CN115804768A

CN115804768A - Application of peucedanum praeruptorum dunn in preparing medicine for treating nerve degenerative disease

Info

Publication number: CN115804768A
Application number: CN202310027678.8A
Authority: CN
Inventors: 郑瑞茂; 赵云
Original assignee: Peking University
Current assignee: Peking University
Priority date: 2023-01-09
Filing date: 2023-01-09
Publication date: 2023-03-17

Abstract

The invention provides an application of peucedanum praeruptorum dunn in preparing a medicine for treating neurodegenerative diseases, belonging to the technical field of biological medicines. The invention provides an application of peucedanum praeruptorum dunn in preparing a medicine for preventing and/or treating neurodegenerative diseases. Experiments prove that the peucedanum praeruptorum dunn can obviously restore the mouse motor dysfunction caused by MPTP, the dopaminergic neuron apoptosis caused by MPTP, the dopaminergic neuron projection in the striatum of the mouse and the neuroinflammation level; can obviously improve the cell survival rate of nerve cells and play a role in protecting neurons.

Description

Application of peucedanum praeruptorum dunn in preparing medicine for treating nerve degenerative disease

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to application of peucedanum praeruptorum dunn B in preparation of a medicine for treating neurodegenerative diseases.

Background

Neurodegenerative diseases are diseases caused by gradual loss of neuronal structures or functions of organisms, and comprise Parkinson diseases, alzheimer diseases, huntington diseases and the like; at present, the etiology of the diseases is not clear, no effective curing means exists, and the quality of life of patients is seriously threatened.

Parkinson's disease is one of the most common neurodegenerative movement disorders worldwide, and it is estimated that 570 people are ill in every 100,000 people in the population 50 years and older worldwide, and increase with age. It is estimated that the global number of PD cases will increase from about 700 million in 2015 to about 1300 million in 2040. China is the country with the most PD patients in the world, and the number of the patients accounts for more than half of the number of the PD patients in the world. According to global disease burden studies, neurological diseases are currently the major source of disability, while parkinson's disease is the fastest growing of these diseases (prevalence, disability rate and mortality rate, normalized by age). Therefore, the research on the pathological mechanism and the treatment method of PD has great medical and social significance.

The main pathological feature of PD is the progressive degenerative loss of Dopaminergic (DA) neurons in the substantia nigra pars compacta (SNpc), with the presence of eosinophilic insoluble aggregates called Lewy Bodies (LBs) in the remaining neuronal cytoplasm. Degeneration loss of dopaminergic neurons, degeneration of dopaminergic pathways of the substantia nigra-striatum (striatum), significant reduction of striatal dopamine transmitter level, and positive correlation between the degree of reduction of dopamine transmitter and the severity of patient symptoms; formation of lewy's body, the intracellular pathological marker of the remaining dopaminergic neurons, the main component of which is α -synuclein (α -synuclein). The etiology of parkinson's disease is not completely understood, and is currently thought to be related to a variety of factors, such as genetic factors, environmental factors, oxidative stress, excitotoxicity, mitochondrial dysfunction, apoptosis, and the like.

The clinical diagnosis of the Parkinson disease at the present stage mainly depends on the medical history, clinical symptoms and physical signs, and the change of the tyrosine hydroxylase level of substantia nigra and striatum is detected in an animal model. Among them, tyrosine Hydroxylase (TH) is a rate-limiting enzyme for biosynthesis of catecholamine-type active substances, and plays an important role in regulation of dopamine biosynthesis, and therefore, the activity and expression amount of the Tyrosine Hydroxylase (TH) in organisms, particularly in substantia nigra and striatum, are changed to directly influence the biosynthesis of L-dopamine. Thus, tyrosine hydroxylase levels are a common indicator of changes in dopaminergic neuronal levels in the substantia nigra, striatum. In a mouse Parkinson disease model, MPTP is mainly used for inducing the apoptosis of dopaminergic neurons in substantia nigra and simulating the clinical Parkinson disease onset symptoms.

Peucedanum Praeruptorin B is an important compound isolated from Peucedanum praeruptorum and has been reported to have various biochemical and pharmacological activities. Peucedanum praeruptorum dunn can improve diet-induced obesity in a mouse model by inhibiting SREBPs to play a role in reducing blood lipid; it can also exert anti-inflammatory and anti-tumor properties, has the ability of inhibiting the metastasis of human cervical cancer cells, and has no obvious toxicity.

At present, no relevant research report is found about the application of peucedanum praeruptorum dunn in preventing Parkinson.

Disclosure of Invention

In view of this, the present invention aims to provide an application of peucedanum praeruptorum in the preparation of a medicament for treating neurodegenerative diseases.

The invention provides an application of peucedanum praeruptorum dunn in preparing a medicine for preventing and/or treating neurodegenerative diseases.

Preferably, the neurodegenerative disease includes at least one of the following diseases: parkinson's disease, alzheimer's disease and Huntington's disease.

The invention provides an application of peucedanum praeruptorum B in preparing a medicine for preventing and/or treating neuroinflammation.

Preferably, the medicament has the effect of reducing the level of Glial Fibrillary Acidic Protein (GFAP) positive cells and/or ionic calcium binding adaptor protein 1 (Iba 1) positive cells.

The invention provides an application of peucedanum praeruptorum dunn in preparing neuroprotective medicine.

Preferably, the neuroprotection comprises restoration of dopaminergic neuron projection in the striatum or restoration of dopaminergic neuron apoptosis.

The invention provides application of peucedanum praeruptorum dunn in preparation of a medicine for promoting dopamine synthesis.

Preferably, the promotion of dopamine synthesis is achieved by increasing tyrosine hydroxylase levels in the substantia nigra pars compacta or striatum.

Preferably, the structural formula of the peucedanum praeruptorum dunn is shown as a formula I;

preferably, the dosage form of the medicament comprises at least one of: tablet, powder, granule, capsule, oral liquid and sustained release preparation.

The invention provides an application of peucedanum praeruptorum dunn in preparing a medicine for preventing and/or treating neurodegenerative diseases. Experiments show that the medicine peucedanum praeruptorum dunn can obviously recover the mouse motor dysfunction caused by MPTP; can remarkably recover dopaminergic neuron apoptosis caused by MPTP; can remarkably recover dopaminergic neuron projection in mouse striatum; can significantly reduce the level of neuroinflammation; can obviously improve the cell survival rate of nerve cells, thereby achieving the effect of protecting neurons.

Drawings

FIG. 1 shows the change of behavioral indexes of mice in each group 7 days after injection of Peucedanum praeruptorum dunn (or DMSO);

FIG. 2 shows the morphology of tyrosine hydroxylase positive neurons in substantia nigra pars compacta after 7 days of injection of Peucedanum praeruptorum dunn (or DMSO) in each group of mice;

FIG. 3 shows the change of striatum tyrosine hydroxylase level 7 days after injection of Peucedanum praeruptorum B (or DMSO) in each group of mice;

FIG. 4 shows GFAP positive cell levels in substantia nigra pars compacta 7 days after injection of Peucedanum praeruptorin (or DMSO) in each group of mice;

FIG. 5 shows the Iba1 positive cell level in substantia nigra pars compacta 7 days after injection of Peucedanum praeruptorin (or DMSO) in each group of mice.

Detailed Description

In the invention, the structural formula of the peucedanum praeruptorum dunn is preferably shown as formula I; from biotech, inc. of Dou Pufei D.

In the present invention, the neurodegenerative disease preferably includes at least one of the following diseases: parkinson's disease, alzheimer's disease and huntington's disease. In the present example, the effect of administering peucedanum praeruptorum is understood by taking parkinson's disease as an example. The experimental result shows that the peucedanum praeruptorum dunn can remarkably recover the mouse dyskinesia caused by MPTP; can remarkably recover dopaminergic neuron apoptosis caused by MPTP; can remarkably recover dopaminergic neuron projection in mouse striatum; can significantly reduce the level of neuroinflammation; can obviously improve the cell survival rate of nerve cells. Therefore, the peucedanum praeruptorum dunn has the function of treating neurodegenerative diseases.

In the present invention, the drug preferably has an effect of reducing the level of Glial Fibrillary Acidic Protein (GFAP) -positive cells and/or ionic calcium binding adaptor protein 1 (Iba 1) -positive cells. The occurrence of neuroinflammation mainly comprises inflammatory reactions such as microglial activation and astrocyte activation. GFAP and Iba1 are markers of astrocytes and microglia, respectively, whose abnormal activation indicates an increase in the level of neuroinflammation. Experiments show that after a Parkinson disease model is constructed, compared with a normal group of mice, the ratio of GFAP positive cells to Iba1 positive cells is increased, and after the Peucedanum praeruptorum B is administrated to the model mice, the ratio of GFAP positive cells to Iba1 positive cells of the model group mice can be effectively reduced, so that the Peucedanum praeruptorum B can reduce the neuroinflammation reaction level by inhibiting the activation of microglia and the activation of astrocytes, and realize the treatment of neuroinflammation.

The invention provides an application of peucedanum praeruptorum dunn in preparing a neuroprotective medicament.

In the present invention, the neuroprotection preferably comprises restoration of dopaminergic neuron projection in the striatum or restoration of dopaminergic neuron apoptosis. Tyrosine Hydroxylase (TH) is a rate-limiting enzyme for synthesizing dopamine in brain, the level of the tyrosine hydroxylase is high or low and represents the dopamine synthesizing capacity of a mouse, and therefore the tyrosine hydroxylase is used as an index of the dopamine secreting capacity of the mouse. Experimental results show that the peucedanum praeruptorum dunn group can obviously recover dopaminergic neuron apoptosis caused by MPTP, and meanwhile, the dopaminergic neuron projection in mouse striatum in the recovery model group is obviously reduced.

In the present invention, the promotion of dopamine synthesis is preferably achieved by increasing the level of tyrosine hydroxylase in the substantia nigra pars compacta or striata. The dosage form of the medicament preferably comprises at least one of: tablet, powder, granule, capsule, oral liquid and sustained release agent.

The following examples are provided to illustrate the application of peucedanum praeruptorum dunn in the preparation of drugs for treating neurodegenerative diseases, but they should not be construed as limiting the scope of the present invention.

The mice used in the examples were: male SPF grade C57 BL/6 mice, 7-8 weeks old, were purchased from the department of Experimental animal sciences of the department of medicine, beijing university. C57 BL/6 mouse feeding conditions: the ambient temperature is 22 +/-0.5 ℃, and the light and the shade are alternated for 12 hours/12 hours.

All experimental data are expressed as mean ± sem, # P < 0.05, # P < 0.01, # P < 0.001, n =10.

Example 1

Method for treating Parkinson's disease by using peucedanum praeruptorum dunn

Establishing an MPTP (Multi-Point Transmission protocol) induced Parkinson disease model: after the mice are adaptively raised for 1 week, C57 BL/6 mice with the weight of more than 22g are randomly selected, 30mg/kg MPTP solution is injected into the abdominal cavity, 1 time is injected for 1 day, and 5 days are continuously injected.

The MPTP injection group (hereinafter referred to as the model group) was randomly divided into 2 groups, which were a model + control solvent group (referred to as the model group) and a model + peucedanum praeruptorin group (referred to as the administration group). Wherein, each group of mice is injected with a control solvent (dimethyl sulfoxide DMSO) and Peucedanum praeruptorum B (50 mg/kg) respectively in the abdominal cavity based on the weight of the mice; normal group injected with control solvent (dimethylsulfoxide, DMSO); the volume of the solvent injected in each group was the same and was 25. Mu.l.

And (3) detecting the change of the behavioral indexes of each group of the medicaments and the control solvent after the medicaments and the control solvent are injected once a day and continuously injected for 7 days.

And (3) detection of the behavioral indexes:

after the MPTP-induced Parkinson disease model is established, the method is carried out on the 4 th day (3 days before material drawing) of the injection of each group of medicaments, the detection and the training of the behavioral indexes of the mice are carried out in a quiet and proper environment, and each experiment is detected 3 times a day so as to eliminate the interference of other factors on the behavioral indexes.

1. Balance beam experiment

1. The mice were moved to the test room and habituated for 1h.

2. A set of round rods of 100cm length and 10mm, 20mm and 30mm diameter, respectively, and square rods of 5mm, 15mm and 30mm width, respectively, were prepared.

3. Two platforms 50cm above the ground were constructed, 80cm apart, with one platform holding a dark room. And overlapping the widest rod between the two platforms and fixing the widest rod.

4. The mouse head is placed on a platform on the opposite side of the dark room forwards, the mouse is stimulated to walk forwards with strong light or noise behind the mouse, and the time required for the mouse to reach the dark room and the number of times of hind limb skidding are recorded.

5. The wide rod is replaced by the narrow rod, and the testing steps are the same.

6. The same batch of mice was tested 5 times a day for three consecutive days and the average was taken.

2. Pole climbing experiment

1. The mouse home cage was moved to the test room and habituated for 1h.

2. Preparing a round rod with the length of 50-60 cm and the diameter of 1-1.5 cm, wrapping the round rod with gauze for skid prevention, and vertically standing the round rod in a mouse home cage.

3. The mouse was placed head up on top of the rod and the time required to turn it head down and to reach the bottom of the rod was recorded.

4. The same batch of mice was tested 5 times a day for three consecutive days, and the average was taken.

3. Hind limb grip test

1. The mouse home cage was moved to the test room and habituated for 1h.

2. The mouse tail is grabbed and lifted from the middle part, the activity state of the hind limbs of the mouse is recorded by a camera for 20s.

3. Scoring according to the hindlimb activity of the mice: in the 0 minute-test process, the hind limbs are in a natural open state; 1 minute-one hind limb appears to be gripped or both hind limbs are gripped slightly; 2 minutes-both hind limbs are held tightly most of the time but still flexible; 3 minutes-both sides of the hind limb are completely gripped without flexibility.

4. The same batch of mice was tested 3 times a day for three consecutive days, and the average was taken.

4. Rolling rod experiment

1. The mouse home cage was moved to the test room and habituated for 1h.

2. The mice were placed on the test instrument (rotating rod) at an initial speed of 4rpm, gradually accelerated by 1rpm every 8s and no further acceleration after increasing the speed to 40rpm at 5min.

3. The time the mouse dropped from the rod was recorded.

4. The same batch of mice was tested 3 times a day, each time at 20min intervals, for three consecutive days, and the average was taken.

Results of the experiment

The results are shown in FIG. 1, with data presented as mean. + -. SEM standard error.

The balance beam test result data is shown in table 1. As can be seen from table 1, the mouse has a decreased ability to exercise in balance as compared with the normal group, and the ability to exercise in balance can be significantly improved as compared with the normal group after the peucedanum praeruptorum dunn is treated.

TABLE 1 balance beam test results

The climbing pole test result data are shown in table 2. As can be seen from Table 2, the pole-climbing time of the model group mice was significantly prolonged compared to the normal group; after the mice in the administration group are administrated, the rod climbing time of the mice can be obviously reduced, and the praeruptorin can effectively improve the movement capability of the mice in the model group.

TABLE 2 Pole climbing test results

Hindlimb grip reflex result data are shown in table 3. As can be seen from Table 3, compared with the normal group, the hindlimb gripping reflex time of the model group xiao Hu is obviously prolonged, and the hindlimb gripping time of the model group mice can be obviously reduced after the peucedanitin is administrated to the model group mice, which indicates that the peucedanitin can effectively improve the exercise capacity of the model group mice.

TABLE 3 hind limb grip reflex results

The results of the rotarod experiments are shown in table 4. As can be seen from Table 4, compared with the normal group, the rod turning time of the model group xiao Hu is obviously shortened, and after the peucedanib is administrated to the model group mice, the rod turning time of the model group mice can be obviously improved, which indicates that the peucedanib can effectively improve the movement ability of the model group mice.

TABLE 4 results of the bar rotation experiment

In conclusion, compared with the normal group, the behavioral indexes of the model group are obviously reduced, obvious dyskinesia occurs, and the Parkinson model is successfully constructed; the model + Peucedanum praeruptorum dunn group can remarkably recover mice motor dysfunction caused by MPTP (figure 1).

Example 2

Protection effect of peucedanum praeruptorum dunn on nigra and striatum dopaminergic neurons

After the Parkinson disease model was successfully established according to the method of example 1, the grouping and administration method of mice were the same as that of example 1, and the tyrosine hydroxylase level of substantia nigra pars compacta of each group of mice was detected by immunofluorescence technique 7 days after intraperitoneal injection of each group of drugs and control solvent (the injection dose was the same as that of example 1).

The detection steps of the immunofluorescence technique are as follows:

1. mice were perfused and then brains were removed, 4% PFA (paraformaldehyde) fixed for 24-48 hours, dehydrated with a 30% sucrose gradient at 20%. Embedding with OCT embedding medium, and freezing the section.

2. After slicing, the sections were plated and washed 3 times with PBS for 5min each.

3. And (5) carrying out permeabilization treatment. Permeabilized for 30min with 0.5% TritonX-100 (1 XPBS).

4. And (3) antigen retrieval, namely immersing the tablets into a sodium citrate solution, carrying out 30min at 98 ℃, taking out and cooling to room temperature.

PBS wash 3 times, each for 5min.

6. Adding proper amount of goat serum working solution for sealing dropwise. Incubate at room temperature for 15min, decant serum, and do not wash.

7. A primary antibody. Adding a proper amount of primary antibody dropwise, and incubating for 12h at 4 ℃.

PBS wash 3 times, each time for 5min.

9. And (4) secondary antibody. And adding a proper amount of secondary antibody (GFP fluorescence labeling) dropwise, and incubating for 2h at room temperature.

PBS wash 3 times, each for 5min.

11. And (6) sealing the sheet.

12. And (5) storing. The sealed slices are put into a film box for storage at room temperature or 4 ℃.

13. And (6) shooting.

Tyrosine Hydroxylase (TH) is the rate-limiting enzyme for dopamine synthesis in brain, and the level of the tyrosine hydroxylase represents the dopamine synthesis capacity of mice, so the tyrosine hydroxylase is often used as an index of the dopamine secretion capacity of the mice. The immunofluorescence experiment result is shown in fig. 2, and the result proves that compared with the normal group, the number of TH positive neurons of the substantia nigra pars compacta of the mice in the model group is remarkably reduced, namely, the obvious apoptosis condition of dopaminergic neurons occurs. While the model + peucedanum praeruptorin group was able to significantly restore dopaminergic neuron apoptosis caused by MPTP (fig. 2).

In addition, it was also confirmed that dopaminergic neuron projection was significantly reduced in the striatum of the model group mice, and that peucedanum praeruptorin was able to significantly recover this condition (fig. 3).

Example 3

The pharmaceutical composition of the invention treats neuroinflammation

The occurrence and development of neuroinflammation is now considered to be one of the relevant factors in the pathogenesis of neurodegenerative diseases, and the role of neuroinflammation in promoting the mediation of progressive neuronal death in the course of neurodegenerative diseases has been widely demonstrated. The occurrence of neuroinflammation mainly comprises inflammatory reactions such as microglial cell activation and astrocyte activation. Further, these central inflammatory cells interact through multiple mechanisms to produce proinflammatory cytokines, amplify inflammatory signals, produce neurotoxins that act directly on neurons, and ultimately lead to the development of neurodegenerative diseases.

After the Parkinson's disease model was successfully established according to the method of example 1, the grouping and administration method of mice were the same as that of example 1. The positive cells of the substantia nigra pars compacta GFAP and the Iba1 of each group of mice are detected by an immunofluorescence technique. Among them, GFAP and Iba1 are markers of astrocytes and microglia, respectively, and their abnormal activation indicates an increase in the level of neuroinflammation. See example 2 for specific steps of immunofluorescence techniques.

The results of the experiment are shown in table 5 and fig. 4 to 5.

TABLE 5 detection results of positive cells of substantia nigra pars compacta GFAP and Iba1 of mice in each group

The result proves that the peucedanum praeruptorum dunn can obviously reduce neuroinflammation.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Claims

1. Application of Peucedanum praeruptorum dunn in preparing medicine for preventing and/or treating neurodegenerative diseases is provided.

2. The use of claim 1, wherein the neurodegenerative disease comprises at least one of: parkinson's disease, alzheimer's disease and Huntington's disease.

3. Application of peucedanum praeruptorum dunn in preparing medicine for preventing and/or treating neuroinflammation.

4. The use of claim 3, wherein the medicament has the effect of reducing the level of glial fibrillary acidic protein positive cells and/or ionic calcium binding adaptor 1 positive cells.

5. Application of Peucedanum praeruptorum dunn in preparing neuroprotective medicine is provided.

6. The use of claim 5, wherein the neuroprotection comprises restoration of dopaminergic neuron projection in the striatum or restoration of dopaminergic neuron apoptosis.

7. Application of peucedanum praeruptorum dunn in preparation of medicine for promoting dopamine synthesis.

8. The use according to claim 7, wherein the promotion of dopamine synthesis is by increasing tyrosine hydroxylase levels in the substantia nigra pars compacta or striatum.

9. The use of any one of claims 1-8, wherein the peucedanum praeruptorin has a structural formula shown in formula I;

10. the use according to any one of claims 1 to 8, wherein the medicament is in a dosage form comprising at least one of: tablet, powder, granule, capsule, oral liquid and sustained release agent.