Recovery article

Application of peptide MOTS-c in preparation of drug for ...

Application of peptide MOTS-c in preparation of drug for treating Parkinson's disease Download PDF Info Publication number CN113876929A CN113876929A CN202111389409.3A CN202111389409A CN113876929A CN 113876929 A CN113876929 A CN 113876929A CN 202111389409 A CN2

By Recovery & Performance Peptides Editorial Team Published January 2, 2026

Application of peptide MOTS-c in preparation of drug for treating Parkinson's disease Download PDF

Info

Publication number CN113876929A CN113876929A CN202111389409.3A CN202111389409A CN113876929A CN 113876929 A CN113876929 A CN 113876929A CN 202111389409 A CN202111389409 A CN 202111389409A CN 113876929 A CN113876929 A CN 113876929A Authority CN China Prior art keywords mots peptide parkinson rat rats Prior art date 2021-11-22 Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.) Granted Application number CN202111389409.3A Other languages Chinese (zh) Other versions CN113876929B (en Inventor 赛燕肖靖淞叶枫程晋张启夫单耀辉但国蓉张玺赵远鹏邹仲敏 Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.) Army Medical University Original Assignee Third Military Medical University TMMU Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.) 2021-11-22 Filing date 2021-11-22 Publication date 2022-01-04 2021-11-22 Application filed by Third Military Medical University TMMU filed Critical Third Military Medical University TMMU 2021-11-22 Priority to CN202111389409.3A priority Critical patent/CN113876929B/en 2022-01-04 Publication of CN113876929A publication Critical patent/CN113876929A/en 2024-02-23 Application granted granted Critical 2024-02-23 Publication of CN113876929B publication Critical patent/CN113876929B/en Status Active legal-status Critical Current 2041-11-22 Anticipated expiration legal-status Critical

Images

Classifications

- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
- A61K38/10—Peptides having 12 to 20 amino acids
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
- A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
- A61P25/16—Anti-Parkinson drugs

Landscapes

Health & Medical Sciences (AREA)
Life Sciences & Earth Sciences (AREA)
Bioinformatics & Cheminformatics (AREA)
Engineering & Computer Science (AREA)
Pharmacology & Pharmacy (AREA)
General Health & Medical Sciences (AREA)
Chemical & Material Sciences (AREA)
Animal Behavior & Ethology (AREA)
Medicinal Chemistry (AREA)
Public Health (AREA)
Veterinary Medicine (AREA)
Biomedical Technology (AREA)
Neurology (AREA)
Epidemiology (AREA)
Neurosurgery (AREA)
General Chemical & Material Sciences (AREA)
Psychology (AREA)
Chemical Kinetics & Catalysis (AREA)
Dermatology (AREA)
Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
Organic Chemistry (AREA)
Gastroenterology & Hepatology (AREA)
Immunology (AREA)
Proteomics, Peptides & Aminoacids (AREA)
Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Peptides Or Proteins (AREA)

Abstract

The invention relates to application of a peptide MOTS-c in preparation of a drug for treating Parkinson, and the peptide MOTS-c can reduce the level of brain oxidative stress injury in a rotenone-induced Parkinson rat animal model, protect dopaminergic neurons of a substantia nigra striatum of a rat midbrain, reduce the loss of the dopaminergic neurons, obviously improve the dopamine level of the striatum, and effectively improve the neurobehavioral symptoms of the Parkinson rat. Therefore, the peptide MOTS-c has important application significance in preparing anti-Parkinson drugs.

Description

Application of peptide MOTS-c in preparation of drug for treating Parkinson's disease

Technical Field

The invention relates to the technical field of medicines, and in particular relates to application of peptide MOTS-c in preparation of a medicine for treating Parkinson's disease.

Background

Parkinson's Disease (PD) is the second most common neurodegenerative disease worldwide, and its incidence increases with age, seriously affecting the health and quality of life of the elderly. The main pathological features of PD are the loss of the nigral dopaminergic neurons of the midbrain and the formation of Lewy bodies (Lewy bodies), with the decrease of striatal dopamine, the common clinical manifestations of resting tremor, muscular rigidity, bradykinesia, abnormal posture and gait, etc. appear, the symptoms are progressive, and in severe cases, the patient cannot take care of his own lives. At present, the etiology of the disease is not completely clear, and the disease is mainly considered to be the result of the mutual interaction of multiple factors such as heredity, environment and aging, and particularly the environmental factor plays an important role in sporadic PD pathogenesis. A large number of epidemic diseases and small animal experimental researches find that pesticide rotenone is related to the disease attack of PD, and a simulated PD animal model can be successfully constructed by a rotenone infected rat. Among the established pathogenesis of PD, oxidative stress is an important factor leading to impairment of dopaminergic neurons of the nigrostriatal body, and plays an important role in the pathogenesis of PD.

At present, PD is mainly based on drug therapy, and the common dopamine precursor levodopa can enter the brain through a blood brain barrier and supplement endogenous dopamine deficiency after being metabolized into dopamine so as to relieve the symptoms of PD. However, most patients have gradually declined curative effect after long-term administration, and various motor complications (including switching phenomenon, motility disorder and the like) appear, and the treatment difficulty is further increased. Although various methods of combination medication are adopted clinically to prolong the use time of the medicine, the good clinical treatment effect is still not achieved. Therefore, the search for new drugs for treating PD is an important research direction in neuroscience. With the deep understanding of the pathogenesis of PD, starting from multiple links, the emphasis on protecting dopaminergic neurons also becomes one of the research and development ideas of new medicaments for treating PD.

Mitochondrial Derived Peptides (MDPs) are bioactive short peptides encoded by open reading frames of Mitochondrial DNA (mtDNA), participate in the regulation of metabolic homeostasis of the body, and exert a wide range of physiological effects in the human body. The existing research indicates that MOTS-c, one of three endogenous MDPs known at present, can be involved in regulating the biomedical functions of mitochondria, influence the metabolic process mediated by the mitochondria and is an important target point for researching related diseases of the mitochondria. In addition, the peptide MOTS-c can protect cells under metabolic stress conditions, participate in processes such as glycolipid metabolism and the like, and play multiple roles in regulating insulin resistance, improving lipid deposition, regulating bone metabolism and the like. Moreover, the polypeptide drug has the advantages of high efficiency, high selectivity, wide action target, low potential toxicity and the like, so that the peptide MOTS-c has great research value and application prospect.

However, the mechanism by which the peptide MOTS-c acts has not been fully elucidated, and there is no report on the use of the peptide MOTS-c in the treatment of Parkinson's disease.

Disclosure of Invention

The invention aims to provide application of peptide MOTS-c in preparation of drugs for treating Parkinson, the peptide MOTS-c has a remarkable effect in treating Parkinson induced by rotenone, can reduce the oxidative damage level of the midbrain of a rat, protect dopamine neurons, remarkably reduce the loss of striatum dopamine neurons, improve the striatum dopamine content, further improve the neurobehavioral symptoms of the rat, and provide a new choice for drugs for treating Parkinson's disease.

The technical scheme of the invention is as follows:

the application of the peptide MOTS-c in preparing a medicament for treating Parkinson's disease.

The Parkinson is induced by rotenone.

The amino acid sequence of the peptide MOTS-c is shown in SEQ.ID.NO. 1.

The drug is a protective agent for dopaminergic neurons.

The drug increases striatal dopamine levels.

The medicine is administrated by an intraperitoneal injection mode.

The dosage of the medicine is 3-5 mg/kg/day.

The invention uses rotenone to induce a Parkinson rat model, the rotenone is administrated in a gradient way, the injection dosage of the first stage is 2mg/kg, and the administration lasts for 3 days. The second phase injection dose is 1mg/kg, and the administration lasts for 7 days. The third phase injection dose is 0.5mg/kg, and the administration lasts 20 days. The total administration time is 30 days, and a rotenone induced rat Parkinson animal model is established. The treatment group is administered to the model group by intraperitoneal MOTS-c injection for 30 days at a dose of 3-5 mg/kg.

The animal experiments of the applicant prove that the peptide MOTS-c can obviously improve the neurobehavioral symptoms of rotenone-induced Parkinson rats. The peptide MOTS-c can reduce the oxidative damage level in the midbrain of a Parkinson rat, further protect dopamine neurons of the rat, reduce the loss of striatal dopamine neurons, and obviously improve the content of dopamine in striatal bodies. In the aspect of the behaviourology, the autonomous exploration behavior and the movement coordination condition of the rat in a new environment are obviously improved. The suggestion that the peptide MOTS-c provides a new idea for treating PD.

Drawings

FIG. 1 is a graph showing the results of measuring the level of oxidative damage of the brain in rats, in which,

a is the detection result of the content of MDA (malondialdehyde); panel B is the result of measurement of GSH (glutathione) content; panel C shows the results of SOD (superoxide dismutase) activity (note: P <0.05, model vs. control;. P <0.01, model vs. control;. P <0.05, treatment vs. model)

FIG. 2 shows the results of HE staining of rat striatum, wherein the position indicated by the arrow is the nucleus.

FIG. 3 shows the results of rat striatal immunohistochemistry, wherein A is the result of striatal immunohistochemical staining (TH) for each group; b is the statistic of positive areas of TH in each group (note: P <0.05, model group compared with control group;. P <0.01, model group compared with control group;. P <0.05, treatment group compared with model group).

FIG. 4 detection results of rat striatal Western blot, in which TH is dopamine synthesis rate-limiting enzyme tyrosine hydroxylase, PSD95 is postsynaptic membrane marker, and SYP is presynaptic membrane marker.

FIG. 5 shows the results of the open field experiment, wherein A is the total distance of movement of the rat; b is a heat map of the rat's motion trajectory; c is the standing times of the hind limbs of the rat; d is the time to immobility (Note: P <0.05, model vs control;. P < 0.01; treatment vs model).

FIG. 6 shows the results of the rotarod experiment, wherein: p <0.05, model group compared to control group; p <0.01, model group compared to control group; # P <0.05, treatment group compared to model group.

Detailed Description

Materials and reagents

1. Animal(s) production

Healthy SD male rats (5-7 weeks old) weighing 220-270 g were purchased from animal center of China civil liberation army military medical university.

2. Reagent

Other reagents were commercially available analytical pure products.

3. Main instrument equipment

Second, the embodiment

Example 1 animal treatment

The peptide MOTS-c is synthesized by Wuhan Baiyixin biotechnology limited, the purity is more than 95%, and the amino acid sequence is shown in SEQ.ID.NO. 1:

Met Arg Trp Gln Glu Met Gly Tyr Ile Phe Tyr Pro Arg Lys Leu Arg。

SD male rats 44 with body weight of 220-270 g are kept in an animal room with indoor temperature of 23 + -2 deg.C, during which the rats are provided with free water and food. Rats were randomly divided into 3 groups: control, model and treatment groups. Model group (gradient dose subcutaneous injection): the first phase injection dose was 2mg/kg for 3 days. The second phase injection dose is 1mg/kg, and the administration lasts for 7 days. The third phase injection dose is 0.5mg/kg, and the administration lasts 20 days. The total administration time is 30 days, and a rotenone induced rat Parkinson animal model is established. Treatment groups: MOTS-c (3-5mg/kg) was injected intraperitoneally for 30 days while the model group was administered. After the treatment, the behavioral indexes of the rats including open field experiments and rotating rod experiments are detected. After the behavioral testing was completed, the rats were sacrificed under anesthesia, and the striatum (a portion fixed with paraformaldehyde and a portion quick frozen with liquid nitrogen) was rapidly isolated on ice and the remaining brain tissue was collected.

Example 2 Experimental testing of neurobehavioral testing of groups of rats

1) Open field experiment: the open field experiment is a method for evaluating the autonomous behavior of an experimental animal in a new environment and exploring behavior and tensity so as to evaluate whether the activity of the animal is normal and uniform. After adjusting the software and system parameters, the rats were placed in an open field reaction chamber and allowed to move freely for 10 minutes, and the system recorded all the movement records of the rats. After the experiment is finished, the software records and analyzes parameters such as the moving distance, the moving track, the hind limb standing times and the like (before each experiment, the reaction box needs to be wiped by alcohol to eliminate the influence of odor on the animals).

2) Rotating rod type fatigue tester experiment: the method is used for evaluating the balance force, the movement coordination, the central inhibition and the like of the animal by measuring the staying time of the animal on the roller. Half an hour before the start of the experiment, rats were placed in the laboratory and acclimated. The instrument rotation parameters were set so that it rotated at a uniform speed of 12 rpm. The experimental animals were placed on the roller in sequence and the duration of time each rat could remain in equilibrium on the roller was recorded, with a maximum duration of no more than 3 minutes.

EXAMPLE 3 rat striatal dopamine content (high resolution liquid chromatography-mass spectrometry)

Rat striatum was weighed and weight recorded, 1mL of methanol (containing 0.1% formic acid) was added precisely, vortexed for 1min, and homogenized for 3 min. Centrifuging at 14000rpm for 10min, and sampling the supernatant for analysis. Chromatography Waters T3 (150X 2.1mm, 3 μm). The flow rate is 0.3mL/min, the aqueous phase is 0.1% formic acid aqueous solution, the organic phase is 0.1% formic acid acetonitrile, the needle washing solution is methanol, and the temperature of the column incubator is 35 ℃.

Example 4 immunohistochemical staining

Striatum tissues were fixed with 4% paraformaldehyde, dehydrated, embedded in paraffin, sectioned, and 5 μm thick. Tissue sections were dewaxed and hydrated, antigen repaired, endogenous catalase blocked, antibody hybridization (TH antibody), DAB color development, counterstaining, dehydration, transparency and microscopic examination after mounting. Further scanning images by using a tissue slice digital scanner, automatically reading a tissue measurement area by using sevier image analysis software, and firstly, performing positive grade division: negative no coloration,

count

0 point; weak positive light yellow is counted as 1 point, and medium positive brown yellow is counted as 2 points; the strong positive brown color is counted as 3 points, and the ratio of the positive areas in the measurement area and the number of the reaction positive areas are finally analyzed and calculated.

Example 5HE staining

Striatum tissues were fixed with 4% paraformaldehyde, dehydrated, embedded in paraffin, sectioned, and 5 μm thick. And (3) after dewaxing and hydrating the slices, performing hematoxylin-eosin staining, hematoxylin staining nuclei, hydrochloric acid ethanol differentiation, eosin staining cytoplasm, and sealing the slices after transparentization according to the HE kit instruction. Striatal structure and lesion changes were observed under an optical microscope and photographed.

Example 6 immunoblotting

Extracting total protein from striatum tissues; carrying out protein concentration determination by using a BCA method; then, SDS-polyacrylamide gel electrophoresis, membrane transfer, blocking, primary antibody incubation overnight (4 degrees), corresponding species secondary antibody incubation (room temperature), chemiluminescence solution development. After photographing, grey value analysis was performed using Image J software.

Example 7 measurement of the content of GSH, MDA and SOD in rat brain tissue

GSH, MDA and SOD detection kits are purchased from Shanghai Biyuntian biotechnology limited company, and the operation steps are carried out according to the kit specification.

Results of three

1. Peptide MOTS-c can relieve the oxidative stress injury of midbrain of rotenone parkinsonian rats

The results show that the MDA content in the brains of the rats in the treatment group is reduced, and the GSH content and the SOD activity are improved, which indicates that the peptide MOTS-c has obvious relieving effect on the oxidative stress damage level in the brains of the rotenone parkinsonism rats (see figure 1).

2. Peptide MOTS-c for alleviating rotenone parkinsonian striatal dopamine neuron injury in rats

The HE staining results (see FIG. 2) show that the striatum tissue morphology of the treated group is basically regular, the cell morphology is close to normal although retraction is carried out, and the phenomenon of nucleus shrinkage is obviously improved. The peptide MOTS-c is shown to have obvious protective effect on striatal neurons of rotenone rats.

3. Peptide MOTS-c can reduce loss of striatal dopamine neurons in Parkinson rats

As shown in the figure (figure 3, figure 4), the positive coloration of striatum TH of the rat in the model group is obviously reduced, and meanwhile, the expression level of striatum TH protein is reduced, which indicates that dopaminergic neurons in the striatum of the Parkinson rat have obvious loss, and the positive coloration is also an important mark of PD. The number of TH positive cells and TH protein expression level of striatum of rats in the treatment group are obviously improved. In addition, the expression levels of the synaptic markers PSD95 and SYP of the striatal neurons in the treated group are remarkably increased compared with those in the model group, and the results indicate that MOTS-c has obvious protective effect on striatal dopamine neurons.

4. Peptide MOTS-c can increase striatal dopamine content of Parkinson rats

The results in table 1 show that the striatum DA content of the model group is significantly reduced, further illustrating the success of model replication; the peptide MOTS-c can obviously improve the striatum dopamine level.

Table 1: determination result of DA content in striatum of 3 groups of rats

(Note: P <0.05, model group compared to control group;. P <0.01, model group compared to control group;. P <0.05, treatment group compared to model group)

5. Peptide MOTS-c can improve exploration capacity of rotenone Parkinson rats

The open field experiment result is shown in fig. 5, the hind limb standing times of the model group rats are reduced, the movement distance is shortened, the rest time is increased, the autonomous exploration behavior capacity of the model group animals in a new environment is remarkably reduced, and the successful replication of the Parkinson model is also prompted. The indexes of the treatment groups are improved to a certain extent, and the fact that the administration of the peptide MOTS-c can improve the capability of autonomously exploring behaviors of the parkinsonian rat caused by rotenone in a new environment is suggested.

6. Peptide MOTS-c can improve the motor coordination capacity of rotenone parkinsonian rats

The rats in the model group have obvious motor coordination disorder and poor limb movement, the time of the rats moving on the rotating rod is obviously shortened, and even individual rats are extremely uncoordinated and difficult to continuously move on the rotating rod. The movement coordination capacity of the rats in the treatment group is obviously improved, and the time of activity on the rotating rod is obviously prolonged. The peptide MOTS-c was suggested to have a positive effect on the alleviation of motor coordination in Parkinson's rats (see FIG. 6).

In conclusion, the invention establishes a rotenone induced Parkinson rat model, and simultaneously provides peptide MOTS-c for intervention. The experimental result shows that the peptide MOTS-c can relieve the oxidative stress injury level of the brain of the Parkinson rats, protect dopamine neurons, reduce the loss of the dopamine neurons, increase the striatal dopamine content and improve the neurobehavioral symptoms of the Parkinson rats. Therefore, the peptide MOTS-c has obvious protective effect on the Parkinson rats. The peptide MOTS-c can be applied to the preparation of medicaments for treating the Parkinson disease.

Sequence listing

<110> China people liberation army, military and medical university

Application of <120> peptide MOTS-c in preparation of drug for treating Parkinson's disease

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 16

<212> PRT

<213> Artificial Synthesis (Artificial Synthesis)

<400> 1

Met Arg Trp Gln Glu Met Gly Tyr Ile Phe Tyr Pro Arg Lys Leu Arg

1 5 10 15

Claims (7)

1. The application of the peptide MOTS-c in preparing a medicament for treating Parkinson's disease.

2. Use according to claim 1, characterized in that: the Parkinson is induced by rotenone.

3. Use according to claim 1, characterized in that: the amino acid sequence of the peptide MOTS-c is shown in SEQ.ID.NO. 1.

4. Use according to claim 1, characterized in that: the drug is a protective agent for dopaminergic neurons.

5. Use according to claim 1, characterized in that: the drug can increase striatal dopamine content.

6. Use according to claim 1, characterized in that: the medicine is administrated by intraperitoneal injection.

7. Use according to claim 1, characterized in that: the dosage of the medicine is 3-5 mg/kg/day.

CN202111389409.3A 2021-11-22 2021-11-22 Application of peptide MOTS-c in preparation of medicament for treating parkinsonism Active CN113876929B (en)

Priority Applications (1)

Application Number	Priority Date	Filing Date	Title
CN202111389409.3A CN113876929B (en)	2021-11-22	2021-11-22	Application of peptide MOTS-c in preparation of medicament for treating parkinsonism

Applications Claiming Priority (1)

Application Number	Priority Date	Filing Date	Title
CN202111389409.3A CN113876929B (en)	2021-11-22	2021-11-22	Application of peptide MOTS-c in preparation of medicament for treating parkinsonism

Publications (2)

Publication Number	Publication Date
CN113876929A true CN113876929A (en)	2022-01-04
CN113876929B CN113876929B (en)	2024-02-23

Family

ID=79015472

Family Applications (1)

Application Number	Title	Priority Date	Filing Date
CN202111389409.3A Active CN113876929B (en)	2021-11-22	2021-11-22	Application of peptide MOTS-c in preparation of medicament for treating parkinsonism