Application of DOR agonist in preparation of medicine for regulating and controlling phosphorylation level of Tau protein
By using DOR agonists such as UFP-512 to inhibit the over-phosphorylation of Tau protein and regulate the expression of related protein kinases and cyclin, the problem of difficult to control over-phosphorylation of Tau protein in the prior art is solved, and the Alzheimer Effective prevention and treatment of Silky disease.
Patent Information
- Application Number
- CN202411678544.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-22
- Publication Date
- 2025-02-14
AI Technical Summary
The prior art has not yet successfully developed drugs that can effectively reduce the hyperphosphorylation of Tau protein, leading to the treatment difficulties of anti-degenerative neurological diseases, especially Alzheimer's disease.
DOR agonists, such as UFP-512, are used to inhibit the hyperphosphorylation of Tau protein and regulate the expression levels of related protein kinases and cyclin to achieve the purpose of preventing and treating Alzheimer's disease.
DOR agonists can effectively prevent and treat Alzheimer's disease by inhibiting the hyperphosphorylation of Tau protein and regulating the expression of related protein kinases and cyclin.
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Figure CN119424647A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the field of medical technology, and in particular to the use of a DOR agonist in preparing a drug for regulating the phosphorylation level of a Tau protein. Background Art
[0002] Alzheimer's disease (AD) is a progressive neurodegenerative disease. One of its typical pathological features is the formation of neurofibrillary tangles due to hyperphosphorylation of intracellular Tau protein, which causes damage to neuronal structure and function. In addition, this Tau lesion also occurs in the pathological process of other degenerative neurological diseases.
[0003] Tau protein is a major microtubule-associated protein present in neurons, and its biological function is to promote microtubule polymerization and stabilize microtubule structure. Hyperphosphorylation of Tau protein causes it to lose the above biological functions and is prone to aggregate to form double helical filaments and neurofibrillary tangles, leading to the collapse of neuronal microtubule structure, damage to normal axonal transport, damage to neuronal function, and ultimately brain neuron degeneration. The imbalance of protein kinases and phosphatases is considered to be the main cause of Tau protein hyperphosphorylation. Protein kinases such as cyclin-dependent protein kinase 5 (CDK5), glycogen synthase kinase-3β (GSK-3β), mitogen-activated protein kinase (MAPK) and adenosine monophosphate-activated protein kinase (AMPK) play an important role in Tau protein hyperphosphorylation.
[0004] Anti-Tau targeted drugs are being developed, mainly targeting gene silencing therapy targeting Tau protein, regulating Tau protein post-translational modification, Tau-based immunotherapy, etc., but there are no successful cases so far. Therefore, developing drugs that target Tau protein and reduce its hyperphosphorylation is an important goal for preventing and treating degenerative neurological diseases, especially AD. Summary of the invention
[0005] The object of the present invention is to provide the use of a DOR agonist in the preparation of a drug for regulating the phosphorylation level of Tau protein, wherein the DOR agonist can inhibit the hyperphosphorylation of Tau protein, thereby achieving the effect of preventing and / or treating Alzheimer's disease.
[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:
[0007] The present invention provides an application of a DOR agonist in the preparation of a drug for regulating the phosphorylation level of a Tau protein. The DOR agonist can inhibit the excessive phosphorylation of a Tau protein.
[0008] Preferably, the DOR agonist is UFP-512.
[0009] The present invention also provides a reagent capable of inhibiting the hyperphosphorylation of Tau protein, wherein the active ingredient of the reagent includes the above-mentioned DOR agonist.
[0010] The present invention also provides the use of the reagent in preparing medicines for treating and / or preventing degenerative nervous system diseases.
[0011] The present invention also provides the use of a DOR agonist in preparing a drug for regulating the expression levels of protein kinases CDK5 and AMPK. The DOR agonist can inhibit the expression levels of protein kinases CDK5 and AMPK.
[0012] The present invention also provides the use of the DOR agonist in the preparation of a drug for regulating the expression levels of cell cycle proteins Cyclin D1 and Cyclin B1. The DOR agonist can inhibit the expression levels of cell cycle proteins Cyclin D1 and Cyclin B1.
[0013] The present invention also provides the use of the DOR agonist in the preparation of a drug for regulating the expression level of the apoptotic protein c-caspase 3. The DOR agonist can inhibit the expression level of the apoptotic protein c-caspase 3.
[0014] By adopting the above technical solution, the present invention has the following beneficial effects:
[0015] The present invention provides the use of a DOR agonist in the preparation of a drug for regulating the phosphorylation level of Tau protein, and the DOR agonist can inhibit the hyperphosphorylation of Tau protein, thereby achieving the effect of preventing and / or treating Alzheimer's disease. The present invention also provides the use of a DOR agonist in the preparation of a drug for regulating the expression level of protein kinase CDK5, protein kinase AMPK, cell cycle protein Cyclin D1, cell cycle protein Cyclin B1 and apoptosis protein c-caspase 3, and the DOR agonist can inhibit the expression level of the above protein kinases, thereby inhibiting the hyperphosphorylation of Tau protein, thereby also achieving the effect of preventing and / or treating Alzheimer's disease.
[0016] In the examples of the present invention, neuronal cell (HT22 cell) and neuron-like cell (highly differentiated PC12 cell) AD models were established respectively, and both showed that DOR agonists can inhibit the hyperphosphorylation of Tau protein and the expression level of the above-mentioned protein kinases, so as to achieve the effect of preventing and / or treating Alzheimer's disease. BRIEF DESCRIPTION OF THE DRAWINGS
[0017] Figure 1 The effects of different concentrations of DOR agonist UFP-512 on Tau protein hyperphosphorylation ( Figure 1 A in the figure shows the effects of different treatment groups in the highly differentiated PC12 cell AD model, and B shows the effects of different treatment groups in the HT22 cell AD model);
[0018] Figure 2 Effects of different concentrations of DOR inhibitor naltrindole on the hyperphosphorylation level of Tau protein ( Figure 2 A in the figure shows the effect of different concentrations of DOR inhibitor on Tau protein hyperphosphorylation in a highly differentiated PC12 cell AD model, B shows the effect of adding DOR inhibitor alone on Tau protein hyperphosphorylation in a highly differentiated PC12 cell AD model, and C shows the effect of different treatment groups in a HT22 cell AD model);
[0019] Figure 3 Effects of DOR on the expression levels of different protein kinases GSK-3β, CDK5 and AMPK ( Figure 3 A in the figure represents the effect of different treatment groups on the expression of GSK-3 and CDK5 in the highly differentiated PC12 cell AD model, B represents the effect of different treatment groups on the expression of GSK-3 and CDK5 in the HT22 cell AD model, C represents the effect of different treatment groups on the expression of AMPK in the highly differentiated PC12 cell AD model, and D represents the effect of different treatment groups on the expression of AMPK in the HT22 cell AD model);
[0020] Figure 4 The effect of DOR on the expression levels of cell cycle proteins Cyclin D1 and Cyclin B1 ( Figure 4 A in the figure represents the effects of different treatment groups in the highly differentiated PC12 cell AD model, and B represents the effects of different treatment groups in the HT22 cell AD model);
[0021] Figure 5 The effect of DOR on the expression level of apoptotic protein c-caspase 3 ( Figure 5 A in FIG. 1 represents the effects of different treatment groups in a highly differentiated PC12 cell AD model, and B represents the effects of different treatment groups in a HT22 cell AD model). DETAILED DESCRIPTION
[0022] The present invention provides an application of a DOR agonist in preparing a drug for regulating the phosphorylation level of Tau protein.
[0023] In the present invention, the DOR agonist can inhibit the hyperphosphorylation of Tau protein.
[0024] In the present invention, the DOR agonist is preferably UFP-512.
[0025] The present invention also provides a reagent capable of inhibiting the hyperphosphorylation of Tau protein, wherein the active ingredient of the reagent includes the above-mentioned DOR agonist.
[0026] The present invention also provides the use of the reagent in preparing medicines for treating and / or preventing degenerative nervous system diseases.
[0027] In the present invention, the degenerative nervous system disease includes Alzheimer's disease.
[0028] The present invention also provides the use of a DOR agonist in preparing a drug for regulating the expression levels of protein kinases CDK5 and AMPK. The DOR agonist can inhibit the expression levels of protein kinases CDK5 and AMPK.
[0029] The present invention also provides the use of the DOR agonist in the preparation of a drug for regulating the expression levels of cell cycle proteins Cyclin D1 and Cyclin B1. The DOR agonist can inhibit the expression levels of cell cycle proteins Cyclin D1 and Cyclin B1.
[0030] The present invention also provides the use of the DOR agonist in the preparation of a drug for regulating the expression level of the apoptotic protein c-caspase 3. The DOR agonist can inhibit the expression level of the apoptotic protein c-caspase 3.
[0031] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.
[0032] Example 1 Establishment of AD injury model (one)
[0034] PC12 cells (purchased from Shanghai Academy of Sciences) were used to construct AD injury model.
[0035] The specific steps are as follows:
[0036] (1) Cell culture: PC12 cells were cultured in complete culture medium (DMEM high-glucose medium containing 10% fetal bovine serum and 1% penicillin-streptomycin double antibody);
[0037] (2) Cell digestion: discard the old culture medium, and digest the PC12 cells in the culture dish with a coverage rate of 80-90% density with 0.25% trypsin for 3-5 minutes. After the cells become round, add new culture medium to terminate the digestion and collect the cells; centrifuge at 1000 rpm for 5 minutes;
[0038] (3) Plating: Resuspend the centrifuged cells and count the cells according to 15*10 4 Cell number / well (6-well plate) for cell plating;
[0039] (4) Place the cell plate in a constant temperature incubator (37°C, 5% CO2) for culture;
[0040] (5) After 24 hours, the culture medium was replaced with new culture medium containing 50 nM okadaic acid (OA, purchased from MedChem Express, MCE, New Jersey, USA), and the cells were returned to the normal incubator for 24 hours. (two)
[0042] HT22 cells (purchased from Shanghai Academy of Sciences) were used to construct an AD injury model.
[0043] The specific steps are as follows:
[0044] (1) Cell culture: HT22 cells were cultured in complete culture medium (DMEM high-glucose medium containing 10% fetal bovine serum and 1% penicillin-streptomycin double antibody);
[0045] (2) Cell digestion: discard the old culture medium, digest the HT22 cells in the culture dish with a coverage rate of 80-90% density with 0.25% trypsin for 3-5 minutes, and add new culture medium to terminate the digestion after the cells become round, and collect the cells; centrifuge at 1000 rpm for 5 minutes;
[0046] (3) Plating: Resuspend the centrifuged cells and count the cells according to 2*10 4 Cell number / well (24-well plate) for cell plating;
[0047] (4) Virus infection: After 16-24 hours of cell culture, when the cell confluence rate reached 30%-40%, the old culture medium was discarded, and a lentivirus containing an empty plasmid at an MOI of 20 and 5 μL of new culture medium containing Polybrene-plus were added to one well, and a lentivirus containing a P301L-Tau mutant plasmid at an MOI of 20 and 5 μL of new culture medium containing Polybrene-plus were added to another well (the lentivirus was purchased from Heyuan Biotechnology Co., Ltd., Shanghai, China). The cells were placed in a constant temperature incubator (37°C, 5% CO2) for culture, and the cell status was observed. After 6-8 hours, the culture medium was replaced and the culture was continued;
[0048] (5) After 48 hours, observe the fluorescence. When the infection efficiency reaches 70%-80%, transfer the cells into a 12-well plate and continue culturing. After 24 hours, add 4 μg / mL of puromycin.
[0049] (6) After 48 hours, all the cells selected with puromycin in step 5 were seeded in a 6-well plate and complete medium containing 4 ug / mL puromycin was added for further culture;
[0050] (7) After the cell confluence rate of the 6-well plate reaches 80%-90%, WB verification is performed; a control cell line containing an empty plasmid and a stable cell line overexpressing Tau are obtained.
[0051] Example 2
[0052] The obtained PC12 cell AD injury model was divided into four groups, including a cell model group, a cell model + DOR activation group, a cell model + DOR activator + inhibitor group, and a cell model + DOR inhibitor group. A cell control group (i.e., normally cultured PC12 cells) was also set up.
[0053] Cell model group: the okadaic acid modeling group described in Example 1;
[0054] Cell model + DOR activation group: DOR agonist UFP-512 (provided by Professor GIANFRANCO BALBONI, the inventor of the agonist and collaborator of Professor Xia Ying for many years, see the following literature) was added to the new culture medium containing okadaic acid described in Example 1;
[0055] Cell model + DOR activator + inhibitor group: DOR agonist UFP-512 and DOR inhibitor Naltrindole (Nal, McLean Biochemical Technology Co., Ltd., Shanghai, China) were added to the new culture medium containing okadaic acid described in Example 1;
[0056] Cell model + DOR inhibitor group: The DOR inhibitor naltrindole was added to the new culture medium containing okadaic acid as described in Example 1.
[0057] The obtained HT22 cell AD injury model was divided into four groups, including a cell model group, a cell model + DOR activation group, a cell model + DOR activator + inhibitor group, and a cell model + DOR inhibitor group. A cell control group (i.e., HT22 cells infected with a lentivirus expressing an empty plasmid) was also set up.
[0058] Cell model group: HT22 cells infected with the lentivirus expressing the P301L-Tau mutant described in Example 1;
[0059] Cell model + DOR activation group: After culturing the HT22 cell model described in Example 1 for 24 hours, the culture medium was replaced with a new medium containing the DOR agonist UFP-512;
[0060] Cell model + DOR activator + inhibitor group: After culturing the HT22 cell model described in Example 1 for 24 hours, a new culture medium was added with the DOR agonist UFP-512 and the DOR inhibitor naltrindole;
[0061] Cell model + DOR inhibitor group: After culturing the HT22 cell model described in Example 1 for 24 hours, a new culture medium was added with the DOR inhibitor naltrindole.
[0062] (I) Effect of DOR agonist UFP-512 on Tau hyperphosphorylation
[0063] The cells in the culture medium of the above cell model + DOR activation group were treated with different concentrations (1, 5, 10 μM) of DOR agonist UFP-512.
[0064] (1) After 24 h of culture, the cells were digested with 0.25% trypsin for 3-5 minutes until the cells became round; complete medium was added to terminate the digestion, and the cells were centrifuged at 1500 rpm for 5 min;
[0065] (2) Add PBS to resuspend the cells and centrifuge at 1500 rpm for 5 min;
[0066] (3) After adding RIPA containing protease inhibitors and phosphatase inhibitors, the cells were lysed on ice for 20-30 min, centrifuged at 12000 rpm for 10 min, and the supernatant was extracted to obtain the total cell protein of each group;
[0067] (4) Quantification was performed using a BCA kit (purchased from Bio-Tech Biotech Co., Ltd., Shanghai, China).
[0068] Finally, Western blot was used to detect the phosphorylation levels of different sites of Tau protein in the cell model group and the cell model + DOR activation group.
[0069] The results are as follows Figure 1 As shown in the figure, in the AD injury model constructed by highly differentiated PC12 cells, the hyperphosphorylation of Tau protein decreased with the increase of DOR agonist concentration; in the AD injury model constructed by HT22 cells, different concentrations of DOR agonists reduced the hyperphosphorylation of Tau protein, among which 5μM DOR agonist UFP-512 had a relatively good reducing effect.
[0070] (II) Effect of DOR inhibitor naltrindole on Tau protein hyperphosphorylation
[0071] The cells were treated with different concentrations (1, 5, 10 μM) of DOR inhibitor naltrindole in the corresponding culture medium of the above-mentioned cell model + DOR activation group, cell model + DOR activator + inhibitor group, and cell model + DOR inhibitor group, respectively. The concentration of DOR agonist UFP-512 in the PC12 cell model was 10 μM, and the concentration of DOR agonist UFP-512 in the HT22 cell model was 5 μM. The total protein of each group of cells was extracted and quantified by BCA method (the steps were the same as (a)). Then Westernbolt was used to detect the phosphorylation levels of different sites of Tau protein in the cell model group, cell model + DOR activation group, cell model + DOR activator + inhibitor group, and cell model + DOR inhibitor group.
[0072] The results are as follows Figure 2 As shown in the figure, in the AD injury model constructed by highly differentiated PC12 cells, different concentrations of DOR inhibitor naltrindole significantly increased the phosphorylation levels of different sites of Tau protein; and compared with the cell model group, the addition of 1μM naltrindole alone had no effect on the phosphorylation level of Tau protein. In the AD injury model constructed by HT22 cells, 1μM DOR inhibitor naltrindole significantly increased the phosphorylation levels of different sites of Tau protein.
[0073] (III) Effects of DOR on the expression of different protein kinases
[0074] Based on the above-mentioned method of "the concentration of DOR agonist UFP-512 in PC12 cell model is 10μM, the concentration of DOR inhibitor naltrindole is 1μM; the concentration of DOR agonist UFP-512 in HT22 cell model is 5μM, the concentration of DOR inhibitor naltrindole is 1μM", the culture medium of the corresponding groups was treated. The total protein of each group of cells was extracted and quantified by BCA method (the steps are the same as (a)). Then Western bolt was used to detect the effect of DOR on the expression of different protein kinases in different AD injury models.
[0075] The results are as follows Figure 3 As shown in the results, in the AD injury model constructed by highly differentiated PC12 cells, the activation of DOR had no significant effect on the expression of GSK-3β, but significantly downregulated the expression of CDK5 and AMPK, and the use of the DOR inhibitor naltrindole reversed the downregulation of CDK5 and AMPK. In the AD injury model constructed by HT22 cells, the activation of DOR had no significant effect on the expression of GSK-3β, but significantly downregulated the expression of CDK5 and AMPK, and the use of the DOR inhibitor naltrindole reversed the downregulation of CDK5 and AMPK.
[0076] (IV) Effects of DOR on the expression of cell cycle proteins and apoptosis proteins
[0077] Based on the above method of "the concentration of DOR agonist UFP-512 in PC12 cell model is 10μM, the concentration of DOR inhibitor naltrindole is 1μM; the concentration of DOR agonist UFP-512 in HT22 cell model is 5μM, the concentration of DOR inhibitor naltrindole is 1μM", the culture medium of the corresponding groups was treated. The total protein of each group of cells was extracted and quantified by BCA method (the steps are the same as (a)). Then, Western bolt was used to detect the effect of DOR on the expression of cell cycle proteins and apoptosis proteins in different AD injury models.
[0078] The results are as follows Figure 4 As shown in the results, in the AD injury model constructed by highly differentiated PC12 cells, activation of DOR reduced the expression of Cyclin D1 and Cyclin B1, and the use of the DOR inhibitor naltrindole reversed the expression of Cyclin D1, but had no effect on the expression of Cyclin B1. In the AD injury model constructed by HT22 cells, activation of DOR reduced the expression of Cyclin D1 and Cyclin B1, and the DOR inhibitor naltrindole reversed the expression of Cyclin D1 and Cyclin B1.
[0079] The results are as follows Figure 5 As shown, in the AD injury model constructed by highly differentiated PC12 cells, activation of DOR reduced the expression of c-caspase 3, and the DOR inhibitor naltrindole reversed the effect of DOR. In the AD injury model constructed by HT22 cells, activation of DOR reduced the expression of c-caspase 3.
[0080] In summary, the DOR agonist of the present invention can inhibit the hyperphosphorylation of Tau protein; it can also inhibit the expression levels of protein kinase CDK5, protein kinase AMPK, cell cycle protein Cyclin D1, cell cycle protein Cyclin B1 and apoptosis protein c-caspase 3.
[0081] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.
Claims
1. Use of a DOR agonist in the preparation of a drug for regulating the phosphorylation level of Tau protein, characterized in that: DOR agonists can inhibit the hyperphosphorylation of Tau protein.
2. The use according to claim 1, characterized in that: The DOR agonist is UFP-512.
3. A reagent capable of inhibiting the hyperphosphorylation of Tau protein, characterized in that: The active ingredient of the reagent includes the DOR agonist according to claim 1 or 2.
4. Use of the reagent according to claim 3 in the preparation of drugs for treating and / or preventing degenerative nervous system diseases.
5. Use of a DOR agonist in the preparation of a drug for regulating the expression level of protein kinases CDK5 and AMPK, characterized in that: DOR agonists can inhibit the expression levels of protein kinases CDK5 and AMPK.
6. Use of a DOR agonist in the preparation of a drug for regulating the expression level of cell cycle proteins Cyclin D1 and Cyclin B1, characterized in that: DOR agonists can inhibit the expression levels of cell cycle proteins Cyclin D1 and Cyclin B1.
7. Use of a DOR agonist in the preparation of a drug for regulating the expression level of apoptotic protein c-caspase 3, characterized in that: DOR agonists can inhibit the expression level of apoptotic protein c-caspase 3.