CN111329995B - Application of GLP-2 analogue in preparing PD prevention and treatment medicines and PD prevention and treatment medicines - Google Patents
Application of GLP-2 analogue in preparing PD prevention and treatment medicines and PD prevention and treatment medicines Download PDFInfo
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- CN111329995B CN111329995B CN202010100236.8A CN202010100236A CN111329995B CN 111329995 B CN111329995 B CN 111329995B CN 202010100236 A CN202010100236 A CN 202010100236A CN 111329995 B CN111329995 B CN 111329995B
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Abstract
The invention discloses application of GLP-2 analogues in preparing a medicament for preventing and treating PD and a medicament for preventing and treating PD, wherein the medicament for preventing and treating PD is prepared by utilizing an action mechanism that the GLP-2 analogues can protect dopaminergic neuron cells caused by MPP +. Relates to the technical field of PD prevention and treatment medicines, and has the beneficial effects that: GLP-2 analogues can protect dopaminergic neuron cell damage caused by MPP +, and therefore have the potential of being developed into anti-PD drugs.
Description
Technical Field
The embodiment of the invention relates to the technical field of PD prevention and treatment medicines, in particular to application of GLP-2 analogues in preparing PD prevention and treatment medicines and medicines for preventing and treating PD.
Background
Parkinson's Disease (PD) is second only to Alzheimer's Disease, the second largest degenerative Disease of the central nervous system, first described by James Parkinson in 1817 by British physician, and suggested by Charcot in 1892 to refer to this Disease as PD. PD is manifested as blackThe major pathological features are severe degeneration, loss and formation of Lewy Bodies (LB) as inclusion bodies in the cytoplasm of the nucleus. Clinically, the symptoms are mainly static tremor, bradykinesia, muscular rigidity and gait disturbance of posture, and the symptoms can be accompanied by non-motor symptoms such as depression, constipation, sleep disturbance and the like. Human neuroblastoma cells (SH-SYSY,
CRL-2266) has many characteristics of dopaminergic neurons and is therefore widely used as a neuronal cell model for PD studies. Neurotoxin 1-methyl-4-phenylpyridine ion (1-methyl-4-pehnyl-pyridine, MPP +) is an active neurotoxic metabolite of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (l-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, MPTP), can produce a syndrome similar to PD in clinical and pathological terms, and is the most commonly used PD-mimicking toxin. MPTP and MPP + induced PD experimental models greatly promote the research progress of PD.
At present, two methods of stereotactic surgery treatment and drug treatment are clinically adopted for treating PD, the surgery mainly blocks a globus pallidus passage or a striatum globus pallidus passage of a subthalamic nucleus, the drugs mainly focus on anticholinergic drugs and drugs influencing dopaminergic function, but the pathological progress of PD cannot be prevented or reversed, the curative effect is limited, and the side effect is large.
Glucagon-like peptide-2 (GLP-2) is a 33 amino acid polypeptide that is processed by post-transcriptional and post-translational processing of pro-glucagon. Glucagon, when cleaved, releases both glucagon-like peptide-1 (GLP-1) and GLP-2. In recent years, GLP-1 analogs have been used in the treatment of diabetes, such as liraglutide. The GLP-2 analog apraglutide has been approved for the treatment of short bowel syndrome and the like.
Disclosure of Invention
The invention aims to overcome the defects in the prior art, namely to overcome the pathological process of PD, and the embodiment of the invention discloses application of GLP-2 analogues in preparing a medicament for preventing and treating PD and a medicament for preventing and treating PD.
The first aspect of the embodiment of the invention discloses application of GLP-2 analogues in preparing a medicament for preventing and treating PD.
Further, the GLP-2 analogues are derivatives derived from GLP-2 polypeptides.
Preferably, the amino acid sequence of the GLP-2 analog is HGDGSFSDEMSTILDNLATRDFINWLIQTKITD.
The second aspect of the embodiment of the invention discloses a medicament for preventing and treating PD, which is a preparation prepared by taking GLP-2 analogues as active substances.
Preferably, the amino acid sequence of the GLP-2 analog is HGDGSFSDEMSTILDNLATRDFINWLIQTKITD.
Preferably, the preparation is in the form of tablets, capsules, injections, aerosols or oral liquids.
The embodiment of the invention has the beneficial effects that:
PD is a common nervous system degenerative disease of middle-aged and elderly people, is clinically mainly manifested by resting tremor, bradykinesia, muscular rigidity and postural gait disturbance, and can be accompanied by non-motor symptoms such as depression, constipation and sleep disturbance, and the like, and is mainly characterized by progressive death and loss of dopaminergic neurons in the nigrostriatal system. The invention discovers that the GLP-2 analogue can protect dopaminergic neuron cell damage caused by MPP +, thereby having the potential of developing anti-PD drugs.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without making any creative effort based on the embodiments in the present invention, belong to the protection scope of the present invention.
The first aspect of the embodiment of the invention discloses application of GLP-2 analogues in preparing a PD prevention and treatment drug.
It is to be noted that GLP-2 analogs are derivatives derived from GLP-2 polypeptides.
The amino acid sequence of the GLP-2 analogue is HGDGSFSDEMSTILDNLATRDFINWLIQTKITD.
The second aspect of the embodiment of the invention discloses a medicament for preventing and treating PD, which is a pharmaceutically acceptable preparation formulation prepared by taking GLP-2 analogue as an active substance.
The amino acid sequence of the GLP-2 analogue is HGDGSFSDEMSTILDNLATRDFINWLIQTKITD.
The preparation can be tablet, capsule, injection, aerosol or oral liquid.
The application of GLP-2 in PD drugs and the drugs for preventing and treating PD are not researched and reported. We find that GLP-2 can protect SH-SY5Y nerve cells from cell activity reduction, mitochondrial damage, autophagy damage and apoptosis caused by MPP +, so that the GLP-2 has the potential of being developed into a medicament for preventing and treating PD.
Glucagon-like peptide-2 (glp-2), a polypeptide consisting of 33 amino acids, is secreted and synthesized by L cells in the intestinal tract and is also produced in discrete neuronal clusters in the brainstem and hypothalamus. GLP-2 has a short half-life in blood circulation (only about 7min in human body) because dipeptidyl peptidase 4 (DPP-4) hydrolyzes the alanine residue at the second amino acid of the end of GLP-2, and the inventor prolongs the biological half-life in vivo by changing the amino acid position.
The discovery and verification process of the application of the GLP-2 analogue in preparing the PD prevention and treatment medicine is as follows:
1. experimental Material
MPP + was purchased from Sigma; DMEM/F-12 medium and fetal bovine serum were purchased from Gibco; the detection principle of the MTT cell proliferation detection reagent is that exogenous MTT is reduced into water-insoluble purple blue crystal formazan through succinate dehydrogenase in mitochondria of living cells and is deposited in the cells, the formazan can be dissolved by dimethyl sulfoxide (DMSO), and the quantity of the living cells is indirectly reflected by measuring a light absorption value at a wavelength of 570nm by an enzyme-linked immunosorbent detector. In a certain cell number range, the formazan formation amount is in direct proportion to the cell number. The GLP-2 analogue is synthesized by Shanghai Qianzhizao biotechnology limited, and the amino acid sequence is as follows: HGDGSFSDEMSTILDNLATRDFINWLIQTKITD; liraglutide was synthesized by shanghai gahnje biotechnology limited.
Preparation of MPP + solution: 100mg MPP + powder was dissolved in sterile PBS, filtered through a 0.22 μm sterile filter, dispensed and stored in a refrigerator at-20 ℃ with the stock solution (PMA from Sigma) at a concentration of 100nM.
Preparation of GLP-2 analogue and liraglutide solutions: GLP-2 or liraglutide powder was dissolved in sterile double distilled water, and the resulting solution was stored at-20 ℃ in a refrigerator at a stock solution (PMA from Sigma) concentration of 1mM.
2. Experimental method
1. Cell culture
Taking out human neuroblastoma cell SH-SY5Y stored in liquid nitrogen for freezing, rapidly placing in 37 deg.C water bath, gently shaking until cell thawing, centrifuging at 1000rpm for 5min, discarding supernatant, adding small amount of culture medium containing 10% fetal calf serum and 1% double-antibody DMEM/F-12 culture medium, transferring cell into cell bottle, and diluting with CO at 37 deg.C and 5% 2 And culturing under the saturated humidity condition.
2. Preparation of cell model
SH-SY5Y cells in logarithmic growth phase are prepared to have a concentration of 3.0X 10 5 Each/mL cell suspension was inoculated into 100. Mu.L of a 96-well cell plate, and after 24 hours of culture, the plate was replaced with media containing 0.5mM, 1mM, 2mM, and 4mM MPP +, respectively, and culture was continued for 24 hours with 5 wells per well. MTT cell proliferation detection reagent is adopted to detect cell activity, and the minimum MPP + concentration (2 mM) capable of obviously inducing cell damage is selected to prepare a PD cell model.
3. Cell viability assay
SH-SY5Y cells in logarithmic growth phase are prepared to have a concentration of 3.0X 10 5 Each/mL cell suspension was inoculated into a 96-well cell plate at 100. Mu.L per well, and after 12 hours of culture, 50. Mu.L of each drug diluted in a medium (100 nM liraglutide, 1nM GLP-2, 10 nM GLP-2, 100nM GLP-2 and 200nM GLP-2, respectively) was directly added to each experimental group, and 50. Mu.L of each medium was directly added to each of the control group and the model group. After 12h of treatment, 10. Mu.L of MPP + with the appropriate concentration was added to each experimental group to a final concentration of 2mM, and 10. Mu.L of PBS was added to the control group. Each group is provided with 5 multiple holes.
And continuously culturing for 24h according to the above groups, and detecting the cell activity by using an MTT cell proliferation detection reagent. The cell viability was calculated by taking the average of the OD values of 5 replicate wells and the control group was recorded as 100%.
4. Data processing and analysis
The cell survival rate of each group is calculated by EXCEL software, and single factor method analysis among multiple groups is carried out by Prism 7 software One-way ANOVA, and the difference is P <0.05, which has statistical significance.
3. Results of the experiment
Cell viability for each group is shown in the table (note: compare # P <0.05, # P <0.01, # P <0.001 for control group; P <0.05, # P <0.01, # P <0.001 for model group).
TABLE 1 Effect of GLP-2 on SH-SY5Y cell and MPP + -induced SH-SY5Y cell proliferative Activity
Therefore, the results in table 1 show that 100nM (nmol/L for short) GLP-2 analogues can significantly protect MPP + induced cell damage, and have potential for developing anti-PD drugs.
Finally, it should be noted that: although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that modifications may be made to the embodiments or portions thereof without departing from the spirit and scope of the invention.
Claims (2)
1. An application of GLP-2 analogue in preparing medicine for treating MPP + induced dopaminergic neuron injury is characterized in that the GLP-2 analogue is a preparation prepared by taking the GLP-2 analogue as an active substance, and the amino acid sequence of the GLP-2 analogue is HGDGSFSDEMSTILDNLATRDFINWLIQTKITD.
2. The use of a GLP-2 analogue of claim 1 in the manufacture of a medicament for the treatment of MPP + induced dopaminergic neuronal damage, wherein the formulation is in the form of a tablet, a capsule, an injection, an aerosol or an oral liquid.
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104540850A (en) * | 2012-05-03 | 2015-04-22 | 西兰制药公司 | Glucagon-like-peptide-2 (GLP-2) analogues |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104540850A (en) * | 2012-05-03 | 2015-04-22 | 西兰制药公司 | Glucagon-like-peptide-2 (GLP-2) analogues |
Non-Patent Citations (3)
| Title |
|---|
| A GLP-2 Analogue Protects SH-SY5Y and Neuro-2a Cells Against Mitochondrial Damage, Autophagy Impairments and Apoptosis in a Parkinson Model.;Yunfang等;《Drug Res》;20201006;43–50 * |
| GLP-2对LPS诱导的小胶质细胞活化的影响及机制;栗楠;《硕士电子期刊出版信息》;20161115;摘要 * |
| Novel Long-Acting GLP-2 Analogue, FE 203799 (Apraglutide), Enhances Adaptation and Linear Intestinal Growth in a Neonatal Piglet Model of Short Bowel Syndrome with Total Resection of the Ileum;George M. Slim等;《Journal of Parenteral and Enteral Nutrition》;20181231;1-8 * |
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