Application of carrageenan degradation product in preparation of medicine for improving male reproductive power

Abstract

The invention discloses an application of a carrageenan degradation product in preparation of a medicine for improving male reproductive power. According to the method disclosed by the invention, a compound capable of improving sperm motility and male reproductive power is obtained, and a new thought and clue are provided for deeply researching the pathogenesis of oligoasthenospermia and researchers of male reproductive health in the future.

Description

Technical Field

[0001] The invention relates to a carrageenan degradation product which can be used to improve sperm motility and semen density and application thereof in preparing a drug for improving male fertility, belonging to the field of reproductive biology technology and biomedicine. Background Art

[0002] At present, the global infertility rate continues to rise, and now it is close to 15%-20%, becoming the third largest health problem for humans after cancer and cardiovascular and cerebrovascular diseases. Although my country has successively implemented the policies of "two children for single couples", "two children for all couples" and "three children", the number of births in my country has shown a cliff-like decline in recent years, from 17.86 million in 2016 to 9.02 million in 2023, and there will be negative population growth in 2022. The total fertility rate of women is only 1.13, far below the 2.1 required to maintain intergenerational balance. These figures show that my country is facing a severe fertility crisis and an unoptimistic population situation. According to statistics, the proportion of infertile people in my country has increased from 6.9% to 17.6%. Among adults with infertility, the ratio of men to women is roughly the same. However, compared with women, there are many hidden risks in male reproductive health that have not been discussed enough.

[0003] Studies have shown that spermatogenesis disorders are the main cause of male infertility. As the core function of species reproduction, spermatogenesis is an important indicator of male reproductive health. However, due to the influence of multiple factors such as environmental pollution, changes in lifestyle and increased social pressure, the average sperm concentration of men worldwide has dropped by more than 51% from 1973 to 2018. Abnormal sperm health has been called a "silent epidemic" by the international community. So far, there is no effective treatment for male infertility caused by spermatogenesis disorders. There is an urgent need to explore a practical and effective means to improve the fertility of male infertility patients.

[0004] Carrageenan is a member of the seaweed polysaccharide family. Due to its stable composition, it has been used as a thickener and gelling agent and is widely used in the food and cosmetics industries. As a degradation product of carrageenan, carrageenan oligosaccharides have a smaller molecular weight and better solubility. They also have stronger biological activity and show more potential health benefits. They have broad application prospects in the food, medicine, and cosmetics industries. Carrageenan oligosaccharides have high prebiotic activity and can act as prebiotics to change the composition of the microbiome, thereby having a positive impact on intestinal health. In terms of anti-tumor activity, carrageenan oligosaccharides and their derivatives can inhibit tumor growth, and studies related to immunomodulatory activity have shown that carrageenan oligosaccharides have immune activity and can increase the content of immune factors in the body. Summary of the invention

[0005] The purpose of the present invention is to provide an effective treatment method for poor male sperm quality and low fertility, that is, the use of carrageenan degradation products in the preparation of drugs, foods or health products for improving male fertility.

[0006] In the present invention, a male mouse disease model of oligoasthenozoospermia was established by intraperitoneal injection of busulfan, and it was found that the sperm motility and semen density of male mice could be significantly improved after adding carrageenan degradation product carrageenan oligosaccharide.

[0007] Therefore, the present invention provides the use of carrageenan degradation products in the preparation of medicines, foods or health products for improving male fertility.

[0008] Preferably, the carrageenan degradation product is carrageenan oligosaccharide.

[0009] Preferably, the medicine, food or health product for improving male fertility is a medicine, food or health product for improving sperm motility and semen density.

[0010] Preferably, the drug contains an effective amount of carrageenan degradation products and an acceptable carrier.

[0011] The present invention also provides a medicine, food or health product for improving male fertility, which contains an effective amount of carrageenan degradation products.

[0012] The method of the present invention obtains a compound that can be used to improve sperm motility and male fertility, providing new ideas and clues for future in-depth studies on the pathogenesis of oligoasthenozoospermia and for researchers of male reproductive health. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 Conceptual image of the research conducted for this invention.

[0014] Figure 2 The sperm motility test results of three groups of mouse samples.

[0015] Figure 3 The semen density statistics of three groups of mouse samples are shown in Figure 2.

[0016] Figure 4 These are the results of WB detection of germ cell marker proteins in testicular samples of three groups of mice. DETAILED DESCRIPTION

[0017] The method of the present invention will be further described below with reference to the accompanying drawings and through specific embodiments.

[0018] Embodiment 1,

[0019] 1. Experimental animal treatment and reagent preparation

[0020] The present invention uses 3-week-old ICR male mice as experimental subjects. The mice are caged and raised in a clean animal room. They are raised strictly according to standard mouse breeding conditions. The mice are placed in a 12-hour light and 12-hour dark cycle, the room temperature is 23±1°C, the ambient humidity is 50%, and they can drink water and eat freely. All procedures comply with animal ethics standards and experimental specifications. A normal control group (Con), a busulfan treatment group (Bus), and a carrageenan oligosaccharide (purchased from Qingdao Bozhihuili Biotechnology Co., Ltd., production batch number 2023021301KBF) rescue group (Bus+Cos) were set. Specific treatment method for mice: This experiment used 3-week-old ICR male mice. The Con group received a single intraperitoneal injection of normal saline at a dose of 7.5 μL per gram of body weight on the morning of the first day, and then continued to receive a single intragastric gavage of normal saline at a dose of 7.5 μL per gram of body weight every morning on the second day for five weeks; the Bus group received a single intraperitoneal injection of 1 mg / mL busulfan according to body weight on the morning of the first day, and then continued to receive a single intragastric gavage of normal saline at a dose of 7.5 μL per gram of body weight every morning on the second day for five weeks; the Bus+Cos group received a single intraperitoneal injection of 1 mg / mL busulfan according to body weight on the morning of the first day, and then continued to receive a single intragastric gavage of 40 mg / mL carrageenan oligosaccharide according to body weight every morning on the second day for five weeks.

[0021] Preparation of busulfan solution: Dissolve 0.1 g busulfan (B2635, Sigma, USA) in 10 mL DMSO solution, dispense 10 mg / mL busulfan solution into 1.5 mL Eppendorf centrifuge tubes and store at 4°C in dark for later use, dilute 10 times with sterile saline before use (final concentration is 1 mg / mL). Use a 1 mL syringe for intraperitoneal injection to ensure that the mice take in 30 mg busulfan per kg body weight per day.

[0022] Preparation of carrageenan oligosaccharide solution: Dissolve carrageenan oligosaccharide powder in sterile saline and set the concentration to 10 mg / kg. Perform intragastric gavage every morning, with the amount of liquid used for gavage being 0.10 ml / mouse / day for 5 weeks. Ensure that the daily intake of carrageenan oligosaccharide in mice is 10 mg per kilogram of body weight.

[0023] 2. Sample collection

[0024] After 5 weeks of continuous gavage treatment, testicles and semen samples from different treatment groups were collected for comparative testing. After the mice were killed by cervical dislocation, alcohol was sprayed on the abdominal skin of the mice and the testicles were exposed with sterilized surgical scissors. The testicles were placed in a centrifuge tube and frozen at -80℃ for subsequent Western Blot experiments; the mouse epididymis was picked up, sperm was released for in vitro capacitation, and sperm motility data was measured.

[0025] 3. Sperm motility and semen density testing

[0026] Prepare sperm capacitation solution in advance, take α-MEM / F12 and FBS in a volume ratio of 9:1 and mix them evenly, and place them on a 37°C hot plate for preheating. Kill the mice by cervical dislocation, carefully remove the epididymal tail tissue and place it in a 3 cm dish containing 200 μL sperm capacitation solution, cut the epididymal tail with a blade to release fresh sperm and time it for 5 minutes, then draw 20 μL of turbid liquid and dilute it in a new 200 μL sperm capacitation solution and mix it evenly to obtain sperm liquid, and finally draw 4 μL of sperm liquid and drop it on the sperm slide to make the sperm quickly diffuse into the slide, and use SCA sperm quality analyzer to take pictures for sperm motility detection and semen density analysis.

[0027] 4. Western Blot of germ cell marker proteins in testicular tissue samples

[0028] The collected testicular samples were added with an appropriate amount of RIPA tissue lysis buffer, lysed with a tissue grinder, vortexed every 5 minutes, centrifuged at 12000rpm for 1 minute, and repeated this step 3 times. The supernatant was transferred to a 1.5mL centrifuge tube and 1 / 4 of 5×SDS was added. After oscillation and mixing, it was boiled in boiling water for 5 minutes to obtain a protein sample that can be used for subsequent electrophoresis. A 10% separation gel and a 4% concentration gel were prepared for SDS polyacrylamide gel electrophoresis. During the electrophoresis, the pre-stained protein Marker was first run out under 80V constant voltage conditions and then switched to 120V constant voltage until the end; then the wet transfer method was used for membrane transfer, and the antibody was incubated after blocking in TBST blocking solution containing 5% BSA. After the incubation, the bands were chemiluminesced with a luminescent liquid, and the protein expression was analyzed according to the gray value of the bands.

[0029] 5. Results

[0030] Figure 1 This is a conceptual diagram of the research conducted in the present invention. We injected busulfan (1 mg / mL) into three-week-old male mice once to obtain a mouse model of oligoasthenozoospermia. Starting from the second day after the successful establishment of the model, we administered carrageenan oligosaccharides (40 mg / mL) by gavage every morning for five weeks. Finally, we evaluated whether gavage supplementation of carrageenan oligosaccharides for 5 weeks after busulfan injection could improve sperm motility and semen density. Figure 2 The results of sperm motility test of three groups of mice showed that compared with the control group, injection of busulfan (1 mg / mL) caused a significant decrease in sperm motility (P<0.05), but after oral administration of carrageenan oligosaccharide (40 mg / mL) for 5 weeks, the sperm motility of mice was significantly improved compared with the busulfan group (P<0.05). Figure 3The results show that compared with the control group, busulfan injection caused a significant decrease in semen density (P<0.05), but after five weeks of oral administration of carrageenan oligosaccharides, the semen density of mice was significantly higher than that of the busulfan group (P<0.05). Figure 4 The results of WB test for germ cell marker proteins in testicular samples of three groups of mice showed that compared with the control group, the protein level of VASA in the testicular tissue of the busulfan injection group was significantly reduced (P<0.05), but after 5 weeks of oral administration of carrageenan oligosaccharides, the protein level of VASA in the testicular tissue was significantly higher than that of the busulfan group (P<0.05).

[0031] The method of the present invention obtains a compound that can be used to improve sperm motility and male fertility, providing new ideas and clues for future in-depth studies on the pathogenesis of oligoasthenozoospermia and for researchers of male reproductive health.

Claims

1. Application of carrageenan degradation products in the preparation of drugs, foods or health products for improving male fertility.

2. The use according to claim 1, characterized in that: The carrageenan degradation product is carrageenan oligosaccharide.

3. The use according to claim 1 or 2, characterized in that: The medicine, food or health product for improving male fertility is a medicine, food or health product for improving sperm motility and semen density.

4. The use according to claim 1 or 2, characterized in that: The medicine, food or health product contains an effective amount of carrageenan degradation products and an acceptable carrier.

5. A drug, food or health product for improving male fertility, characterized in that: Contains an effective amount of carrageenan degradation products.

6. The drug, food or health product for improving male fertility according to claim 5, characterized in that: The carrageenan degradation product is carrageenan oligosaccharide.

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