CN117379456A - Preparation and application of nano-selenium composite particles - Google Patents
Preparation and application of nano-selenium composite particles Download PDFInfo
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- CN117379456A CN117379456A CN202210784762.XA CN202210784762A CN117379456A CN 117379456 A CN117379456 A CN 117379456A CN 202210784762 A CN202210784762 A CN 202210784762A CN 117379456 A CN117379456 A CN 117379456A
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Classifications
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- A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
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- B—PERFORMING OPERATIONS; TRANSPORTING
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- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
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Abstract
The invention provides a preparation method and application of nano-selenium composite particles. The invention obtains nano selenium composite particles by co-processing and denaturing albumin and ethanol solution, opening the inner space of albumin, sequentially adding selenium compound and reducing agent cystine to perform reduction reaction on albumin carrier. The nano-selenium composite particle can be used for preparing medicines for preventing and treating neurodegenerative diseases and medicines for promoting repair or regeneration of tyrosine hydroxylase positive nerve cells in a substantia nigra region. The invention provides experimental evidence for clinical application of the nano-selenium composite particles, and simultaneously provides a new idea for PD prevention and drug development.
Description
Technical Field
The invention belongs to the field of medical nano materials, and particularly relates to preparation and application of nano selenium composite particles.
Background
Selenium is an important component of the antioxidant enzyme glutathione peroxidase to produce its main biological effect. The lack of selenium has been reported to induce or exacerbate more than 40 related diseases, especially diseases such as AIDS (AIDS), hepatitis B, white myopathy, skeletal muscle and myocardial necrosis (keshan disease), large bone joint disease, male sterility, cardiovascular and cerebrovascular diseases, liver cancer and other malignant tumors, diabetes, apoplexy, alzheimer's disease and other nervous system and immune dysfunction; meanwhile, the proper amount of selenium is added to help to improve the immunity of organisms, and has good inhibiting and repairing effects on pathological states such as aging, alzheimer disease, epilepsy, liver cancer, cardiovascular and cerebrovascular diseases, diabetic complications, AIDS, chronic hepatitis, renal fibrosis, kaempferia, leukemia, thyroid diseases and the like.
Traditional food sources or nutritional supplements include inorganic selenium (sodium selenite, sodium selenate) and organic selenium (yeast selenium, selenomethionine, selenium-enriched bacteria, selenocysteine). Clinical researches show that the safe dosage range of selenium in a human body is very narrow, the daily nutrition recommended intake of selenium is 60 mug, and the oral intake of selenium reaches 200 mug-300 mug, so that the oral liquid has the effect of preventing cancer; however, when selenium is taken daily at a dosage of 819 mug+ -126 mug or higher, a susceptible patient exhibits selenium poisoning, and the maximum daily intake of selenium is currently defined to be 400 mug to 500 mug. Therefore, the selenium supplement is easy to generate toxicity due to excessive selenium supplement, which limits the wide application of the traditional selenium compound in the aspect of clinical disease prevention and treatment, and the existing selenium preparation still has the problems of poor stability, easy aggregation and the like. Therefore, the preparation of the novel nano selenium preparation with good biocompatibility, good stability, high biological activity and low toxicity by adopting the nano technology is still an important scientific problem to be solved urgently, and especially the development and the mechanism research of the nano selenium preparation for preventing and treating brain diseases are important recently.
Parkinson's Disease (PD) is a central nervous system degenerative disease with bradykinesia, increased muscular tension, resting tremor and dysequilibrium as main clinical signs, which is the second most frequent neurodegenerative disease for elderly people in our country. According to the latest investigation, the parkinsonism prevalence rate of men and women in China is 1.7% and 1.6% respectively. With the arrival of the aging of most cities, the number of patients suffering from parkinsonism is in a rapid rising trend, and the prevention and treatment situations are very serious. About 5% of the cases reported show significant genetic susceptibility in PD patients and are also referred to as sporadic PD. However, the exact cause and pathogenesis of parkinsonism are not yet fully elucidated, and effective drug treatment and methods are not yet available. At present, the clinical treatment strategy for parkinsonism still uses exogenous dopamine supplementation to relieve clinical symptoms, but the drug treatment has serious side effects. Although new therapeutic regimens (e.g., deep brain electrical stimulation) and therapeutic drugs continue to emerge, they only partially alleviate their clinical symptoms and fail to prevent disease progression. Therefore, the method has extremely important significance for preventing PD and improving the life quality of PD patients by deeply researching the etiology of PD, exploring new therapeutic targets and developing new PD therapeutic drugs.
Disclosure of Invention
The present invention aims to solve at least one of the technical problems in the prior art described above. To this end, the first aspect of the present invention proposes a method for preparing nano-selenium composite particles.
The second aspect of the invention provides an application of nano-selenium composite particles in preparing medicines for preventing and treating neurodegenerative diseases and/or antagonizing nerve tissue injury.
The third aspect of the invention provides an application of nano-selenium composite particles in preparing a medicament for promoting repair or regeneration of tyrosine hydroxylase positive nerve cells in a substantia nigra region.
According to the first aspect of the invention, the preparation method of the nano-selenium composite particle comprises the following steps:
s1: dissolving albumin in ethanol solution, and stirring to obtain mixed solution;
s2: adding sodium selenite solution and cystine solution, stirring, and purifying to obtain nanometer selenium composite particle.
In the invention, albumin and ethanol solution are processed and denatured together, an albumin internal space is opened, sodium selenite and reducer cystine are sequentially added to carry out reduction reaction on an albumin carrier, and nano-selenium composite particles are obtained.
In some embodiments of the invention, the albumin concentration in the mixture of S1 is 10 mg/mL-30 mg/mL, preferably 15 mg/mL-25 mg/mL.
In some embodiments of the invention, the concentration of the ethanol solution in S1 is 60% to 85%.
In some embodiments of the present invention, the volume ratio of the sodium selenite solution, cystine solution in S2 to the mixed solution in S1 is 1 (8-11): 20-100, preferably 1 (9-10): 30-80.
In some embodiments of the invention, the concentration of the sodium selenite solution in S2 is 1 mM-100 mM, preferably 5mM mM mM-80 mM, more preferably 15 mM-50 mM, and even more preferably 20 mM-40 mM.
In some embodiments of the invention, the cystine solution concentration in S2 is 10 mM-200 mM, preferably 20mM mM mM-150 mM, more preferably 40 mM-100 mM, and even more preferably 50 mM-80 mM.
In some embodiments of the invention, the albumin in S1 is selected from at least one of human serum albumin, recombinant human serum albumin, bovine serum albumin, ovalbumin, donkey serum albumin, transferrin.
In some embodiments of the invention, the stirring time in S1 is 1 to 12 hours, preferably 3 to 8 hours, more preferably 4 to 6 hours.
In some embodiments of the invention, the temperature of the stirring in S1 is between 35 ℃ and 50 ℃.
In some embodiments of the invention, the stirring time in S2 is 1h to 24h, preferably 3h to 20h, further preferably 10h to 15h.
In some preferred embodiments of the invention, the purification in S2 comprises: high speed centrifugation or dialysis removes excess unreacted reagents.
Further, the molecular weight of the dialysis is 8 kD-14 kD, and the dialysis time is 2 days-3 days.
According to a second aspect of the invention, an application of nano-selenium composite particles in preparing medicines for preventing and treating neurodegenerative diseases and/or antagonizing nerve tissue injury is provided.
According to a third aspect of the invention, an application of nano-selenium composite particles in preparing a medicament for promoting repair or regeneration of tyrosine hydroxylase-positive nerve cells in a substantia nigra region is provided.
In some embodiments of the present invention, the above-described nano-selenium composite particles comprise albumin and nano-selenium, the albumin encapsulating the nano-selenium; the nano-selenium composite particles are spherical or spheroid, and have an average particle diameter of 15nm to 100nm, preferably 20nm to 80nm, and more preferably 25nm to 60nm.
In some embodiments of the present invention, the mass of the nano-selenium in the nano-selenium composite particle is 0.01% to 10%, preferably 0.1% to 8%, and more preferably 0.5% to 5% of the weight of the composite particle.
In some embodiments of the invention, the neurodegenerative disease is acute neurodegenerative disease and chronic neurodegenerative disease, preferably chronic neurodegenerative disease.
In some embodiments of the invention, the chronic neurodegenerative disease comprises Alzheimer's Disease (AD), parkinson's Disease (PD), huntington's Disease (HD), amyotrophic Lateral Sclerosis (ALS), spinocerebellar ataxia (SCA), pick's disease.
In some preferred embodiments of the invention, the chronic neurodegenerative disease is parkinson's disease.
In the present inventionIn the Ming dynasty, PD mouse model and MPP are prepared by utilizing neurotoxin MPTP at the same time + The PD cell model is prepared by administering the nano-selenium composite particle solution in a gastric lavage mode in a mouse model and adding nano-selenium composite particles in a cell culture medium to treat the PD cell model, so that the effect of relieving the disease condition of the Parkinson disease is achieved, and a new target and a new way are provided for clinically preventing and treating the Parkinson disease.
In the invention, the nano-selenium composite particles can improve the dyskinesia of the parkinsonism and increase the quantity of the nerve cells in the substantia nigra region and the tyrosine hydroxylase positive nerve cells.
In the invention, the inventor finds that the prepared nano-selenium composite particles can reduce the neurotoxic effect and relieve the parkinsonism.
In some preferred embodiments of the invention, the neural tissue is preferably a neural tissue in the brain nervous system.
In some preferred embodiments of the invention, the effective therapeutic dose of the nano-selenium composite particles is from 0.2mg Se/kg to 0.6mg Se/kg, preferably from 0.3mg Se/kg to 0.5mg Se/kg.
In some embodiments of the invention, the medicament comprises nano-selenium composite particles and pharmaceutically acceptable excipients.
In some embodiments of the invention, the pharmaceutically acceptable excipients include one or a combination of diluents, excipients, flavoring agents.
In some preferred embodiments of the invention, the pharmaceutical dosage form is an oral formulation.
In some more preferred embodiments of the present invention, the oral formulation is selected from any one of granules, capsules, tablets, pills.
The beneficial effects of the invention are as follows:
the nano-selenium composite particle has a simple preparation method, can reduce neurotoxicity effect, relieves parkinsonism disease conditions, and provides a new target and a new way for clinically treating parkinsonism.
Drawings
The invention is further described with reference to the accompanying drawings and examples, in which:
in FIG. 1, A is a transmission electron microscope image (scale bar, 20 nm) of the nano-selenium composite particles prepared by the invention; the curve in the B sequentially corresponds to the particle size distribution curve of Human Serum Albumin (HSA) and nano selenium composite particles (HSA/Se NPs) in the aqueous solution from left to right; c is Zeta potential analysis; d is Fourier transform infrared spectrum analysis; e is circular dichroism spectroscopy; f is the stability of the nano-selenium composite particle in ph=7.4 buffer.
Panel a in fig. 2 shows HE staining of heart, liver, spleen, lung, kidney of mice from left to right; b is the content detection result of the valley ammonia transferase (ALT) in the serum of the mice; c is the content detection result of urea nitrogen (BUN) in blood in the serum of the mice; d is the detection result of the creatinine (Cr) content in the serum of the mice.
In fig. 3, A, B is an open field experiment result, C is a rotating rod experiment result, D is a pole climbing experiment result, and E is a hanging experiment result.
FIG. 4 shows the results of detection of expression of the brain substantia nigra compact tyrosine hydroxylase in mice, wherein A is the results of TH immunohistochemical staining of the substantia nigra compact in mice; b is the statistics of the TH immunohistochemistry of the substantia nigra compact part of the mouse.
FIG. 5 shows the results of detection of expression of mouse striatum tyrosine hydroxylase, wherein A is the results of immunohistochemical staining of mouse striatum TH; b is the statistics of mouse striatum TH immunohistochemistry.
FIG. 6 shows the results of the expression test of tyrosine hydroxylase in the brain tissue of mice, wherein A is the WB result of the TH band in the brain tissue of mice; b is the statistical result of TH band WB in the brain tissue of the mice.
FIG. 7 shows the detection of tyrosine hydroxylase expression in MN9D cell lines, A is the WB result of TH band in MN9D cell lysates; b is the statistical result of TH band WB in MN9D cell lysate.
Detailed Description
The conception and the technical effects produced by the present invention will be clearly and completely described in conjunction with the embodiments below to fully understand the objects, features and effects of the present invention. It is apparent that the described embodiments are only some embodiments of the present invention, but not all embodiments, and that other embodiments obtained by those skilled in the art without inventive effort are within the scope of the present invention based on the embodiments of the present invention. The nano-selenium composite particles used in the following test examples were all exemplified by the nano-selenium composite particles prepared in example 1 unless otherwise specified.
Example 1
The embodiment prepares the nano-selenium composite particle, which comprises the following specific processes:
1000mg of Human Serum Albumin (HSA) was dissolved in 40mL of deionized water containing 75% ethanol, stirred at 40℃for 2 hours, and 1mL of 60 mM Na was added 2 SeO 3 Finally 9ml of cystine at 40mM concentration was added and stirred for 3h until red nano-selenium composite particles (HSA/Se NPs) were produced. The obtained nano selenium composite particles are dialyzed in deionized water by a dialysis bag with MWCO of 8 kD-14 kD for two days at room temperature, so that redundant unreacted reagents including ethanol and oxidized cystine byproducts can be removed, and then the nano selenium composite particles are stored at 4 ℃.
The nano selenium composite particles are digested with nitric acid at 50 ℃ overnight, and the loading efficiency of Se is measured by ICP-OES; observing the morphology of the nano-selenium composite particles by adopting a Transmission Electron Microscope (TEM) and a Dynamic Light Scattering (DLS) technology and evaluating the stability of the nano-selenium composite particles; circular Dichroism (CD) spectroscopy and fourier transform infrared spectroscopy (FT-IR) analysis of the secondary structure of the nano-selenium composite particles; the results are shown in FIG. 1.
In FIG. 1, A is a transmission electron microscope image (scale bar, 20 nm) of the nano-selenium composite particle; the curve in the B sequentially corresponds to the particle size distribution curve of Human Serum Albumin (HSA) and nano-selenium composite particles (HSA/Se NPs) in the aqueous solution from left to right; c is Zeta potential analysis, n=3/group; d is Fourier transform infrared spectrum analysis; e is circular dichroism spectroscopy; f is the stability of the nano-selenium composite particles in ph=7.4 buffer, n=3 times/group, no statistical difference (p > 0.05). Results are expressed as mean ± standard error of the mean.
Analysis of results: as shown in fig. a, the microstructure of the nano-selenium composite particles was observed by Transmission Electron Microscopy (TEM), and their average nano-size was about 20nm. Hydrodynamic detection of laser particle size analyzer (DLS) HSA/Se NPs hydrated particle size (-60 nm) was larger than HSA alone (-9 nm) (panel B); since the nano-selenium nano-particles are hydrated in the aqueous solution, all the hydrated particle sizes are larger than the TEM observation size under the dry condition. The zeta potential of HSA/Se NPs in Panel C is more negative than HSA (-36.6 mV vs-29.1 mV) and these more negative nanoparticles can accelerate intestinal absorption. Meanwhile, the secondary structure of HSA/Se NPs was demonstrated by CD spectroscopy and FT-IR analysis, e.g., CD absorbance of HSA/Se NPs was similar to that of native HSA, FT-IR spectroscopy of HSA/Se NPs was similar to that of HSA, providing direct evidence that HSA and nanoselenium constituted part of HSA/Se NPs (panels D, E). HSA/Se NPs showed good stability in ph=7.4 buffer for 21 days (figure F). The above results demonstrate that nano-selenium composite particles HSA/Se NPs are constructed according to self-assembly design.
Example 2
The embodiment prepares the nano-selenium composite particle, which comprises the following specific processes:
1000mg of Human Serum Albumin (HSA) was dissolved in 50mL of deionized water containing 77% ethanol, stirred at 35℃for 6 hours, and 1mL of 30mM Na was added 2 SeO 3 Finally 9ml of cystine at 30mM concentration was added and 5h was stirred until red nano-selenium composite particles (HSA/Se NPs) were produced. The obtained nano-selenium composite particles are subjected to centrifugation at 18000 Xg for 30min, and can remove excessive unreacted reagents including ethanol and oxidized cystine byproducts, and then are stored at 4 ℃. The hydrated particle size of HSA/Se NPs was measured to be about 80nm by DLS analysis.
Example 3
The embodiment prepares the nano-selenium composite particle, which comprises the following specific processes:
500mg of Human Serum Albumin (HSA) was dissolved in 50mL of deionized water containing 85% ethanol, stirred at 45℃for 5 hours, and 1mL of 20mM Na was added 2 SeO 3 Finally 9ml of cystine at 30mM concentration was added and stirred for 7h until red nano-selenium composite particles (HSA/Se NPs) were produced. Compounding the obtained nano seleniumThe particles were centrifuged at 18000 Xg for 60min to remove excess unreacted reagents, including ethanol and oxidized cystine by-products, and then stored at 4 ℃. The hydrated particle size of HSA/Se NPs was measured to be about 50nm by DLS analysis.
Example 4
The embodiment prepares the nano-selenium composite particle, which comprises the following specific processes:
800mg of Human Serum Albumin (HSA) was dissolved in 30mL of deionized water containing 81% ethanol, stirred at 42℃for 6 hours, and 1mL of 40mM Na was added 2 SeO 3 Finally 9ml of cystine at 50mM concentration was added and stirred for 12h until red nano-selenium composite particles (HSA/Se NPs) were produced. The obtained nano selenium composite particles are centrifuged at 18000 Xg for 60min, and redundant unreacted reagents including ethanol and oxidized cystine byproducts can be removed, and then the nano selenium composite particles are stored at 4 ℃. The hydrated particle size of HSA/Se NPs was measured to be about 90nm by DLS analysis.
Example 5
600mg of Human Serum Albumin (HSA) was dissolved in 30mL of deionized water containing 65% ethanol, stirred at 40℃for 8 hours, and 1mL of 40mM Na was added 2 SeO 3 Finally 9ml of cystine at 50mM concentration was added and stirred for 8h until red nano-selenium composite particles (HSA/Se NPs) were produced. The obtained nano selenium composite particles are dialyzed in deionized water by a dialysis bag with MWCO of 8 kD-14 kD for two days at room temperature, so that redundant unreacted reagents including ethanol and oxidized cystine byproducts can be removed, and then the nano selenium composite particles are preserved at 4 ℃. The hydrated particle size of HSA/Se NPs was measured to be about 50nm by DLS analysis.
Test example 1 biological safety of nano-selenium composite particles
(1) Short-term toxicity of nano-selenium composite particles at non-lethal dose
The 20 mice were randomly divided into two groups of 10 mice each. Control mice were orally administered physiological saline and drug group (HSA/Se NPs) mice were orally administered 1.0mg Se/kg dose of nano-selenium composite particles for 7 consecutive days. After 24h from the last dose, all animals were sacrificed.
(2) Blood and tissue preparation
At the end of each group of experiments, mice were sacrificed due to cervical dislocation. Peripheral blood from the ocular vein was collected in a non-heparinized tube and centrifuged to obtain serum. The mice were perfused with brain, heart, lung, spleen, liver, kidney, and rinsed with cold saline. The tissue samples were stored at-80 ℃ prior to analysis and the blood samples were stored in a 4 ℃ refrigerator prior to analysis.
(3) Biochemical parameters and histopathological analysis
The content of alanine Aminotransferase (ALT), blood Urea Nitrogen (BUN) and creatinine (Cr) in the serum of the mice was determined spectrophotometrically with a kit. Histopathological examination tissue specimens were fixed in paraformaldehyde for more than 8h, and the fixed organs were dehydrated in gradient ethanol and embedded in embedding medium. The tissue sections were HE stained with hematoxylin and eosin and then observed under a light microscope.
Results are shown in FIG. 2, A, from left to right, are HE staining of mice heart, liver, spleen, lung, kidney; b is the content detection result of glutamic pyruvic transaminase (ALT) in the serum of the mouse; c is the content detection result of urea nitrogen (BUN) in blood in the serum of the mice; d is the detection result of the creatinine (Cr) content in the serum of the mice. N=3 times per group, no statistical difference (p > 0.05), the results are expressed as mean ± standard error of the mean.
Analysis of results: as shown in the graph A, the HE staining experiment result shows that compared with the control group, the important mouse organ of the nano-selenium composite particle treatment group has no abnormality of tissue structure and cell morphology, and the nano-selenium composite particle has good biological safety. In the case of liver injury, liver metabolic enzymes such as ALT, AST and LDH are released from the liver into the serum. As shown in FIGS. B, C, and D, after mice orally administrate HSA/Se NPs, ALT, BUN and Cr were expressed at levels in serum that were not different from those of the control group. Experimental results show that HSA/Se NP has good histocompatibility and low toxicity.
Test example 2 preparation and identification of PD mouse model
MPTP preparation of PD mouse model
Dividing mice into four groups of Control groups, nano-selenium composite particle groups, MPTP (1-methyl-4-phenyl-1, 2,3, 6-tetrahydropyridine, neurotoxin) groups and MPTP+nano-selenium composite particle groups, injecting physiological saline into the abdominal cavity of the mice in the Control groups, injecting nano-selenium composite particles into the nano-selenium composite particle groups in a gastric lavage mode, and injecting MPTP 30mg/kg into the abdominal cavity of the MPTP groups; the nano-selenium composite particles are injected into stomach after MPTP of 30mg/kg is injected into the abdominal cavity.
Healthy male C57/bl mice (weight about 25 g-30 g) of 12 weeks old are randomly divided into a Control group, a nano-selenium composite particle group, an MPTP group and an MPTP+nano-selenium composite particle group, and are intraperitoneally injected with 30mg/kg of MPTP 1 time a day for 5 days. The modeling was examined by methods such as animal behaviours at day 4 after injection and immunohistochemical staining after mice were sacrificed.
The detecting content comprises the following steps:
(1) Change stick, pole climbing, hanging and open field experiment to detect mouse behavioural change
Rotating rod experiment: the rotarod experiment was used to evaluate the motor coordination ability of mice. Briefly, all mice were trained for three consecutive days, including 5min at constant speed (10 revolutions per minute), three times per day, and 5min at rest between each training. This training method ensures that all mice get the same training time. The fourth day, the rotameter parameters were set to accelerate from 10r/min to 40r/min within 5min. The residence time of each mouse on the rotarod apparatus was recorded.
Pole climbing experiment: a wooden pole with a round head (the diameter of the round head is 2 cm) with a diameter of 1cm and a length of 50cm is prepared, and gauze is wound around the wooden pole to prevent the mouse from slipping during climbing. After the manufacturing of the behavioural instrument was completed, the mouse tail was grasped by hand and the time required for the mouse to climb from the top to the bottom of the pole was recorded for statistical analysis. Training was continued for 3 days, 3 times per day, and testing was performed on day 4.
Suspension experiment: suspension experiments were used to assess the coordination of the limb movements of mice. A stainless steel bar (diameter 1.5cm, length 30 cm) was fixed to a plexiglass box at a distance of 30cm from the bottom of the box. Each mouse was suspended from an iron bar with the forelimb. The time to fall was recorded for each mouse. Training is carried out three times a day, and rest is carried out for 5min between each training. The test was performed on the fourth day, and each mouse was evaluated three times at 5min intervals. Suspension test scoring was performed by recording the residence time on the iron bars of each mouse.
Open field experiments: the open field experiment included a wall consisting of a white square floor (50 cm. Times.50 cm) and a height of 40 cm. The venue was brightly lit, with a central area (25 cm x 25 cm) and a peripheral area. Each mouse was placed in the center of the instrument and observed for 10min. Behavior parameters of mice were recorded with a video camera and analyzed using an Any-Maze behavior tracking software (Ugo Basile, USA). After each test, the apparatus was rinsed with 75% ethanol.
The results are shown in fig. 3, a and B are open field experimental results, C is a rotating rod experimental result, D is a pole climbing experimental result, E is a hanging experimental result, and p <0.05; * Represents p <0.001.
(2) Immunohistochemical staining of substantia nigra or striata tyrosine hydroxylase (tyrosine hydroxylase, TH) detects changes in dopaminergic neurons.
After the mice were anesthetized and heart perfused, whole brains were taken, dehydrated overnight with 20% 30% sucrose, OCT-embedded, serial coronal sections were performed using a frozen microtome, and brain tissue cut to a thickness of about 10 μm was attached to each slide; placing the cut slide in a 65 ℃ incubator for baking for about 3.5 hours; placing the baked slide in an ultralow temperature refrigerator at-80 ℃ so as to be used for subsequent research; taking out the slide to be dyed from the ultralow temperature refrigerator at the temperature of-80 ℃ and placing the slide at the room temperature for about 5min; baking in a constant temperature oven at 42 ℃ for 10min; then rinsing with PBS at least twice (about 5 min/time); antigen retrieval: placing the slide in a staining jar containing 0.01M citrate buffer (pH=6.0), and carrying out water bath at 98 ℃ for 10-15 min; the slide was placed in a staining jar containing 0.01M citrate buffer until naturally cooled to room temperature (about 30min required); the slides were rinsed 3 times in a staining jar containing 1 XPBS for about 5 minutes each; placing the slice in a staining jar containing 0.3% Triton, and rupture of membranes at 37deg.C for 30min; the slides were rinsed 3 times in a staining jar containing 1 XPBS for about 5 minutes each; the glass sheet was placed in a container containing 3% H 2 O 2 After incubation for 10min at room temperature in the staining jar,rinsing with 1 XPBS for 3 times, 5min each time, and drying the surrounding liquid; then continuing to dropwise add 5% BSA which is diluted and prepared, and sealing for 60min at room temperature; dripping diluted primary antibody, and placing the primary antibody in a wet box at 4 ℃ overnight (16-24 h) or a 37 ℃ constant temperature box for at least 2h; the slides were rinsed 3 times in a staining jar containing 1 XPBS for about 5 minutes each; dripping secondary antibody diluent, placing in an incubator, setting the parameters to be 37 ℃, and incubating for about 1h; the slides were rinsed 3 times in a staining jar containing 1 XPBS for about 5 minutes each and wiped dry; DAB color developer color development: dripping the prepared DAB on a brain slice, observing and detecting the color reaction time under a mirror at room temperature; placing the staining jar containing the glass sheet in running water for rinsing the glass sheet for about 10min; then, continuously counterstaining the glass slide with hematoxylin dye liquor for 1-2 min, and then rinsing with tap water for about 10min; color separation is carried out on the glass slide by using hydrochloric acid alcohol for 1 time, the time is between 10s and 30s (the time is determined according to the decolorization condition), and tap water is used for pickling for about 10 minutes; dehydrating: 70% ethanol 5min, 80% ethanol 5min, 90% ethanol 5min, 95% ethanol I5 min, 95% ethanol II 5min, 100% ethanol I5 min, 100% hundred-ethanol II 5min, xylene I5 min, xylene II 5min; and (5) sealing the sheet with neutral resin and performing microscopic examination. The results are shown in FIG. 4, FIG. 5 and FIG. 6.
FIG. 4 shows the results of expression detection of the brain substantia nigra compact tyrosine hydroxylase in mice, wherein the results of TH immunohistochemical staining of the mouse substantia nigra compact; b is the statistical result of the mouse substantia nigra dense TH immunohistochemistry, p <0.01, p <0.0001.
FIG. 5 shows the results of detection of expression of mouse striatum tyrosine hydroxylase, wherein A is the results of immunohistochemical staining of mouse striatum TH; b is the statistics of the mouse striatum TH immunohistochemistry; * P <0.001 and p <0.0001.
FIG. 6 shows the results of the expression test of tyrosine hydroxylase in the brain tissue of mice, wherein A is the WB result of the TH band in the brain tissue of mice; counting TH (TH) band WB (WB) in brain tissues of the mice B; * Representing p <0.05.
Analysis of results: after modeling according to experimental requirements, the content of the midbrain substantia nigra and the striatum dopaminergic neurons and dopamine in the PD group mice are found to be obviously reduced, the behavioural change of the mice is obvious, and the functional disorder is prominent. TH protein expression was reduced in the PD model. And a significant increase in TH protein expression following treatment with the nano-selenium composite particles indicates that they are therapeutically effective. It was therefore concluded that the nano-selenium composite particles can increase TH protein expression in PD models.
Test example 3 detection of TH protein expression level in PD model
The PD mouse model is obtained after MPTP injection, and then midbrain tissue lysate immunoblotting detection protein lysate TH protein is extracted. PD cell model is used for culturing mouse midbrain dopaminergic neuron cell line MN9D, and dividing cells into Control group, nano selenium composite particle group and MPP + Group and nano-selenium composite particle+MPP + Groups with medium, MPP respectively + (1 mM), solution of nano-selenium composite particles (200 nM), or nano-selenium composite particles+MPP + After the cells are treated for 24 hours by the working solution (the final concentration of the working solution and the working solution are the same as the final concentration), the cell lysate immunoblotting detection protein lysate TH protein is extracted. The results are shown in FIG. 6.
FIG. 7 shows the detection of tyrosine hydroxylase expression in MN9D cell lines, A is the WB result of TH band in MN9D cell lysates; b is TH band WB statistics in MN9D cell lysates, representing p <0.05.
Analysis of results: after modeling according to experimental requirements, the expression of TH in the PD group cell model is found to be significantly reduced, and the significant increase of TH protein expression after treatment with the nano-selenium composite particle indicates that the treatment is recovered. It was therefore concluded that the nano-selenium composite particles can increase TH protein expression in PD cell models.
While the embodiments of the present invention have been described in detail, the present invention is not limited to the above embodiments, and various changes may be made without departing from the spirit of the present invention, within the knowledge of those skilled in the art. Furthermore, embodiments of the invention and features of the embodiments may be combined with each other without conflict.
Claims (10)
1. The preparation method of the nano-selenium composite particles is characterized by comprising the following steps:
s1: dissolving albumin in ethanol solution, and stirring to obtain mixed solution;
s2: adding sodium selenite solution and cystine solution, stirring, and purifying to obtain nanometer selenium composite particle.
2. The method of claim 1, wherein the albumin concentration in the mixture of S1 is 10mg/mL to 30mg/mL.
3. The method of claim 1, wherein the concentration of the ethanol solution in S1 is 60% to 85%.
4. The method according to claim 1, wherein the volume ratio of the sodium selenite solution, the cystine solution in S2 and the mixed solution in S1 is 1 (8-11): 20-100.
5. The method of claim 1, wherein the concentration of the sodium selenite solution in S2 is 1mM to 100mM and the concentration of the cystine solution is 10mM to 200mM.
6. Application of nano-selenium composite particles in preparing medicines for preventing and treating neurodegenerative diseases and/or nerve tissue injury antagonism medicines.
7. Application of nano-selenium composite particles in preparing medicines for promoting repair or regeneration of black matrix region tyrosine hydroxylase positive nerve cells.
8. The use according to claim 6 or 7, wherein the nano-selenium composite particles comprise albumin and nano-selenium, the albumin encapsulating the nano-selenium; the nano selenium composite particles are spherical or spheroid, and the average particle diameter is 15 nm-100 nm.
9. The use according to claim 8, wherein the mass of the nano-selenium in the nano-selenium composite particle is 0.01% -10% of the weight of the composite particle.
10. The use according to claim 8, wherein the therapeutically effective dose of the nano-selenium composite particles is between 0.2mg Se/kg and 0.6mg Se/kg.
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