Construction method and application of Parkinson's disease with qi deficiency and blood stasis syndrome combined with animal model

Abstract

The application belongs to the technical field of animal model construction, and particularly relates to a construction method of a Parkinson's disease (PD) animal model with qi deficiency and blood stasis syndrome and application thereof. The construction method comprises: daily exhaustive training of animals under negative gravity for a first predetermined time, and then daily administration of MPTP after the exhaustive training of the animals under negative gravity for a second predetermined time. The application is based on the theory of traditional Chinese medicine, and an animal model is constructed through a time sequence compound modeling strategy, which can simulate the core pathology of PD (dopaminergic neuron injury) and reproduce the dynamic evolution process of the traditional Chinese medicine syndrome of qi deficiency and blood stasis (energy metabolism disorder and microcirculation disorder).

Description

Technical Field

[0001] This application belongs to the field of animal model construction technology, specifically involving the construction method and application of an animal model combining the symptoms and disease patterns of Parkinson's disease with Qi deficiency and blood stasis syndrome. Background Technology

[0002] Parkinson's disease (PD) is a common neurodegenerative disease affecting middle-aged and elderly people. Its core pathological features are the progressive loss of dopaminergic neurons in the substantia nigra and the formation of Lewy bodies. Animal models are indispensable tools for studying its pathogenesis and screening drugs. Currently, the mainstream technical approaches for constructing PD animal models include neurotoxin-induced models (such as animal models constructed by injecting drugs such as MPTP, 6-OHDA, and rotenone into animals), genetic engineering models (such as introducing human A53T mutant α-Syn gene or other genes into the animal genome), and disease-symptom combined models (such as adding factors of "syndrome" based on the disease model through methods of etiology in traditional Chinese medicine, such as starvation and fatigue).

[0003] However, while the animal models constructed using these existing methods provide important tools for PD research, they still have the following limitations: (1) Ignoring TCM “syndrome”: The classic PD model only focuses on “disease” (i.e., damage to dopaminergic neurons) and ignores the existence of TCM “syndrome”. It is difficult to reflect the complex pathological state of “deficiency of the root and excess of the branch” that is common in PD patients (especially middle-aged and elderly patients), resulting in a large deviation between the drug screening results based on such models and the clinical efficacy. (2) The disease-symptom combination model lacks theoretical guidance and dynamic simulation: The existing disease-symptom combination model is mostly a mechanical and static superposition of two or more modeling factors, lacking the guidance of systematic TCM theory. In clinical practice, PD is a dynamic process that starts from the decline of organ function, gradually produces pathological products and forms a vicious cycle. However, the existing model cannot simulate the dynamic and progressive pathological evolution process of clinical PD patients. (3) The evaluation system is too simple: the evaluation of existing models is mostly focused on behavioral and histopathological examinations. There is a lack of an objective and multidimensional quantitative indicator system for evaluating “disease” and “syndrome”, which makes it difficult to scientifically verify the “syndrome attribute” of the model. Moreover, existing technologies do not pay enough attention to objective indicators that can reflect the essence of “syndrome” in traditional Chinese medicine.

[0004] Therefore, a new method for constructing an animal model of Parkinson's disease is needed, which follows the pathogenesis evolution law of "deficiency as the root, stagnation as the manifestation, and mutual binding of deficiency and stasis" in the theory of "deficiency and stagnation of qi". It adopts a temporal composite modeling strategy of "deficiency first and then toxicity" and is verified by combining a multidimensional evaluation system. Summary of the Invention

[0005] The problem this application aims to solve is to provide a method for constructing and applying an animal model that combines the symptoms of Qi deficiency and blood stasis in Parkinson's disease. Based on traditional Chinese medicine theory, an animal model can be constructed through a temporal composite modeling strategy that can both simulate the core pathology of PD (damage to dopaminergic neurons) and reproduce the dynamic evolution of the TCM syndrome of "Qi deficiency and blood stasis" (energy metabolism disorder and microcirculation disorder).

[0006] To address the above problems, this application provides the following solution: On the one hand, this application provides a method for constructing an animal model of Parkinson's disease with qi deficiency and blood stasis syndrome, the method comprising: subjecting the animal to weight-bearing exhaustion training daily for a first predetermined time period, and then administering MPTP to the animal after subjecting the weight-bearing exhaustion training daily for a second predetermined time period.

[0007] In the above technical solution, the animal undergoes exhaustion training after being subjected to heavy loads, which can simulate the pathogenesis of "exhaustion of qi" in traditional Chinese medicine and establish the pathological basis of qi deficiency. During the second predetermined time, the animal is injected with the neurotoxin MPTP at the same time as the exhaustion training, which can simulate the invasion of "toxic evil" into the brain network, directly trigger the core pathological changes of dopaminergic neuron damage, and interact with the existing "qi deficiency" state, inducing and aggravating the qi and blood circulation disorder, and finally forming a "stagnation" pathological state with "blood stasis" as the main manifestation.

[0008] Furthermore, the weight-bearing exhaustion training includes applying a weight to the animal's tail and then swimming in water at 22-28°C until exhaustion.

[0009] For example, the preferred water temperature for swimming is 25±1℃, and the water depth is ≥30 cm.

[0010] For example, the load is 3% to 8% of the animal's body weight.

[0011] Preferably, the load is 5% of the animal's body weight.

[0012] In the above technical solutions, a load of less than 3% is unlikely to effectively induce a state of "qi deficiency", while a load of more than 8% can easily lead to rapid drowning or excessive stress in animals.

[0013] For example, the state of exhaustion includes an animal whose head is submerged in water for more than 10 seconds and who has lost the ability to float on its own or who is unable to right itself within 5 seconds after being retrieved.

[0014] In some embodiments, the first predetermined time is 7 to 10 days.

[0015] Preferably, the first scheduled time is 7 days.

[0016] In some embodiments, the second predetermined time is 7 to 10 days.

[0017] Preferably, the second scheduled time is 7 days.

[0018] In the above technical solution, the total modeling cycle is 14 to 20 days, preferably 14 days. If the modeling cycle is too short (<14 days), it is easy to cause insufficient "qi deficiency" or insufficient "toxic damage". If the cycle is too long (>20 days), it is easy to cause animal tolerance or chronic MPTP toxicity, thereby increasing the risk of non-specific damage.

[0019] In some embodiments, the dosage of MPTP is 25-35 mg / kg / day.

[0020] In some embodiments, the MPTP is administered via intraperitoneal injection.

[0021] On the one hand, this application provides an animal model of Parkinson's disease with qi deficiency and blood stasis syndrome obtained by the above method.

[0022] On the one hand, this application provides the application of the animal model of Parkinson's disease with qi deficiency and blood stasis syndrome obtained by the above method, or the application of the above animal model of Parkinson's disease with qi deficiency and blood stasis syndrome in screening or preparing drugs for treating Parkinson's disease.

[0023] In addition, successful model validation requires a multi-dimensional evaluation system that combines macro and micro "disease-symptom" assessments. This system includes the following four dimensions: (1) Macroscopic syndrome evaluation: The indicators of "Qi deficiency" (such as body temperature, weight, activity ability, hunchback, clumsiness, mental state, etc.) and "blood stasis" (such as nails, extremities, tail blood vessels, tongue color, color and shape of sublingual veins, etc.) are quantified. The verification criteria are: ① total score ≥ 8 points, ② the time of appearance of the "Qi deficiency" dimension score is significantly earlier than that of the "blood stasis" dimension score; (2) Behavioral evaluation: The motor function of experimental animals was evaluated through a series of behavioral experiments. The verification criteria were: the animal model showed serious motor impairment, such as a significant reduction in total distance and average speed in the open field experiment, a significant shortening of time on the rod in the twirling rod experiment, and a significant prolongation of time to turn around and climb down in the pole climbing experiment. (3) Histopathological evaluation: The degree of damage to dopaminergic neurons in the substantia nigra-striatum was detected. The verification criteria were: the number of tyrosine hydroxylase (TH) positive neurons in the substantia nigra, the number of surviving neurons stained with Nissl stain, and the density of TH positive fibers in the striatum were all significantly reduced in the animal model, reflecting the body's energy metabolism disorder. (4) Molecular biological evaluation: By detecting specific molecular indicators in peripheral serum and brain tissue, objective biological evidence of "qi deficiency" and "blood stasis" syndromes is obtained. ① When verifying "qi deficiency", the serum energy metabolism indicators (such as ATP / ADP ratio and cAMP / cGMP ratio) of the animal model are significantly reduced. ② When verifying "blood stasis", the serum microcirculation indicators (such as TXA2 / PGI2 ratio) are detected. The verification standard is: the TXA2 / PGI2 ratio of the animal model is significantly reduced, reflecting the imbalance of vasomotor function and platelet aggregation tendency. ③ When verifying the microscopic mechanism of "retention", the neuroinflammatory level of the substantia nigra tissue is detected. The verification standard is: the mRNA expression of glial cell markers (such as Iba1 and GFAP) and pro-inflammatory factors (such as IL-1α, IL-1β, IL-6, TNF-α, etc.) in the animal model should be significantly upregulated, and the compensatory upregulation of anti-inflammatory factor IL-10 is blocked, forming a pro-inflammatory-anti-inflammatory imbalance.

[0024] This application has the following beneficial effects: (1) This application’s animal model is the first to systematically reproduce the TCM syndrome characteristics of “Qi deficiency and blood stasis” in PD at the animal level. The model not only presents the typical pathological change of loss of TH positive neurons in the substantia nigra, but more importantly, after dynamic evaluation through macroscopic syndrome scale, it can be confirmed that this application has successfully simulated the dynamic evolution process of “Qi deficiency leading to blood stasis” from “fatigue” (Qi deficiency) to “purple veins under the tongue” (blood stasis) in clinical PD patients, providing a more clinically relevant experimental vehicle for evaluating the “formula-syndrome correspondence” advantage of Qi-tonifying and blood-activating TCM. (2) Unlike existing static superposition models, the sequential design of the model in this application, which is "deficiency first and then toxicity", reproduces the cascade pathological process of "deficiency-stasis-deficiency-stasis intermingling" in experimental animals for the first time. Through dynamic analysis of syndromes, it was found that the "qi deficiency" dimension score increased significantly on the second day, while the "blood stasis" dimension score did not appear until the eighth day. This finding provides direct experimental evidence for the theory of "deficiency qi retention". Attached Figure Description

[0025] Figure 1 This is a schematic diagram of the temporal modeling strategy for the animal model of Parkinson's disease with Qi deficiency and blood stasis syndrome, which is based on the present application.

[0026] Figure 2 The results of TCM syndrome evaluation for the model in this application are as follows: A~E represent the dynamic changes in the total TCM syndrome score of each group of mice during the modeling period (n=12, Mean±SD); F represents the comparison of the total TCM syndrome score of each group of mice on day 14 of modeling; G represents the score of the "Qi deficiency" dimension summarized based on the syndrome scale on day 14 of modeling; H represents the score of the "blood stasis" dimension summarized based on the syndrome scale on day 14 of modeling; the above data are expressed as Mean±SD (n=12).

[0027] Figure 3 The results are the behavioral evaluation results of the model in this application. Among them, A is the quantitative analysis of the open field test, which, from left to right, are: total distance traveled, average speed of movement, distance of activity in the central area, and time spent in the central area; B is the representative movement trajectory diagram of the open field test; C is the test result of the rotarod test; D is the test result of the pole climbing test; E is the score result of the suspension test. The above data are expressed as Mean±SD (n=6).

[0028] Figure 4 The images show the immunohistochemical staining results of TH in the substantia nigra and striatum of the model in this application. A represents representative TH immunohistochemical images, with the top row showing the substantia nigra (SN) region and the bottom row showing the striatum (STR) region, scale bar = 200 μm. B shows a semi-quantitative analysis of TH-positive neuron counts in the substantia nigra region; three non-contiguous sections were selected from each mouse, and the number of TH-positive cells was counted, with the average value taken. C shows a semi-quantitative analysis of the integrated optical density (Total IOD) of TH-positive fiber terminals in the striatum region; the same area of ​​the striatum region was selected from each section, and the Total IOD value was measured. All data are expressed as Mean ± SD (n = 4).

[0029] Figure 5 The images show the Nissl staining results of the substantia nigra of the model in this application. A is a representative Nissl staining image, with the top row being 200× and the scale bar being 100 μm, and the bottom row being 400× and the scale bar being 50 μm. B is a semi-quantitative analysis of the number of surviving neurons in the substantia nigra region. Three non-contiguous slices were selected from each mouse, and Nissl bodies were counted in the same region of each slice, and the average value was taken. The above data are expressed as Mean±SD (n=4).

[0030] Figure 6 The following are the Western Blot results of TH and DAT protein expression in the substantia nigra of the model in this application. In this data, A represents the representative bands of TH, DAT and internal reference GAPDH in Western Blot; B is the semi-quantitative analysis of the relative expression level of TH protein (normalized with CON group as 1.0); C is the semi-quantitative analysis of the relative expression level of DAT protein (normalized with CON group as 1.0). All data are expressed as Mean ± SD (n=6).

[0031] Figure 7The Western Blot results for the expression of glial cell activation markers (GFAP, Iba1) in the model of this application are shown. In the figure, A represents the representative bands of GFAP, Iba1 and internal control GAPDH in Western Blot; B is the semi-quantitative analysis of the relative expression level of GFAP protein (normalized with CON group as 1.0); C is the semi-quantitative analysis of the relative expression level of Iba1 protein (normalized with CON group as 1.0). All data are expressed as Mean ± SD (n=6).

[0032] Figure 8 The results of RT-qPCR for the mRNA expression of substantia nigra inflammatory factors (IL-1α, IL-1β, IL-6, TNF-α, IL-10) in the model of this application are shown. In the figure, A represents the relative mRNA expression level of IL-1α; B represents the relative mRNA expression level of IL-1β; C represents the relative mRNA expression level of IL-6; D represents the relative mRNA expression level of TNF-α; and E represents the relative mRNA expression level of IL-10. All data were detected by RT-qPCR, with GAPDH as an internal control and CON group as 1.0 for standardization. The data are expressed as Mean ± SD (n=5).

[0033] Figure 9 The results of ELISA detection of peripheral serum energy metabolism (ATP / ADP, cAMP / cGMP) and microcirculation (TXA2 / PGI2) indicators in the model of this application are shown. Wherein, A is ATP content; B is ADP content; C is cAMP content; D is cGMP content; E is TXA2 content; F is PGI2 content; G is ATP / ADP ratio; H is cAMP / cGMP ratio; I is TXA2 / PGI2 ratio. The serum samples were detected by ELISA, and the data are expressed as Mean ± SD (n=6). Detailed Implementation

[0034] The technical solutions in some embodiments of this application will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of this application, and not all embodiments. Based on the embodiments provided in this application, all other embodiments obtained by those skilled in the art are within the scope of protection of this application.

[0035] Unless the context otherwise requires, throughout the specification and claims, the term "comprising" is interpreted as open and encompassing, that is, "including, but not limited to".

[0036] In describing some embodiments, the expressions "at least one of A, B and C" and "at least one of A, B or C" may be used, both of which have the same meaning and include the following combinations of A, B and C: only A, only B, only C, a combination of A and B, a combination of A and C, a combination of B and C, and a combination of A, B and C.

[0037] Example 1: Construction of a mouse model of Parkinson's disease with Qi deficiency and blood stasis syndrome Eight-week-old SPF-grade male C57BL / 6N mice were acclimatized for one week and then randomly divided into four groups (n=12 / group). The results were then compared with... Figure 1 The diagram shown illustrates the mouse modeling process, and the treatment of each group during the modeling process is as follows.

[0038] Blank control group (CON): Normal feeding from day 1 to day 14, and intraperitoneal injection of sterile saline with an equal volume of MPTP daily from day 8 to day 14.

[0039] Qi deficiency and blood stasis syndrome group (ES): From day 1 to 14, weight-bearing exhaustive swimming was performed every morning; from day 8 to 14, an equal volume of normal saline was injected into the peritoneum in the afternoon.

[0040] PD model group (MPTP): Normal feeding from day 1 to day 7, and intraperitoneal injection of MPTP (30 mg / kg / day) every afternoon from day 8 to day 14.

[0041] PD Qi Deficiency and Blood Stasis Syndrome Group (MPTP+ES, Target Model Group): From day 1 to 14, the same weight-bearing exhaustive swimming was performed every morning as in the ES group; from day 8 to 14, the same MPTP intraperitoneal injection was performed every afternoon as in the MPTP group.

[0042] In the above treatment, the parameters for weight-bearing exhaustion swimming were as follows: swimming tank diameter 35 cm, height 60 cm, water temperature 25±1℃, water depth ≥30 cm. Lead sheets equal to 5% of the mouse's body weight were wrapped around the mouse's tail. Exhaustion criteria: the mouse's head was submerged for more than 10 seconds and it lost the ability to float independently, or it could not right itself within 5 seconds after being retrieved. The swimming time to exhaustion was recorded daily. MPTP injection protocol: MPTP was prepared as a 3 mg / mL working solution with physiological saline and injected intraperitoneally at a volume of 10 mL / kg, once daily for 7 consecutive days.

[0043] Example 2: Macroscopic Symptom Evaluation in Animal Models Scoring was conducted at fixed time points on days 0, 2, 4, 6, 8, 10, 12, and 14 after modeling to capture the temporal differences in the scores of the "Qi deficiency" and "blood stasis" dimensions. Scoring was performed independently by two trained but uninformed researchers using the "Parkinson's Disease Qi Deficiency and Blood Stasis Syndrome Mouse Syndrome Evaluation Scale" shown in Table 1. The scale covers general physical signs (body temperature, weight, feces), behavioral characteristics (movement, limbs, muscles, arched back, huddling), and physical features (mental state, nails, extremity color, tail blood vessels, body hair, tongue color, sublingual veins). The evaluation results are as follows: Figure 2 As shown in Table 2.

[0044] Table 1. Symptom Evaluation Scale for Parkinson's Disease with Qi Deficiency and Blood Stasis Syndrome in Mice

[0045] Note: Detailed criteria for the syndrome scale assessment: ① Total score < 8 points: normal; ② Total score 8~15 points: mild Qi deficiency and blood stasis syndrome; ③ Total score 16~30 points: moderate Qi deficiency and blood stasis syndrome; ④ Total score > 31 points: severe Qi deficiency and blood stasis syndrome.

[0046] Table 2. Dynamic changes of the total score of TCM syndrome over time

[0047] Note: ◎: compared with the CON group; #: compared with the MPTP group. Data are expressed as Mean ± SD (n=12), all P < 0.05.

[0048] The results showed that on day 14 of modeling, the CON group had the lowest score (3.00±0.74 points), while the MPTP+ES group had the highest score (26.42±0.36 points), significantly higher than the MPTP group (20.25±0.81 points) and the ES group (16.63±0.43 points) (P <0.0001). Dynamic analysis showed that the "Qi deficiency" dimension score in the target model group increased significantly from day 2, while the "blood stasis" dimension score did not appear until day 8, successfully reproducing the dynamic process of "blood stasis caused by deficiency".

[0049] Example 3: Model Behavioral Validation Behavioral tests were conducted on the mice obtained after modeling, including the open field test, rotarod test, and pole climbing test. The test results are as follows: Figure 3 As shown.

[0050] Open field test: Mice were placed in a 40 cm × 40 cm open field box, and their movement trajectory was recorded over 10 minutes using ANY-MAZE software.

[0051] Rotor experiment: The rotor was rotated at a constant speed of 25 r / min, and the time the mouse stayed on the rotor was recorded. Each mouse was tested 3 times and the average value was taken.

[0052] Pole climbing experiment: Place the mouse head up on the top of the pole (60 cm long and 1 cm in diameter) and record the total time it takes for it to turn around and for its hind limbs to contact the platform. Each mouse was tested 5 times and the average value was taken.

[0053] The results showed that the total distance traveled and the average speed of the target model group (MPTP+ES) were significantly lower than those of the MPTP group (P<0.0001); the time spent on the pole was significantly shorter than that of the MPTP group (P=0.002); and the total time spent climbing the pole was significantly longer than that of the MPTP group (P=0.0001).

[0054] Example 4: Validation of Model Histopathology After anesthetizing each group of model mice with sodium pentobarbital, four mice from each group were used for cardiac perfusion fixation (4% paraformaldehyde). Brain tissue was then collected, embedded in paraffin, sectioned, and subjected to TH immunohistochemical staining of the substantia nigra and striatum, as well as Nissl staining of the substantia nigra. The results are as follows: Figure 4 As shown, three non-contiguous slices were taken from each group, and images were acquired from the same region on each slice. The number of TH-positive neurons and Nissl-stained surviving neurons were counted, and the integrated optical density (IOD) of TH-positive fibers was analyzed using Image-Pro Plus software. The results are as follows. Figure 5 As shown.

[0055] The results showed that the number of TH-positive neurons in the substantia nigra and the IOD of TH-positive fibers in the striatum in the target model group (MPTP+ES) were significantly lower than those in the MPTP group (P<0.0001). Nissl staining showed that the number of morphologically intact neurons in the substantia nigra region in the target model group was significantly less than that in the MPTP group (P=0.0029).

[0056] Example 5: Model Molecular Biological Validation Six mice were used in each model group. Substantia nigra tissue was rapidly separated, and total protein was extracted. TH and DAT were detected by Western blotting (e.g., ...). Figure 6 As shown), Iba1 and GFAP (as shown) Figure 7 Protein expression (as shown) was analyzed (using GAPDH as an internal control). In addition, total RNA was extracted from the substantia nigra tissue of five mice in each group, and the mRNA expression of inflammatory factors (IL-1α, IL-1β, IL-6, TNF-α, IL-10) was detected by RT-qPCR (using GAPDH as an internal control). Figure 8 (As shown). Simultaneously, blood was collected from the retroocular venous plexus of 6 mice in each group, and serum was separated for ELISA detection of ATP, ADP, cAMP, cGMP, TXB2, and PGI2. The ratios of ATP / ADP, cAMP / cGMP, and TXB2 / PGI2 were calculated (e.g., ...). Figure 9 (As shown) The results showed that, regarding TH and DAT protein expression, the target model group had the lowest TH and DAT protein expression levels in the substantia nigra, significantly lower than the MPTP group (P<0.0001). This indicates that the "Qi deficiency" state further exacerbated MPTP-induced dopamine synthesis dysfunction, and the superposition of Qi deficiency and blood stasis syndromes intensified the damage to dopaminergic nerve endings. In addition, there was no significant difference in TH expression between the ES group and the CON group (P=0.0542), but DAT expression was significantly lower than that in the CON group (P<0.0001), suggesting that the "Qi deficiency" state may selectively affect dopamine transporter function rather than directly damage neuronal cell bodies. Regarding glial cell activation markers, the target model group had the highest Iba1 and GFAP protein expression levels in the substantia nigra, significantly higher than the MPTP group (P<0.0001), indicating that the target model group induced the strongest glial cell activation response. Regarding inflammatory factor expression, the target model group showed the highest levels of pro-inflammatory factors IL-1β, TNF-α, IL-1α, and IL-6 in the substantia nigra. The mRNA expression level was the highest, and the upregulation of the anti-inflammatory factor IL-10 expression was significantly lower in the target model group than in the MPTP group (P<0.0001), indicating a severe pro-inflammatory / anti-inflammatory imbalance. For energy metabolism indicators, the ATP / ADP ratio and cAMP / cGMP ratio in the target model group were significantly lower than those in the MPTP and ES groups (P<0.0001). For microcirculation indicators, the TXA2 / PGI2 ratio in the target model group was significantly higher than that in the MPTP and ES groups (P<0.0001).

[0057] The above description is merely a preferred embodiment of the present invention. It should be understood that the present invention is not limited to the forms disclosed herein and should not be construed as excluding other embodiments. It can be used in various other combinations, modifications, and environments, and can be altered within the scope of the concept described herein through the above teachings or related technologies or knowledge. Modifications and variations made by those skilled in the art that do not depart from the spirit and scope of the present invention should be within the protection scope of the appended claims.

Claims

1. A method for constructing an animal model of Parkinson's disease with Qi deficiency and blood stasis syndrome, characterized in that... The construction method includes: subjecting the animal to weight-bearing exhaustion training daily for a first predetermined time period, and then administering MPTP to the animal after subjecting the animal to weight-bearing exhaustion training daily for a second predetermined time period.

2. The construction method according to claim 1, characterized in that, The weight-bearing exhaustion training involves applying a weight to the animal's tail and then swimming in water at 22-28°C until exhaustion.

3. The construction method according to claim 2, characterized in that, The load is 3% to 8% of the animal's body weight.

4. The construction method according to claim 2, characterized in that, The state of exhaustion includes an animal whose head has been submerged for more than 10 seconds and who has lost the ability to float on its own, or who, after being pulled out of the water, is unable to right itself within 5 seconds.

5. The construction method according to any one of claims 1 to 4, characterized in that, The first scheduled time is 7 to 10 days.

6. The construction method according to any one of claims 1 to 4, characterized in that, The second scheduled time is 7 to 10 days.

7. The construction method according to claims 1-6, characterized in that, The dosage of MPTP is 25-35 mg / kg / day.

8. The construction method according to claim 7, characterized in that, The MPTP can be administered via intraperitoneal injection.

9. An animal model of Parkinson's disease with qi deficiency and blood stasis syndrome obtained by the construction method described in any one of claims 1 to 8.

10. The construction method as described in any one of claims 1 to 8 or the application of the animal model of Parkinson's disease with qi deficiency and blood stasis syndrome as described in claim 9 in screening or preparing drugs for treating Parkinson's disease.

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