Application of a nano-metal sulfide in the preparation of drugs for treating diseases caused by brain nerve damage

Abstract

This invention belongs to the field of nerve damage repair drugs, specifically involving the application of nano-metal sulfides in the preparation of drugs for treating diseases caused by brain nerve damage. Its advantages are: it provides a new approach to preparing nerve damage repair drugs; the materials are readily available, low in cost, and easy to prepare.

Description

Technical Field

[0001] This invention belongs to the field of nerve damage repair drugs, and relates to the application of nano-metal sulfides in the preparation of drugs for treating diseases caused by brain nerve damage. Background Technology

[0002] The complexity of the nervous system prevents many drugs from reaching the brain, and the limited self-repair capabilities of nerve cells contribute to the irreversibility and progressive nature of many neurological diseases, ultimately leading to severe consequences, huge economic losses, and social burdens.

[0003] Nanomaterials, such as iron(III) oxide (Fe3O4), possess peroxide-mimicking enzyme activity, which can enhance the efficiency of hydrogen peroxide in generating free radicals and thus improve bactericidal effects. The generated free radicals can effectively degrade proteins, nucleic acids, and polysaccharides. Currently, nanoparticles are commonly used as highly effective antibacterial agents.

[0004] There is no publicly available information on the application of nano-sulfides as nerve repair drugs.

[0005] This invention provides a novel application for nano-metal sulfides, offering a new approach to treating related diseases and addressing problems in existing technologies. Summary of the Invention

[0006] The technical problem to be solved by this invention is to provide a new use for nano-metal sulfides and to offer a solution for treating nerve damage.

[0007] This invention discloses the application of a nano-metal sulfide in the preparation of drugs for treating diseases caused by brain nerve damage.

[0008] Furthermore, the nano-metal sulfide is a nano-metal sulfide compound or composition.

[0009] Furthermore, the nano-metal sulfide is a compound M consisting of metal ions bonded to polysulfide bonds represented by general formula (I). x S n :

[0010]

[0011] Or a composition of the compound, wherein n takes any value from 2, 3, 4, or 5.

[0012] Preferably, n = 2 or 3.

[0013] Preferably, the nano-metal sulfide is a disulfide M comprising a metal. x S2 and metal trisulfides M x A mixture of S2 and S3, wherein, by amount of substance, S2:S3 = 1:1-5.

[0014] Furthermore, the amount of the nano-metal sulfide added is 0.1-10 mg / kg.

[0015] Preferably, the amount of the nano-metal sulfide added is 2.5 mg / kg.

[0016] Preferably, the dosage concentration of the nano-metal sulfide is 0.5 mg / mL.

[0017] Furthermore, the nano-metal sulfide is administered via at least one of oral, intravenous, or intramuscular injection.

[0018] Furthermore, the particle size of the nano-metal sulfide particles is 10-100 nm.

[0019] Preferably, the nano-metal sulfide is nano-iron sulfide Fe with a certain particle size and shape. 1-y The mixture is S, wherein S has a valence of -2 and y has a valence of 0.1-0.2.

[0020] Preferably, the nano-metal sulfide is prepared by a method comprising the following steps:

[0021] Step 1: Dissolve the iron source in water or a non-aqueous solvent in a certain proportion to obtain reaction solution A;

[0022] Step 2: Add an alkaline solution to reaction solution A and stir to dissolve it, thus obtaining reaction solution B;

[0023] Step 3: Add the sulfur source to reaction solution B and stir to dissolve it, thus obtaining reaction solution C;

[0024] Step 4: Heat the reaction solution C to prepare a mixture of nano-iron sulfide.

[0025] Furthermore, step 4 includes heating and drying the reaction solution.

[0026] Furthermore, the heating temperature in step 4 is 100-500℃, preferably 200-400℃; the heating time is 1-48h, preferably 12-24h.

[0027] Furthermore, the iron source is selected from ferrous iron and / or ferric iron, preferably at least one of ferric chloride, ferrous sulfate, ferric nitrate, and ferric bromide.

[0028] Further, the non-aqueous solvent is at least one of alcohol solvents, ether solvents, ketone solvents, hydrocarbon solvents, and ester solvents; preferably, the non-aqueous solvent is an alcohol solvent; more preferably, the alcohol solvent is at least one of ethylene glycol, glycerol, ethanol, and polyethylene glycol.

[0029] Furthermore, the alkaline solution is selected from sodium acetate, sodium citrate, sodium bicarbonate, sodium carbonate, sodium hydroxide, potassium hydroxide, and NH4. + At least one of the following, preferably a solution of sodium acetate, sodium citrate and / or sodium bicarbonate.

[0030] Further, the sulfur source is selected from sulfur-containing compounds; sulfur-containing compounds are divided into organic sulfur-containing compounds and inorganic sulfur-containing compounds. Preferably, the sulfur-containing compound is selected from one or more of sulfur-containing amino acids and thioether compounds; more preferably, the sulfur-containing amino acid is selected from one or more of L-cysteine, cystine, and GSH (glutathione), and the thioether compound may be an unsaturated thioether compound, especially at least one of allyl methyl sulfide, diallyl sulfide, allyl methyl disulfide, diallyl disulfide, allyl methyl trisulfide, diallyl trisulfide, and allyl ethyl sulfide.

[0031] Furthermore, the molar ratio of the iron source to water or non-aqueous solvent is 1:1-1000; the final concentration of the alkaline solution is 0.01-1 mol / L; and the final concentration of the added sulfur source is 0.01-10 mol / L.

[0032] Further, the molar ratio of the iron source to the sulfur source is 1:0.02-5, preferably 1:0.05-2, more preferably 1:0.06-1, more preferably 1:0.1-0.8, more preferably 1:0.2-0.6, and more preferably 1:0.3-0.5.

[0033] Further, the weight ratio of the iron source to the sulfur source is 1:0.1-10; the weight ratio of the iron source:water or non-aqueous solvent:alkaline solution:sulfur source is 0.1-1:10-100:0.1-1:0.1-1, preferably 0.5-1:20-80:0.5-1:0.5-1, more preferably 0.5-1:30-60:0.5-1:0.5-1.

[0034] The beneficial effects of this invention are as follows:

[0035] 1. It provides a new approach to preparing drugs for nerve injury repair;

[0036] 2. The materials are readily available, low in cost, and easy to prepare; Attached Figure Description

[0037] Figure 1 Application Example 1: Results of in vitro protective effect test on damaged neurons;

[0038] Figure 2 Application Example 2: Results of in vitro protective effect test on damaged neurons;

[0039] Figure 3Application Example 3: Results of in vitro protective effect test on damaged neurons;

[0040] Figure 4 Application Example 4: Results of in vitro protective effect test on damaged neurons;

[0041] Figure 5 Application Example 5: Results of in vitro protective effect test on damaged neurons;

[0042] Figure 6 Application Example 6: Results of in vitro protective effect test on damaged neurons;

[0043] Figure 7 Figure 1 shows the results of the test on the effect of Application Example 1 on the expression of Tuj1 protein in vivo.

[0044] Figure 8 Figure 2 shows the results of the test on the effect of Example 2 on the expression of Tuj1 protein in vivo;

[0045] Figure 9 Figure 3 shows the results of the test on the effect of Example 3 on the expression of Tuj1 protein in vivo;

[0046] Figure 10 Figure 4-6 shows the results of the test on the effect of Tuj1 protein expression in vivo.

[0047] Figure 11 Figure 1 shows the results of the test on the effect of Application Example 1 on the expression of GFAP protein in vivo.

[0048] Figure 12 Figure 1 shows the results of the test on the effect of Application Example 2 on the expression of GFAP protein in vivo.

[0049] Figure 13 Figure 3 shows the results of the test on the effect of Example 3 on the expression of GFAP protein in vivo.

[0050] Figure 14 Figure 4-6 shows the results of the test on the effect of GFAP protein expression in vivo.

[0051] Figure 15 Application Example 1: Ataxia Adjustment Test Rotary Bar Test Results (Figure);

[0052] Figure 16 Application Example 2: Ataxia Regulation Test Rotary Bar Test Results (Figure 2)

[0053] Figure 17 Application Example 3: Ataxia Adjustment Test Rotary Bar Test Results Graph;

[0054] Figure 18 Application Example 4-6: Ataxia Adjustment Test Rotary Bar Test Results Graph;

[0055] Figure 19Application Example 1: Ataxia Adjustment Test Flip Test Results (Figure);

[0056] Figure 20 Application Example 2: Ataxia Adjustment Test Flip Test Results (Figure 2)

[0057] Figure 21 Application Example 3: Ataxia Adjustment Test Flip Test Results (Figure);

[0058] Figure 22 Application Example 4-6: Ataxia Adjustment Test Flip Test Results (Figure);

[0059] Figure 23 The structural diagram of degenerative nerve cells in a brain slice corresponding to Case 1, using Fluoro Jade B staining technique; Detailed Implementation

[0060] The specific embodiments of the present invention will be further described below with reference to the examples. The following examples are only used to illustrate the technical embodiments of the present invention more clearly, and should not be used to limit the scope of protection of the present invention.

[0061] The materials and instruments selected for the embodiments and application examples of this invention are as follows:

[0062] Laboratory animals:

[0063] C57BL6 / J (SPF grade, male, 8-10 months old, weight 22±3g) was purchased from Spaford Animal Company.

[0064] Reagents:

[0065] MTT powder was purchased from Solarbio.

[0066] Fluoro Jade B dye was purchased from Merck;

[0067] Tuj1 and GFAP primary antibodies were purchased from CST.

[0068] Anhydrous ethanol was purchased from Amlogic; ECL luminescent solution was purchased from Millipore.

[0069] The primary antibody diluent was purchased from Beyotime.

[0070] Goat Anti-Mouse IgG(H&L)-HRP Conjugated was purchased from Baiyijie.

[0071] OCT embedding agent was purchased from Sakura;

[0072] Eosin and hematoxylin staining solutions were purchased from Solarbio.

[0073] TUNEL staining solution was purchased from Beyotime.

[0074] equipment:

[0075] CO2 incubator model: Thermo-BB15;

[0076] Microplate reader model: MR-96A;

[0077] Inverted fluorescence microscope model: Leica DMiL;

[0078] Rotating rod device model: Zhishu Duobao DB024;

[0079] model of cryostat slicer: Leica 1950;

[0080] Chemiluminescence imaging system model: Tonon 4800;

[0081] Data processing software: GraphPad Prism6.

[0082] Example 1

[0083] The nano-metal sulfide was prepared using a method comprising the following steps:

[0084] Step 1: Dissolve ferric chloride and ethylene glycol in a molar ratio of 1:237 to obtain reaction solution A;

[0085] Step 2: Add sodium acetate to reaction solution A and stir to dissolve it, to obtain reaction solution B. The concentration of sodium acetate in reaction solution B is 0.7 mol / L.

[0086] Step 3: Add L-cysteine ​​to reaction solution B and stir to dissolve it, to obtain reaction solution C. The concentration of L-cysteine ​​in reaction solution C is 0.04 mol / L.

[0087] Step 4: Heat reaction solution C to 200℃ and react for 12 hours to prepare a mixture of nano-iron sulfide.

[0088] Application Example 1

[0089] The nano-iron sulfide mixture prepared in Example 1 was used to prepare a drug for the recovery of acute ethanol poisoning. To illustrate its beneficial effects, the following tests were performed:

[0090] Protective effect of nano-metal sulfides on nerve cells damaged by ethanol in vitro

[0091] The MTT assay was used to detect cell viability in vitro. SH-SY5Y neuroblastoma cells were evenly seeded at a density of 5E6 cells per well in 96-well plates (100 μL per well). After 4 hours of incubation in a 5% CO2 incubator at 37°C, the original culture medium was removed, and fresh culture medium containing 500 mM ethanol was added to induce damage to the SH-SY5Y neuroblastoma cells. Simultaneously, six concentrations of nano-ferric sulfide (0, 7, 9, 11, 13, and 15 μg / mL) were added, and the plates were returned to the incubator for another 24 hours. The next day, the culture medium was removed, and 100 μL of 1 mg / mL MTT solution was added. The plates were incubated for another 4 hours, the MTT solution was removed, and 150 μL of DMSO was added to dissolve formazan. The absorbance at 490 nm was measured using a microplate reader.

[0092] The experimental results are attached. Figure 1 Adding 500mM ethanol causes more than 40% of neuroblastoma cells to die. However, after administering the nano-iron sulfide mixture, the survival rate of neuroblastoma cells showed a dose-dependent relationship with the concentration of the nano-iron sulfide mixture, and the survival rate of neuroblastoma cells increased by more than 10%, indicating that the nano-iron sulfide mixture can improve the survival rate of damaged nerve cells in vitro.

[0093] Protective effect of nano-metal sulfides on damaged nerve cells after acute ethanol poisoning

[0094] Fluoro Jade B staining was used to stain degenerative neurons in brain slices, which appeared bright green. SPF-grade 9-10 week old C57BL / 6J mice were used in this animal experiment. Mice were administered a 0.5 mg / mL nano-iron sulfide mixture at a dose of 2.5 mg / kg via gavage. Ten minutes later, 25% ethanol was administered intraperitoneally at a dose of 2.2 g / kg. Cardiac perfusion was performed 3 hours later. The perfused brains were carefully removed to avoid damaging the brain tissue. The brains were soaked overnight in 4% PFA, then for one day each in 20% and 30% sucrose solutions, and finally for 2-3 days in 40% sucrose solution. Brain slices with a thickness of 15 μm were obtained from frozen sections. For FJB staining: the slices were immersed in anhydrous ethanol for 5 minutes, then rinsed in 70% ethanol for 2 minutes, then in 30% ethanol for 2 minutes, and finally in distilled water for 2 minutes. The sections were then incubated in a 0.06% potassium permanganate solution for 30 min. After rinsing with running water for 2 min, the sections were transferred to a 0.0004% Fluoro Jade B Chemicon solution dissolved in 0.1% acetic acid. The sections were washed three times with distilled water, 1 min each time. After clarifying in xylene for 2 min, the sections were covered with DPX non-fluorescent mounting medium. Visualization was performed under a Nikon Eclipse E600 fluorescence microscope using a 520 nm fluorescein filter set.

[0095] The experimental results are attached. Figure 7 The scare bar is 100μm. (From the attached document...) Figure 2 The results showed that the acute ethanol group had the most degenerative neurons, while the treatment group given the nano-iron sulfide mixture had a reduced number of degenerative neurons, indicating that the nano-iron sulfide mixture can protect against neuronal damage in the brain caused by acute ethanol poisoning.

[0096] Western blotting was used to detect the expression of Tuj1 protein, a marker of mature neuronal cells. SPF-grade C57BL / 6J mice aged 9–10 weeks were used in this animal experiment. Mice were administered a 0.5 mg / mL nano-iron sulfide mixture at a dose of 2.5 mg / kg via gavage. Ten minutes later, 25% ethanol was administered intraperitoneally at a dose of 2.2 g / kg. Three hours later, the heart was perfused, and total protein was extracted using standard methods. A 12% SDS-PAGE gel was prepared to separate the protein samples. Proteins were transferred to PVDF membranes using wet transfer, blocked with 5% skim milk for 1 hour, incubated with Tuj1 primary antibody for 1 hour, washed three times with TBST for 5 minutes each time, incubated with secondary antibody for 1 hour, washed three times with TBST for 5 minutes each time, and then exposed to light using a chemiluminescence imaging system for imaging.

[0097] The experimental results are attached. Figure 11 Tuj1 expression in the acute ethanol group was significantly lower than that in the normal group, while Tuj1 expression in the treatment group given the nano-iron sulfide mixture was increased compared to the acute ethanol control group (Figure 2).

[0098] The alleviating effect of nano-metal sulfides on brain inflammation induced by acute ethanol poisoning

[0099] When inflammation occurs in the brain, astrocytes are overactivated, producing a series of pro-inflammatory factors, thereby inducing inflammation. Western blotting was used to detect the expression of GFAP protein, a marker of astrocyte activation. SPF-grade 9-10 week old C57BL / 6J mice were used in this animal experiment. Mice were administered a 0.5 mg / mL nano-iron sulfide mixture at a dose of 2.5 mg / kg via gavage. Ten minutes later, 25% ethanol was administered intraperitoneally at a dose of 2.2 g / kg. Three hours later, cardiac perfusion was performed, and total protein was extracted using standard methods. Protein samples were separated using a 12% SDS-PAGE gel. Proteins were transferred to PVDF membranes using a wet transfer method. The membranes were blocked with 5% skim milk for 1 hour, incubated with GFAP primary antibody for 1 hour, washed three times with TBST for 5 minutes each time, incubated with secondary antibody for 1 hour, washed three times with TBST for 5 minutes each time, and then developed with a chemiluminescence imaging system for exposure and imaging.

[0100] The experimental results are attached. Figure 15 The GFAP expression in the acute ethanol control group (Comparative Example 2) was higher than that in the normal group. The GFAP expression in the treatment group given the nano-iron sulfide mixture was lower than that in the acute ethanol control group (Comparative Example 2), indicating that the nano-iron sulfide mixture has a relieving effect on brain inflammation induced by acute ethanol poisoning.

[0101] Improvement of cerebellar ataxia following acute ethanol poisoning by nano-metal sulfides

[0102] The ameliorative effect of nano-iron sulfide mixture on cerebellar ataxia was investigated using flipping and balance tests. Eight to ten-week-old C57BL / 6J mice were used in this experiment. The 0.5 mg / mL nano-iron sulfide mixture was administered to ethanol-intoxicated mice via gavage at a dose of 2.5 mg / kg. Flipping and balance tests were performed 30 minutes later.

[0103] Flipping experiment: Place the mouse in the center of a 25×30cm wire mesh, which is 50cm above the ground. Quickly flip the wire mesh and measure the time it takes for the mouse to climb to the top.

[0104] The experimental results are attached. Figure 19 In the flipping test, rats poisoned by ethanol had difficulty climbing from the center to the top of the wire mesh and were prone to falling off. The time spent on the suspended vertical wire mesh was reduced. However, the time spent on the wire mesh by rats given the nano-iron sulfide mixture was increased compared to the rats given only ethanol, indicating that the nano-iron sulfide mixture can improve the balance ability of rats poisoned by ethanol.

[0105] Spinner Test: The performance of the spinning rod was evaluated on a suspension rod of an accelerating spinning rod device (diameter: 3 cm). The spinning rod device was accelerated at a constant rate of 1 to 23 rpm for 300 seconds. Mice were trained continuously for 5 days and placed on the rod for 3 trials. The time of each trial was recorded. The trial ended when the mouse fell off the spinning rod or when 300 seconds had elapsed. A 180-second rest period was allowed between each trial.

[0106] The experimental results are attached. Figure 23 In the rotarod test, the treadmill time of ethanol-poisoned mice was reduced by half compared to the normal group. However, the treadmill time of mice given the nano-iron sulfide mixture was significantly increased compared to the ethanol-only group, indicating that the nano-iron sulfide mixture can improve the treadmill ability of ethanol-poisoned mice.

[0107] Application Example 2-4

[0108] Compared with Application Example 1, Application Examples 2-4 differ only in the specific selection of the nano-metal sulfide mixture; the parameters are shown in Table 1.

[0109] Table 1 Selection of Nanometal Sulfides - 1

[0110]

[0111] Application Example 5-6

[0112] Compared with Application Example 1, Application Examples 4-6 differ only in the specific selection of the nano-metal sulfide; the parameters are shown in Table 2.

[0113] Table 2 Selection of Nanometal Sulfides - 2

[0114]

[0115] To further illustrate the beneficial effects of this invention and to explore the influence of different material selections on the effects, comparative examples 1 and 2 were set up. Comparative example 1 consisted of normal cells and mice of the same type (normal group). Comparative example 2 was the same as comparative example 1, except that no nano-metal sulfide was added (poisoning control group).

[0116] The test experiments of Example 1 were repeated in Comparative Examples 1-2 and Application Examples 2-6. The test results are shown in Table 3:

[0117] Table 3 Summary of Test Results

[0118]

[0119]

[0120] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. The application of a nano-iron sulfide mixture as a preparation of a drug for the recovery of acute ethanol poisoning, characterized in that, The nano-iron sulfide mixture protects nerve cells damaged after acute ethanol poisoning; The preparation method of the nano-iron sulfide mixture includes the following steps: Step 1: Dissolve ferric chloride and ethylene glycol in a molar ratio of 1:237 to obtain reaction solution A; Step 2: Add sodium acetate to reaction solution A and stir to dissolve it, to obtain reaction solution B. The concentration of sodium acetate in reaction solution B is 0.7 mol / L. Step 3: Add L-cysteine ​​to reaction solution B and stir to dissolve it, to obtain reaction solution C. The concentration of L-cysteine ​​in reaction solution C is 0.04 mol / L. Step 4: Heat reaction solution C to 200℃ and react for 12 hours to prepare a mixture of nano-iron sulfide.

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