Antioxidant nano material as well as preparation method and application thereof

Abstract

The invention discloses an antioxidant nano material as well as a preparation method and application thereof, and belongs to the technical field of material preparation. On the basis of the reduction reaction of GSH and selenium-containing substances, oxidation of selenium is effectively protected by utilizing a protective atmosphere, and it is ensured that the selenium element is combined with GSH in a stable valence state. Finally, a novel selenium nano material (GSH-Se) is synthesized, and the GSH-Se has antioxidant ability and GSH-Px-like activity, can relieve oxidative stress induced primary yak follicular granular cell and mouse ovary steroid hormone synthesis disorder, cell apoptosis and mitochondrial division fusion abnormality, and can be used for preparing a novel selenium nano material (GSH-Se). Meanwhile, the GSH-Se can regulate and control steroid hormone synthesis and cell apoptosis of the yak follicular granular cells by inducing mitochondrial fusion, and a new thought is provided for application of the GSH-Se to improvement of female reproductive performance.

Description

Technical Field

[0001] The present invention belongs to the technical field of material preparation, and in particular relates to an antioxidant nanomaterial and a preparation method and application thereof. Background Art

[0002] Yak breeding is the primary source of income for farmers and herders in the plateau region. Currently, the low reproductive performance of yaks severely restricts the development of the yak farming industry. Oxidative damage caused by various etiologies is one of the main pathogenic mechanisms. Granulosa cells are the largest cell population in the ovary and participate in multiple processes such as follicular development, maturation, and apoptosis, making them crucial for female reproductive capacity.

[0003] Research has shown that selenium is closely linked to the reproductive performance of cattle. Selenium plays a crucial role in improving animals' stress resistance, immune function, and overall health, as well as reproductive capacity. Selenium possesses significant antioxidant properties, protecting the body from oxidative damage and serving as a key component of the antioxidant system. Whether it is selenium or a selenium complex, its antioxidant activity is crucial for its effectiveness. Currently, a variety of selenium sources are used clinically, including sodium selenite, nanoselenium, and yeast-derived selenium, most of which rely primarily on elemental selenium for their effectiveness. Inorganic selenium materials, such as SeNPs, are primarily synthesized by chemical reduction methods (e.g., sodium selenite reduction with ascorbic acid) or microbial synthesis. These materials are prone to aggregation and oxidation, further losing their nano-effects, and exhibit low biocompatibility and limited targeting. Organic compounds, such as selenocystine, are complex to synthesize, requiring multiple purification steps, resulting in low yields, easy decomposition in the body, and a short half-life. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide an antioxidant nano material and a preparation method and application thereof, so as to solve the technical problem of the complex synthesis process of selenium-containing nano materials.

[0005] To achieve the above object, the present invention adopts a technical solution of providing a method for preparing an antioxidant nanomaterial, comprising the following steps: S1. In a protective atmosphere, a peptide substance, a selenium-containing solution, and a reducing agent are mixed, and then reacted at 35-40° C. for 2.5-3.5 hours to obtain a reaction solution; the ratio of the peptide substance, the selenium-containing solution, and the reducing agent is 10-30 mg: 0.5-1.5 mL: 1.5-2.5 mL; S2. The reaction solution is dialyzed for 10 to 14 hours, and the dialyzate is freeze-dried to obtain antioxidant nanomaterials.

[0006] On the basis of the above technical solution, the present invention can also be improved as follows: Furthermore, the protective atmosphere gas is nitrogen.

[0007] Furthermore, the peptide substance is a small molecule peptide substance containing a thiol group, the solute of the selenium-containing solution is a water-soluble selenium salt, and the reducing agent is an ascorbic acid (Vc) solution.

[0008] Furthermore, the peptide substance is reduced glutathione (GSH), and the selenium-containing solution is a sodium selenite (Na2SeO3) solution.

[0009] Furthermore, the concentration of the selenium-containing solution is 40-60 mM, and the concentration of the ascorbic acid solution is 90-110 mM.

[0010] Furthermore, the ratio of the peptide substance, the selenium-containing solution and the ascorbic acid solution is 20 mg: 1 mL: 2 mL.

[0011] Furthermore, the molecular weight cut-off of the dialysis bag used for dialysis is 150-250 Da.

[0012] Furthermore, the dialysis process also includes changing water every 3 to 5 hours.

[0013] Furthermore, the freeze-drying temperature is -90~-70°C and the time is 22-26h.

[0014] The invention also discloses an antioxidant nano material prepared by the preparation method.

[0015] The invention also discloses the application of the antioxidant nano material in preparing a preparation for treating ovarian antioxidant.

[0016] The beneficial effects of the present invention are: 1. The present invention is based on the reduction reaction of GSH and selenium-containing substances, supplemented by Vc as a reducing agent. During the reaction, the protective atmosphere effectively protects the oxidation of selenium, ensuring that the selenium element combines with GSH in a stable valence state. The synthesis method is simple, efficient, easy to operate, and suitable for industrial production. It also avoids the use of toxic reagents and conforms to the concept of green chemistry.

[0017] 2. Selenium in GSH-Se exists in a high-valent state, indicating that GSH-Se exerts its antioxidant effect by scavenging ROS; and GSH-Se nanoparticles can be excited by 380nm laser and produce strong light emission at 430nm.

[0018] 3. GSH-Se has antioxidant capacity and GSH-Px-like activity, which can alleviate oxidative stress-induced steroid hormone synthesis disorders, cell apoptosis, and mitochondrial fission and fusion abnormalities in primary yak follicular granulosa cells and mouse ovaries. At the same time, GSH-Se can regulate steroid hormone synthesis and cell apoptosis in yak follicular granulosa cells by inducing mitochondrial fusion, providing new ideas for the application of GSH-Se in improving female reproductive performance. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Transmission electron microscopy (TEM) image of GSH-Se; Figure 2 is the particle size distribution diagram of GSH-Se; Figure 3 is the FT-IR analysis result of GSH-Se; Figure 4 is the XPS analysis result of GSH-Se; Figure 5 is the XPS analysis result of Se 3d; Figure 6 for fluorescence analysis of GSH-Se; Figure 7 This is the result of DPPH scavenging by GSH-Se; Figure 8 This is the result of GSH-Se clearing ABTS; Figure 9 It is the GSH-Px-like enzyme activity of GSH-Se; Figure 10 The effect of GSH-Se on the activity of oxidatively damaged yak follicle granulosa cells; Figure 11 The effect of GSH-Se on ROS in yak follicle granulosa cells; Figure 12 The changes of selenium content in granulosa cells of yak follicles; Figure 13 Effects of GSH-Se on progesterone (P4) secretion in oxidatively damaged yak follicle granulosa cells; Figure 14 Effects of GSH-Se on estradiol (E2) secretion in oxidatively damaged yak follicle granulosa cells; Figure 15 This is a Western blot image of the effect of GSH-Se on steroid hormone synthase in oxidatively damaged yak follicle granulosa cells; Figure 16 The effect of GSH-Se on the apoptosis rate of oxidatively damaged yak follicle granulosa cells; Figure 17 Effects of GSH-Se on apoptotic proteins in oxidatively damaged yak follicle granulosa cells; Figure 18 The effect of GSH-Se on mitochondrial ROS (mtROS) in oxidatively damaged yak follicle granulosa cells; Figure 19 The effect of GSH-Se on mitochondrial membrane potential in oxidatively damaged yak follicle granulosa cells; Figure 20 The effect of GSH-Se on the number of mitochondria in granulosa cells of yak follicles damaged by oxidation; Figure 21 The effect of GSH-Se on ATP production in oxidatively damaged yak follicle granulosa cells; Figure 22 The effect of GSH-Se on mitochondrial fusion and fission regulatory proteins in yak follicle granulosa cells damaged by oxidation; Figure 23 For GSH-Se hemolysis experiment; Figure 24 HE staining results of mouse ovary; Figure 25 To count the number of follicles at each level; Figure 26 The changes in the activity of the antioxidant enzyme SOD in mice; Figure 27 The changes in the activity of antioxidant enzyme GSH-Px in mice; Figure 28 The changes in the activity of antioxidant enzyme MDA in mice; Figure 29 is the secretion of serum estradiol (E2) in mice; Figure 30 is the secretion of mouse serum progesterone (P4); Figure 31 Effects on steroid hormone synthesis regulatory proteins in mouse ovaries; Figure 32 This is a Western blot image of apoptosis proteins in mouse ovarian cells; Figure 33 This is a Western blot image of mouse ovarian mitochondrial fission and fusion proteins. DETAILED DESCRIPTION

[0020] The specific embodiments of the present invention are described below to facilitate understanding of the present invention by those skilled in the art. In the examples, where specific conditions are not specified, the experiments were carried out under conventional conditions or conditions recommended by the manufacturer. Reagents or instruments used where the manufacturer is not specified are conventional products that can be purchased commercially. However, it should be understood that the present invention is not limited to the scope of the specific embodiments. It is obvious to those skilled in the art that various modifications are within the spirit and scope of the present invention as defined and determined by the appended claims, and all inventions and creations utilizing the present invention are protected.

[0021] Example 1 A method for preparing an antioxidant nanomaterial comprises the following steps: S1. In a nitrogen atmosphere, dissolve 20 mg of reduced glutathione (GSH) in 20 mL of distilled water. After complete dissolution, add the mixture to a three-necked flask under nitrogen and stir. Maintain the temperature at 37°C and the rotation speed at 450 rpm / min. Then, quickly add 1 mL of 50 mM sodium selenite (Na2SeO3) solution dropwise. Then, add 2 mL of 100 mM ascorbic acid (Vc) solution dropwise while stirring. Finally, react at 37°C for 3 h to obtain a reaction solution. S2. The reaction solution was dialyzed using a dialysis bag with a molecular weight cutoff of 200 Da for 12 hours, during which the water was replaced every 4 hours. The dialysate was freeze-dried at -80°C for 24 hours to obtain the antioxidant nanomaterial (GSH-Se).

[0022] Example 2 A method for preparing an antioxidant nanomaterial comprises the following steps: S1. In a nitrogen atmosphere, dissolve 10 mg of reduced glutathione (GSH) in 20 mL of distilled water. After complete dissolution, add the mixture to a nitrogen-protected three-necked flask and stir. Maintain the temperature at 37°C and the rotation speed at 300 rpm / min. Then, quickly add 1.5 mL of a 40 mM sodium selenite (Na2SeO3) solution dropwise. Then, add 1.5 mL of a 110 mM ascorbic acid (Vc) solution dropwise while stirring. Finally, react at 35°C for 3.5 h to obtain a reaction solution. S2. The reaction solution was dialyzed using a dialysis bag with a molecular weight cutoff of 250 Da for 10 hours, during which the water was replaced every 3 hours. The dialysate was freeze-dried at -70°C for 26 hours to obtain the antioxidant nanomaterial (GSH-Se).

[0023] Example 3 A method for preparing an antioxidant nanomaterial comprises the following steps: S1. In a nitrogen atmosphere, dissolve 30 mg of reduced glutathione (GSH) in 20 mL of distilled water. After complete dissolution, add the mixture to a nitrogen-protected three-necked flask and stir. Maintain the temperature at 37°C and the rotation speed at 600 rpm / min. Then, quickly add 0.5 mL of a 60 mM sodium selenite (Na2SeO3) solution dropwise. Then, add 2.5 mL of a 90 mM ascorbic acid (Vc) solution dropwise while stirring. Finally, react at 40°C for 2.5 h to obtain a reaction solution. S2. The reaction solution was dialyzed using a dialysis bag with a molecular weight cutoff of 150 Da for 14 hours, during which the water was replaced every 5 hours. The dialysate was freeze-dried at -90°C for 22 hours to obtain the antioxidant nanomaterial (GSH-Se).

[0024] The GSH-Se used in the following experiments is the antioxidant nanomaterial (GSH-Se) prepared in Example 1.

[0025] Experimental Example 1 Morphological structure of GSH-Se 1. The morphology of GSH-Se was characterized by transmission electron microscopy (TEM). The image results are as follows: Figure 1 As shown. Figure 1 It can be seen that GSH-Se is composed of spherical aggregates with a particle diameter of about 102.5±39.3nm ( Figure 2 ), with a small particle size and uniform distribution, indicating that its cell membrane penetration ability may be enhanced, enabling effective delivery in subsequent experiments.

[0026] 2. The structure and elemental composition of GSH-Se were analyzed using Fourier transform infrared spectroscopy (FT-IR) and X-ray photoelectron spectroscopy (XPS). Figure 3-Figure 6 shown.

[0027] The FT-IR analysis results of GSH-Se are as follows Figure 3 As shown in the figure, the results show that the peak of the key energy group of GSH-Se is shifted compared with GSH and Na2SeO3, indicating that GSH-Se forms a chemical bond with GSH and Na2SeO3. Figure 4 and Figure 5 As shown, the Se 3d orbital peak of GSH-Se is at 60.28 eV, indicating that the valence state of Se in GSH-Se is SeO4 2- .like Figure 6 As shown, GSH-Se can emit fluorescence under the excitation of light of a specific wavelength, with the excitation wavelength (Ex) being 380 nm and the emission wavelength (Em) being 430 nm, indicating that GSH-Se has fluorescence.

[0028] Experimental Example 2: Free Radical Scavenging Ability of GSH-Se The free radical scavenging ability of GSH-Se was tested using DPPH. Figure 7 As shown in the figure, as the concentration of GSH-Se increases, the inhibition rate gradually increases, indicating that the ability of GSH-Se to scavenge DPPH is stronger. When the concentration of GSH-Se is 400 μg / mL, the inhibition rate reaches 52.6%.

[0029] ABTS was used to detect the free radical scavenging ability of GSH-Se. Figure 8 As shown, the inhibition rate gradually increased with increasing GSH-Se concentration, indicating that GSH-Se's ability to scavenge ABTS is enhanced. At a GSH-Se concentration of 200 μg / mL, the inhibition rate reached 97.78%. These results demonstrate that GSH-Se has antioxidant properties.

[0030] Experimental Example 3 Antioxidant enzyme activity of GSH-Se and GSH-Px DTNB was used as a colorimetric reagent and GSH was used as a substrate to detect the GSH-Px-like enzyme activity of GSH-Se. Figure 9 As shown in the figure, the GSH-Px enzyme activity also gradually increased with the increase of material concentration, indicating that GSH-Se has GSH-Px-like activity.

[0031] Experimental Example 4 Effect of GSH-Se on oxidative stress-induced yak follicular granulosa cell dysfunction Primary isolated yak follicular granulosa cells were used as the research subjects. An in vitro oxidative stress model was established by treating the cells with H2O2. 200 μM H2O2 for 24 hours was the optimal concentration and duration for establishing the in vitro oxidative stress model. Different concentrations of GSH-Se were used for different treatment times. The experimental groups were divided into the following groups: control, H2O2, medium (30 μM) GSH-Se, high (60 μM) GSH-Se, low (15 μM) GSH-Se + H2O2, medium (30 μM) GSH-Se + H2O2, and high (60 μM) GSH-Se + H2O2.

[0032] 1. Effect of GSH-Se on the activity of oxidatively damaged yak follicular granulosa cells like Figure 10 As shown, after pretreatment of oxidatively damaged yak follicle granulosa cells with GSH-Se at three concentrations (15μM, 30μM, and 60μM) for 12h and 24h, CCK8 results showed that the cell viability in the H2O2 group was significantly reduced compared with the control group. Compared with the H2O2 group, the cell viability was significantly increased after pretreatment with GSH-Se at concentrations of 30μM and 60μM for 12 / 24h (p<0.05 or p<0.01).

[0033] 2. Effect of GSH-Se on ROS in oxidatively damaged yak follicular granulosa cells The effects of GSH-Se on ROS in yak follicle granulosa cells damaged by oxidative stress were determined by pretreatment with GSH-Se at concentrations of 15 μM, 30 μM, and 60 μM for 12 h / 24 h. The ROS in cells were observed using DHE staining, and ROS were stained red. Figure 11 As shown in the figure, compared with the control group, the red fluorescence in the H2O2 group was significantly increased. Compared with the H2O2 group, the red fluorescence in each GSH-Se combined treatment group was significantly reduced, among which 30μM and 60μM GSH-Se pretreatment had the best effect.

[0034] 3. Effect of GSH-Se on the selenium content of oxidatively damaged yak follicle granulosa cells like Figure 12 As shown in the figure, compared with the control group, the selenium content in each GSH-Se treatment group increased significantly except for the 30 μM GSH-Se group, and increased with the increase of exposure concentration and time (p<0.05 or p<0.01).

[0035] 4. Effect of GSH-Se on steroid hormone synthesis in oxidatively damaged yak follicle granulosa cells like Figure 13 and Figure 14 As shown in the results, compared with the control group, the secretion of progesterone (P4) and estradiol (E2) in the H2O2 group was significantly reduced (p < 0.05 or p < 0.01), indicating that oxidative stress can inhibit the secretion of estradiol in yak follicular granulosa cells. Compared with the H2O2 group, the secretion of E2 in the GSH-Se+H2O2 group was significantly increased (p < 0.01), but there was no significant change in the secretion of P4.

[0036] 5. Mechanism of GSH-Se regulating hormone secretion The synthesis of steroid hormones is not regulated by a single enzyme but requires the combined action of multiple enzymes. To explore whether GSH-Se regulates hormone secretion through steroid hormone synthase, the enzyme proteins and genes related to steroid hormone synthesis in yak follicular granulosa cells were detected. The results are as follows: Figure 15 Western blotting results showed that compared with the control group, GSH-Se treatment for 12 / 24 h significantly increased StAR protein expression (p<0.05), while the H2O2 group significantly decreased the protein levels of 3β-HSD1, CYP19A1, CYP11A1, and StAR. When GSH-Se was pretreated for 12 h, the 60 μM GSH-Se+H2O2 group significantly increased the protein expression of 3β-HSD1 compared with the H2O2 group. When GSH-Se was pretreated for 24 h, the 30 μM GSH-Se+H2O2 group significantly increased the protein expression of 3β-HSD1, CYP19A1, and CYP11A1, while the 60 μM GSH-Se+H2O2 group significantly increased the protein expression of 3β-HSD1, CYP11A1, and StAR compared with the H2O2 group.

[0037] 6. Effect of GSH-Se on apoptosis of oxidatively damaged yak follicular granulosa cells Flow cytometry was used to detect cell apoptosis rate. Figure 16 Compared with the control group, the apoptosis rate of cells in the H2O2 group was significantly increased (p<0.01); compared with the H2O2 group, the apoptosis rate of cells in each GSH-Se+H2O2 group was significantly decreased (p<0.05 or p<0.01).

[0038] like Figure 17As shown in the data, compared with the control group, the protein expressions of pro-apoptotic proteins Bax and Cleaved-caspase3 in the H2O2 group were significantly increased, and the protein expression of anti-apoptotic protein Bcl-2 was significantly decreased; compared with the H2O2 group, when GSH-Se was pretreated for 12 h, the protein expression of Bcl-2 was significantly increased, and when GSH-Se was pretreated for 24 h, the protein expression of Bax and Cleaved-caspase3 was significantly decreased, and decreased with the increase of GSH-Se concentration, while the protein expression of Bcl-2 was significantly increased, and increased with the increase of GSH-Se concentration.

[0039] In summary, compared with H2O2, the GSH-Se treatment groups showed a significant decrease in ROS levels, a significant increase in estradiol secretion, a significant increase in the protein levels of the steroidogenic enzymes 3β-HSD1, CYP19A1, CYP11A1, and StAR, a decrease in cell apoptosis, a significant decrease in the expression levels of the pro-apoptotic proteins BAX and Cleaved-caspase3, and a significant increase in the expression level of the anti-apoptotic protein Bcl-2. These results indicate that GSH-Se can alleviate oxidative stress-induced impairment of steroidogenicity and increased apoptosis in granulosa cells.

[0040] Experimental Example 5 Effect of GSH-Se on mitochondrial damage induced by oxidative stress in yak follicular granulosa cells 1. Effect of GSH-Se on ROS in mitochondria of yak follicle granulosa cells damaged by oxidation GSH-Se was used to treat H2O2-induced granulosa cells. MitoSox Red staining was used to detect mtROS in yak follicle granulosa cells. Figure 18 As shown in the data, compared with the control group, there was no significant difference in the fluorescence intensity of the GSH-Se group, and the mtROS fluorescence intensity of the H2O2 group increased significantly; compared with the H2O2 group, when GSH-Se was pretreated for 12 / 24 h, the mtROS fluorescence intensity decreased, the content decreased significantly, and decreased with increasing concentration.

[0041] 2. Effect of GSH-S on mitochondrial membrane potential of oxidatively damaged yak follicle granulosa cells JC-1 is an ideal fluorescent probe widely used to measure mitochondrial membrane potential. It can be used to measure mitochondrial membrane potential in cells, tissues, or purified mitochondria. When the mitochondrial membrane potential is high, JC-1 aggregates in the mitochondrial matrix, forming aggregates that produce red fluorescence. When the mitochondrial membrane potential is low, JC-1 cannot aggregate in the mitochondrial matrix and forms monomers, producing green fluorescence. Increased green fluorescence indicates a decrease in mitochondrial membrane potential. A decrease in membrane potential indicates mitochondrial dysfunction, a key factor in pathological processes such as apoptosis and oxidative stress.

[0042] like Figure 19 As shown in the data, compared with the control group, the JC-1 monomer content in the H2O2 group was significantly increased (p<0.01); compared with the group, the JC-1 monomer content was significantly decreased after 24h of GSH-Se pretreatment (p<0.05 or p<0.01).

[0043] 3. Effect of GSH-Se on the number of mitochondria in oxidatively damaged yak follicle granulosa cells Mito Tracker green fluorescent probe was used to detect the number and morphology of mitochondria in yak follicle granulosa cells. Figure 20 Compared with the control group, the number of mitochondria in the H2O2 group decreased, some mitochondria shrank, and the fluorescence intensity decreased; compared with the H2O2 group, the fluorescence intensity of each GSH-Se treatment group after 24 hours of pretreatment was significantly enhanced.

[0044] 4. Effect of GSH-Se on ATP in oxidatively damaged yak follicle granulosa cells like Figure 21 As shown, compared with the control group, the ATP content in the H2O2 group was significantly decreased (p<0.01); when GSH-Se was pretreated for 12 hours, the ATP content in the 60μΜ GSH-Se treatment group was significantly increased compared with the H2O2 group (p<0.05); when GSH-Se was pretreated for 24 hours, the ATP content in each GSH-Se treatment group was significantly increased compared with the H2O2 group (p<0.01).

[0045] 5. Effects of GSH-Se on mitochondrial fission and fusion in oxidatively damaged yak follicle granulosa cells Mitochondrial fission and fusion may be the key to affecting the function of granulosa cells. Figure 22As shown, the results of western blotting showed that compared with the control group, the expression of Mfn1 protein in the 30 μM GSH-Se group treated for 24 h was significantly increased, the expression of mitochondrial fusion proteins Mfn1 and Mfn2 proteins in the H2O2 group was significantly decreased, and the expression of mitochondrial fission proteins Drp1 and Fis1 proteins was significantly increased; when GSH-Se was pretreated for 12 h, the expression of Fis1 protein in the 30 μM GSH-Se+H2O2 group was significantly decreased compared with the H2O2 group; when GSH-Se was pretreated for 24 h, compared with the H2O2 group, the levels of Mfn1 and Mfn2 proteins in each GSH-Se treatment group were significantly increased (p<0.01), and the expression of Drp1 and Fis1 proteins were significantly decreased (p<0.05 or p<0.01).

[0046] Experimental Example 6 Effects of GSH-Se on Oxidative Damage in Mouse Ovaries 3-NPA, as an inhibitor of mitochondrial electron transport chain complex II, increases ROS and disrupts energy metabolism, driving mitochondrial dynamics toward fission, leading to fragmentation and functional loss. In this study, 48 female ICR mice were treated with intraperitoneal injection of 3-nitropropionic acid (3-NPA) to establish an in vivo oxidative stress model. GSH-Se (1 mg / kg) was then administered for intervention. The mice were divided into the following groups: control, GSH-Se (1 mg / kg), 3-NPA (12.5 mg / kg), and GSH-Se (1 mg / kg) + 3-NPA (12.5 mg / kg). Each experimental group was treated for 14 consecutive days.

[0047] 1. GSH-Se hemolysis test In order to verify the biosafety of GSH-Se in vivo, hemolysis test was performed to further evaluate the in vivo toxicity. Figure 23 As shown in the results, when the GSH-Se concentration reached 37.5 μg / mL, the hemolysis rate was approximately 1.05%; when the GSH-Se concentration reached 300 μg / mL, the hemolysis rate was 1.79%, less than 5%. Therefore, it can be seen that GSH-Se has good biosafety in vivo, providing support for further in vivo therapeutic experiments.

[0048] 2. Pathological changes of mouse ovarian tissue The results of HE staining of mouse ovaries are as follows Figure 24As shown, the black arrow indicates the primordial follicle, the red arrow indicates the primary follicle, the white arrow indicates the secondary follicle, the blue arrow indicates the corpus luteum, and the yellow arrow indicates the atretic follicle. The ovarian tissue structure of the mice in the control group and the GSH-Se group was normal, and the ovarian parenchyma was divided into the central medulla and the peripheral cortex; the primordial follicles, primary follicles, secondary follicles, atretic follicles and corpus luteum were visible in the cortex. In the secondary follicles, obvious oocytes, follicular cavity, granulosa layer and corona radiata were visible, among which the granulosa cells were densely arranged and distributed around the follicular cavity. The phenomenon of follicular atresia increased in the 3-NPA group, among which the oocytes were deformed, the granulosa cells were loosely arranged, and they fell off into the follicular cavity, and the nuclei were condensed and the follicular wall collapsed. The ovarian results of mice treated with GSH-Se (GSH-Se+3-NPA group) were closer to normal than those in the 3-NPA group. The results of follicles at all levels were statistically shown to be ( Figure 25 Compared with the control group, the number of primordial follicles in the 3-NPA group was significantly decreased (p<0.01), while the number of atretic follicles was significantly increased (p<0.01). No significant differences were observed in the number of primary follicles, secondary follicles, or corpora lutea between the two groups. After GSH-Se treatment, the number of primordial follicles was significantly increased (p<0.05), while the number of atretic follicles was significantly decreased (p<0.05) compared with the 3-NPA group.

[0049] 3. Changes in oxidative stress indicators in mouse ovaries like Figure 26-Figure 28 As shown in the results, compared with the control group, the activities of GSH-Px and SOD in the 3-NPA group were significantly decreased (p<0.01), and the activity of MDA was significantly increased (p<0.05). Compared with the 3-NPA group, the activities of SOD and GSH-Px in the GSH-Se+3-NPA group were significantly increased (p<0.05 or p<0.01), and the activity of MDA was significantly decreased (P<0.05). This indicates that GSH-Se can alleviate 3-NPA-induced oxidative damage and protect ovarian function.

[0050] 4. Changes in ovarian steroid hormone synthesis in mice like Figure 29 and Figure 30 As shown, compared with the control group, the secretion of E2 in the GSH-Se group was significantly increased (p<0.01), and the secretion of E2 and P4 in the 3-NPA group was significantly decreased (p<0.01); compared with the 3-NPA group, the secretion of E2 and P4 in the GSH-Se+3-NPA group was significantly increased (p<0.05 or p<0.01).

[0051] Western blotting was used to examine the effect of GSH-Se on steroid hormone synthesis in mice with oxidative damage. Figure 32As shown in the data, compared with the control group, the protein expressions of 3β-HSD1, CYP11A1, CYP19A1 and StAR in the 3-NPA group were significantly decreased; compared with the 3-NPA group, the protein expressions of CYP11A1, CYP19A1 and StAR in the GSH-Se+3-NPA group were significantly increased.

[0052] 5. Changes in mouse ovarian cell apoptosis like Figure 32 As shown in the results, compared with the control group, the protein expressions of pro-apoptotic proteins Bax and Cleaved-caspase3 in the 3-NPA group were significantly increased, and the protein expression of anti-apoptotic protein Bcl-2 was significantly decreased; compared with the 3-NPA group, the protein expressions of pro-apoptotic proteins Bax and Cleaved-caspase3 in the GSH-Se+3-NPA group were significantly decreased.

[0053] 6. Changes in mitochondrial fission and fusion in mouse ovaries like Figure 33 As shown in the data, compared with the control group, the expressions of mitochondrial fusion proteins Mfn1 and Mfn2 in the 3-NPA group were significantly decreased, and the expressions of mitochondrial fission proteins Drp1 and Fis1 were significantly increased; compared with the 3-NPA group, the expression of mitochondrial fusion protein Mfn2 in the GSH-Se+3-NPA group was significantly increased, and the expressions of mitochondrial fission proteins Drp1 and Fis1 were significantly decreased.

[0054] In summary, this study used a hydrothermal method to synthesize a nanomaterial GSH-Se with in vitro antioxidant capacity. The results showed that GSH-Se can alleviate steroid hormone synthesis disorders and cell apoptosis caused by oxidative stress in yak follicular granulosa cells and mouse ovaries. GSH-Se mainly improves the function of yak follicular granulosa cells by promoting mitochondrial fusion function, providing new ideas for the application of the new selenium source supplement GSH-Se in improving female reproductive performance.

Claims

1. A method for preparing an antioxidant nanomaterial, characterized in that: The following steps are involved: S1. In a protective atmosphere, a peptide substance, a selenium-containing solution, and a reducing agent are mixed, and then reacted at 35-40° C. for 2.5-3.5 hours to obtain a reaction solution; the ratio of the peptide substance, the selenium-containing solution, and the reducing agent is 10-30 mg: 0.5-1.5 mL: 1.5-2.5 mL; S2. The reaction solution is dialyzed for 10 to 14 hours, and the dialyzate is freeze-dried to obtain antioxidant nanomaterials.

2. The method for preparing the antioxidant nanomaterial according to claim 1, wherein: The protective atmosphere is nitrogen.

3. The method for preparing the antioxidant nanomaterial according to claim 1, wherein: The peptide substance is a small molecule peptide substance containing a thiol group, the solute of the selenium-containing solution is a water-soluble selenium salt, and the reducing agent is an ascorbic acid solution.

4. The method for preparing the antioxidant nanomaterial according to claim 3, wherein: The peptide substance is reduced glutathione, and the selenium-containing solution is a sodium selenite solution.

5. The method for preparing the antioxidant nanomaterial according to claim 3, wherein: The concentration of the selenium-containing solution is 40-60 mM, and the concentration of the ascorbic acid solution is 90-110 mM.

6. The method for preparing the antioxidant nanomaterial according to claim 1, wherein: The ratio of the peptide substance, the selenium-containing solution and the ascorbic acid solution is 20 mg: 1 mL: 2 mL.

7. The method for preparing the antioxidant nanomaterial according to claim 1, wherein: The molecular weight cut-off of the dialysis bag used for the dialysis is 150-250 Da.

8. The method for preparing the antioxidant nanomaterial according to claim 1, wherein: The freeze-drying temperature is -90 to -70°C, and the time is 22 to 26 hours.

9. An antioxidant nanomaterial, characterized in that: The method is prepared according to any one of claims 1 to 8.

10. Use of the antioxidant nanomaterial according to claim 9 in the preparation of a preparation for treating ovarian antioxidant disease.

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