Use of alginate oligosaccharides as a tissue radioprotector

Abstract

The application belongs to the field of plant-derived oligosaccharides, and particularly relates to application of alginate oligosaccharide as a tissue radiation protection agent. The application relates to application of alginate oligosaccharide as a radiation protection agent for reproductive tissues of female mammals or fish. The application utilizes the application of alginate oligosaccharide in protecting radiation-induced reproductive damage, and particularly provides application of alginate oligosaccharide in protecting radiation-induced reproductive damage in zebrafish and mice, utilizes the natural, safe and efficient biological activity of alginate oligosaccharide, realizes accurate protection of radiation-induced reproductive damage, and simultaneously expands the application of marine oligosaccharide in the field of radiation-induced reproductive damage protection.

Description

Technical Field

[0001] This invention belongs to the field of plant-derived oligosaccharides, specifically relating to the application of alginate oligosaccharide as a tissue radiation protectant. Background Technology

[0002] Alginate is an acidic linear polysaccharide derived from brown algae, composed of α-L-guluronic acid (G) and β-D-mannuronic acid (M) linked by 1,4-glycosidic bonds. Alginate oligosaccharides (AOS) are degradation products of alginate, linear oligomers with a degree of polymerization less than 10, and are widely found in the cell walls of brown algae (such as kelp, giant kelp, and Sargassum). Due to their well-defined structure, high water solubility, safety, stability, and high added value and physiological activities, such as antioxidant, immunomodulatory, and antitumor activities, alginate oligosaccharides have broad application prospects in biomedicine, food industry, and other fields.

[0003] With the development of technologies such as aerospace activities and tumor radiotherapy, the side effects of ionizing radiation are increasing. The reproductive system, as a target organ highly sensitive to ionizing radiation, is extremely vulnerable to its attack, especially the female reproductive system. Damage to the female reproductive system often leads to various problems such as ovarian dysfunction, decreased egg quality, impaired fertility, and endocrine disorders, seriously threatening reproductive health and even causing developmental abnormalities and reduced viability in offspring. Clinical studies have confirmed that pelvic radiotherapy in patients with cervical and rectal cancer inevitably exposes ovarian tissue to radiation, leading to decreased ovarian reserve, impaired follicle development, and impaired fertility. Older women are more sensitive to radiation damage. Currently, amifostine is a representative clinical radiation protectant, but it requires intravenous administration shortly before radiotherapy, and the administration process and conditions are highly demanding. It is also frequently accompanied by adverse reactions such as hypotension, nausea, and vomiting, affecting patient tolerance and compliance, thus limiting its further application. Developing natural, safe, and highly effective novel radiation protectants has become a current research hotspot and urgent need in the field.

[0004] On the one hand, ionizing radiation can directly act on DNA, causing single-strand and double-strand breaks. On the other hand, ionizing radiation can rapidly generate reactive oxygen species (ROS) through the radiolysis of water molecules, further inducing oxidative stress, mitochondrial dysfunction, and inflammatory responses. Studies have shown that functional sugars such as galactooligosaccharides (GOS), inulin, and fructooligosaccharides (FOS) have the ability to scavenge free radicals and alleviate oxidative stress. GOS and inulin can scavenge DPPH and ABTS free radicals. Specifically, GOS can reduce the accumulation of ROS and lipid peroxidation levels in the colonic mucosa, improve the oxidative stress state of the colonic mucosa, and protect the intestinal epithelial barrier function. Inulin can protect the contractile function of human colonic smooth muscle cells by alleviating oxidative stress in the colonic mucosa. Fructooligosaccharides have the ability to scavenge ·OH free radicals, which can increase the activity of antioxidant enzymes in the intestinal mucosa and reduce the level of inflammatory factors, thereby alleviating damage to intestinal epithelial function. However, the ability of functional sugars to scavenge free radicals is not equivalent to their protective effect against ionizing radiation. For example, a study evaluated the protective effect of a mixture of inulin and fructooligosaccharides against acute radiation enteritis in patients who underwent abdominal radiotherapy after gynecological tumor surgery. The results showed that the mixture of inulin and fructooligosaccharides did not exhibit any protective effect against radiation damage. Alginate oligosaccharides are a class of acidic linear oligosaccharides composed primarily of uronic acid units. They contain abundant carboxyl groups and can form unsaturated double bonds at non-reducing ends. This structural characteristic gives them the ability to scavenge free radicals, but whether they can protect against ionizing radiation damage requires further investigation. Summary of the Invention

[0005] The purpose of this invention is to provide an application of alginate oligosaccharide as a tissue radiation protectant.

[0006] To achieve the above-mentioned objectives, the present invention adopts the following technical solution: Application of an alginate oligosaccharide as a tissue radiation protectant.

[0007] The application of the alginate oligosaccharide as a radiation protectant for the reproductive tissues of female mammals or fish.

[0008] The radiation is ionizing radiation, including X-ray ionizing radiation with doses of 15 Gy and 10 Gy.

[0009] The radiation protection agent is for protecting female mammals or fish from reproductive damage, wherein reproductive damage includes one or more of the following: decreased egg-laying capacity, increased embryo lethality / teratogenicity, and decreased hatchability.

[0010] Furthermore, radiation exposure can lead to ovarian dysfunction, endometrial damage, impaired follicle development, decreased egg quality, reduced fertility, and damage to offspring.

[0011] The female mammals are humans, rats, and mice, and the fish are zebrafish.

[0012] The alginate oligosaccharide is a degradation product of alginate, mainly comprising oligosaccharide components formed by α-L-guluronic acid and β-D-mannuronic acid linked by 1,4-glycosidic bonds, wherein the degree of polymerization of the oligosaccharide components is 2-5.

[0013] The dosage of the alginate oligosaccharide is 50-300 mg / L, preferably 300 mg / L and 50 mg / kg, and the administration method is to dissolve it in the daily feeding water and by gavage.

[0014] Compared with the prior art, the present invention has the following advantages: This invention utilizes alginate oligosaccharides in the protection against radiation-induced reproductive damage, particularly in the protection against radiation-induced reproductive damage in zebrafish and mice. Leveraging the natural, safe, and highly effective bioactivity of alginate oligosaccharides, it achieves precise protection against radiation-induced reproductive damage, while also expanding the application of marine oligosaccharides in the field of radiation-induced reproductive damage protection. Specifically: 1. This invention utilizes zebrafish and mouse models to verify the protective effect of alginate oligosaccharides against radiation-induced reproductive damage. Zebrafish offer advantages such as a short reproductive cycle, high egg production, convenient administration, and easy observation of reproductive damage, making them suitable for rapid and efficient preliminary evaluation of the radiation-protective effect of alginate oligosaccharides. The mouse reproductive system structure, physiological function, and radiation damage response are similar to those of humans, allowing for further verification of the protective effect of alginate oligosaccharides against radiation-induced reproductive damage. Combining zebrafish and mouse models improves the efficiency and reliability of the protective effect evaluation and enhances the scientific rigor of the experimental results.

[0015] 2. This invention uses alginate oligosaccharides, a food-grade raw material with good biocompatibility and no toxic side effects, to protect tissues and cells from radiation-induced reproductive damage. Compared to existing hormone-based protective drugs and chemical antioxidants, it does not interfere with the body's normal endocrine metabolism, can be used long-term, and has a greater safety advantage. Furthermore, its raw materials are widely available, its preparation process is mature, it is easy to scale up production, effectively control production costs, and facilitate subsequent industrialization and application, thus balancing practicality and economy.

[0016] 3. The alginate oligosaccharide administration method described in this invention is simple and convenient, requiring no complicated administration equipment or operation. For fish, it can be administered via water exposure, i.e., dissolved in the daily feeding water. For mammals, it can be flexibly adjusted to various administration methods such as oral, gavage, and intraperitoneal injection, adapting to different experimental subjects and application scenarios. Moreover, the dosage is mild, further improving the feasibility of its clinical application and industrialization. Attached Figure Description

[0017] Figure 1 This is an ESI-MS mass spectrum of alginate oligosaccharides in negative ion mode.

[0018] Figure 2 The effect of alginate oligosaccharides on the cumulative spawning of female zebrafish after radiation. NC, blank control group; RAD, radiation group; AOS, alginate oligosaccharide treatment group. * P <0.05 indicates that the difference between groups is statistically significant.

[0019] Figure 3 The effects of alginate oligosaccharides and galactooligosaccharides on the average daily spawning of female zebrafish after radiation. NC, blank control group; RAD, radiation group; AOS, alginate oligosaccharide treatment group; GOS, galactooligosaccharide treatment group. P <0.05,** P <0.01, *** P <0.001 indicates that the difference between groups is statistically significant.

[0020] Figure 4 The effect of alginate oligosaccharides on the malformation rate of zebrafish offspring after radiation. NC, blank control group; RAD, radiation group; AOS, alginate oligosaccharide treatment group. P <0.05 indicates that the difference between groups is statistically significant.

[0021] Figure 5 The effect of alginate oligosaccharides on AMH mRNA expression levels in ovarian tissue of radiation-damaged mice. NC, blank control group; RAD, radiation group; AOS, alginate oligosaccharide treatment group. P <0.05 indicates that the difference between groups is statistically significant.

[0022] Figure 6 To investigate the effects of alginate oligosaccharides on the pathological changes of ovarian tissue in radiation-damaged mice. NC, blank control group; RAD, radiation group; AOS, alginate oligosaccharide treatment group. Detailed Implementation

[0023] The following examples further illustrate specific embodiments of the present invention. It should be noted that the specific embodiments described herein are merely for illustration and explanation and are not intended to limit the scope of the present invention.

[0024] This invention discloses alginate oligosaccharides that significantly increase the cumulative and daily egg production of female zebrafish after radiation exposure, reversing the radiation-induced decline in egg production capacity. Simultaneously, it significantly reduces the rate of embryonic malformations in offspring after radiation exposure and improves the quality of embryonic development. Mouse experiments have shown that alginate oligosaccharides can improve radiation-induced ovarian reserve decline and alleviate ovarian tissue pathological damage in mice. This invention discloses that alginate oligosaccharides have a clear protective effect against reproductive damage in female zebrafish after radiation exposure and can improve radiation-induced ovarian damage and ovarian reserve decline in mice. It can be used to prepare drugs, health products, or functional preparations to protect against female reproductive damage caused by ionizing radiation. It has advantages such as readily available raw materials, high safety, significant effects, and ease of industrialization, and has broad application prospects in radiation medicine and reproductive health protection.

[0025] Example 1 1. Experimental Materials Wild-type zebrafish, AB strain, 5 months old; C57BL / 6J mice, female, 5-6 weeks old; alginate oligosaccharide; galactooligosaccharide; E3 culture medium. All materials used in this experiment were commercially available.

[0026] 2 Experimental Methods 2.1 ESI-MS analysis of alginate oligosaccharides 20 mg of alginate oligosaccharide sample was dissolved in 2 mL of water-methanol mixture. The sample was analyzed by ESI-MS in negative ion mode using an LCQ Fleet electrospray ionization mass spectrometer (Thermo Fisher Scientific, USA).

[0027] 2.2 Feeding and Grouping of Zebrafish Zebrafish were housed in a fish house with a 14-hour light / 10-hour dark alternating light cycle. The zebrafish rearing system maintained a freshwater circulation at (28±1)℃, with pH adjusted to 7-7.8 and conductivity adjusted to 500-550 μS / cm. The zebrafish were fed fresh brine shrimp twice daily and acclimatized for 14 days before the experiment. Five-month-old zebrafish from the same batch were randomly divided into four groups using a random number table method based on different treatment methods. (1) Control group (NC group): They were raised in fresh water without alginate oligosaccharide for 7 days, and then received 0 Gy sham irradiation under the same conditions as the radiation group (only placed in the radiation environment but without actual radiation exposure). After sham irradiation, they were raised in fresh water without alginate oligosaccharide for 2 days.

[0028] (2) Radiation group (RAD group): They were raised in fresh water without alginate oligosaccharide for 7 days, and then received a single whole-body irradiation of 15 Gy at a radiation dose rate of 1 Gy / min. After irradiation, they were raised in fresh water without alginate oligosaccharide for 2 more days.

[0029] (3) Alginate oligosaccharide + radiation group (AOS group): Zebrafish were kept in freshwater with a concentration of 300 mg / L alginate oligosaccharide for 7 days, with 2 / 3 of the volume of the freshwater solution of the same concentration of alginate oligosaccharide replaced daily. Zebrafish were irradiated according to the method of the radiation group, and then kept in freshwater with the same concentration of alginate oligosaccharide for 2 days.

[0030] (4) Galacto-oligosaccharide + Radiation Group (GOS Group): Zebrafish were kept in freshwater with a concentration of 300 mg / L galacto-oligosaccharide for 7 days, with 2 / 3 of the volume of the galacto-oligosaccharide freshwater solution of the same concentration replaced daily. Zebrafish were irradiated according to the method of the radiation group, and then kept in freshwater with the same concentration of galacto-oligosaccharide for 2 days.

[0031] 2.3 Reproductive testing Spawning rate and deformity rate: After radiation treatment, each group was reared for another 2 days, followed by a 6-day continuous spawning test. Spawning occurred at dawn, and eggs were collected 1 hour after fertilization and thoroughly rinsed to remove debris. Six females were used per group, with a female-to-male ratio of 1:2. The number of eggs laid was counted to assess the effect of radiation on zebrafish spawning rate. Simultaneously, the zebrafish were cultured in E3 medium for 54 hours, and the deformity rate was recorded to assess the effect of radiation on offspring development. Major deformities included cardiac hemorrhage, pericardial edema, yolk sac edema, and spinal curvature.

[0032] 2.4 Feeding and Grouping of Mice SPF-grade mice were selected and housed in an animal facility. The temperature was maintained at 23-25℃, relative humidity at 40%-60%, and the day / night cycle at 12 h / 12 ​​h. Feed, water, and bedding were prepared according to SPF-grade animal husbandry requirements. After 7 days of acclimatization, the mice were randomly divided into 3 groups using a random number table. (1) Control group (NC group): Sterile water was administered by gavage daily for 7 consecutive days, followed by 5 days of 0 Gy sham irradiation (being placed in a radiation environment but without actual radiation exposure), during which gavage was continued. After sham irradiation, gavage treatment continued for 9 days; (2) Radiation group (RAD group): Sterile water was administered by gavage daily for 7 consecutive days, followed by X-ray irradiation of the mouse ovaries for 5 days, with a total cumulative dose of 10 Gy, during which gavage was continued. After irradiation, gavage treatment continued for 9 days; (3) Alginate oligosaccharide + radiation group (AOS group): Alginate oligosaccharide was dissolved in sterile water and administered by gavage at a dose of 50 mg / kg daily for 7 consecutive days. Then, the mouse ovaries were irradiated with X-rays for 5 days, with a total cumulative dose of 10 Gy, and gavage was continued during the period. After irradiation, gavage treatment continued for 9 days.

[0033] 2.5 Real-time quantitative PCR RNA was extracted from mouse ovarian tissue using the TransZol Up Plus RNA Kit, and its concentration and purity were determined using a NanoDrop spectrophotometer. The extracted RNA was reverse transcribed into cDNA using the HiScriptIV RT SuperMix reverse transcription kit. The resulting cDNA was then subjected to RT-qPCR using the Taq Pro UniversalSYBR qPCR Master Mix kit. Experiments were performed under ice bath conditions, with three replicates per sample, using 2... -ΔΔCT The method calculates relative gene expression levels.

[0034] 2.6 Ovarian tissue staining After fixation, mouse ovarian tissue was washed with 75% alcohol, dehydrated with graded ethanol, cleared with xylene, impregnated with paraffin, and embedded to prepare paraffin sections with a thickness of 4-5 μm, which were then transferred onto glass slides. Subsequently, the sections were dewaxed, hydrated, and washed before H&E staining, with hematoxylin used for nuclear staining and eosin used for cytoplasmic contrast staining. After staining, the sections were dehydrated with ethanol, cleared with xylene, and mounted with neutral resin. Finally, they were observed and images were acquired under an optical microscope.

[0035] 3 Experimental Results 3.1 ESI-MS Analysis Results The results are as follows Figure 1 As shown, in negative ion mode, a series of characteristic ion peaks of alginate oligosaccharides were detected in the alginate oligosaccharide sample, mainly distributed in the DP2-5 range, indicating that the sample is mainly composed of alginate oligosaccharides.

[0036] 3.2 Cumulative number of eggs laid Figure 2 It can be seen that the cumulative number of eggs laid in the control group showed an increasing trend over 6 days, and was significantly higher than that in the radiation group on the 6th day. P <0.05 indicates that radiation inhibited the spawning ability of zebrafish, causing reproductive damage. The cumulative spawning in the alginate oligosaccharide group was significantly higher than that in the radiation group ( P <0.05, which not only reversed radiation-induced oviposition inhibition, but also significantly exceeded the normal level in the control group ( P The value was <0.05, demonstrating that alginate oligosaccharides can effectively protect against radiation damage to the spawning ability of female zebrafish.

[0037] 3.3 Average daily egg production Figure 3 It can be seen that the average daily egg production in the radiation group was less than that in the control group, indicating that radiation has an inhibitory effect on the spawning capacity of zebrafish. However, the average daily egg production in the galactooligosaccharide group was significantly lower than that in the control group ( P <0.01) and radiation group (P <0.05 indicates that galactooligosaccharides cannot enhance the spawning capacity of zebrafish after radiation and do not have a protective effect against ionizing radiation. It is noteworthy that the alginate oligosaccharide group had significantly higher levels than the radiation group ( P The concentration of alginate oligosaccharides was <0.05%, which was higher than that of the control group, indicating that alginate oligosaccharides can improve the reproductive capacity of radiated zebrafish.

[0038] 3.4 Offspring deformity rate Figure 4 It can be seen that the rate of birth defects in offspring of the radiation group was significantly higher than that of the control group. P <0.05 indicates that radiation not only damages the parental reproductive system but also leads to developmental abnormalities in offspring. Pretreatment with alginate oligosaccharides significantly reduced the offspring malformation rate compared to the radiation group. P The value was <0.05, and there was no significant difference compared with the control group, proving that alginate oligosaccharides can effectively improve radiation-induced intergenerational damage, improve offspring development quality, and reduce the risk of embryonic malformations.

[0039] 3.5 Relative expression levels of mouse ovarian mRNA Figure 5 It can be seen that, compared with the control group, the relative expression level of AMH mRNA in the ovarian tissue of the radiation group mice was significantly reduced. P <0.05), the expression level of AMH increased significantly after intervention with alginate oligosaccharides, indicating that radiation can cause damage to ovarian reserve function in mice, while intervention with alginate oligosaccharides improves ovarian reserve function in irradiated mice.

[0040] 3.6 Pathological changes in mouse ovaries Figure 6 The pathological analysis revealed that, compared with the radiation group, the ovarian tissue damage in the alginate oligosaccharide group was significantly reduced, with reduced interstitial gland hyperplasia and fibrosis-like changes, and some recovery of granulosa cell morphology. This indicates that alginate oligosaccharide has an ameliorative effect on radiation-induced ovarian tissue phenotypic damage in mice.

[0041] In summary, alginate oligosaccharides not only improve radiation-induced decreased ovulation capacity and reduce offspring malformation rates in zebrafish, but also improve radiation-induced ovarian reserve decline in mice and alleviate ovarian tissue pathological damage. This indicates that alginate oligosaccharides have a clear protective effect against reproductive damage caused by ionizing radiation and can be used to prepare radiation-protective agents for reproductive damage caused by ionizing radiation.

Claims

1. The application of an alginate oligosaccharide as a tissue radiation protectant.

2. The application according to claim 1, characterized in that, The application of the alginate oligosaccharide as a radiation protectant for the reproductive tissues of female mammals or fish.

3. The application according to claim 2, characterized in that, The radiation is ionizing radiation, including X-ray ionizing radiation with radiation doses of 10 Gy and 15 Gy.

4. The application according to claim 2, characterized in that, The radiation protection agent is for protecting female mammals or fish from reproductive damage, wherein reproductive damage includes one or more of the following: decreased egg-laying capacity, increased embryo lethality / teratogenicity, and decreased hatchability.

5. The application according to claim 2, characterized in that, The female mammals are humans, rats, and mice, and the fish are zebrafish.

6. The application according to claim 1 or 2, characterized in that, The alginate oligosaccharide is a degradation product of alginate, mainly comprising oligosaccharide components formed by α-L-guluronic acid and β-D-mannuronic acid linked by 1,4-glycosidic bonds, and the degree of polymerization of the oligosaccharide is 2-5.

7. The application according to claim 6, characterized in that, The dosage of the alginate oligosaccharide is 50-300 mg / L, and the administration method is by dissolving it in the feeding water and by gavage.

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