CN111346216A - Composition for treating cerebral apoplexy and application thereof - Google Patents
Composition for treating cerebral apoplexy and application thereof Download PDFInfo
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- CN111346216A CN111346216A CN202010122676.3A CN202010122676A CN111346216A CN 111346216 A CN111346216 A CN 111346216A CN 202010122676 A CN202010122676 A CN 202010122676A CN 111346216 A CN111346216 A CN 111346216A
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Abstract
The invention provides an application of TDL23 peptide in preparing a medicament for treating ischemic cerebrovascular diseases, and an application of TDL23 peptide and Conservative Dopaminergic Neurotrophic Factor (CDNF) in treating cerebral apoplexy. The invention provides a new targeted medicine for clinical prevention and treatment of cerebral apoplexy, and proves the potential huge application of the TDL23 peptide and CDNF composition in clinical prevention and treatment of ischemic cerebral apoplexy.
Description
Technical Field
The invention relates to the field of biological medicines, and particularly relates to a composition for treating cerebral apoplexy and application thereof.
Background
Neurotrophic factors (NTFs) are polypeptides or protein factors produced by the body and capable of promoting the survival, growth and differentiation of nerve cells, regulating the survival of neurons in the development process, preventing the death of mature neurons after injury, and promoting the repair and axon regeneration of neurons, regulating synaptic plasticity, neurotransmitter transmission and other nervous system protection functions. Currently known neurotrophic factors include: conservative Dopaminergic Neurotrophic Factor (CDNF), Nerve Growth Factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3 ), neurotrophin-4 (NT-4), glial-derived neurotrophic factor (GDNF), and the like. Different neurotrophic factors act on different specific types of subpopulations of neurons. Because of their trophic effects on neurons, neurotrophic factors are not only potential therapeutic factors for neurodegenerative diseases including parkinson's disease, alzheimer's disease, lateral sclerosis, but also for neurotrauma such as spinal cord injury.
TDL23 peptide is TDL23 peptide derived from extracellular matrix phosphoglycoprotein (MEPE) and containing 23 amino acids, and can be used as polypeptide for bone metabolism related application, and is beneficial to bone formation and cartilage formation.
Stroke can cause endoplasmic reticulum stress. The cerebral apoplexy is one of the most common clinical diseases, has the characteristics of high morbidity, disability rate and mortality rate, and seriously threatens the health of human beings, wherein the ischemic cerebral apoplexy accounts for 70-80 percent of the total cerebral apoplexy. Because the pathogenesis of the stroke is different from that of the diseases such as Parkinson's disease, Alzheimer's disease, lateral sclerosis and the like, no effective method is available clinically at present for treating the stroke, and the reconstruction and recovery of the nerve function are still the difficult problems of treating the stroke.
The use of combinations of TDL23 and CDNF for the treatment of stroke is not known in the prior art.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a medicament for treating cerebral apoplexy.
In order to achieve the purpose, the invention adopts the technical scheme that: the application of TDL23 peptide in preparing medicine for treating ischemic cerebrovascular disease.
Preferably, the TDL23 peptide has an amino acid sequence as set forth in SEQ ID NO: 1 is shown.
Preferably, the ischemic cerebrovascular disease is stroke.
Preferably, the pharmaceutical composition further comprises an aqueous solution and one or more pharmaceutically acceptable excipients, additives, carriers or adjuvants.
The invention also provides a pharmaceutical composition for treating cerebral apoplexy, which comprises TDL23 peptide and Conservative Dopaminergic Neurotrophic Factor (CDNF).
Preferably, the TDL23 peptide has an amino acid sequence as set forth in SEQ ID NO: 1 is shown.
Preferably, the pharmaceutical composition further comprises an aqueous solution and one or more pharmaceutically acceptable excipients, additives, carriers or adjuvants.
The invention has the beneficial effects that: the inventor of the application finds the application of the TDL23 peptide and CDNF composition in treating ischemic stroke through research. The application provides a new targeted drug for clinical prevention and treatment of cerebral apoplexy, and proves the potential huge application of the TDL23 peptide and CDNF composition in clinical prevention and treatment of ischemic cerebral apoplexy.
Drawings
FIG. 1 shows the purity of HPCL, a TDL23 peptide synthesized by the invention.
FIG. 2 is a graph showing the mortality of different groups of MCAO rats in example 2.
FIG. 3 is a graph of mortality for different groups of neurological scores in example 2.
FIG. 4 is a photograph of cerebral infarct size of MCAO rats of different groups in example 2 (wherein A is a model control group, B is TDL23 group, C is CDNF group, and D is TDL23+ CDNF group).
FIG. 5 is a graphical representation of the cerebral infarct size of different groups of MCAO rats in example 2.
FIG. 6 is a schematic representation of the serum biochemical indicator MDA of MCAO rats of different groups in example 2.
Detailed Description
In order to more concisely and clearly demonstrate technical solutions, objects and advantages of the present invention, the following detailed description of the present invention is provided with reference to specific embodiments and accompanying drawings.
EXAMPLE 1 Synthesis of polypeptide TDL23
TDL23 peptide entrusts Shanghai soaring biotechnology Limited company, adopts conventional solid phase technology to synthesize, and synthetic peptide purity is > 95% (see figure 1), synthesizes 500mg, and its amino acid sequence is: TDLQERGDNDISPFSGDGQPFKD are provided.
CDNF was purchased from R & D company, lot number: RRG 0319061.
Example 2 therapeutic Effect of TDL23 and CDNF composition on the rat ischemic stroke model of MCAO
1. Experimental methods
Molding: 70 male SPF SD rats of 6-7 weeks old are selected, the weight of the rats is 190.2-218.2 g, after the rats are fasted and water is not forbidden for 12 hours, the rats are inhaled and anesthetized with 2% isoflurane, and the rats are fixed on an operating table in a supine position. The neck skin was cut open, the subcutaneous tissue and muscles were bluntly isolated, the Common Carotid Artery (CCA) was exposed, and the Internal Carotid Artery (ICA) and External Carotid Artery (ECA) continued to be free, with a ligature placed on each of the ICA and ECA. Cutting a small opening at 45 degrees in an oblique way by using an eye scissors (about 2mm away from the bifurcation of the common carotid artery) on CCA, introducing a embolus line, ligating a suspended line which is reserved in the common carotid artery to reduce bleeding, adjusting the direction and the angle of the embolus line, slightly pushing the embolus line, inserting the embolus line into the ICA, loosening an artery clamp on the ICA, and continuously inserting the embolus line until the start part of MCA (stopping when resistance is met) is reached, wherein the length is about 18-20 mm, and the middle cerebral artery blood supply interruption (MCAO) is formed. And (5) tightening the fixing thread by the double knots, suturing the skin after confirming no bleeding, exposing the tail part of the fixing thread out of the skin, and fixing the thread. After 1.5h, the plug thread is softly and slowly drawn out by about 10mm from the tail part of the plug thread exposed out of the skin, blood flow reperfusion is carried out, and the residual tail end of the plug thread is cut off; and (5) returning the animals to cages for breeding.
Grouping: after the animals were awake, the animals were scored for neurological function, and the rats with successful models were selected and randomly divided into 5 groups by body weight, which were a sham operation control group, a model control group, a TDL23 group, a CDNF group, and a TDL23+ CDNF group, respectively, and the number of animals in each group is shown in table 1.
Administration: the thread plug is pulled out to recover the blood flow, and after 1.5h of reperfusion, the corresponding medicines are respectively given to each group for treatment. TDL23 group was injected via tail vein; the CDNF group adopts intracerebral injection, the intracerebral injection is carried out by means of a brain locator, the CDNF group is injected into three cortical parts distributed in the middle cerebral artery in the brain, and the coordinates of the CDNF group positioned on the skull in a three-dimensional way are as follows: AP (before and after bregma): +1.2mm (site 1), -0.3mm (site 2), -1.8mm (site 3); ML (left and right meet): +5.5 mm; DV (cranial (dura) plane down): -3.5 mm. mu.L of CDNF was injected using a 10. mu.L Hamilton syringe and a 30G blunt needle, and the needle was lifted 2mm at the time of injection. Syringe infusion rate: each site was 0.5. mu.L/min, 1. mu.L/spot. The needle was slowly removed 2 minutes after completion of each injection. The test substances, the dose and the frequency of administration are shown in Table 1.
TABLE 1 rat MCAO model groups dosing regimen
Evaluation index
General observations were: the overall state of the animals is observed, and after 24 hours of treatment, the mortality rate is counted.
And (3) nerve function scoring: before grouping and after 24 hours of treatment, neurological grading is carried out according to animal motor and other behavioral manifestations. Grading standard:
0 point no symptoms of nerve damage;
1 minute of mild neurological impairment, incomplete extension of the contralateral forepaw or delayed/absent pain retraction;
2, dividing the degree of focal neurological deficit, and turning to the outside;
3, pouring the severe focal neurological deficit to the opposite side;
walking was not spontaneous in 4 minutes, and the level of consciousness decreased or lost.
Biochemical determination of serum blood: after 24 hours of treatment, 3.5ml/kg of 10% chloral hydrate was injected intraperitoneally to anesthetize the test animals, and the abdominal aorta was bled until death due to blood loss. Centrifuging blood at 3000/r for 10min, separating serum, and storing at 4 deg.C. The MDA (malondialdehyde) kit (Shanghai Biyuntian biotechnology limited company, specification: 100T) is adopted to measure the MDA content in serum.
Cerebral infarction volume measurement: after blood is taken, 6 rats in each group are randomly taken, brains are taken out, liquid nitrogen is placed into the rats for fixation for 5-7 s, the rats are taken out and placed in a mold, 4 knives are coronal-incised after visual crossing, the 1 st knife is arranged between the anterior pole of the brain and the visual crossing connecting line, the 2 nd knife is arranged at the visual crossing position, the 3 rd knife is arranged at the funnel handle position, the 4 th knife is arranged between the funnel handle and the posterior leaf tail pole, the thickness is about 2mm, brain slices are quickly placed in phosphoric acid buffer solution of 2% triphenyltetrazolium chloride (TTC) to be incubated for 30min in a dark place at 37 ℃, and the brain slices are turned over once every 3-5 min in. The digital camera shoots and the Image pro-Plus Image analysis software is used for analysis to calculate the percent of cerebral infarction. The normal tissue was stained red, and the cerebral infarction tissue was white.
Data statistics
Data statistics were performed using GraphpadPrism software and all measurements were expressed as means plus minus standard deviation (x ± s). The comparison of the means between groups is carried out by One-Way analysis of variance (One-Way ANOVA), two-by-two comparison of the means between groups, and t-test or Dunnett's T3.
2. Results of the experiment
2.1 Effect of TDL23 and CDNF composition on mortality of MCAO rats
After 24h of treatment, the mortality of each treatment group is shown in fig. 2, and it can be seen that, compared with the model control group, the TDL23, CDNF, TDL23+ CDNF treatment group all significantly reduced the mortality of MCAO rats, and the TDL23+ CDNF group had more significant effects.
2.2 Effect of TDL23 and CDNF composition on the neural function of MCAO rats
After 24h of treatment, compared with the model control group (2.89 +/-0.67), the peptide TDL23 treatment group (1.86 +/-0.63), the CDNF treatment group (1.23 +/-0.57) and the TDL23+ CDNF treatment group (0.64 +/-0.35) all reduced the neurological score of MCAO rats, and obvious statistical differences (TDL23 group P < 0.05, CDNF group P < 0.05, TDL23+ CDNF group P < 0.01) suggest that the peptides TDL23, CDNF, TDL23+ CDNF treatment can reduce the neurological score of MCAO rats. Compared with the TDL23 group and the CDNF group, the TDL23+ CDNF group was reduced more and statistically significant (P < 0.05), suggesting that TDL23 and CDNF may synergistically reduce the neurological score of MCAO rats, as shown in fig. 3.
2.3 Effect of TDL23 and CDNF composition on the cerebral infarct size of MCAO rats
After modeling, compared with a sham operation group, the cerebral infarction area of the MCAO rat of the model control group is obviously increased (P is less than 0.01). After 24h of treatment, compared with a model control group (32.15 +/-5.38), the peptide TDL23 treatment group (19.27 +/-5.39), the CDNF treatment group (15.76 +/-6.53) and the TDL23+ CDNF treatment group (8.27 +/-3.46) all reduced the cerebral infarction area of MCAO rats, and obvious statistical differences (TDL23 group P is less than 0.05, CDNF group P is less than 0.05, TDL23+ CDNF group P is less than 0.01) suggest that the peptide TDL23, CDNF, TDL23+ CDNF treatment can reduce the cerebral infarction area of the MCAO rats. Compared with the TDL23 group and the CDNF group, the TDL23+ CDNF group is reduced more and has obvious statistical significance (P < 0.05 or P < 0.01), and the TDL23 and the CDNF can synergistically reduce the cerebral infarction area of MCAO rats, as shown in figures 4 and 5.
2.4 Effect of TDL23 and CDNF composition on MCAO rat serum biochemical index MDA
After the model is made, compared with a false operation group, the serum biochemical index MDA concentration of the model control group is obviously increased (P is less than 0.01). After 24h of treatment, compared with a model control group (1.86 +/-0.57), the peptide TDL23 treatment group (1.15 +/-0.27), the CDNF treatment group (0.96 +/-0.28) and the TDL23+ CDNF treatment group (0.38 +/-0.17) all reduced the MDA level of MCAO rats, and obvious statistical differences (TDL23 group P is less than 0.05, CDNF group P is less than 0.05, TDL23+ CDNF group P is less than 0.01) suggest that the peptide TDL23, CDNF and TDL23+ CDNF treatment can reduce the MDA level in the blood serum of the MCAO rats. Compared with the TDL23 group and the CDNF group, the TDL23+ CDNF group is reduced more and has statistical significance (P is less than 0.05), and TDL23 and CDNF can synergistically reduce the MDA level in the serum of MCAO rats, and the figure 6 shows that.
The research results are combined, and the conclusion can be drawn that the TDL23 and CDNF composition has obvious synergistic treatment effect on a rat MCAO model and is likely to be developed into a novel drug combination for treating ischemic stroke.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
SEQUENCE LISTING
<110> Guangzhou Zhicheng medical science and technology Limited
<120> composition for treating cerebral apoplexy and application thereof
<130>2.12
<160>1
<170>PatentIn version 3.3
<210>1
<211>23
<212>PRT
<213> Artificial Synthesis
<400>1
Thr Asp Leu Gln Glu Arg Gly Asp Asn Asp Ile Ser Pro Phe Ser Gly
1 5 10 15
Asp Gly Gln Pro Phe Lys Asp
20
Claims (7)
- An application of TDL23 peptide in preparing the medicines for treating ischemic cerebrovascular disease is disclosed.
- 2. The use of claim 1, wherein the TDL23 peptide has an amino acid sequence as set forth in SEQ ID NO: 1 is shown.
- 3. The use of claim 1, wherein the ischemic cerebrovascular disease is stroke.
- 4. The composition of claim 3, wherein the pharmaceutical composition further comprises an aqueous solution and one or more pharmaceutically acceptable excipients, additives, carriers, or adjuvants.
- 5. A pharmaceutical composition for treating stroke, comprising TDL23 peptide and a conserved dopaminergic neurotrophic factor.
- 6. The composition of claim 5, wherein the TDL23 peptide has an amino acid sequence as set forth in SEQ ID NO: 1 is shown.
- 7. The composition of claim 4, wherein the pharmaceutical composition further comprises an aqueous solution and one or more pharmaceutically acceptable excipients, additives, carriers, or adjuvants.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20030166239A1 (en) * | 2000-02-29 | 2003-09-04 | Brown Thomas A. | Mammalian osteoregulins |
| US20080145412A1 (en) * | 2006-04-17 | 2008-06-19 | Acologix, Inc. | Method of promoting angiogenesis |
| CN107149673A (en) * | 2017-05-18 | 2017-09-12 | 山东大学 | Applications of the neurotrophic factor CDNF in ICVD medicine is prepared |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20030166239A1 (en) * | 2000-02-29 | 2003-09-04 | Brown Thomas A. | Mammalian osteoregulins |
| US20080145412A1 (en) * | 2006-04-17 | 2008-06-19 | Acologix, Inc. | Method of promoting angiogenesis |
| CN107149673A (en) * | 2017-05-18 | 2017-09-12 | 山东大学 | Applications of the neurotrophic factor CDNF in ICVD medicine is prepared |
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