CN111374986A

CN111374986A - Application of vitamin B12 as α -synuclein aggregation inhibitor in preparation of medicines, health-care products or foods

Info

Publication number: CN111374986A
Application number: CN201811621728.0A
Authority: CN
Inventors: 刘夫锋; 王英; 贾龙刚; 陈贝贝; 路福平
Original assignee: Tianjin University of Science and Technology
Current assignee: Tianjin University of Science and Technology
Priority date: 2018-12-28
Filing date: 2018-12-28
Publication date: 2020-07-07

Abstract

The invention provides an application of vitamin B12 as a α -synuclein aggregation inhibitor in preparation of medicines, health products or foods, and belongs to the technical field of medicines, health products or foods.an application of vitamin B12 in preparation of medicines, health products or foods for preventing and/or treating diseases characterized by α -synuclein aggregation and precipitation is provided.

Description

Application of vitamin B12 as α -synuclein aggregation inhibitor in preparation of medicines, health-care products or foods

Technical Field

The invention belongs to the technical field of medicines, health-care products or foods, and particularly relates to an application of vitamin B12 as a α -synuclein aggregation inhibitor in preparation of medicines, health-care products or foods.

Background

Parkinson's Disease (PD) is a common degenerative disease of the nervous system of the elderly. The main clinical features are muscle tremor, myotonia and bradykinesia. It was found that the majority of parkinson's syndrome patients have major lesions in the midbrain substantia nigra and striatum, within which Lewy bodies (Lewy) are present.

To date, three synuclein proteins, named α -synuclein, β -synuclein, and gamma-synuclein, were found, wherein only α -synuclein (α -synuclein, α -Syn) contains NAC domain and participates in the formation of Lewy bodies, while gene point mutation and amplification due to α -Syn aggregation can cause familial Parkinson's disease, α -Syn is an abundant presynaptic protein, which is composed of 140 highly soluble amino acids, has a molecular weight of 19kDa, is usually localized at the presynaptic end of neurons, is expressed in the central nervous system, and contains a conserved lipid binding domain, and participates in synaptosomal transport.

More and more studies have shown that the α -Syn monomer is not toxic, but its aggregates are cytotoxic through various modes of action, causing neuronal degeneration and even apoptosis, and increased activity of glial cells in the brain, thereby triggering inflammatory responses in vivo.

Since α -Syn is closely related to PD, how to prevent α -Syn aggregation becomes a hot issue of current researchers' interest.three approaches and approaches to PD from the point of view of α -Syn generation and conformational transition can be used to 1) stabilize the native monomeric state of amyloid, 2) target different intermediates on the amyloid pathway and block their conversion to fibrils, 3) alter the aggregation pathway to promote the generation of non-amyloid-like protein aggregates, the most safe and reliable approach to PD treatment is now to stabilize α -Syn initial structure and inhibit its aggregation, since α -Syn itself has the physiological functions necessary for the organism.

In recent years, with the deep research of PD, the small molecules in food are gradually paid attention to researchers due to the characteristics of small side effect, strong adaptability to human bodies, suitability for long-term taking and the like, so that the effective α -Syn aggregation inhibitor is screened from the food and is an effective way for developing new PD medicines.

Disclosure of Invention

The invention provides an application of vitamin B12 in preparing a α -synuclein aggregation inhibitor, and the application of the vitamin B12 in preparing medicines, health-care products or foods can effectively prevent PD.

The invention provides an application of vitamin B12 in preparing α -synuclein aggregation inhibitor.

Further, use of vitamin B12 in the preparation of a medicament, health product or food for the prevention and/or treatment of a disease characterized by aggregation and precipitation of α -synuclein.

Further, the disease is parkinson's syndrome.

Further, vitamin B12 is present as an aqueous dispersion.

Further, vitamin B12 was present as an aqueous dispersion of vitamin B12 at a concentration of 2.5-100. mu.M.

Further, vitamin B12 was present as an aqueous dispersion of vitamin B12 at a concentration of 5-50. mu.M.

The application of the vitamin B12 in inhibiting α -synuclein aggregation has the following advantages:

the vitamin B12 provided by the invention can effectively inhibit α -Syn aggregation, effectively prevent PD from generating and delay PD development, effectively inhibit toxicity of α -Syn aggregate to cells, inhibit aggregation of α -Syn and the conformational change process thereof, and is an ideal medicament for treating and preventing Parkinson.

Drawings

The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate an embodiment of the invention and, together with the description, serve to explain the invention and not to limit the invention. In the drawings:

FIG. 1 is a ThT fluorescence plot of cultures incubated with different concentrations of vitamin B12 and α -Syn for different periods of time in example 1 of the present invention.

FIG. 2 is a graph showing the cytotoxicity of cultures on PC12 after co-culture of vitamin B12 and α -Syn for 5 days in example 2.

FIG. 3 is a graph showing the secondary structure changes of the cultures after co-cultivation of vitamin B12 and α -Syn for 6 days in example 3.

FIG. 4 is a graph showing the staining of FDA/PI cells of the culture after co-culturing 100 μm vitamin B12 with α -Syn for 7 days in example 4 of the present invention.

FIG. 5 is an Atomic Force Microscope (AFM) photograph of a culture after co-culturing 100. mu.M vitamin B12 with α -Syn for 7d in example 5 of the present invention.

Detailed Description

It should be noted that the embodiments and features of the embodiments may be combined with each other without conflict.

The embodiment of the invention provides application of vitamin B12 in preparing α -synuclein aggregation inhibitor.

Vitamin B12 has the following structural formula:

vitamin B12, VB12 for short, also called cobalamin, is a vitamin indispensable to human body, is the only vitamin containing metal elements, is mainly present in animal food and can be obtained by a biosynthesis method, in the prior art, vitamin B12 is mainly used for preparing medicines, food additives, feed additives and the like and is used for supplementing vitamin B12 which is lacked in normal organism growth, the application provides a vitamin B12 as α -synuclein aggregation inhibitor in the preparation of medicines, health products or food, α -synuclein is rich presynaptic protein which is composed of 140 highly soluble amino acids and has the molecular weight of 19kDa, is usually positioned at the presynaptic end of neurons and is expressed in the central nervous system, the vitamin B α -synuclein is involved in synuclein transportation, and VB12 is provided to effectively inhibit the aggregation of α -synuclein and provides a new thought for the research of the α -synuclein inhibitor.

In one embodiment of the invention, VB12 is used for preparing a medicine, a health-care product or food for preventing and/or treating diseases characterized by α -synuclein aggregation and precipitation, and the VB12 serving as a food, a health-care product micromolecule, a small toxic and side effect and the like is fully utilized, and is respectively used for the new development of the medicine, the health-care product or the food as a α -synuclein aggregation inhibitor.

In a preferred embodiment of the invention, VB12 is used as a α -synuclein aggregation inhibitor in the preparation of medicines, and VB12 has small toxic and side effects, so that patients can adapt faster and the side effects are low when the VB is used for the development of medicines.

Furthermore, the use of VB12 as a α -synuclein aggregation inhibitor in the preparation of medicaments for preventing and/or treating Parkinson's syndrome (PD) is a common degenerative disease of the nervous system of middle-aged and elderly people, most of Parkinson's syndrome patients have main lesions in the substantia nigra media and striatum, Lewy bodies (Lewy) exist in the PD, α -Syn contains an NAC structural domain and participates in the formation of Lewy bodies, and the genetic point mutation and amplification of α -Syn can cause the occurrence of familial Parkinson's disease.

Specifically, VB12 can be present in an aqueous dispersion. In other words, VB12 can be administered in the form of aqueous dispersion, such as injection, injection solution, soft capsule, beverage, oral liquid, etc.

Specifically, VB12 was present as an aqueous dispersion of VB12 at a concentration of 2.5-100. mu.M. Preferably, VB12 is present in an aqueous VB12 dispersion at a concentration of 5-50. mu.M. Specifically, it may be 2.5. mu.M, 5. mu.M, 20. mu.M, 25. mu.M, 40. mu.M, 50. mu.M, 80. mu.M, 100. mu.M, or the like.

In a preferred embodiment of the invention, VB12 is used as a α -synuclein aggregation inhibitor in the preparation of health care products or foods, and preferably VB12 is used as a α -synuclein aggregation inhibitor in the preparation of health care products or foods for preventing Parkinson's disease.

The use of VB12 to inhibit α -synuclein aggregation is further described below with reference to specific examples.

Example 1Varying concentrations of VB12 and α -Syn Co-cultivation the intensity of Thioflavin (ThT) fluorescence of the cultures after different periods of time

Firstly, constructing a recombinant α -Syn protein escherichia coli strain from a topic group at the early stage, performing induced expression and purification to finally obtain α -Syn protein with the purity of more than 95%, freeze-drying, storing at-20 ℃ to obtain purified and freeze-dried α -Syn, adding TBS buffer solution into purified and freeze-dried α -Syn, performing ultrasonic treatment for 10min to fully dissolve the protein, centrifuging at the speed of 16000Xg for 20min to remove aggregated protein, then taking 75% of supernatant, and measuring the protein concentration by using an instrument to obtain α -Syn protein mother liquor.

Next, 16mg of ThT was weighed and dissolved in 50mL of Tris-HCl buffer (TBS, Tris buffered saline, pH 5.0, Tris buffer concentration 20mM, NaCl concentration 150mM) to obtain a ThT mother liquor with a concentration of 1 mM.

Then, 1.4mg of VB12 was weighed out and dissolved in 1mL of membrane-passing water to obtain a 1mM VB12 mother solution, which was stored in the dark.

The α -Syn mother liquor and VB12 were diluted in a gradient and added to a ThT solution to obtain α -Syn solutions with VB12 final concentrations of 0. mu.M, 25. mu.M, 50. mu.M and 100. mu.M, respectively (in this case, α -Syn concentration in the solution is 50. mu.M and ThT concentration is 250. mu.M), and the four solutions were incubated in situ at 37 ℃.

The fluorescence intensity of the fluorescent material at the excitation wavelength of 440nm and the emission wavelength of 480nm is measured, the emission slit width is 5nm, the scanning speed is 100nm/min, and the scanning results are average values of 3 times. The fluorescence intensities at 440nm and 480nm were plotted against time, and the results are shown in FIG. 1.

As can be seen from FIG. 1, the ThT fluorescence diagram of α -Syn is an S-shaped curve, the ThT fluorescence intensity of α -Syn is obviously reduced after the addition of VB12 in a lag phase (0-24 h), a rapid growth phase (24-120 h) and a stable plateau phase (>120h), the reduction degree of the ThT fluorescence is in direct proportion to the concentration of VB12, the higher the concentration of VB12 is, the stronger the fluorescence inhibition effect is, thus VB12 effectively inhibits the aggregation of α -Syn, and VB12 effectively inhibits the aggregation of α -Syn.

Example 2MTT method is used for detecting cytotoxicity of VB12 and α -Syn at different concentrations for 5 days on PC12

The cell used in the cytotoxicity test was a murine adrenal chromaffin tumor cell line (PC 12).

First, poorly differentiated cells were cultured in a medium containing 10% fetal bovine serum and 1% penicillin-streptomycin, and Neuronal Growth Factor (NGF) was added to the medium to a final concentration of 50ng/mL and 5% CO₂Culturing at 37 deg.C for 3 days, observing that PC12 cells grow longer protrusions under the induction of NGF to become highly differentiated cells, adding 0.25% pancreatin solution to the culture flask to digest the highly differentiated cells, diluting the cells with RPMI-1640 medium containing 10% fetal bovine serum and 1% penicillin-streptomycin at an appropriate concentration, and diluting with 5 × 10⁴cell/well cell concentrations were added to 96-well plates at 90. mu.L per well. 5% CO₂And culturing at 37 ℃ for 24 h.

Then, α -Syn solutions were prepared at 50. mu.M α -Syn solution and VB12 at 5. mu.M, 10. mu.M and 50. mu.M, respectively (in this case, α -Syn concentration in the solutions was 50. mu.M), wherein α -Syn was treated in the same manner as in example 1, and cultured at 37 ℃ for 5 days.

Mixing the aged α -Syn with α -Syn solution containing VB12 at different concentrationsAdding into 96-well plate containing PC12 cultured for 24 hr, 10 μ L/well, blank control well without VB12 and α -Syn solution, adding PBS buffer solution 10 μ L/well, adding VB12 to 5 μ M, 5 μ M, 10 μ M, and α -Syn to 5 μ M, i.e. α -Syn: VB12 are 2:1, 1:1, and 1:2, respectively, and culturing with 5% CO in incubator₂After culturing at 37 ℃ for 48 hours, 10. mu.L/well of MTT solution was added so that the final concentration of MTT in the medium was 0.5 mg/mL. 5% CO₂The culture was continued at 37 ℃ for 4 hours.

Removing the solution in a 96-well plate, adding 100 mu L of DMSO into each well, culturing at 37 ℃ for 10min, and detecting the light absorption value at 570nm, taking the wells without α -Syn and VB12 in the culture medium as blank control groups, marking the cell activity as 100%, and then taking the blank control groups as control groups to calculate the cell survival rate of the drug adding group (figure 2). in the experiment, 6 multiple wells are arranged in each VB12 concentration gradient, as can be seen from figure 2, when only α -Syn exists alone, the cell survival rate is 58%, and after adding VB12(2.5 mu M, 5 mu M and 10 mu M) with different concentrations, the cell survival rate is improved to 62%, 68% and 75%, the higher the concentration of the added VB12 is, the stronger the toxicity relieving effect is, which indicates that VB12 can effectively inhibit the cytotoxicity generated by α -Syn.

Example 3Secondary structure changes of cultures at different concentrations of VB12 and α -Syn after cocultivation for different periods of time

First, 500. mu.L of α -Syn solution (in this case, α -Syn concentration in the solution is 50. mu.M) containing VB12 at 0. mu.M, 25. mu.M, 50. mu.M and 100. mu.M, respectively, was prepared, wherein α -Syn was treated in the same manner as in example 1 and cultured at 37 ℃ for 5 days.

And secondly, adding 500 mu L of culture solution into a CD detection pool with an optical path of 0.1mm for detection, wherein the wavelength scanning range is 190-260 nm, the bandwidth is 2nm, the scanning speed is 100nm/min, the experimental result is the average value of three times of scanning, and the result is shown in figure 3.

As can be seen from FIG. 3(a), neither the protein solution nor the inhibitor-added solution showed any significant β -sheet structure at 0 days of culture.

As can be seen from FIG. 3(b), after 6d of culture, only the protein control group formed a distinct β -sheet structure, and after 25 μ M of VB12 was added, the β -sheet degree of the protein was significantly reduced, and after 50 μ M or 100 μ M of VB12 was added, the protein was shifted from β -sheet structure to α -helix, indicating that VB12 could inhibit the structural shift of the protein and inhibit aggregation of the protein.

Example 4Detection of cytotoxicity of cultures to PC12 after VB12 and α -Syn co-culture for 7 days by FDA/PI mixed double staining

Prepare stock solution, weigh FDA 5mg, add to 15mL centrifuge tube. Add 1mL of acetone, shake, wrap around tinfoil and label, store at-4 deg.C. Weigh PI 1mg and add to a 15mL centrifuge tube. Add l mL of PBS buffer, shake, wrap around tinfoil and label, store at-4 ℃ cryogenically. Prepare working solution, take 10mL PBS to centrifuge tube, add FDA stock solution 20 u L. The final FDA concentration of 50. mu.L PI stock was 10. mu.g/mL and the final PI concentration was 5. mu.g/mL. Wrapping the tinfoil and marking, and storing at-4 deg.C.

PC12 cells differentiated in the same manner as in example 2 were collected at a density of 5 × 10⁴cells/mL were diluted and added to 6-well plates, 2mL per well. 5% CO₂And culturing at 37 ℃ for 24 h.

α -Syn solution (α -Syn concentration in the solution is 50 μ M) of VB12 solution with concentration of 0 μ M and 100 μ M respectively is prepared, wherein α -Syn is treated in the same manner as in example 1, and cultured at 37 ℃ for 7 d.

The aged α -Syn solution of α -Syn, VB12 was added to a 6-well plate containing PC12 which had been cultured for 24 hours at 200. mu.L/well, 200. mu.L/well of PBS buffer was added to a blank control well, and cells were cultured in an incubator at 5% CO2 at 37 ℃ for 48 hours and then subjected to FDA/PI mixed double staining.

The culture medium is slowly and lightly sucked out, PBS is used for light washing for 2 times and sucked out, the staining solution is added into the slow wall pasting to be shaded and stained for 1-2min, and the PBS is used for observation by a fluorescence microscope after light washing (figure 4). the result can be shown in figure 4. compared with a control group, α -Syn can reduce the number of the survival cells which can be stained by FDA and increase the number of the apoptosis cells which can be stained by PI, and the apoptosis caused by α -Syn can be obviously reduced after VB12 is added, and the cytotoxicity is very low.

Example 5Atomic Force (AFM) profile of cultures after co-cultivation of 100 μm VB12 and α -Syn for 7 days

α -Syn molecules were treated in the same manner as in example 1, and α -Syn solution containing 100 μm VB12 was prepared, the final concentration of α -Syn in the solution was 100 μm. the above solution was cultured at 37 ℃ and 200 rpm.

Taking 100 mu L of α -Syn culture solution under different culture time, performing ultrasonic treatment for 10min, fixing a round iron sheet in a clean culture dish by using a double-sided adhesive tape, fixing a mica sheet on the round iron sheet by using the double-sided adhesive tape, continuously sticking the mica sheet three times by using a transparent adhesive tape, removing the unclean layer of the mica sheet, then dropping a 20 mu L of ultrasonically-treated sample on the mica sheet by using a 20 mu L pipette gun, after 5-10min, slowly washing 1mL of membrane-passing water by using the pipette gun, naturally drying, observing by using an atomic force microscope, detecting the voltage at 100KV, selecting an image with the amplification scale of 5 mu m, and observing, wherein the result is shown in figure 5.

As can be seen from FIG. 5, after VB12 is added, the aggregation speed and the aggregate morphology of α -Syn are changed, the protein cultured for 0d does not form fibers but exists in the state of protein particles, as shown in FIG. 5(a), when the culture time is 7d, a large number of fiber aggregates appear when α -Syn is cultured alone, as shown in FIG. 5(b), and when the VB12 group is added, small oligomers are taken as main components and small short rod-shaped aggregates are accompanied, as shown in FIG. 5(c), the existence of VB12 changes the morphology of α -Syn aggregates and prevents the fibers from being transformed.

Example 6: VB12 preparation for health products

The weight portions of the components are (each portion is 0.01 g): VB 121 parts, vitamin C10 parts, vitamin H10 parts, ferrous sulfate 5 parts and zinc oxide 1 part.

When in use, 1L of water is added for taking with water.

When the mixed solution prepared by using the health product and α -Syn is used for fluorescence intensity test, compared with a control group (a solution formed by the health product without VB12 and α -Syn), the fluorescence intensity of α -Syn is obviously reduced after VB12 is added, which shows that VB12 effectively inhibits the aggregation of α -Syn.

Example 7: VB12 for preparing beverage

The weight portions are as follows: VB 120.01 parts, citric acid 50 parts, glucose 25 parts and water 1000 parts.

A health beverage is prepared by dissolving the active ingredients, mixing, stirring at 85 deg.C for 1h, filtering, and filling all the ingredients into bottles for sterilization.

The mixed solution of the drink and α -Syn is used for fluorescence intensity test, and compared with the solution formed by not adding VB12 and α -Syn, the fluorescence intensity of α -Syn solution is obviously reduced after VB12 is added, which shows that VB12 effectively inhibits the aggregation of α -Syn.

α -Syn aggregation inhibitor is a major focus OF development OF new PD therapeutics by inhibiting α -Syn aggregation, thereby preventing or treating PD generation, and α -Syn aggregation inhibitor has also been reported in THE related literature as having an important effect on THE treatment or prevention OF PD, VOL.285, NO.20, pp.41-14954, May 14,2010, and it is obvious that VB12 is used for preparing drugs for inhibiting α -Synuclein aggregation and applying it to α -Syn aggregation and cytotoxicity inhibition experiments, it is obvious that THE relevant technical matters are described by better implementation, and THE invention can be combined with other technical matters within THE scope OF THE present invention, and THE invention is a method for achieving THE same effect as THE technical matters OF THE present invention.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.

Claims

1. Use of vitamin B12 in the preparation of α -synuclein aggregation inhibitor.

2. Use of vitamin B12 in the manufacture of a medicament, nutraceutical or food product for the prevention and/or treatment of diseases characterized by the aggregation and precipitation of α -synuclein.

3. Use according to claim 1, characterized in that the disease is parkinson's syndrome.

4. Use according to claim 1, characterized in that vitamin B12 is present in an aqueous dispersion.

5. Use according to claim 1, characterized in that vitamin B12 is present in an aqueous dispersion of vitamin B12 at a concentration of 2.5-100 μ Μ.

6. Use according to claim 1, wherein vitamin B12 is present in an aqueous dispersion of vitamin B12 at a concentration of 5-50 μ Μ.