Application of protein nano-selenium in preparation of medicine for preventing and treating ionizing radiation injury

Abstract

The invention belongs to the technical field of medicines, and discloses application of protein nano-selenium in preparation of a medicine for preventing and treating ionizing radiation injury. HSA-SeNPs with higher stability and safety than Se NPs is utilized, and the HSA-SeNPs has a more excellent effect of repairing peripheral blood cells caused by radiation injury than Se NPs, can inhibit spleen and myeloid tissue cell apoptosis, and can be used as a medicine for treating acute radiation injury diseases. The HSA-SeNPs prepared by the invention not only can be taken orally, but also can be used for intravenous injection and local administration.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, and specifically relates to the use of a protein nano-selenium in the preparation of drugs for preventing and treating ionizing radiation damage. Background Technology

[0002] Ionizing radiation (IR) refers to radiation produced by energy that ionizes atoms or molecules; nuclear radiation is a special form of ionizing radiation. The development of current nuclear technology applications is accompanied by increasing exposure risks, making research on protection against ionizing radiation damage an urgent scientific issue. Based on the duration of ionizing radiation exposure, drugs can be classified as radiation protectants, radiation relievers, and radiation therapies. Clinically commonly used drugs for the prevention and treatment of ionizing radiation include amifostine and hematopoietic growth factors (including various colony-stimulating factors, stem cell factors, thrombopoietin, and erythropoietin), but existing drugs suffer from problems such as narrow therapeutic indices, high toxicity, rapid metabolism, and high cost. Developing a highly effective radiation damage treatment drug with fewer side effects is both urgently needed and of significant social importance.

[0003] Selenium (Se) is an essential trace element that cannot be synthesized in the body and must be obtained from external sources. Selenium exists in both inorganic and organic forms. Studies have shown that Se has biological effects in protecting against radiation damage, and it is closely related to anti-oxidative stress, DNA protection, inhibition of apoptosis, and immune regulation. However, due to its low solubility, low bioavailability, and narrow safe dosage range after ingestion, high doses or long-term use can easily lead to accumulation and toxicity in the body. In recent years, red elemental Se, namely selenium nanoparticles (Se NPs), prepared using nanotechnology, has shown higher bioactivity, better bioavailability, and lower toxicity compared to organic selenium (such as SeIV, SeVI) and inorganic selenium. Se NPs can scavenge ROS generated by IR, prevent DNA damage and apoptosis, and increase the sensitivity of tumor cells to radiation; however, Se NPs still have drawbacks such as easy aggregation and easy oxidation. Summary of the Invention

[0004] In order to overcome the shortcomings and deficiencies of the prior art, the present invention aims to provide the use of protein nano-selenium in the preparation of drugs for preventing and treating ionizing radiation damage.

[0005] The objective of this invention is achieved through the following technical solution: The use of a protein nano-selenium in the preparation of drugs to prevent and treat ionizing radiation damage.

[0006] The protein nano-selenium is used as an inhibitor of apoptosis in spleen and bone marrow tissue cells in the preparation of drugs to prevent and treat ionizing radiation damage.

[0007] The preparation steps of the protein nano-selenium include: reducing human serum albumin with a protein reducing agent at 37°C, then adding a sodium selenite solution containing chitosan or a sodium selenate solution containing chitosan, stirring the reaction at room temperature or 4°C for 3-12 hours, and removing unreacted substances by dialysis or centrifugation to obtain protein nano-selenium particles.

[0008] The protein reducing agent is tris(2-carboxyethyl)phosphine.

[0009] The protein nano-selenium was prepared according to the following steps: Human serum albumin was dissolved in a phosphate buffer solution containing a protein reducing agent at a pH of 5.0–9.0, with a final concentration of human serum albumin of 0.01–200 mg / mL and a final concentration of the protein reducing agent of 0.1–60 mM; the mixture was reacted at 37°C for 90–110 min with stirring to obtain a homogeneous protein solution with expanded spatial structure; then, a sodium selenite solution or a sodium selenate solution containing chitosan was added to the homogeneous protein solution with expanded spatial structure, with a final concentration of chitosan of 0.05–5 mg / mL and a final concentration of sodium selenite or sodium selenate of 0.1–30 mM; the mixture was stirred at room temperature or 4°C for 3–12 h to obtain a crude protein nano-selenium solution; the crude protein nano-selenium solution was placed in a dialysis bag and dialyzed overnight with PBS solution at 0–20°C, or the crude protein nano-selenium solution was centrifuged at 8000–30000 rpm for 10–60 minutes. After repeatedly resuspending in deionized water and centrifuging several times, protein nano-selenium was finally obtained.

[0010] The dialysis bag has a dialysis molecule rejection rate of not less than 1000.

[0011] The principle of this invention: Based on the good stability of albumin under certain temperature and pH conditions, and its advantages such as non-toxicity, low immunogenicity, and biodegradability, this invention constructs orally biodegradable protein-bound selenium nanoparticles (also known as protein-bound selenium nanoparticles, protein-bound elemental selenium nanoparticles, or HSA-SeNPs) using protein self-assembly technology. Compared to elemental selenium nanoparticles (SeNPs), HSA-SeNPs exhibit higher stability and bioactivity. In a mouse model of ionizing radiation, HSA-SeNPs demonstrated significantly superior radiation protection efficacy compared to traditional SeNPs. HSA-SeNPs showed stronger protective activity in key indicators such as improving survival rate, maintaining the integrity of hematopoietic system function, and inhibiting tissue cell apoptosis. HSA-SeNPs also have potential value as a drug for the prevention and treatment of radiation damage.

[0012] The present invention has the following advantages and beneficial effects compared with the prior art: This invention utilizes a substance with higher stability and safety than Se NPs, and exhibits superior repair of radiation-damaged peripheral blood cells, while also inhibiting apoptosis in spleen and bone marrow tissue cells. It can be used as a drug for the treatment of acute radiation injury. The HSA-SeNPs prepared by this invention can be administered orally, intravenously, and topically. Attached Figure Description

[0013] Figure 1 Figures show the preparation and characterization of HSA-SeNPs and Se NPs; where A is the stability diagram of HSA-SeNPs and Se NPs; and B and C are TEM images (scale, 200 nm) of HSA-SeNPs and Se NPs.

[0014] Figure 2 This is a safety assessment chart for HSA-SeNPs; where A is the mouse tissue weight chart; B is the mouse white blood cell count chart; C is the mouse platelet count chart; D is the mouse alanine aminotransferase level chart; E is the mouse aspartate aminotransferase level chart; F is the mouse creatinine level chart; and G is the mouse urea level chart.

[0015] Figure 3 These are graphs showing the establishment of the mouse model and the relationship between radiation dose and mortality. A shows a photograph of each mouse anesthetized with Avertin (mice)-tribromoethanol, 0.0125 mL / intraperitoneal injection; B shows a photograph of mice receiving a single radiation dose of 5 mice; C shows a photograph of the mice before radiation; D shows a photograph of the mouse model successfully established after a single X-ray irradiation of 4 Gy-2 Gy / min; E shows the relationship between radiation dose of 4 Gy-7.5 Gy and mouse survival rate.

[0016] Figure 4 This is a graph evaluating the protective effect of HSA-SeNPs at the median lethal radiation dose; where A is the survival rate curve of mice in different treatment groups from day 1 to 28, B is the body weight curve of mice in different treatment groups from day 1 to 28, C is the spleen index of mice in different treatment groups on day 28, D is the spleen morphology image of mice in different treatment groups on day 28, E is the blood leukocyte count analysis of mice in different treatment groups from day 1 to 28, F is the blood platelet count analysis of mice in different treatment groups from day 1 to 28, G is the blood lymphocyte count analysis of mice in different treatment groups from day 1 to 28, H is the blood hemoglobin count analysis of mice in different treatment groups from day 1 to 28, I is the blood erythrocyte count analysis of mice in different treatment groups from day 1 to 28, and J is the blood neutrophil count analysis of mice in different treatment groups from day 1 to 28.

[0017] Figure 5The images show the improvement in tissue damage caused by HSA-SeNPs at the median lethal radiation dose. A represents a spleen tissue section on day 28 in the control group; B represents a spleen tissue section on day 28 in the model group; C represents a spleen tissue section on day 28 in the amifostine group; D represents a spleen tissue section on day 28 in the group that received oral SeNPs at a dose of 0.5 mg / kg / d after radiation; E represents a spleen tissue section on day 28 in the group that received oral SeNPs at a dose of 1 mg / kg / d; F represents a spleen tissue section on day 28 in the group that received oral HSA-SeNPs at a dose of 0.5 mg / kg / d; G represents a spleen tissue section on day 28 in the group that received oral HSA-SeNPs at a dose of 1 mg / kg / d; H represents a bone marrow tissue section on day 28 in the control group; I represents a bone marrow tissue section on day 28 in the model group; J represents a bone marrow tissue section on day 28 in the group that received oral SeNPs at a dose of 1 mg / kg / d; and K represents a bone marrow tissue section on day 28 in the group that received oral HSA-SeNPs at a dose of 1 mg / kg / d.

[0018] Figure 6 This is a graph evaluating the protective effect of HSA-SeNPs at total lethal radiation doses; where A is the survival curve of mice in different treatment groups from day 1 to 8, B is the weight change curve of mice in different treatment groups from day 1 to 7, C is the spleen index of mice in different treatment groups on day 8, D is the spleen morphology image of mice in different treatment groups on day 8, E is the blood leukocyte count analysis of mice in different treatment groups on day 8, F is the blood platelet count analysis of mice in different treatment groups on day 8, G is the blood lymphocyte count analysis of mice in different treatment groups on day 8, H is the blood hemoglobin count analysis of mice in different treatment groups on day 8, I is the blood erythrocyte count analysis of mice in different treatment groups on day 8, and J is the blood neutrophil count analysis of mice in different treatment groups on day 8.

[0019] Figure 7 This is a TUNEL staining assay of apoptosis in mouse spleen tissue on day 8 after total lethal dose radiation. A is the blank group, B is the model group, C is the amifostine group, D is the group treated with 0.3 mg / kg / d of oral SeNPs, E is the group treated with 0.6 mg / kg / d of oral SeNPs, F is the group treated with 0.3 mg / kg / d of oral HSA-SeNPs, and G is the group treated with 0.6 mg / kg / d of oral HSA-SeNPs.

[0020] Figure 8This is a graph showing the expression of Bcl-2 protein in the spleen tissue of mice on day 8 after total lethal irradiation; where A is the blank group, B is the model group, C is the amifostine group, D is the group that received oral administration of SeNPs at a dose of 0.3 mg / kg / d, E is the group that received oral administration of SeNPs at a dose of 0.6 mg / kg / d, F is the group that received oral administration of HSA-SeNPs at a dose of 0.3 mg / kg / d, and G is the group that received oral administration of HSA-SeNPs at a dose of 0.6 mg / kg / d.

[0021] Figure 9 This is a graph showing the expression of Bax protein in the spleen tissue of mice on day 8 after total lethal irradiation; where A is the blank group, B is the model group, C is the amifostine group, D is the group that received oral SeNPs at a dose of 0.3 mg / kg / d, E is the group that received oral SeNPs at a dose of 0.6 mg / kg / d, F is the group that received oral HSA-SeNPs at a dose of 0.3 mg / kg / d, and G is the group that received oral HSA-SeNPs at a dose of 0.6 mg / kg / d.

[0022] Figure 10 This is a graph showing the expression of p53 protein in the spleen tissue of mice on day 8 after total lethal irradiation; where A is the blank group, B is the model group, C is the amifostine group, D is the group that received oral administration of SeNPs at a dose of 0.3 mg / kg / d, E is the group that received oral administration of SeNPs at a dose of 0.6 mg / kg / d, F is the group that received oral administration of HSA-SeNPs at a dose of 0.3 mg / kg / d, and G is the group that received oral administration of HSA-SeNPs at a dose of 0.6 mg / kg / d. Detailed Implementation

[0023] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto.

[0024] Example 1: Preparation and characterization of elemental selenium nanoparticles (Se NPs) and protein selenium nanoparticles (HSA-SeNPs) 1. Method (1) Preparation of elemental selenium nanoparticles (Se NPs) Weigh 736.8 mg of glutathione (GSH) and 480 mg of human serum albumin (BSA) and pour them into 22.92 mL of water. After stirring evenly, add 103.2 mg of Na2SeO3 solution and finally add 1 mol / L NaOH solution. Stir evenly to prepare a red elemental selenium nanoparticle (Se NPs) solution with a concentration of 1520 μg / mL.

[0025] (2) Preparation of protein nano-selenium (HSA-SeNPs) Human serum albumin was dissolved in a phosphate-buffered saline (PBS) solution containing tris(2-carboxyethyl)phosphine (TCEP) at pH 5.0, with a final albumin concentration of 10 mg / mL and a final TCEP concentration of 20 mM. The mixture was reacted at 37 °C for 100 min with stirring to obtain a homogeneous protein solution with expanded spatial structure. Then, a sodium selenite solution containing chitosan was added to the homogeneous protein solution with expanded spatial structure, with a final chitosan concentration of 0.05 mg / mL and a final sodium selenite concentration of 5 mM. The mixture was stirred at 4 °C for 12 h to obtain a crude protein nano-selenium solution. The crude protein nano-selenium solution was placed in a dialysis bag and dialyzed overnight in PBS solution at 0-20 °C to obtain the final human serum albumin nano-selenium (HSA-SeNPs) stock solution.

[0026] (3) Characterization of elemental selenium nanoparticles (Se NPs) and protein selenium nanoparticles (HSA-SeNPs) To verify the stability of HSA-SeNPs, the prepared Se NPs and HSA-SeNPs were dispersed in PBS and left at room temperature for 28 days. To determine the morphology and size of the nanoparticles, the appearance and size of HSA-SeNPs and Se NPs were detected using a transmission electron microscope (FEI Tecnai G212, USA).

[0027] 2. Conclusion After the Se NPs and HSA-SeNPs prepared above were dispersed in PBS and left at room temperature for 28 days, the stability of HSA-SeNPs was better than that of Se NPs. Figure 1 According to transmission electron microscopy analysis, both HSA-SeNPs and Se NPs exhibit a quasi-spherical morphology. The particle size of Se NPs is approximately 80 nanometers, while the particle size of HSA-SeNPs is approximately 20 nanometers. Figure 1 (BC).

[0028] Example 2: Safety assessment of the prepared protein-based selenium nanoparticles (HSA-SeNPs) 1. Method Twenty-five male C57BL / 6 mice, 4-6 weeks old, weighing 20±2g, were used in a clean-grade environment with a 1:1 day-night cycle, maintaining indoor humidity at 44–65% and room temperature at 22–26 °C. Mice were provided with normal food and water for one week of acclimatization. The mice were then randomly divided into five groups: PBS group, 1 mg / kg Se NPs group (Se NPs-1), 4 mg / kg Se NPs group (Se NPs-4), 1 mg / kg HSA-SeNPs group (HSA-SeNPs-1), and 4 mg / kg HSA-SeNPs group (HSA-SeNPs-4). The Se NPs and HSA-SeNPs prepared in Example 1 were administered via gavage daily for 14 days. Two weeks later, blood samples were collected from the mouse eyeballs, and WBC and PLT levels were measured using a blood cell analyzer. ALT, AST, CREA, and UREA levels were measured using a biochemical analyzer. Heart, liver, spleen, lung, kidney, and brain tissues from the mice were collected and weighed.

[0029] 2. Conclusion The results showed that HSA-SeNPs did not cause changes in the weight of the heart, liver, spleen, lungs, kidneys, and brain in mice. In contrast, high doses of Se NPs increased liver weight, suggesting that Se NPs may pose a risk of hepatotoxicity at higher doses. Figure 2 Hematological analysis showed that HSA-SeNPs had no significant effect on hematological parameters such as white blood cells and platelets, while both low and high doses of SeNPs reduced platelet counts in mice, suggesting that SeNPs may have adverse effects on the hematopoietic system. Figure 2 The levels of alanine aminotransferase and aspartate aminotransferase in the SeNPs were significantly higher than those in the normal group after oral administration of 4 mg / kg, further confirming the hepatotoxicity of SeNPs. Figure 2 The results of renal function tests showed that HSA-SeNPs did not produce nephrotoxicity even at high doses, while high doses of SeNPs showed abnormalities in creatinine and urea levels, indicating significant nephrotoxicity. Figure 2 (FG). Therefore, compared with Se NPs, HSA-SeNPs showed good safety, with no significant effect on mouse body weight, hematological parameters, liver function, and kidney function.

[0030] Example 3: Establishment of a mouse model and study on the relationship between radiation dose and mortality: 1. Method Ninety male C57BL / 6 mice, 4-6 weeks old, weighing 20±2g, were used in a clean-grade environment with a 1:1 day-night cycle, maintaining indoor humidity at 45–65% and room temperature at 22–26 °C. The mice were provided with normal food and water and allowed to acclimatize for one week. The mice were then randomly divided into 9 groups of 10 mice each: a control group, a 4Gy group, a 4.5Gy group, a 5Gy group, a 5.5Gy group, a 6Gy group, a 6.5Gy group, a 7Gy group, and a 7.5Gy group. Except for the control group, all other mice were anesthetized by intraperitoneal injection of Avertin (mouse)-tribromoethanol, 0.0125 mL / g, followed by a single irradiation. The modeling conditions were as follows: Equipment: Varian Clinacix; total X-ray doses: 4 Gy, 4.5 Gy, 5 Gy, 5.5 Gy, 6 Gy, 6.5 Gy, 7 Gy, and 7.5 Gy; dose rate: 2 Gy / min; energy: 6 MV; modeling environment: atmospheric pressure -984 hPa × 10, T -20℃; the mortality of mice was observed and recorded 28 days after radiation.

[0031] in conclusion Mice were anesthetized and laid flat on an acrylic plate to receive different total doses of whole-body irradiation, ensuring uniform irradiation. Figure 3 (AB). Observe the morphology of the mice, including general signs such as fur, feces, and mental state. After irradiation, the mice obviously showed a relatively reduced intake of water and food, slow movement, lethargy, slightly poor fur luster, and a slightly disheveled state. Occasionally, diarrhea occurred. The mice huddled together. It was preliminarily determined that the mouse radiation model was successfully established. Figure 3 The CD). Based on the statistical results of the survival of mice in different treatment groups (total dose: 4–7.5 Gy) within 28 days after radiation ( Figure 3 E (Table 1) can determine the median lethal dose (LD50) in mice. 50 The total lethal dose (LD50) is 6 Gy. 100 The value is 7.5 Gy.

[0032] Table 1. Mortality statistics of mice in different treatment groups on day 28 after radiation.

[0033] Example 4: Evaluation of the protective effect of protein nano-selenium (HSA-SeNPs) at the median lethal dose. 1. Method Forty-two male C57BL / 6 mice, 4-6 weeks old, weighing 20±2g, were used in this experiment. They were kept in a clean-grade environment with a 1:1 day-night cycle, maintaining indoor humidity at 44–65% and room temperature at 22–26 ℃. Mice were provided with normal food and water and allowed to acclimatize for one week. After this period, the mice were randomly divided into seven groups of six mice each: a control group, a model group, an amifostine group (as a positive control), a 0.5 mg / kg Se NPs group (Se NPs-0.5), a 1 mg / kg Se NPs group (Se NPs-1), a 0.5 mg / kg HSA-SeNPs group (HSA-SeNPs-0.5), and a 1 mg / kg HSA-SeNPs group (HSA-SeNPs-1). Except for the normal control group, all mice in the other groups were anesthetized by intraperitoneal injection of avertin (mouse)-tribromoethanol, 0.0125 mL / g, followed by a single irradiation. Based on the median lethal radiation dose obtained in Example 3, the modeling conditions were set as follows: total dose: 6 Gy, dose rate: 2 Gy / min, energy level: 6 MV. Using Se NPs and HSA-SeNPs prepared in Example 1, mice were administered the drugs via gavage after fasting for 6-10 hours daily (fasting but not water restriction) for 28 days. The control group and model group were administered the same volume of physiological saline via gavage daily. Mouse weight and mortality were recorded. On day 28, blood was collected from the eyeballs for complete blood cell analysis, and spleen and bone marrow tissues were collected to observe cell morphological changes.

[0034] 2. Conclusion The results showed that at the median lethal radiation dose (6 Gy), the HSA-SeNPs group significantly improved the survival rate of mice up to day 28 compared with the Se NPs group, and reduced the degree of weight loss after irradiation, demonstrating a superior survival benefit. Figure 4 The spleen index was improved in the HSA-SeNPs group compared with the model group (*P<0.05), while SeNPs did not show a significant improvement in the spleen index after irradiation. Further observations of spleen morphology and size suggest that HSA-SeNPs can inhibit radiation-induced spleen atrophy, which may help maintain the body's immune function. Figure 4 The CD). Hematological results showed that multiple blood cell parameters in the HSA-SeNPs group mice increased over time, and the overall recovery was better than that in the Se NPs group, with the most significant improvement observed in the 1 mg / kg HSA-SeNPs group. Analysis on day 28 after irradiation showed that the white blood cell count was significantly higher in the 0.5 and 1 mg / kg HSA-SeNPs groups than in the model group (**p<0.01), while no statistically significant difference was observed in the Se NPs group ( Figure 4In terms of platelet count, only the 1 mg / kg HSA-SeNPs group showed a significant improvement (**p<0.01), while the SeNPs group showed no significant improvement (E); Figure 4 Regarding lymphocyte recovery, both doses of HSA-SeNPs showed significant increases (****p<0.0001), with the 0.5 mg / kg SeNPs group and the 1 mg / kg SeNPs group showing **p<0.01 and ***p<0.001, respectively. Figure 4 Regarding hemoglobin levels, the SeNPs group showed a restorative effect (**p<0.01), but the HSA-SeNPs group showed a stronger enhancing effect (***p<0.001, ****p<0.0001). Figure 4 Although the red blood cell count did not reach statistical significance between groups, the HSA-SeNPs group had a higher count than the SeNPs group (H). Figure 4 In terms of neutrophil count, the 1 mg / kg HSA-SeNPs group was significantly higher than the model group (**p<0.01), while there was no significant difference in the SeNPs group (I). Figure 4 In conclusion, under median lethal dose irradiation conditions, HSA-SeNPs not only exhibited superior survival benefits compared to Se NPs, but also demonstrated a more comprehensive and stronger improvement effect on radiation-induced multi-lineage hematologic cytopenia, suggesting that they possess better radiation damage protection.

[0035] Example 5: Protein-based selenium nanoparticles (HSA-SeNPs) improve tissue damage at the median lethal radiation dose. method Based on the mouse spleen tissue, femur and tibia obtained in Example 4, the femur and tibia were fixed in fixative at 4°C for 24 hours, washed once with PBS, soaked in decalcification solution, and fixed in a rotating shaker at room temperature for decalcification for 30 days, with the decalcification solution being changed every 2 days. Spleen tissue and decalcified femur and tibia were fixed and dehydrated using a fully automated dehydrator (dehydration time: 75% ethanol 2 h, 85% ethanol 1 h, 95% ethanol 1 h, 100% ethanol I 20 min, 100% ethanol II 20 min, 100% ethanol III 20 min, 100% ethanol IV 20 min, clearing agent I 25 min, clearing agent II 30 min, paraffin I 30 min, paraffin II 1 h, paraffin III 1 h), embedded, and sectioned. The following procedures were then performed: ① Dewaxing to water: Dewaxing solution I 30 min, dewaxing solution II 30 min, anhydrous ethanol I 5 min, anhydrous ethanol II 5 min, 95% ethanol 5 min, 85% ethanol 5 min, 75% ethanol 5 min, rinsing with running tap water for 5 min; ② Hematoxylin staining for 5-10 min, rinsing with running tap water until colorless; ③ Differentiation with hydrochloric acid alcohol solution for approximately 3 minutes. 4. Rinse with tap water; 5. Blue with weakly alkaline aqueous solution, rinse with tap water; 6. Immerse in alcohol-soluble eosin for 3 min; 7. Dehydrate with gradient alcohols, clear with a clearing agent, and mount with neutral resin. The SQS-600P slide scanning imaging system manufactured by Shenzhen Shengqiang Technology Co., Ltd. was used to acquire images of the slides. Each slide was first observed at low magnification, and then 100x and 400x microscopic images were acquired.

[0036] in conclusion The results showed that the spleen structure of mice in the blank control group was intact, with no fibrous connective tissue proliferation observed in the capsule and trabecular areas, and the cells were morphologically intact and densely distributed. Figure 5 In contrast, the white pulp region of the spleen in the model group mice showed significant atrophy and a significant reduction in cell number, exhibiting a typical pathological state of radiation damage. Figure 5 (B). In the treatment groups, both the 0.5 mg / kg and 1 mg / kg HSA-SeNPs treatment groups improved spleen tissue structure to a certain extent, showing an increase in cell number compared to the model group, a reduction in white pulp atrophy, and a certain degree of relief of spleen tissue damage. The improvement effect of the 1 mg / kg HSA-SeNPs treatment group was comparable to that of the positive drug amifostine group. Figure 5 The C, F, and G values ​​were also present. Secondly, compared to the Se NPs group at the same dose, the HSA-SeNPs group showed better recovery of cell number and improvement in white pulp structure, demonstrating a more significant effect in improving spleen tissue damage. Figure 5 DG). Bone marrow histological results further showed that the cells in the bone marrow cavity of the blank group mice were intact and tightly arranged, and the morphology of blood cells of all lineages was normal and evenly distributed.Figure 5 The bone marrow tissue of mice in the radiation model group showed severe radiation-induced pathological changes, including significant emptiness of the bone marrow cavity, a significant reduction in the total number of cells, sparse cell arrangement, extensive infiltration of adipocytes, and disordered structure, suggesting that their hematopoietic function was severely suppressed. Figure 5 The 1 mg / kg HSA-SeNPs significantly improved the degree of bone marrow injury compared to the model group, as evidenced by a significant increase in cell number, enhanced structural integrity, and more compact cell arrangement. Figure 5 The same dose of Se NPs showed limited improvement, with the bone marrow cavity remaining relatively empty and cell count recovery not significant. Figure 5 (J). In summary, HSA-SeNPs exhibited more significant radiation protection effects than SeNPs in both spleen and bone marrow tissues.

[0037] Example 6: Evaluation of the protective effect of protein nano-selenium (HSA-SeNPs) at total lethal radiation dose. 1. Method This experiment used 84 C57BL / 6 mice, half male and half female, aged 4-6 weeks, weighing 20±2g, clean grade, and kept in a 1:1 day-night cycle environment, with indoor humidity maintained at 44–65% and room temperature at 22–26 ℃. The mice were given normal food and water and were acclimatized for one week. Mice were randomly divided into 7 groups, with 6 males and 6 females in each group: blank group, model group, amifostine group (as positive control), 0.3 mg / kg Se NPs group (Se NPs-0.3), 0.6 mg / kg Se NPs group (Se NPs-0.6), 0.3 mg / kg HSA-SeNPs group (HSA-SeNPs-0.3), and 0.6 mg / kg HSA-SeNPs group (HSA-SeNPs-0.6). Except for the normal group mice, each mouse in the other groups was anesthetized by intraperitoneal injection of Avertin (mouse)-tribromoethanol, 0.0125 mL / g, and then irradiated once. Based on the total lethal radiation dose obtained in Example 4, the modeling conditions were set as follows: total dose: 7.5 Gy, dose rate: 2 Gy / min, energy level: 6 MV. Using the Se NPs and HSA-SeNPs prepared in Example 1, mice were administered the drugs via gavage after fasting for 6-10 hours daily following irradiation (with no restriction on water intake). The control group and model group were administered the same volume of physiological saline via gavage daily for 7 days. Mouse weight and mortality were recorded. On the 8th day after irradiation, blood was collected from the eyeballs for complete blood cell analysis, and the spleen was harvested for weighing, cell morphology observation, and immunohistochemical analysis.

[0038] 2. Conclusion The results showed that, compared with the Se NPs group, the HSA-SeNPs group mice had a higher survival rate up to day 8 and a reduced degree of weight loss after irradiation, demonstrating a superior survival benefit. Figure 6 The spleen index was significantly higher in the HSA-SeNPs group than in the model group (*P<0.05), while SeNPs did not show a significant improvement in the spleen index after irradiation. Further observations of spleen morphology and size suggest that HSA-SeNPs can inhibit radiation-induced spleen atrophy, which may help maintain the body's immune function. Figure 6 CD). Hematological results showed that, compared with the model group, on day 8 after oral administration of HSA-SeNPs at a dose of 0.6 mg / kg / d, multiple blood cell parameters were improved, including white blood cell count (**p<0.01, Figure 6 E), platelets (**p<0.01, Figure 6 F), lymphocytes (*p<0.05, Figure 6 (G), hemoglobin (*p<0.05, Figure 6 H), red blood cells (*p<0.05, Figure 6 The difference in neutrophil count between the groups was statistically significant, indicating that HSA-SeNPs have a clear protective effect on the blood system after radiation exposure. Although the neutrophil count did not show statistical significance between the groups, the HSA-SeNPs group had a higher count than the SeNPs group (I). Figure 6 (J). SeNPs, when taken orally at the same dose, only showed a trend of improvement in radiation-induced thrombocytopenia, without significantly protecting against the decline in other blood cell counts. In conclusion, under high-dose radiation conditions, HSA-SeNPs showed superior survival benefits compared to SeNPs and had a better effect on improving radiation-induced multi-lineage thrombocytopenia, suggesting that they possess better radiation protection.

[0039] Example 7: Effects of selenium protein nanoparticles (HSA-SeNPs) on the expression of apoptosis-related proteins in mouse spleen tissue cells at total lethal radiation doses. 1. Method (1) Immunohistochemistry was used to observe the expression of apoptosis-related proteins Bcl-2, Bax, and P53 in mouse spleen tissue: After treating mice in each group according to the method described in Example 6, spleen tissue was taken from the sacrificed mice and fixed in 10% (w / w) neutral formaldehyde for 30 min. The fixed tissue was aliquoted into correspondingly labeled embedding cassettes, placed in an automatic tissue dehydrator for dehydration, embedded, and sectioned with a section thickness of 3 μM. The sections were baked at 65℃ overnight, cooled to room temperature, and then successively immersed in covered glass jars containing environmentally friendly clearing agent I and environmentally friendly clearing agent II for 15 min, and in covered glass jars containing 100%, 95%, 85%, and 70% alcohol by volume for 2 min each, and rinsed with distilled water 3 times for 5 min each time. The obtained sections were placed in a solution (25 mL of EDTA (pH 8.0) in 500 mL distilled water), boiled in an autoclave for 2.5 min, and then allowed to stand until the water temperature in the autoclave returned to room temperature. They were then washed three times with PBS buffer, 5 min each time. After treatment with 3% H2O2 in the dark for 25 min, they were washed three times with distilled water (containing two drops of Tween-20 per 500 mL), 5 min each time. Primary antibodies Bcl-2, Bax, and P53 (1:100 dilution) were added, and the negative control group was treated with PBS. The sections were incubated at 37°C for 2 h, followed by three washes with PBS buffer, 5 min each time. Biotin-labeled goat anti-mouse IgG was added to each section, and the sections were incubated at 37°C for 30 min, followed by three washes with PBS buffer, 5 min each time. DAB chromogenic reagent was used for development, followed by washing with tap water, counterstaining with hematoxylin, immersing in differentiation solution for 3-4 seconds, clearing, and mounting.

[0040] (2) The expression of apoptotic cells in spleen tissue was detected by the Tunnel method: The procedure was performed according to the instructions for detecting apoptosis in paraffin sections. The main steps were gradient hydration with alcohol, digestion with 2% proteinase K, TUNEL mixture and POD solution (peroxidase labeling reagent) in a humidified chamber at 37°C for 30 min in sequence, washing with PBS for 5 min × 3 after each reaction, DAB staining, counterstaining and mounting.

[0041] (3) Image acquisition: Observation was performed under an upright fluorescence microscope, and photographs were taken for record-keeping. Image-ProPlus 6.0 was used for image analysis and processing, mainly measuring the average optical density value of positive expression cells. Ten 400x magnification fields were randomly selected from each slice for image analysis, with the selected locations being consistent across groups. The higher the number of positive cells, the higher the intracellular immune activity (expression level) of the protein.

[0042] 2. Conclusion (1) TUNEL staining revealed that apoptotic cell nuclei were brown, predominantly round, with scattered triangular or crescent-shaped staining, and were generally scattered. Normal cell nuclei were blue. The number of TUNEL-positive cells was used as an indicator of the degree of DNA damage. Only a very small number of scattered positive cells were observed in the blank group. Compared with the SeNPs group, the number of TUNEL-positive cells in the HSA-SeNPs group was significantly reduced (**p<0.01), indicating that there were fewer apoptotic cells in this group and that it had a better effect on improving DNA damage. Among them, the improvement effect was particularly obvious in mice orally administered HSA-SeNPs at a dose of 0.6 mg / kg / d. Figure 7 (AG).

[0043] (2) In the mechanism of apoptosis regulation, the overexpression of the anti-apoptotic protein Bcl-2 can effectively inhibit the p53-mediated apoptosis pathway. This protein is brown in color and mainly round in shape. The results showed that compared with the SeNPs group, the expression level of Bcl-2 protein in the spleen tissue of mice after irradiation was significantly increased in the HSA-SeNPs group (**p<0.01), indicating that HSA-SeNPs has a significant advantage in promoting the expression of anti-apoptotic proteins. Among them, the HSA-SeNPs group administered at a dose of 0.6 mg / kg / d had the most outstanding effect, and its Bcl-2 expression level was significantly higher than that of the SeNPs group at the same dose, further confirming that HSA-SeNPs is superior to traditional SeNPs in inhibiting radiation-induced apoptosis ( Figure 8 (AG).

[0044] (3) In mouse spleen tissue sections, Bax protein is a key pro-apoptotic protein in the process of apoptosis (programmed cell death). Positive expression of Bax protein is located in the cytoplasm and appears as a deep brown-yellow stain. The results showed that, compared with the SeNPs group, the positive expression level of Bax protein in the spleen tissue of mice in the HSA-SeNPs group was significantly reduced after irradiation (*p<0.05), suggesting that HSA-SeNPs can more effectively inhibit the expression of radiation-induced pro-apoptotic proteins. Figure 9 (AG).

[0045] (4) In mouse spleen tissue sections, p53 upregulated the expression of pro-apoptotic genes such as Bax. In the blank group, only a small amount of scattered p53 protein was positively expressed in the nucleus, appearing as light brown granular deposits in the cell nucleus. After irradiation treatment, compared with the SeNPs group, the expression of p53 protein in the spleen tissue of mice in both HSA-SeNPs groups (0.3 mg / kg / d and 0.6 mg / kg / d) was significantly reduced (*p<0.05). Among them, the inhibitory effect of the 0.6 mg / kg / d dose group was particularly significant. This result indicates that HSA-SeNPs can effectively inhibit radiation-induced upregulation of p53 protein expression and shows a dose-dependent trend, further confirming that its protective effect in regulating DNA damage response pathways is superior to that of traditional SeNPs. Figure 10 (AG).

[0046] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. The use of a protein nano-selenium in the preparation of drugs for preventing and treating ionizing radiation damage.

2. The use of the protein nano-selenium according to claim 1 in the preparation of drugs for preventing and treating ionizing radiation damage, characterized in that: The protein nano-selenium is used as an inhibitor of apoptosis in spleen and bone marrow cells in the preparation of drugs to prevent and treat ionizing radiation damage.

3. The use of the protein nano-selenium according to claim 1 in the preparation of drugs for preventing and treating ionizing radiation damage, characterized in that: The preparation steps of the protein nano-selenium include: reducing human serum albumin with a protein reducing agent at 37°C, then adding a sodium selenite solution containing chitosan or a sodium selenate solution containing chitosan, stirring the reaction at room temperature or 4°C for 3-12 hours, and removing unreacted substances by dialysis or centrifugation to obtain protein nano-selenium particles.

4. The use of the protein nano-selenium according to claim 3 in the preparation of drugs for preventing and treating ionizing radiation damage, characterized in that: The protein reducing agent is tris(2-carboxyethyl)phosphine.

5. The use of the protein nano-selenium according to claim 3 in the preparation of drugs for preventing and treating ionizing radiation damage, characterized in that: The protein nano-selenium was prepared according to the following steps: Human serum albumin was dissolved in a phosphate buffer solution containing a protein reducing agent at a pH of 5.0–9.0, with a final concentration of human serum albumin of 0.01–200 mg / mL and a final concentration of the protein reducing agent of 0.1–60 mM; the mixture was reacted at 37°C for 90–110 min with stirring to obtain a homogeneous protein solution with expanded spatial structure; then, a sodium selenite solution or a sodium selenate solution containing chitosan was added to the homogeneous protein solution with expanded spatial structure, with a final concentration of chitosan of 0.05–5 mg / mL and a final concentration of sodium selenite or sodium selenate of 0.1–30 mM; the mixture was stirred at room temperature or 4°C for 3–12 h to obtain a crude protein nano-selenium solution; the crude protein nano-selenium solution was placed in a dialysis bag and dialyzed overnight with PBS solution at 0–20°C, or the crude protein nano-selenium solution was centrifuged at 8000–30000 rpm for 10–60 minutes. After repeatedly resuspending in deionized water and centrifuging several times, protein nano-selenium was finally obtained.

6. The use of the protein nano-selenium according to claim 5 in the preparation of drugs for preventing and treating ionizing radiation damage, characterized in that: The dialysis bag has a dialysis molecule rejection rate of not less than 1000.

Images