CN118662482A - A preparation method and application of CSAD-VB12/SeMet microsphere hydrogel - Google Patents

Abstract

The invention discloses a preparation method and application of a CSAD-VB12/SeMet microsphere hydrogel, the preparation method comprising the following steps: mixing and dissolving lecithin, cholesterol and vitamin E and evaporating them into a film; dissolving the film into a SeMet solution, ultrasonically treating it, and obtaining a liposome solution; adding the liposome solution into a CSAD-VB12 solution for mixed reaction, and obtaining a CSAD-VB12/SeMet microsphere hydrogel. The invention encapsulates SeMet by liposomes and CSAD-VB12 to prepare a CSAD-VB12/SeMet microsphere hydrogel material, the preparation method is simple, and the preparation effect is good, the CSAD-VB12/SeMet microsphere hydrogel system has good stability, has strong antioxidant capacity, and can alleviate fluorine-induced reproductive damage in male mice.

Description

A preparation method and application of CSAD-VB12/SeMet microsphere hydrogel

Technical Field

The invention relates to the technical field of microsphere hydrosol, and in particular to a preparation method and application of a CSAD-VB12/SeMet microsphere hydrogel.

Background Art

Selenium is an essential trace element for animals and humans, and has antioxidant, anti-inflammatory, chemopreventive and antiviral properties. In nature, selenium exists in the form of organic selenium (selenoamino acids, selenium polysaccharides and selenoethers) and inorganic selenium (selenites, selenides). Inorganic selenium has a narrow safety range, high toxicity, and difficult to control dosage; while organic selenium has low toxicity, less pollution to the environment, and is more effective in absorption, anti-oxidation and tissue accumulation. However, it is unstable when used in premixes and feeds, has low utilization, and is difficult to apply in production practice. Nanoformulations are a drug delivery system that has been widely used in recent years. Compared with other traditional dosage forms, they have the function of improving the oral effect of drugs and are often used to improve drug stability and utilization, control drug release, and reduce drug toxicity.

Liposomes are similar in morphology to cell membranes, have good biocompatibility and biodegradability, and can bind to various substances. They are widely used as delivery systems for a variety of bioactive substances. Sodium alginate is a linear anionic polysaccharide composed of two hexuronic acid residues, β-D-mannuronic acid (M) and α-L-guluronic acid (G). It has high biocompatibility, biodegradability, non-toxicity and non-immunogenicity. The amphiphilic alginate derivatives obtained by hydrophobic modification can self-assemble in aqueous solution to form nanocapsules with core-shell structures. The combination of the two as drug carriers has the characteristics of improving drug encapsulation efficiency and targeting. At present, there have been studies on the use of polysaccharide-encapsulated selenium to prepare nanoparticles, which can improve the biological activity and safety of selenium, but there are still problems such as poor stability and premature release of active ingredients. Therefore, it is necessary to develop a new liposome, sodium alginate-encapsulated selenium microspheres to improve stability and achieve a stable sustained release effect.

Summary of the invention

The technical problem to be solved by the present invention is to provide a preparation method and application of a CSAD-VB12/SeMet microsphere hydrogel, using liposomes and CSAD (amphiphilic cholesterol grafted sodium alginate derivative) containing VB12 (vitamin B12) as carriers to encapsulate SeMet (selenomethionine), and preparing the CSAD-VB12/SeMet microsphere hydrogel material by thin film hydration and self-assembly methods. The prepared CSAD-VB12/SeMet microsphere hydrogel system has good stability, strong antioxidant ability and can alleviate fluoride-induced reproductive damage in male mice.

In order to solve the above technical problems, the present invention provides a method for preparing CSAD-VB12/SeMet microsphere hydrogel, comprising the following steps:

S1, mixing and dissolving lecithin, cholesterol and vitamin E and evaporating them into a film;

S2, dissolving the film in a SeMet solution, and ultrasonically treating the solution to obtain a liposome solution;

S3. Add the liposome solution into the CSAD-VB12 solution for mixed reaction to obtain CSAD-VB12/SeMet microsphere hydrogel.

The present invention adopts lecithin and cholesterol as main raw materials to prepare liposomes. There are two hydrophobic hydrocarbon chains and a hydrophilic group in the lecithin molecule. The hydrophilic groups face the water phases on both sides. The hydrophobic hydrocarbon chains are mutually associated to form a bilayer to form the liposomes. Cholesterol plays the role of a liposome membrane fluidity regulator for the liposomes. Vitamin E is added to maintain the integrity of the liposomes, keep the stability thereof, and is conducive to enhancing the antioxidant performance thereof. SeMet is encapsulated by the liposomes and CSAD-VB12 to prepare a CSAD-VB12/SeMet microsphere hydrogel material. The CSAD-VB12/SeMet microsphere hydrogel system has good stability and strong antioxidant capacity.

Furthermore, the concentration of the SeMet solution is 1-10 g/L, and the concentration of CSAD-VB12 in the CSAD-VB12 solution is 8-16 g/L.

Furthermore, the volume ratio of the liposome solution to the CSAD-VB12 solution is 1:(1-2).

Furthermore, in S3, the mixing reaction is carried out under magnetic stirring for 12-24 hours.

Furthermore, in S2, the ultrasonic treatment time is 0.5-1 h, and before the ultrasonic treatment, the magnetic stirring is performed for 1-2 h.

Furthermore, in S1, the evaporation is specifically: rotary evaporation at 25-35°C for 5-6 hours, followed by evaporation in a vacuum drying oven for 3-4 hours.

Furthermore, in S1, the mixed dissolving solvent is anhydrous ethanol.

Furthermore, the preparation method of the SeMet solution is: dissolving the rich yeast and trypsin in 100 mmol/L tris(hydroxymethyl)aminomethane buffer (pH 7.0-8.0), reacting on a 35-40°C water bath shaker for 24-48 hours, centrifuging at 3000-5000 r/min for 0.5-1 hour, removing the supernatant, and filtering with a 0.22-0.45 μm needle filter to obtain a SeMet solution.

Furthermore, the mass ratio of the added amount of lecithin to SeMet in the SeMet solution is (12-18):1.

Furthermore, the mass ratio of lecithin to cholesterol is (10-5):1.

Further, the preparation method of the CSAD-VB12 solution is as follows: sodium alginate and anhydrous p-toluenesulfonic acid are dissolved in anhydrous dimethyl sulfoxide, stirred at 50-70°C for 0.5-1 h, N,N'-dicyclohexylcarboximide, 4-dimethylaminopyridine and chloroform containing cholesterol are added, magnetic stirring is performed at room temperature for 24-48 h, the precipitate is washed with anhydrous ethanol, and then dried in a vacuum drying oven at 30-50°C for 24-48 h to obtain CSAD; CSAD is dissolved in anhydrous dimethyl sulfoxide as reagent one for standby use, VB12 and N,N'-carbonyldiimidazole are dissolved in anhydrous dimethyl sulfoxide, stirred at room temperature in a nitrogen atmosphere for 1-2 h as reagent two, reagent one and reagent two are mixed, stirred in a nitrogen atmosphere at room temperature in the dark for 24-48 h, acetone is added for precipitation, and centrifuged at 3000-4000 r/min for 0.5-1 h to obtain CSAD-VB12, the precipitate was dissolved in distilled water, and the pH was adjusted to 6-8 to obtain a CSAD-VB12 solution.

Furthermore, the mass ratio of CSAD to VB12 is (10-18):1.

The preparation principle of the CSAD-VB12/SeMet microsphere hydrogel of the present invention is as follows: there are two relatively long hydrophobic hydrocarbon chains and one hydrophilic group in the lecithin molecule, an appropriate amount of phospholipids is added to the buffer solution, the phospholipid molecules are arranged in a directional manner, the hydrophilic groups thereof face the aqueous phases on both sides, the hydrophobic hydrocarbon chains are mutually associated to form a bilayer, and a liposome is formed, and the additive cholesterol is an amphiphilic substance, which is mixed with the phospholipids to adjust the fluidity of the bilayer, reduce the permeability of the liposome membrane, and make the liposome more stable. Sodium alginate is a linear anionic polysaccharide composed of two hexuronic acid residues, β-D-mannuronic acid (M) and α-L-guluronic acid (G), and under the action of two catalysts, N, N'-dicyclohexylcarboximide and dimethylaminopyridine, the carboxyl group thereof is chemically modified by an esterification reaction with cholesterol, thereby improving the hydrophobicity of sodium alginate, and the cholesterol ester of sodium alginate can self-aggregate in an aqueous solution to form nanoparticles, thereby further encapsulating SeMet.

The second aspect of the present invention provides a CSAD-VB12/SeMet microsphere hydrogel prepared by the preparation method described in the first aspect, wherein the particle size of the CSAD-VB12/SeMet microsphere is 900-1200 nm.

Furthermore, the storage temperature of the CSAD-VB12/SeMet microsphere hydrogel is 0-8°C.

The third aspect of the present invention provides the use of the CSAD-VB12/SeMet microsphere hydrogel described in the second aspect in a preparation for alleviating reproductive damage caused by fluoride poisoning.

Furthermore, the dosage of CSAD-VB12/SeMet microsphere hydrogel in the relief preparation is 0.4-0.6 mg/kg•bw.

Beneficial effects of the present invention:

The present invention encapsulates SeMet by liposomes and CSAD-VB12 to prepare a CSAD-VB12/SeMet microsphere hydrogel material. The preparation method is simple and has good preparation effect. The CSAD-VB12/SeMet microsphere hydrogel system has good stability and strong antioxidant ability.

The main raw materials of the invention, lecithin, cholesterol and sodium alginate, are cheap and easily available, and have the advantages of anti-oxidation, high biocompatibility, high biodegradability, no immunogenicity and low toxicity.

The CSAD-VB12/SeMet microsphere hydrogel prepared by the present invention can effectively inhibit the oxidative stress of the reproductive system of male mice induced by high fluoride, and antagonize the reproductive toxicity of fluoride by promoting sperm production; it increases the serum selenium content, and significantly increases the glutathione peroxidase 4, total cholesterol and testosterone contents, thereby alleviating the reproductive damage induced by fluoride in male mice.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a scanning electron microscope image of SeMet in Example 1;

Fig. 2 is a scanning electron microscope image of the liposome in Example 1;

FIG3 is a scanning electron microscope image of the CSAD-VB12/SeMet microsphere hydrogel of Example 1;

FIG4 is a particle size analysis diagram of SeMet, liposomes and CSAD-VB12/SeMet in Example 1;

FIG5 is a graph showing the encapsulation efficiency of SeMet, liposomes and CSAD-VB12/SeMet at 4° C. for 30 days in Example 1;

FIG6 is a graph showing the selenium release rate of the liposomes in simulated gastric fluid and simulated intestinal fluid in Example 1;

FIG7 is a graph showing the selenium release rate of CSAD-VB12/SeMet microsphere hydrogel in simulated gastric fluid and simulated intestinal fluid of Example 1;

FIG8 is a graph showing the DPPH radical scavenging rates of SeMet, liposomes and CSAD-VB12/SeMet in Example 1, wherein A: SeMet, B: liposomes, C: CSAD-VB12/SeMet;

FIG9 is a graph showing the ABTS radical scavenging rates of SeMet, liposomes and CSAD-VB12/SeMet in Example 1, wherein A: SeMet, B: liposomes, and C: CSAD-VB12/SeMet;

FIG10 is the experimental results of sperm deformity rate in each group in Example 2;

FIG11 is the experimental results of sperm motility of each group in Example 2;

FIG12 is the experimental results of sperm density of each group in Example 2;

FIG13 is a graph showing the serum selenium content of each group of mice in Example 2;

FIG14 is a HE staining image of the testicles of each group of mice in Example 2 (200×);

FIG15 is a HE staining image of the testicles of each group of mice in Example 2 (400×);

Figure 16 is the content of ROS in the testicles of each group of mice in Example 2;

Figure 17 is the content of MDA in the testicles of each group of mice in Example 2;

Figure 18 is the content of GSH-PX in the testicles of each group of mice in Example 2;

Figure 19 is the content of CAT in the testicles of each group of mice in Example 2;

Figure 20 is the content of T-AOC in the testicles of each group of mice in Example 2;

Figure 21 is the content of SOD in the testicles of each group of mice in Example 2;

Figure 22 is the content of glutathione peroxidase 4 in the testicles of each group of mice in Example 2;

Figure 23 is the total cholesterol content in the testicles of each group of mice in Example 2;

Figure 24 shows the testosterone content in the testicles of each group of mice in Example 2.

DETAILED DESCRIPTION

The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.

Example 1. This example relates to a method for preparing a CSAD-VB12/SeMet microsphere hydrogel, comprising the following steps:

0.6 g of lecithin and 0.1 g of cholesterol were added to 10 mL of anhydrous ethanol, and then 26 µL of vitamin E was added. The mixture was subjected to constant temperature rotary evaporation at 30°C for 5-6 h, and then placed in a vacuum drying oven for further evaporation for 3-4 h. The film was dissolved with 5 mL of 8 g/L SeMet solution, and the liposome solution was prepared after magnetic stirring for 1 h and ultrasonication for 30 min.

1 g of sodium alginate was dissolved in 38 mL of anhydrous dimethyl sulfoxide, and 0.324 g of anhydrous p-toluenesulfonic acid was weighed and mixed, and stirred at 60°C for 30 min. 0.4 g of N,N'-dicyclohexylcarboximide, 0.475 g of 4-dimethylaminopyridine and 2 mL of chloroform containing 0.66 g of cholesterol were added, and magnetic stirring was performed at room temperature for 24 h. Finally, 200 mL of anhydrous ethanol was added to wash the precipitate, and then dried in a vacuum drying oven at 40°C for 24 h to obtain CSAD.

Dissolve 0.5 g CSAD in 20 mL anhydrous dimethyl sulfoxide for later use (reagent one). Meanwhile, dissolve 0.3 g VB12 and 0.04 g N,N'-carbonyldiimidazole in 10 mL anhydrous dimethyl sulfoxide (reagent two). Stir for 1 h in a nitrogen atmosphere at room temperature. Mix reagent one and reagent two and stir for 24 h in a nitrogen atmosphere at room temperature in the dark. Add 400 mL acetone to precipitate. Centrifuge at 3500 r/min for 20 min to obtain CSAD-VB12. Dissolve the precipitate with distilled water and adjust the pH to 7 to prepare a CSAD-VB12 solution.

The liposome solution was dropped into the CSAD-VB12 solution (volume ratio 1:1) and magnetically stirred for 12 h to obtain the CSAD-VB12/SeMet microsphere hydrogel.

The molecular formulas of the lecithin and cholesterol are C ₄₂ H ₈₀ NO ₈ P and C ₂₇ H ₄₆ O, and their molecular weights are 758.06 and 386.65, respectively. The molecular formula of the sodium alginate is C ₆ H ₇ NaO ₆ , and the M/G ratio is 1:1. The lecithin, cholesterol, and sodium alginate are purchased from MacLean Reagent, Solebold Technology Co., Ltd., and MacLean Reagent, with item numbers L812367, C8280, and S817374, respectively.

The SeMet solution was prepared by adding 60 mg of trypsin to 0.1 g of selenium-enriched yeast and dissolving it in 5 mL of 100 mmol/L Tris buffer (pH=7.6), reacting it on a 37°C water bath shaker for 24 h, centrifuging it at 4000 r/min for 30 min, removing the supernatant, and filtering it with a 0.45 μm needle filter.

Test Example 1

(1) The morphology and particle size of SeMet, liposomes, and CSAD-VB12/SeMet prepared in Example 1 were characterized by scanning electron microscopy and nanoparticle size analyzer.

Take an appropriate amount of SeMet, liposomes, and CSAD-VB12/SeMet samples prepared in this example, wash them twice with PBS, discard the liquid, wash them once with 4% (w/v) sucrose solution, discard the sucrose solution, and dehydrate them with 30%, 50%, 70%, 80%, 90%, 95%, and 100% anhydrous ethanol for 10 minutes each. Gently stick the sample on the conductive glue, critical point dry, vacuum spray, and finally select a suitable position and appropriate magnification for observation under an electron microscope (FEI, USA). Then use Nano Measure software to count the particle size of SeMet, liposomes, and CSAD-VB12/SeMet.

As shown in Figures 1, 2, and 3, in the scanning electron microscope images of SeMet, liposomes, and CSAD-VB12/SeMet, SeMet and CSAD-VB12/SeMet are relatively regular spheres, CSAD-VB12/SeMet is evenly distributed, the system is stable, and liposomes are irregular clusters. As shown in Figure 4, after statistics, it was found that the particle sizes of SeMet, liposomes, and CSAD-VB12/SeMet were 275.33 ± 13.57 nm, 828.83 ± 30.02 nm, and 1244.33 ± 20.27 nm, respectively. The particle size of the microspheres increased step by step, indicating the existence of liposomes and CSAD-VB12/SeMet microspheres, proving that liposomes and CSAD-VB12 successfully encapsulated SeMet.

(2) The encapsulation efficiency and release rate of the liposomes prepared in Example 1 and CSAD-VB12/SeMet were studied.

Take 2 mL of liposomes and CSAD-VB12/SeMet and centrifuge them in an ultrafiltration tube (4000 r/min, 10 min). The liquid in the outer tube of the ultrafiltration tube is digested with nitric acid to a clear and transparent solution, then passed through a 0.22 μm syringe filter, and the selenium content is detected by ICP-MS. The encapsulation efficiency of liposomes and CSAD-VB12/SeMet is calculated as follows:

Encapsulation efficiency = (W total selenium – W free selenium) / W total selenium × 100%

5 mL of liposomes and CSAD-VB12/SeMet were mixed with 15 mL of simulated gastric juice and simulated intestinal juice, respectively, and reacted at a constant temperature of 150 rpm on a shaker at 37°C. Every 1 h, 2 mL of the mixed solution was centrifuged in an ultrafiltration tube (4000 r/min, 10 min). The liquid in the outer tube of the ultrafiltration tube was added with 6 mL of nitric acid and placed on an electric furnace for digestion. After that, it was filtered with a 0.22 µm needle filter and the selenium content was detected by ICP-MS. The formula for calculating the selenium release rate is:

Selenium release rate = W released/W total selenium × 100%

As shown in Figure 5, the encapsulation efficiency of freshly prepared liposomes and CSAD-VB12/SeMet was 67.42% and 91.94%, respectively. After being stored at 4°C in the dark for 30 days, the encapsulation efficiency of CSAD-VB12/SeMet was always higher than that of liposomes, indicating that the modification of liposomes by CSAD-VB12 can significantly improve the encapsulation efficiency of liposomes. Figures 6 and 7 are the release rates of liposomes and CSAD-VB12/SeMet in simulated gastric fluid and simulated intestinal fluid, respectively, from 0 to 7 h. The release of selenium in the simulated gastric fluid environment was significantly less than that in the simulated intestinal fluid, indicating that both have strong acid resistance and can maintain a relatively complete structure in the gastric environment, while being released in large quantities in the simulated intestinal fluid.

(3) The in vitro antioxidant capacity of SeMet, liposomes and CSAD-VB12/SeMet prepared in Example 1 was determined.

DPPH free radical scavenging rate was determined. According to the instructions of DPPH, SeMet, liposomes and CSAD-VB12/SeMet were appropriately diluted and then operated in a 96-well plate. The DPPH free radical scavenging rates of SeMet, liposomes and CSAD-VB12/SeMet microspheres with the same concentration were compared. The calculation formula is:

DPPH free radical scavenging rate % = [(1-(A determination-A control) ÷ A blank) × 100]%

ABTS free radical scavenging rate was determined. According to the ABTS instructions, SeMet, liposomes and CSAD-VB12/SeMet were appropriately diluted and then operated in a 96-well plate. The ABTS free radical scavenging rates of SeMet, liposomes and CSAD-VB12/SeMet at the same concentration were compared. The calculation formula is:

ABTS free radical scavenging rate % = [A blank - (A determination - A control)] ÷ A blank × 100%

The results are shown in Figure 8. The DPPH radical scavenging rates of SeMet, liposomes and CSAD-VB12/SeMet at the same concentration were (12.86 ± 1.75)%, (39.95 ± 1.74)% and (71.86 ± 1.99)%, respectively. As shown in Figure 9, the ABTS radical scavenging rates of SeMet, liposomes and CSAD-VB12/SeMet microsphere hydrogel at the same concentration were (16.01 ± 0.53)%, (75.99 ± 1.60)% and (77.11 ± 6.36)%, respectively, indicating that liposomes and CSAD-VB12/SeMet can significantly improve the in vitro antioxidant activity of SeMet, and CSAD-VB12/SeMet has better antioxidant performance.

Example 2: The CSAD-VB12/SeMet microsphere hydrogel prepared in Example 1 is used as a preparation for alleviating male reproductive damage caused by fluorine poisoning.

By establishing a fluorine poisoning model, the improvement effect of oral administration of CSAD-VB12/SeMet microsphere hydrogel prepared in Example 1 on fluorine-induced reproductive damage in male mice was verified, including statistics of basic sperm indicators, determination of serum selenium content, pathological histological observation of testicular tissue, and detection of oxidative stress indicators, glutathione peroxidase 4, total cholesterol and testosterone in testicular tissue. The chronic fluorine poisoning model was established using 150 mg/L NaF in drinking water, and the dose of CSAD-VB12/SeMet microsphere hydrogel used for oral administration was 0.5 mg/kg•bw. The specific grouping and treatment are as follows: 90 three-week-old male C57BL6J mice weighing approximately 20±5g were randomly divided into six groups, 15 in each group, provided by the Experimental Animal Center of Shanxi Medical University (Taiyuan, Shanxi), and pellet feed and bedding were purchased from Sibefu Biotechnology Co., Ltd. The grouping is as follows:

Control group (C): mice were given free access to distilled water and intragastrically administered with normal saline.

Fluoride group (F): Mice freely drank 150 mg/L sodium fluoride (NaF) solution and were gavaged with normal saline.

Se group (Se): mice were intragastrically administered with SeMet solution at a dose of 0.5 mg/kg·bw.

F + Se group (FSe): mice were given SeMet solution orally at a dose of 0.5 mg/kg·bw daily, under the premise that they freely drank 150 mg/L sodium fluoride solution.

Group B (B): Mice were intragastrically administered with CSAD-VB12/SeMet microsphere hydrogel solution at a dose of 0.5 mg/kg·bw.

F+B group (FB): The mice were given CSAD-VB12/SeMet microsphere hydrogel solution at a dose of 0.5 mg/kg·bw daily by gavage under the premise that the mice drank 150 mg/L sodium fluoride solution freely.

Test Example 2

The bar graphs were statistically analyzed using Graphpad Prism 9 and IBM SPSS Statistics. The results were expressed as mean ± standard error (mean ± SEM). Turkey test was performed after univariate analysis. In the experimental results, univariate analysis (ANOVA) was used for significant difference analysis. Compared with group C, *P < 0.05 indicated a significant difference compared with the control group, and **P < 0.01 indicated an extremely significant difference compared with the control group; #P < 0.05 indicated a significant difference compared with the fluoride group, and ##P < 0.01 indicated an extremely significant difference compared with the fluoride group.

(1) Statistics of basic sperm indices: Epididymis and vas deferens from six random mice in each group were removed of excess fat and placed in EP tubes containing 990 μL of 37°C saline for sperm counting.

Sperm density and sperm motility: Cut the epididymis and vas deferens into pieces with tissue scissors, incubate in a 37°C water bath for 15 min, use a pipette to slowly pipette 10 uL of semen suspension into the gap between the cover glass and the red blood cell counting plate, observe under an electron microscope, and record a 10 s video. Count the number of live and dead sperm in 5 random squares and calculate according to the following formula.

Sperm density = (number of live sperm in 5 squares + number of dead sperm in 5 squares)/5 × 25 × 104 × dilution factor

Sperm motility = number of live sperm in 5 squares/(number of live sperm in 5 squares + number of dead sperm in 5 squares) × 100%

Sperm deformity rate: use a pipette to absorb 10 μL of the above semen suspension and drop it on a clean glass slide, and spread it evenly with another clean glass slide. After drying, fix it in methanol solution for 5 min, dry it in a ventilated place at room temperature, and count 500 sperms under a microscope, record the number of normal sperm and the number of deformed sperm, and calculate according to the following formula.

Sperm abnormality rate = number of sperm abnormalities/500 × 100%

As shown in Figure 10, fluoride at a concentration of 150 mg/L caused a very significant increase in the sperm deformity rate of mice. After mice drank NaF freely and were gavaged with SeMet aqueous solution and CSAD-VB12/SeMet microsphere hydrogel at a dose of 0.5 mg/kg·bw, their sperm deformity rates were significantly reduced compared with the F group, indicating that organic selenium before and after encapsulation has an improvement effect on fluoride-induced sperm quality, among which CSAD-VB12/SeMet microsphere hydrogel has a better improvement effect.

Figures 11 and 12 show that fluoride at a concentration of 150 mg/L caused a significant decrease in sperm motility and sperm density in mice. For mice that freely drank NaF and were gavaged with CSAD-VB12/SeMet microsphere hydrogel at a dose of 0.5 mg/kg·bw, sperm motility increased significantly. In addition, the sperm motility in the FB group increased significantly, indicating that CSAD-VB12/SeMet microspheres were more effective than SeMet in improving sperm motility and were more conducive to spermatogenesis.

(2) Determination of serum selenium content in mice.

Microwave digestion-inductively coupled plasma mass spectrometry (ICP-MS) was used to determine the selenium content in serum. 50 μL of mouse serum sample was placed in a conical flask, 6 mL of nitric acid was drawn into the conical flask, and the flask was placed on an electric heating stove for digestion for 3-5 min. After cooling, the liquid was transferred to a 10 mL syringe, filtered with a 0.22 μm syringe filter, and stored in a 5 mL EP tube for determination on an ICP-MS instrument (iCAP QC, Thermo, USA).

The results are shown in Figure 13. CSAD-VB12/SeMet microsphere hydrogel significantly increased the selenium content in serum, indicating that CSAD-VB12/SeMet microsphere hydrogel can effectively promote the body's absorption of selenium.

(3) Effect of CSAD-VB12/SeMet microsphere hydrogel system on the testicular tissue morphology and structure of fluorine poisoning mice.

HE staining (observation of mouse testicular tissue pathological morphology): The sections of the above six groups of mice were placed in a 60°C baking machine for 1 hour to melt the wax. Dewax with xylene I and II for 10 minutes each; hydration: mixed solution xylene: anhydrous ethanol (1:1) for 10 minutes; anhydrous ethanol I and II for 2 minutes each; 95% alcohol I and II for 2 minutes each; 90%, 80%, 70%, 50% alcohol for 2 minutes each; distilled water for 2 minutes; hematoxylin staining for 5 minutes; tap water flushing for 10 minutes; differentiation solution for 1 minute; tap water blueing for 5 minutes; dehydration: gradient alcohol for 2 minutes each; eosin staining for 5 seconds; 95% alcohol I and II for 2 minutes each, anhydrous ethanol I and II for 5 minutes each; xylene is transparent, and neutral resin is added to evenly seal the slides.

Figure 14 is HE staining of mouse testis (n=3,200 × ), scale bar=100 μm, Figure 15 is HE staining of mouse testis (n=3,400 × ), scale bar=50 μm. The results showed that high fluoride can damage the testicular tissue structure, thereby affecting spermatogenesis and fertility. Supplementation of 0.5 mg/kg·bw SeMet and CSAD-VB12/SeMet can largely alleviate fluoride-induced testicular damage in mice.

(4) Determination of the content of oxidative stress indicators.

100 mg of testicular tissue was taken from the six groups of mice, and then 900 μL of PBS solution was added to each 100 mg of testicular tissue, homogenized in an ice bath in a homogenizer, transferred to a 1.5 mL EP tube, centrifuged at 3000 rpm and 4°C for 15 min, and the supernatant was taken. After measuring the protein concentration of each homogenate with a BCA protein concentration kit, the activity and content of the indicators were determined according to the instructions of the ROS, MDA, GSH-Px, CAT, T-AOC, and SOD kits.

Figures 16, 17, 18, 19, 20, and 21 respectively show the contents of ROS, MDA, GSH-Px, CAT, T-AOC, and SOD in the testes of mice. The MDA and ROS of mice in group F were significantly increased compared with those in the control group, and the activities of GSH-Px and SOD were significantly decreased in the testes of mice in group F, respectively, indicating that fluoride caused testicular damage by destroying the antioxidant system. Compared with group F, 0.5 mg/kg·bw SeMet and CSAD-VB12/SeMet significantly reduced the contents of MDA and ROS, and significantly increased the activities of GSH-Px and SOD. The Se level in the body will affect the activity of the selenium-containing enzyme GSH-Px, which can indirectly inhibit the reduction of testicular antioxidant capacity caused by fluoride, among which the FB group had a better effect.

Therefore, high-dose NaF can cause oxidative stress in mice by increasing the production of reactive oxygen species and reducing the activity of antioxidant enzymes, leading to testicular damage and male reproductive toxicity. Both SeMet and CSAD-VB12/SeMet can alleviate fluoride-induced testicular damage by scavenging ROS and increasing the activity of antioxidant enzymes, and CSAD-VB12/SeMet has a better effect.

(5) Determination of the contents of glutathione peroxidase 4 (GPX-4), total cholesterol (T-CHO) and testosterone (T).

100 mg of testicular tissue was taken from the six groups of mice, and then 900 μL of PBS solution was added to each 100 mg of testicular tissue, homogenized in an ice bath in a homogenizer, transferred to a 1.5 mL EP tube, centrifuged at 3000 rpm and 4°C for 15 min, and the supernatant was taken. After measuring the protein concentration of each homogenate with a BCA protein concentration kit, standard operations were performed according to the kit instructions for mouse-related indicators.

Figures 22, 23, and 24 show the contents of glutathione peroxidase 4, total cholesterol, and testosterone in mouse testes, respectively. GPX-4 is related to chromatin condensation during spermatogenesis and is a component of mature sperm. It is essential for spermatogenesis and has the function of preventing lipid peroxidation, which is particularly critical for improving the antioxidant properties of testes. T is indispensable for male development and is related to spermatogenesis and the formation of the blood-testis barrier. T-CHO is a precursor of T, and its synthesis requires the participation of T-CHO. Compared with group C, the contents of GPX-4, T-CHO, and T in group F mice were significantly reduced. In this experiment, compared with group F, 0.5 mg/kg·bw SeMet and CSAD-VB12/SeMet significantly increased the contents of GPX-4 and T-CHO, and CSAD-VB12/SeMet had a better effect. 0.5 mg/kg·bw CSAD-VB12/SeMet significantly increased the content of T.

Therefore, fluoride induced male reproductive toxicity by downregulating the contents of GPX-4, T-CHO, and T, leading to damage to the sperm structure of mice and decreased testicular antioxidant capacity, while 0.5 mg/kg·bw SeMet and CSAD-VB12/SeMet upregulated the contents of GPX-4 and T-CHO in the testes of mice, thereby ensuring the integrity of mouse sperm and the antioxidant capacity of mouse testes, and providing a nutritional basis for normal spermatogenesis. Among them, 0.5 mg/kg·bw CSAD-VB12/SeMet upregulated the content of T, had a greater impact on T synthesis, and had a better effect on alleviating fluoride-induced sperm damage.

The above-described embodiments are only preferred embodiments for fully illustrating the present invention, and the protection scope of the present invention is not limited thereto. Equivalent substitutions or changes made by those skilled in the art based on the present invention are within the protection scope of the present invention. The protection scope of the present invention shall be subject to the claims.

Claims (10)

1. The preparation method of CSAD-VB12/SeMet microsphere hydrogel is characterized by comprising the following steps:

s1, mixing and dissolving lecithin, cholesterol and vitamin E, and evaporating into a film;

s2, dissolving the film into a SeMet solution, and performing ultrasonic treatment to obtain a liposome solution;

s3, adding the liposome solution into CSAD-VB12 solution, and mixing and reacting to obtain CSAD-VB12/SeMet microsphere hydrogel.

2. The method for preparing CSAD-VB12/SeMet microsphere hydrogel according to claim 1, wherein the concentration of the SeMet solution is 1-10 g/L and the concentration of CSAD-VB12 in the CSAD-VB12 solution is 8-16 g/L.

3. The method for preparing CSAD-VB12/SeMet microsphere hydrogel according to claim 2, wherein the volume ratio of the liposome solution to CSAD-VB12 solution is 1: (1-2).

4. The method for preparing CSAD-VB12/SeMet microsphere hydrogel according to claim 1, wherein in S1, the evaporation is specifically: rotary evaporation is carried out for 5-6h at 25-35 ℃, and then vacuum drying box evaporation is carried out for 3-4h.

5. The method for preparing CSAD-VB12/SeMet microsphere hydrogel according to claim 1, wherein the method for preparing the SeMet solution comprises the following steps: dissolving sun-enriched yeast and trypsin into a tris buffer solution, and reacting at 35-40 ℃ to obtain a SeMet solution.

6. The method for preparing CSAD-VB12/SeMet microsphere hydrogel according to claim 1, wherein the mass ratio of the added amount of lecithin to the SeMet in the SeMet solution is (12-18): 1.

7. The method for preparing CSAD-VB12/SeMet microsphere hydrogel according to claim 1, wherein the mass ratio of lecithin to cholesterol is (10-5): 1.

8. A CSAD-VB12/SeMet microsphere hydrogel prepared by the method of any one of claims 1-7, wherein the particle size of the CSAD-VB12/SeMet microsphere is 900-1200 nm.

9. Use of the CSAD-VB12/SeMet microsphere hydrogel of claim 8 in a preparation for alleviating reproductive lesions caused by fluorosis.

10. The use according to claim 9, wherein the CSAD-VB12/SeMet microsphere hydrogel is used in an amount of 0.4-0.6 mg/kg.