of 1 alpha-synuclein in the gut

Mice (4 x108 CFU/mouse) were given enteric colonization by citrobacter (c.r DBS 770).

Alpha-synuclein is a soluble protein expressed in the central nervous system presynaptic and perinuclear, is closely related to the pathogenesis and related dysfunction of Parkinson's disease, is a main component of Lewy bodies, and can be used for evaluating neurodegenerative disease models.

By immunofluorescent staining of alpha-synuclein, changes in aggregation positions of the alpha-synuclein before and after C.R DBS770 infection were observed in an imaging system, and the level of model construction was assessed.

The experimental result shows that the optical property of abnormal aggregation of the alpha-synuclein after C.R DBS770 infection appears, and the fluorescence intensity is statistically calculated, which shows that the fluorescence intensity of the alpha-synuclein also has significant change. (results are shown in FIG. 3)

2 metabolic conversion of dopamine transmitter in brain

Dopamine (DA), homovanillic acid (HVA) and dihydroxyphenylacetic acid (DOPAC) are pathological changes of Parkinson's disease, the main pathological changes of Parkinson's disease are degeneration and death of midbrain substantia nigra Dopamine (DA) nervous, and the level changes of DOPAC, DA and HVA in striatum. Through detection of the three indexes, the DA is obviously reduced, the DOPAC and the HVA are obviously increased, and the metabolic conversion change of the dopamine transmitter in the brain is obvious. (see FIG. 4 for results)

Example three c.r DBS770 in conjunction with MPTP enhancement can exacerbate parkinson disease progression

In order to prove the synergistic effect of the C.R and the MPTP, the group setting is carried out, the administration control is carried out to establish an animal model, TH positive cells in the substantia nigra are marked by an immunohistochemical method, the cell morphology is observed from a dyeing result, and the cell number marking result is compared, so that the C.R DBS770 infection can remarkably aggravate the loss of dopaminergic neurons in the substantia nigra induced by singly using the MPTP. (see fig. 5) the immunoblotting of TH-characteristic protein shows that, compared with the level of control beta-actin maintained to be consistent, c.r can obviously enhance the reduction of TH-characteristic protein when MPTP is used alone, and has statistically significant difference (see fig. 6) by detecting the mRNA levels of inflammatory factors) IL-6, IL-1 beta, iNOS, CD86, CD68 and COX2 to represent the transcription level, and c.r DBS770 infection can obviously induce or aggravate neuroinflammation in cooperation with MPTP and has statistically significant difference (see fig. 7). The level of neurodegenerative diseases can be reflected by three aspects of climbing behavior test, rod turning behavior test and hanging behavior test, and the C.R DBS770 infection can remarkably induce the motor dysfunction of mice in three aspects by detection and cooperation with MPTP (see figure 8).

Research results show that the DBS770 and MPTP have a remarkable synergistic effect when used together, and are mainly reflected in excessive activation of microglia and astrocytes in substantia nigra-striatum of a PD affected pathway in brain, aggravation of loss of dopaminergic neurons in substantia nigra pars compacta and dopaminergic nerve endings in striatum, more obvious reduction of dopamine transmitter level in striatum and obvious motor coordination disorder of mice.

Group arrangement

Claims (13)

1. A method for establishing an animal model of Parkinson's disease is characterized by comprising the step of carrying out mammal colonization by adopting mouse Citrobacter.

2. The method of claim 1, further wherein the colonization is intestinal colonization.

3. The method of claim 2, further comprising intragastric administration of a suspension of Citrobacter murine.

4. The method of claim 3, wherein the dose administered is 3-5x10 ⁸ CFU/only, preferably 4x10 ⁸ CFU/only.

5. The method of claim 1, further comprising injecting with 1-methyl-4-phenyl-1,2,3.6-tetrahydropyridine (MPTP).

6. The method according to claim 5, wherein the MPTP is administered at a dose of 30-50mg/kg, preferably 40mg/kg.

7. The method of claim 5, wherein the MPTP is administered by intraperitoneal injection.

8. The method according to claim 6, further wherein the MPTP is performed 5-7 days, preferably 6 days after the gavage of the Citrobacter murine suspension.

9. The Citrobacter murine suspension of claim 3, formulated in normal saline.

10. The method of claim 1, wherein the mammal is a mouse or rat.

11. The method of claim 1 or 5, further comprising the step of identifying the obtained mammal.

12. Use of a mammal obtained with the method according to claims 1-11 as a model of parkinson's disease; or for screening drugs for preventing and/or treating Parkinson's disease caused by intestinal infection.

13. Use of Citrobacter murine, characterized in that they are (a) used for the preparation of animal models of Parkinson's disease; and/or (b) is used for preparing or screening medicaments for treating the Parkinson's disease caused by intestinal infection.

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