CN111700908A

CN111700908A - Application of nano metal sulfide in preparation of nerve injury repair drug

Info

Publication number: CN111700908A
Application number: CN202010444427.6A
Authority: CN
Inventors: 薛雪; 毛明星
Original assignee: Nankai University
Current assignee: Nankai University
Priority date: 2020-05-22
Filing date: 2020-05-22
Publication date: 2020-09-25

Abstract

The invention belongs to the field of nerve injury repair medicines, and particularly relates to application of nano metal sulfide in preparation of a nerve injury repair medicine. The beneficial effects are that: provides a new idea for preparing the nerve injury repairing medicine; the material is easy to obtain, the cost is low, and the preparation is easy.

Description

Application of nano metal sulfide in preparation of nerve injury repair drug

Technical Field

The invention belongs to the field of nerve injury repair medicines, and relates to application of nano metal sulfide in preparation of a nerve injury repair medicine.

Background

The complexity of the nervous system prevents many drugs from entering the brain, and the nerve cells have limited self-repair capacity, which results in irreversible and progressive disease of many nervous systems, ultimately leading to severity of the results, enormous economic losses and social burden.

The nano material represented by ferroferric oxide has peroxide mimic enzyme activity, so that the efficiency of generating free radicals by hydrogen peroxide can be improved, and the sterilization effect is enhanced. The generated free radicals can effectively degrade proteins, nucleic acids and polysaccharides. Nanoparticles are currently commonly used as highly effective antimicrobial agents.

No data disclose the use of nanosulfides as neurorestorative agents.

The invention provides a new application of nano metal sulfide, provides a new idea for treating related diseases and solves the problems in the prior art.

Disclosure of Invention

The invention aims to solve the technical problem of providing a new application of nano metal sulfide and providing a thought for treating nerve injury.

The invention discloses application of nano metal sulfide in preparing a nerve injury repair medicine.

Further, the nano metal sulfide is a nano metal sulfide compound or a composition.

Further, the nano metal sulfide is a compound M formed by combining metal ions and polysulfide bonds shown in a general formula (I)_xS_n：

Or combinations of said compounds, wherein n is any of 2, 3, 4, 5.

Preferably, n is 2 or 3.

Preferably, the nano metal sulfide adopts disulfide M comprising metal_xS₂Trisulfide with metals M_xS₃In which S is the amount of the substance₂：S₃＝1:1-5。

Further, the addition amount of the nano metal sulfide is 0.1-10 mg/kg.

Preferably, the addition amount of the nano metal sulfide is 2.5 mg/kg.

Preferably, the administration concentration of the nano metal sulfide is 0.5 mg/mL.

Further, the usage mode of the nano metal sulfide is at least one of oral administration, intravenous injection and intramuscular injection.

Further, the particle size of the nano metal sulfide particles is 10-100 nm.

Preferably, the nano metal sulfide adopts nano iron sulfide Fe with certain particle size and particle shape_1-yA mixture of S, wherein said S is-2 valent and said y is 0.1 to 0.2.

Preferably, the nano metal sulfide is prepared by a method comprising the following steps:

step 1, dissolving an iron source and a water or non-aqueous solvent in proportion to obtain a reaction solution A;

step 2, adding an alkaline solution into the reaction solution A, and stirring to dissolve the alkaline solution to obtain a reaction solution B;

step 3, adding a sulfur source into the reaction liquid B, and stirring to dissolve the sulfur source to obtain a reaction liquid C;

and 4, heating the reaction solution C to prepare the nano iron sulfide mixture.

Further, the step 4 includes a step of heating and drying the reaction solution.

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

Further, 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.

Further, the non-aqueous solvent is at least one of an alcohol solvent, an ether solvent, a ketone solvent, a hydrocarbon solvent and an ester solvent; 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.

Further, the alkaline solution is selected from sodium acetate, sodium citrate, sodium bicarbonate, sodium carbonate, sodium hydroxide, potassium hydroxide, NH₄ ⁺Preferably, a solution of sodium acetate, sodium citrate and/or sodium bicarbonate is used.

Further, the sulfur source is selected from sulfur-containing compounds; the sulfur-containing compounds are classified 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 acid and thioether compound; more preferably, the sulfur-containing amino acid is selected from one or more of L-cysteine, cystine, GSH (glutathione), and the thioether compound may be an unsaturated thioether compound, in particular at least one of allyl methyl sulfide, diallyl sulfide, allyl methyl disulfide, diallyl disulfide, allyl methyl trisulfide, diallyl trisulfide, and allyl ethyl sulfide.

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

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

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

The invention has the beneficial effects that:

1. provides a new idea for preparing the nerve injury repairing medicine;

2. the material is easy to obtain, the cost is low, and the preparation is easy;

drawings

FIG. 1 is a graph of the results of a test of the protective effect of in vitro damaged neurons of application example 1;

FIG. 2 application example 2 test results of the protective effect of in vitro damaged neurons;

FIG. 3 application example 3 test results of the protective effect of in vitro damaged neurons;

FIG. 4 is a graph of the results of a test of the protective effect of in vitro damaged neurons of application example 4;

FIG. 5 application example 5 test results of protective effect of in vitro damaged neurons;

FIG. 6 is a graph of the results of a test of the protective effect of in vitro damaged neurons of application example 6;

FIG. 7 is a graph showing the results of a test for the effect of application example 1 on Tuj1 protein expression in vivo;

FIG. 8 is a graph showing the results of a test for the effect of application example 2 on Tuj1 protein expression in vivo;

FIG. 9 is a graph showing the results of a test for the effect of application example 3 on Tuj1 protein expression in vivo;

FIG. 10 is a graph showing the results of tests for the effect of application examples 4-6 on Tuj1 protein expression in vivo;

FIG. 11 is a graph showing the results of an assay for the effect of application example 1 on GFAP protein expression in vivo;

FIG. 12 is a graph showing the results of a test for the effect of application example 2 on GFAP protein expression in vivo;

FIG. 13 is a graph showing the results of the test for the effect of application example 3 on GFAP protein expression in vivo;

FIG. 14 is a graph showing the results of tests for the effects of application examples 4 to 6 on GFAP protein expression in vivo;

FIG. 15 is a graph of the results of the ataxia modulation test using the example 1 rotor test;

FIG. 16 is a graph of the results of the ataxia modulation test using the example 2 rotarod test;

FIG. 17 application example 3 ataxia regulation test bar test results chart;

FIG. 18 is a graph of the results of the application example 4-6 ataxia modulation test rotor test;

FIG. 19 is a graph of the results of the upset test using the ataxia modulation test of example 1;

FIG. 20 is a graph of the results of the upset test using the ataxia modulation test of example 2;

FIG. 21 application example 3 ataxia regulation test flip test results chart;

FIG. 22 is a graph of the results of the upset test using the ataxia modulation test of examples 4-6;

FIG. 23 shows a schematic diagram of a fluorescent Jade B staining test for degenerative nerve cells in brain slice of example 1 using the Fluoro Jade B staining technique;

Detailed Description

The following examples are given to illustrate the technical examples of the present invention more clearly and should not be construed as limiting the scope of the present invention.

The materials and instruments of the embodiment and the application example of the invention are selected as follows:

experimental animals:

c57BL6/J (SPF grade, male, 8-10 months of age, body weight 22. + -.3 g) was purchased from Spbefu animals Inc.

Reagent:

MTT powder was purchased from solibao corporation;

fluoro Jade B dye from Merck;

tuj1, GFAP primary antibody purchased from CST;

absolute ethanol was purchased from anlilong; ECL luminescence was purchased from Millipore;

the primary anti-dilution solution is purchased from Biyun Tian;

goat Anti-Mouse IgG (H & L) -HRP Conjugated from Berolingjie;

OCT embedding medium is purchased from oriental cherry;

eosin and hematoxylin staining solutions were purchased from Solebao;

TUNEL staining solution was purchased from petunia;

equipment:

CO₂the model of the incubator: Thermo-BB 15;

the type of the microplate reader: MR-96A;

model of inverted fluorescence microscope: leica DMiL;

the model of the rod rotating instrument: DB024 for improving intelligence and mouse;

the type of the freezing microtome is as follows: leica 1950;

chemiluminescence imaging system model: tonon 4800;

data processing software: GraphPad Prism 6.

Example 1

The nano metal sulfide is prepared by adopting a method comprising the following steps:

step 1, dissolving ferric chloride and ethylene glycol according to a molar ratio of 1:237 to obtain a reaction solution A;

step 2, adding sodium acetate into the reaction liquid A, and stirring to dissolve the sodium acetate to obtain reaction liquid B, wherein the concentration of the sodium acetate in the reaction liquid B is 0.7 mol/L;

step 3, adding L-cysteine into the reaction liquid B, and stirring to dissolve the L-cysteine to obtain reaction liquid C, wherein the concentration of the L-cysteine in the reaction liquid C is 0.04 mol/L;

and 4, heating the reaction liquid C to 200 ℃, and reacting for 12 hours to prepare the nano iron sulfide mixture.

Application example 1

The nano iron sulfide mixture prepared in example 1 is used for preparing an acute poisoning recovery medicine for ethanol. In order to illustrate the beneficial effects, the following tests are specially made:

protection of ethanol-damaged nerve cells in vitro by nano metal sulfide

The in vitro cell survival rate is detected by adopting an MTT method. SH-SY5Y neuroblastoma cells were plated evenly in 96-well plates at a cell density of 5E6, 100. mu.L per well. Placing the cell plate in an incubator containing 5% CO2, culturing for 4h at 37 ℃, then sucking out the original culture medium, adding a fresh culture medium containing 500mM ethanol to cause injury to SH-SY5Y neuroblastoma, simultaneously adding six concentrations of nano iron sulfide mixtures of 0, 7, 9, 11, 13 and 15 mu g/mL, and placing the cell plate back in the incubator to continue culturing for 24 h. The following day, the medium was aspirated, 100. mu.L of a 1mg/mL MTT solution was added, the medium was further incubated in an incubator for 4 hours, the MTT was aspirated, 150. mu.L of DMSO was added to dissolve formazan, and absorbance at 490nm was detected using a microplate reader.

The experimental results are shown in the attached figure 1: the addition of 500mM ethanol can cause more than 40% of the neuroblastoma cells to die, and after the nano iron sulfide mixture is given, the survival rate of the neuroblastoma cells and the concentration of the nano sulfide mixture show a dose-dependent relationship, and the survival rate of the neuroblastoma cells is improved by more than 10%, which shows that the nano iron sulfide mixture can improve the survival rate of damaged nerve cells in vitro.

Protection of damaged nerve cells after acute alcohol poisoning by nano metal sulfide

And (3) adopting a Fluoro Jade B staining technology to stain the degenerative nerve cells in the brain slice, wherein the degenerative neurons are bright green. The animal experiment selects SPF-grade C57BL/6J mice of 9-10 weeks old. The nano iron sulfide mixture with the concentration of 0.5mg/mL is administered into the body of a mouse by a gastric lavage method according to the dose of 2.5mg/kg, and after 10min, 25 percent ethanol is administered into the body of the mouse by an intraperitoneal injection method according to the dose of 2.2 g/kg. Cardiac perfusion was performed after 3 h. Taking out the perfused rat brain, and taking care to avoid injuring brain tissues. Soaking with 4% PFA overnight, soaking with 20% sucrose solution and 30% sucrose solution for one day, respectively, and soaking with 40% sucrose solution for 2-3 d. The brain slices with a thickness of 15 μm were cut out using a frozen section. FJB staining: the slices were immersed in absolute ethanol for 5min, then rinsed in 70% ethanol for 2min, rinsed in 30% ethanol for 2min, and finally kept in distilled water for 2 min. The sections were then incubated in 0.06% potassium permanganate solution for 30 min. Rinsing with running water for 2min, the sections were transferred to a 0.0004% Fluoro Jade B Chemicon) solution dissolved in 0.1% acetic acid. Washing with distilled water for 1min for 3 times. Clarified in xylene for 2min and covered with DPX non-fluorescent mounting medium. Visualization was performed under a Nikon Eclipse E600 fluorescence microscope using a 520nm fluorescein filter set.

The experimental results are shown in the attached figure 7: scare bar was 100 μm. It can be seen from fig. 2 that the acute ethanol group had the most degenerative neurons, while the treatment group administered with the nano iron sulfide mixture had a reduced number of degenerative neurons, indicating that the nano iron sulfide mixture could protect neurons in the brain from acute alcoholism.

The expression of a mature neuron cell marker Tuj1 protein is detected by using a Western Blot technology. The animal experiment selects SPF-grade C57BL/6J mice of 9-10 weeks old. The nano iron sulfide mixture with the concentration of 0.5mg/mL is administered into the body of a mouse by a gastric lavage method according to the dose of 2.5mg/kg, and after 10min, 25 percent ethanol is administered into the body of the mouse by an intraperitoneal injection method according to the dose of 2.2 g/kg. Perfusing heart after 3 hr, and extracting total protein by conventional method. 12% SDS-PAGE gels were prepared and protein samples were separated. Transferring the protein to a PVDF membrane by a wet transfer method, sealing with 5% skimmed milk for 1h, incubating with Tuj1 primary antibody for 1h, washing the membrane with TBST for 3 times, 5min each time, adding secondary antibody for incubation for 1h, washing the membrane with TBST for 3 times, 5min each time, dripping a developing solution, and exposing and imaging with a chemiluminescence imaging system.

The experimental results are shown in the attached figure 11: the expression of the acute ethanol group Tuj1 is obviously lower than that of the normal group, and the expression of the Tuj1 of the treatment group given with the nano iron sulfide mixture is increased compared with that of the acute ethanol comparative example 2.

Relieving effect of nano metal sulfide on brain inflammation induced by acute alcoholism

When inflammation occurs in the brain, astrocytes are over-activated to produce a series of pro-inflammatory factors, thereby inducing inflammation. The expression of the astrocyte activation marker GFAP protein is detected by using a Western Blot technology. The animal experiment selects SPF-grade C57BL/6J mice of 9-10 weeks old. The nano iron sulfide mixture with the concentration of 0.5mg/mL is administered into the body of a mouse by a gastric lavage method according to the dose of 2.5mg/kg, and after 10min, 25 percent ethanol is administered into the body of the mouse by an intraperitoneal injection method according to the dose of 2.2 g/kg. Perfusing heart after 3 hr, and extracting total protein by conventional method. 12% SDS-PAGE gels were prepared and protein samples were separated. Transferring the protein to a PVDF membrane by a wet transfer method, sealing with 5% skimmed milk for 1h, incubating with a GFAP primary antibody for 1h, washing the membrane with TBST for 3 times, 5min each time, adding a secondary antibody for incubation for 1h, washing the membrane with TBST for 3 times, 5min each time, dripping a developing solution, and exposing and imaging with a chemiluminescence imaging system.

The experimental results are shown in the attached figure 15: the GFAP expression of the acute ethanol comparative example 2 is higher than that of the normal group, and the GFAP expression of the treatment group given with the nano iron sulfide mixture is reduced compared with that of the acute ethanol comparative example 2, so that the nano iron sulfide mixture has a relieving effect on brain inflammation induced by acute alcoholism.

Improvement of cerebellar ataxia after acute alcohol poisoning by nano metal sulfide

The improvement effect of the nano iron sulfide mixture on cerebellar ataxia is tested by adopting a turnover experiment and a balance experiment. The mice used in the experiment are C57BL/6J 8-10 weeks old. The nano iron sulfide mixture with the concentration of 0.5mg/mL is administrated into an ethanol poisoning mouse by a stomach filling method according to the dose of 2.5mg/kg, and a turning experiment and a balance experiment are carried out after 30 min.

Turning over the experiment: the mouse was placed in the center of an iron net of 25X 30cm, which was 50cm high from the ground, and the iron net was quickly turned over to measure the time for the mouse to climb to the top.

The experimental results are shown in figure 19: in the overturning test, the rats poisoned by the ethanol are difficult to climb from the center to the top of the iron net and are easy to fall off from the iron net, the time on the suspended vertical iron net is reduced, and the time of the rats given the nano iron sulfide mixture on the iron net is improved compared with the rats given the ethanol group only, which shows that the nano iron sulfide mixture can improve the balance capability of the rats poisoned by the ethanol.

Rod turning experiment: the performance of the spinning rod was evaluated on a suspension rod of an accelerated spinning rod set (diameter: 3cm) which was accelerated at a constant rate of 1 to 23rpm for 300 seconds. Mice were trained for 5 consecutive days and placed on rods for 3 trials. The time of each trial was recorded. When the mouse falls off the rotating rod or the time reaches 300 seconds, the test is finished. A rest of 180 seconds was allowed between each trial.

The experimental results are shown in figure 23: the exercise time of the rats poisoned by the ethanol in the rod rotating test is reduced by one time compared with that of the rats in the normal group, and the exercise time of the rats given with the nano iron sulfide mixture is obviously improved compared with that of the rats given with the ethanol group only, which shows that the nano iron sulfide mixture can improve the exercise capacity of the rats poisoned by the ethanol.

Application examples 2 to 4

Application examples 2-4 are compared with application example 1, only the specific selection of the nano-metal sulfide mixture is different, and the parameters are shown in table 1:

TABLE 1 selection of Nano Metal sulfides-1

Application examples 5 to 6

Application examples 4-6 are compared with application example 1, only the specific selection of nano metal sulfide is different, and the parameters are shown in table 2:

TABLE 2 selection of Nano Metal sulfides-2

To further illustrate the beneficial effects of the present invention, the effects of different material selections on the effects were explored, and comparative examples 1-2 were specifically set, with comparative example 1 being the same class of normal cells and mice (normal group). Comparative example 2 compared to comparative example 1, only the nanometal sulfide was not added (poisoned control).

The test experiments of application example 1 were repeated for comparative examples 1 to 2 and application examples 2 to 6, and the test results are shown in table 3:

TABLE 3 summary of test results

Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. An application of nano metal sulfide in preparing the medicine for repairing nerve damage is disclosed.

2. The use of the nano-metal sulfide as claimed in claim 1 for preparing a nerve injury repair medicament, wherein the nano-metal sulfide is added in an amount of 0.1-10 mg/kg.

3. The use of nano-metal sulfide as claimed in claim 1 for preparing a medicament for repairing nerve damage, wherein the nano-metal sulfide is added in an amount of 2.5 mg/kg.

4. The use of the nanometal sulfide of claim 1 for the preparation of a medicament for the repair of nerve damage, wherein the nanometal sulfide is administered at a concentration of 0.5 mg/mL.

5. The use of the nano-metal sulfide as claimed in claim 1 for preparing a nerve injury repair medicament, wherein the nano-metal sulfide is used in at least one of oral administration, intravenous injection and intramuscular injection.

6. The use of the nano-metal sulfide of claim 1 for the preparation of a medicament for repairing nerve damage, wherein the nano-metal sulfide particles have a particle size of 10 to 100 nm.

7. The use of the nano-metal sulfide of claim 1 for preparing a medicament for repairing nerve damage, wherein the nano-metal sulfide is a nano-metal sulfide compound or composition.

8. The use of the nanometal sulfide of any one of claims 1 to 7 for the preparation of a medicament for repairing nerve damage, wherein the nanometal sulfide is a compound M in which a metal ion is bonded to a polysulfide bond of the general formula (I)_xS_n：

Or combinations of said compounds, wherein n is any of 2, 3, 4, 5.

9. The use of the nanometal sulfide of claim 8 for the preparation of a medicament for repairing nerve damage wherein n is 2 or 3.

10. The method of claim 8The application of the nano metal sulfide in preparing the nerve injury repairing medicine is characterized in that the nano metal sulfide adopts disulfide M containing metal_xS₂Trisulfide with metals M_xS₃In which S is the amount of the substance₂：S₃＝1:1-5。