Application of odd-number carbon saturated medium-long chain fatty acid in preparation of medicine for treating polyglutamine diseases

Abstract

The invention belongs to the field of medicines, and particularly relates to application of odd-carbon saturated medium and long-chain fatty acid in preparation of a medicine for treating polyglutamine diseases. Research results of the invention show that the heptadecanoic acid can significantly delay and inhibit the aggregation of polyglutamine (polyQ) disease proteins, and can significantly prolong the survival time of AM141 nematodes at the same time, and the prolonging effect is about 10%. Therefore, the heptadecanoic acid is completely possible to be used as a raw material to prepare related medicines, so as to treat polyQ diseases. In addition, the heptadecanoic acid is clear in component, low in price and easy to obtain, and is more beneficial to benefiting the public.

Description

Technical Field

[0001] The invention belongs to the field of medicine, and specifically relates to the application of an odd-numbered carbon saturated medium-chain fatty acid in the preparation of a medicine for treating polyglutamine diseases. Background Art

[0002] Polyglutamine (polyQ) diseases are a series of neurodegenerative diseases caused by the expansion of the CAG trinucleotide sequence encoding glutamine in other unrelated genes. The formation of protein aggregates has been observed in many in vitro and in vivo systems with polyQ proteins, so protein aggregates are widely considered to be the pathological characteristics of polyQ-mediated diseases. The clinical manifestations of these neurodegenerative diseases include involuntary muscle twitching, cognitive impairment, mental abnormalities and other symptoms, but there is still a lack of effective clinical treatment.

[0003] Therefore, based on this, the technical solution of the present invention is proposed. Summary of the invention

[0004] In order to solve the problems existing in the prior art, the present invention provides an application of an odd-numbered carbon saturated medium-chain fatty acid in the preparation of a drug for treating polyglutamine diseases.

[0005] Preferably, the odd-numbered carbon saturated medium-chain fatty acid is heptadecanoic acid.

[0006] Preferably, the drug is a drug that inhibits polyglutamine protein aggregation.

[0007] Preferably, the drug is a drug that prolongs the survival time of AM141 nematodes.

[0008] Preferably, the drug is a preparation prepared by using odd-numbered carbon saturated medium-chain fatty acids as raw materials and adding medically acceptable excipients.

[0009] Preferably, the preparations include but are not limited to oral preparations, injection preparations and spray preparations.

[0010] The beneficial effects of the present invention are:

[0011] The research results of the present invention show that heptadecanoic acid can significantly delay and inhibit the aggregation of polyQ proteins, and can significantly prolong the survival time of AM141 nematodes, with a prolongation effect of about 10%. Therefore, heptadecanoic acid is entirely possible to be used as a raw material to prepare related drugs to treat polyQ diseases. In addition, heptadecanoic acid has a clear composition, is cheap, and is easily available, which is more beneficial to the general public. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0013] Figure 1 It is a statistical graph showing the change of the number of aggregated proteins over time in the experimental group (heptadecanoic acid) and the blank control group.

[0014] Figure 2 These are fluorescent photos taken during the experiment of "observing the aggregation and counting of polyQ proteins" (heptadecanoic acid experimental group and blank control group).

[0015] Figure 3 It is a statistical graph showing the change of the number of aggregated proteins over time in the experimental group (octadecanoic acid) and the blank control group.

[0016] Figure 4 These are fluorescent photos taken during the experiment of "observing the aggregation and counting of polyQ proteins" (octadecanoic acid experimental group and blank control group).

[0017] Figure 5 It is a statistical graph showing the change of the survival rate of AM141 nematodes over time in the experimental group (heptadecanoic acid) and the blank control group (n in the figure represents the number of normal deaths, the same below).

[0018] Figure 6 It is a statistical graph showing the change of the survival rate of AM141 nematodes over time in the experimental group (octadecanoic acid) and the blank control group. DETAILED DESCRIPTION

[0019] To make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be described in detail below. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other implementation methods obtained by ordinary technicians in this field without creative work belong to the scope of protection of the present invention.

[0020] Example

[0021] Appropriate pathological models can help researchers better solve practical problems. This example uses Caenorhabditis elegans when selecting a polyQ pathological model. Caenorhabditis elegans is transparent throughout its body, allowing observers to easily observe fluorescent proteins. Therefore, with the help of this characteristic, this example uses a pathological model that expresses a fusion protein of 40 glutamines (Q40) and yellow fluorescent protein (YFP) in muscle cells, namely the AM141 [unc-54p::Q40::YFP] strain of nematodes in the experiment.

[0022] After adding various compounds to the culture medium of AM141 nematodes, this example evaluates whether the compounds have therapeutic effects by observing the number of fluorescent protein aggregates and the survival time of AM141 nematodes.

[0023] (I) Preparation of Nematode Growth Medium (NGM)

[0024] Autoclave agar medium (16 g Agar, 1.84 g peptone, 800 mL H2O), add 800 μL of each of 1 mol / L CaCl2, 5 mg / mL cholesterol solution, and 1 mol / LMgSO4 solution, 20 mL of sterile buffer A (108.3 g KH2PO4, 35.6 g K2HPO4, 112.8 g NaCl, 1 L H2O), and 160 μL of nystatin suspension (1 g Nystatin powder, 20 mL 75% sterile ethanol) in a clean bench with a pipette, mix well, and dispense 3 mL of Nystatin into a 3.5 cm sterile culture dish with a pump. The constant medium content in the plate reduces the need to refocus the microscope when switching from one plate to another. Before use, place the plate at room temperature for 1 to 2 days to allow the agar to solidify and excess water to evaporate.

[0025] (II) Preparation of LB liquid medium and solid plate medium

[0026] Sterilize LB liquid culture medium (4 g peptone, 2 g yeast extract, 2 g NaCl, 400 mL H2O) by high pressure, cool to room temperature, and then refrigerate at 4°C for later use.

[0027] Sterilize LB solid medium (4g peptone, 2g yeast extract, 2g NaCl, 4.8g Agar, 400mL H2O) by high pressure, and use a pipette to dispense 8mL of the medium into a 6cm sterile Petri dish in a clean bench. After the agar solidifies, place it upside down in a 4℃ refrigerator for later use.

[0028] (III) Cultivation of Escherichia coli

[0029] In a clean bench, the Escherichia coli BW25113 stored at -80°C was transferred to the LB solid plate using the four-zone streak method and cultured in an incubator at 37°C overnight.

[0030] In the clean bench, a single colony of BW25113 was transferred from the LB plate into 2 mL of autoclaved LB liquid medium using a sterile inoculation loop and grown overnight in a shaker at 220 rpm and 37°C.

[0031] (IV) Preparation of bacterial plates

[0032] Add 100 μL of bacterial solution to the prepared NGM medium and blow it in an ultra-clean workbench for about 1 hour to evaporate the excess water in the bacterial solution.

[0033] (V) Preparation of compound solution

[0034] Weigh 10 mg of heptadecanoic acid and octadecanoic acid on a 1 / 10,000 balance, transfer the powders to sterile 15 mL centrifuge tubes, add 10 mL of corn oil, cover and wrap with tin foil, and place in a mixer to mix overnight.

[0035] (VI) Preparation of sterile synchronized AM141 nematodes

[0036] The life cycle of C. elegans includes the embryonic stage, four larval stages (L1 to L4) and the adult stage. Synchronization takes advantage of the fact that after C. elegans develops to L1, its growth will stagnate if food is scarce. When a large number of eggs appear on the culture medium of C. elegans, they can be prepared for collection. First, rinse the nematodes and eggs with 3 mL of sterile water and collect them into a sterile centrifuge tube. Repeat the operation once to ensure that all the C. elegans and eggs on the plate are collected; centrifuge at 3000 rpm for 1 min and discard the supernatant; add 5 mL of lysis buffer (30 mL H2O, 4 mL 5 mol / L NaOH, 5 mL commercially available bleach), cover and shake vigorously for about 50 times, centrifuge at 3000 rpm for 30 s, repeat once; until no obvious complete C. elegans can be observed under the microscope; add 10 mL of sterile water for washing, centrifuge at 3000 rpm for 30 s, discard the supernatant, repeat twice; add 10 mL of sterile M9 solution (3 g KH2PO4, 6 g Na2HPO4, 0.5 g NaCl, 1 g NH4Cl, 1 L H2O), centrifuge at 3000 rpm for 30 s, discard the supernatant, repeat once; finally, add 5 mL of M9 solution and rotate and culture at 20°C for 24 to 48 hours.

[0037] (VII) Compound feeding AM141 nematodes

[0038] After synchronization, add AM141 nematodes to the NGM plate with bacterial solution, and then add 1 mL of the prepared heptadecanoic acid liquid and octadecanoic acid liquid respectively. The blank control group is added with 1 mL of corn oil and cultured at a constant temperature of 20°C.

[0039] (VIII) Observation and counting of polyQ protein aggregation

[0040] AM141 nematodes were cultured at 20°C until they became adults. On D3, D4, and D5, about 20 AM141 nematodes were placed on a coverslip with anesthetic (10 mmol / L levamisole) and then covered with a slide with a 2% Agarose plate. Each AM141 nematode was photographed under a fluorescence microscope, and the aggregated proteins in the photos were counted using Image J software. 1 mg / mL heptadecanoic acid can significantly delay and inhibit the aggregation of polyQ proteins. The specific effects are shown in the attached Figure 1 , 2 1mg / mL octadecanoic acid has no inhibitory effect on polyQ protein aggregation. See the attached Figure 3 , 4 .

[0041] Statistical method: Data were compared by t-test, error bars used the standard error of the mean (SEM), *P<0.05, **P<0.01, ns means not significant (no significant difference).

[0042] (IX) Observe and record the survival time of AM141 nematodes

[0043] AM141 nematodes were cultured at 20°C and transferred to a new bacterial plate on D1 of adulthood to ensure that the AM141 nematodes had enough food. After the transfer was completed, 1 mL of the prepared heptadecanoic acid, octadecanoic acid liquid and blank control were added to each group. The transfer operation was repeated on D3, D5, D7, D9, D13, and D17, and the newly prepared liquid was added. From D2, AM141 nematodes were observed every day to see if there were any deaths. The AM141 nematodes were gently straightened. If they were not dead and their muscles were still active, the worm body would bend until all AM141 nematodes died and the experiment ended. 1 mg / mL heptadecanoic acid can significantly prolong the survival time of AM141 nematodes by 13.3%. The specific effects are shown in the attached Figure 5 The original data are shown in Table 1. 1 mg / mL octadecanoic acid has no effect on prolonging the survival time of AM141 nematodes. The specific results are shown in Appendix Figure 6, the original data are shown in Table 2. Statistical methods: The data were compared by Log-rank (Mantel-Cox) test, and the median survival time was used to calculate the extension effect.

[0044] Table 1

[0045]

[0046]

[0047] Table 2

[0048]

[0049]

[0050] The above is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art can easily think of changes or substitutions within the technical scope disclosed by the present invention, which should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention should be based on the protection scope of the claims.

Claims

1. Application of odd-numbered carbon saturated medium-chain fatty acids in the preparation of drugs for treating polyglutamine diseases.

2. The use according to claim 1, characterized in that: The odd-numbered carbon saturated medium-chain fatty acid is heptadecanoic acid.

3. The use according to claim 1 or 2, characterized in that: The drug is a drug for inhibiting the aggregation of polyglutamine proteins.

4. The use according to claim 1 or 2, characterized in that: The drug is a drug for prolonging the survival time of AM141 nematodes.

5. The use according to claim 1 or 2, characterized in that: The medicine is a preparation prepared by taking odd-carbon saturated medium-chain fatty acids as raw materials and adding medically acceptable auxiliary materials.

6. The use according to claim 5, characterized in that: The preparation is an oral preparation.

7. The use according to claim 5, characterized in that: The preparation is an injection preparation.

8. The use according to claim 5, characterized in that: The preparation is a spray preparation.

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