CN113018305A - Application of compound in preparation of medicine for treating Alzheimer's disease - Google Patents
Application of compound in preparation of medicine for treating Alzheimer's disease Download PDFInfo
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- CN113018305A CN113018305A CN201911341541.XA CN201911341541A CN113018305A CN 113018305 A CN113018305 A CN 113018305A CN 201911341541 A CN201911341541 A CN 201911341541A CN 113018305 A CN113018305 A CN 113018305A
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Abstract
The invention provides application of a compound in preparation of a medicine for treating Alzheimer's disease, and belongs to the field of biological medicines. The compound provided by the invention can obviously reduce the expression of SH-SY5Y/APPswe cell beta-Amyloid Precursor Protein (APP) and A beta1‑42Can be applied to the preparation of the medicine for treating the Alzheimer disease.
Description
Technical Field
The invention belongs to the field of biological medicines, and relates to an application of a compound in preparation of a medicine for treating Alzheimer's disease.
Background
Myrobalan, a dried mature fruit of myrobalan Terminalia chebula retz, family quisqualaceae. Distributed in Yunnan province and the like. Has the effects of astringing intestines to check diarrhea, astringing lung to relieve cough, reducing pathogenic fire and relieving sore throat. It is commonly used for chronic diarrhea and dysentery, hematochezia and rectocele, cough and dyspnea due to lung deficiency, chronic cough, pharyngalgia and hoarseness. The chebulanic acid is one of the most important chebulan tannin components, and the prior research shows that the chebulan acid has the activities of antioxidation, antibiosis, antivirus, anti-inflammation and the like.
Senile dementia is a serious cognitive dysfunction, and Alzheimer's Disease (AD) is one of the most common diseases causing dementia in senile diseases, and accounts for 75 percent of all dementia patients[1]. Alzheimer's disease is a heterogeneous and pathophysiologically very complex neurodegenerative disease. In many cases, the disease is polygenic (sporadic AD), and in few cases, the disease is monogenic (0.5% autosomal dominant AD), and the clinical manifestations of AD are accompanied by cognitive impairment and dysfunction, and the ability to live independently is lost. Cognitive dysfunction increases with age and its incidence increases with age[2,3]. At present, the morbidity of Chinese AD patients is greatly increased along with the aging of Chinese population, which is a great choice in the medical field of the elderlyOne of war. The excavation of effective preventive or therapeutic drugs has become one of the major jobs of medical workers today. At present, Abeta in cerebrospinal fluid1-42Has been widely considered as a core biomarker for detecting the neuropathological characteristics of AD in living bodies, and the APP protein obtains Abeta by shearing two kinds of shearing enzymes, namely beta-secretase and gamma-secretase1-42Protein[4]。
Human neuroblastoma cells (SH-SY5Y) stably transfected with Swedish mutant amyloid precursor protein (APPsw), namely SH-SY5Y/APPsw (abbreviated as SA cells), can highly express APP protein, and generate A beta after being sheared by specific enzyme1-42Can simulate Abeta in AD pathological model1-42The process of metabolism, the model has been used for years for anti-AD drug screening and the research of the metabolic mechanism thereof[5]. Compound priming by decreasing APP protein expression and Abeta1-42The compound is evaluated to have anti-AD effect on the basis of an SA cell model, and the more obvious the reduction effect is, the more obvious the anti-AD effect is.
The invention utilizes SH-SY5Y/APPswe cells to inhibit APP protein expression and Abeta of a compound1-42The generation screening shows that the compound of the invention can obviously reduce APP protein expression and Abeta1-42The compound has obvious effect of resisting Alzheimer disease, so the invention aims to provide the application of the compound in preparing the medicine for resisting Alzheimer disease.
Reference to the literature
[1]Dubois B,Feldman HH,Jacova C,Cummings JL,DeKosky ST,Barberger-Gateau P,et al.Revising the definition of Alzheimer’s disease:a new lexicon.Lancet Neurol 2010；9:1118–27.
[2]Kumar,A.,Singh,A.,&Ekavali.(2015).A review on Alzheimer's disease pathophysiology and its management:an update.Pharmacol Rep,67(2),195-203.doi:10.1016/j.pharep.2014.09.004
[3]Ikonomovic,M.D.,Mi,Z.,&Abrahamson,E.E.(2017).Disordered APP metabolism and neurovasculature in trauma and aging:Combined risks for chronic neurodegenerative disorders.Ageing Res Rev,34,51-63.doi:10.1016/j.arr.2016.11.003
[4]Blennow K,Zetterberg H.The past and the future of Alzheimer’s disease fluid biomarkers.J Alzheimers Dis 2018；62:1125–40
[5]Masters,C.L.,Bateman,R.,Blennow,K.,Rowe,C.C.,Sperling,R.A.,&Cummings,J.L.(2015).Alzheimer's disease.Nat Rev Dis Primers,1,15056.doi:10.1038/nrdp.2015.56
The invention content is as follows:
the invention aims to disclose application of a compound shown in the formula (I) or a pharmaceutically acceptable salt in preparing a medicament for treating Alzheimer's disease.
The invention also discloses application of the compound shown in the formula (I) as an effective component in preparing a medicament for treating the Alzheimer disease.
Preferably, the compound is added with pharmaceutically acceptable carriers and/or auxiliary materials, and can be prepared into any one dosage form of tablets, capsules, granules, powder, pills, oral liquid, injection and suspension.
Preferably, the cell administration dose of the compound is 10-80 ug/ml.
The invention takes the human neuroblastoma cell of high expression Swedish mutant amyloid precursor protein as an Alzheimer disease research model for experiment, and the result shows that the compound provided by the invention can obviously inhibit the APP expression level in SA cells and can also inhibit A beta1-42Protein generation, thereby being applied to the preparation of the medicine for inhibiting the Alzheimer disease.
The technical solution of the present invention is further described in detail with reference to the following specific examples, but the scope of the present invention is not limited to the following examples.
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FIG. 1 Effect of Compounds on SA cell APP, BACE1 protein expression
Detailed Description
Medicine terminalia fruitAcid, also known as myrobalan tannic acid, english name: chebulinic acid; CAS No.: 18942-26-2; molecular formula C41H32O27(ii) a The molecular weight is 956.68g/mol, and the myrobalic acid is purchased from Doctoreis Biotechnology Co.
Example A creation and culture of an AD cell model, SA cells
SH/SY5Y cells were purchased from ATCC (American Type Culture Collection) and used as a model for evaluating the anti-AD activity of compounds by constructing SA cells, which are cell lines highly expressing APPswe and stably transforming Swedish mutant amyloid precursor protein. The SA cell complete culture medium condition is DMEM medium containing 10% fetal calf serum and 1% double antibody. When the cell density reaches about 90%, removing the culture medium, adding 3ml of 0.01mol/L PBS, washing for 2 times, removing the cells, adding 1ml of pancreatin, digesting for 2min, allowing the cells to fall off from the culture dish, centrifugally collecting the cells, adding 4ml of culture medium, uniformly blowing, counting the cells, and adjusting to obtain the cell density of 1 × 106Cell suspension in ml.
Examples Effect of two Compounds on SA cell APP protein expression
By using SH-SY5Y/APPsw cell line (namely SA cell line), the cell line highly expresses APP/swe protein, and by investigating the activity of the drug on cells, the safe concentration range of the drug action is screened, and after the drug is used in the safe range to act on the cells, if the drug can reduce the expression of the APP protein, the drug has a therapeutic effect on the high expression of the APP/swe mutant protein.
SA cell drug administration treatment and protein extraction
9.5mg of the compound powder was weighed and dissolved in 1ml of DMSO to obtain a compound stock solution having a concentration of 9.5 mg/ml. Mu.l of the stock solution was added with 990. mu.l of the complete cell culture medium and mixed well to obtain a compound solution with a concentration of 95. mu.g/ml, 168. mu.l, 84. mu.l and 21. mu.l of the compound solution with a concentration of 95. mu.g/ml were added with the complete cell culture medium to 4ml, respectively, to obtain compound solutions with concentrations of 4.0. mu.g/ml (H), 2.0. mu.g/ml (M) and 0.5. mu.g/ml (L), respectively.
After 500. mu.l of the cell suspension of example was supplemented with 1.5ml of complete medium, 6-well plates were placed in the cell incubator overnight until the cells were completely adherent. According to the result of the activity inhibition effect of the compound on SA cells, the compound is selected to be administered in a drug concentration gradient of 0.5 mug/ml (L), 2.0 mug/ml (M), 4.0 mug/ml (H), and the influence of the compound on the SA cell APP protein expression is examined after 48 hours of treatment. After the treatment time is over, collecting cells, adding a certain volume of cell lysate to lyse the cells, centrifuging at 12000g for 10min to collect supernatant, adding 5 Xloading Buffer metal bath for denaturation at 100 ℃ for 10min, detecting expression variable bands of APP protein by SDS-PAGE electrophoresis, analyzing the relative gray values of the bands of each treatment group by using Image J software after collecting the result, and evaluating the protein expression level by comparing the relative gray values with the values of the non-administration groups.
SDS-PAGE electrophoresis
Preparing an electrophoresis buffer solution, a membrane transfer buffer solution and 1 XTSST, adding the electrophoresis buffer solution into an electrophoresis tank, wherein the protein loading amount is 15 mu g; starting the power supply of the electrophoresis apparatus, firstly, applying 80V voltage to the concentrated gel for about 30min to enable each protein sample to reach the same starting line; then regulating the voltage value to 120V, and ending electrophoresis; cutting the glue of the target tape, taking a PVDF film with a proper size, activating with methanol for 30s, making a sandwich structure, and performing film conversion, wherein the pressure is limited to 100V under the low-temperature condition for 1h 45 min; after the film is transferred, sealing the film by using 5 percent of skimmed milk powder for 1 hour at room temperature; then primary antibody was incubated at 4 ℃ with APP (CST, 2452S) diluted 1:2000 with 5% skimmed milk powder and GAPDH (Proteintech, 10494-1-AP) diluted 1:2000 with TBST; after washing, incubating the secondary antibody (Proteintech, 10285-1-AP) for 1h at room temperature; after washing, exposure was measured, and the obtained results were analyzed by Image J.
The experimental results are shown in fig. 1, the WB technology is used to detect the change of APP protein expression in SA cells under different concentrations of compounds, and the results show that under 48H treatment conditions, the APP protein expression levels in the compound (M, 2 μ g/ml) and the compound in the compound (H, 4 μ g/ml) are respectively reduced by 37.6% and 55.2% compared with the APP protein expression level in the blank group, and the experimental results show that the compound can reduce the APP protein expression.
EXAMPLE three Compounds on SA cells A β1-42Generating influences
Use of high expression APP/swe muteinsThe SA cell line of (1), which completely simulates the metabolic process of A beta and leads to A beta due to the increase of APP protein1-42If the drug is capable of reducing A beta after the drug acts on the cell1-42Protein expression indicates that the medicine has a therapeutic effect on AD.
SA cell administration treatment and sample Collection
After 10. mu.l of the cell suspension of example I was supplemented with 90. mu.l of complete medium, a 96-well plate was placed in the cell incubator overnight until the cells were completely adherent. According to the result of the activity inhibition effect of the compound on SA cells, the compound is selected to be administered with the drug concentration gradient of 0.5 mug/ml (L), 2.0 mug/ml (M), 4.0 mug/ml (H), the drug is treated for 48 hours, and the compound is examined on the SA cells Abeta1-42An influence is generated. After the treatment time is over, collecting cell culture medium of each treatment group, centrifuging at 4 deg.C for 10min at 5000g, collecting supernatant, and detecting A beta according to ELISA kit (enzyme immunoassay, 1907H)1-42The content of (a).
The results of the experiments are shown in Table 2, and SA cells A beta are detected by ELISA technology under the treatment of different concentrations of compounds1-42The change was generated, and it was found that under 48h treatment conditions, each of the compound treatment groups was compared to the blank group of Abeta1-42The production amount is respectively reduced by 15.6%, 37.5% and 54.3%, and the experimental result shows that the compound can inhibit A beta1-42The amount of production of (c).
TABLE 1 SA cells treated with Compounds A β1-42Generating changes
In conclusion, the compound of the invention can obviously inhibit APP protein expression level and A beta simultaneously when acting on SA cells1-42The product shows higher anti-Alzheimer's disease activity, and can be applied to the preparation of medicaments for treating Alzheimer's disease.
Claims (4)
1. The application of the compound shown as the formula (I) or pharmaceutically acceptable salt in preparing the medicine for treating the Alzheimer disease.
2. The application of the compound shown as the formula (I) as an effective component in preparing a medicament for treating Alzheimer's disease.
3. The use of claim 1 or 2, wherein the compound is formulated with pharmaceutically acceptable carriers and/or excipients, and can be made into any dosage form of tablets, capsules, granules, powders, pills, oral liquids, injections, and suspensions.
4. The use according to claim 1 or 2, wherein the compound is administered to the cells at a dose of 10-80 ug/ml.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105142632A (en) * | 2012-06-27 | 2015-12-09 | 阿马曾提斯公司 | Enhancing autophagy or increasing longevity by administration of urolithins or precursors thereof |
| CN109549961A (en) * | 2017-09-25 | 2019-04-02 | 兰州大学 | A kind of application of Chinese medical extract in the drug of preparation treatment Alzheimer's disease |
| US20190336555A1 (en) * | 2016-11-11 | 2019-11-07 | Laila Nutraceuticals | Synergistic dietary supplement compositions for improving brain health |
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2019
- 2019-12-24 CN CN201911341541.XA patent/CN113018305A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105142632A (en) * | 2012-06-27 | 2015-12-09 | 阿马曾提斯公司 | Enhancing autophagy or increasing longevity by administration of urolithins or precursors thereof |
| US20190336555A1 (en) * | 2016-11-11 | 2019-11-07 | Laila Nutraceuticals | Synergistic dietary supplement compositions for improving brain health |
| CN109549961A (en) * | 2017-09-25 | 2019-04-02 | 兰州大学 | A kind of application of Chinese medical extract in the drug of preparation treatment Alzheimer's disease |
Non-Patent Citations (2)
| Title |
|---|
| AFSHARI, AMIR R.等: "A review on potential mechanisms of Terminalia chebula in Alzheimer\'s disease", 《ADVANCES IN PHARMACOLOGICAL SCIENCES》, pages 1 - 14 * |
| 陈小玉等: "诃子神经保护作用的药效物质基础", 《时珍国医国药》, vol. 23, no. 10, pages 2425 - 2427 * |
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