Use of uridine-5'-monophosphate and its complex for the preparation of a medicament for enhancing the proliferative capacity of male germ cells

Abstract

The application belongs to the technical field of medicine, and discloses application of uridine-5' monophosphate and a compound thereof in preparation of a medicine for enhancing the proliferation ability of male reproductive cells. It is found for the first time that the uridine-5' monophosphate has the proliferation ability on the male reproductive cells, and the proliferation effect is best when the concentration of the uridine-5' monophosphate is 10 μM, the proliferation effect on the mouse TM3 cells is increased by 52.87% compared with a control, and the proliferation effect on the mouse TM4 cells is increased by 53.57%. In addition, the uridine-5' monophosphate has a synergistic ability when being compounded with xylonic acid lactone, and further improves the proliferation effect on the TM3 cells, thereby laying a foundation for treatment of male reproductive system diseases in the future.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, and in particular relates to the application of uridine-5' monophosphate and its complexes in the preparation of drugs that enhance the proliferative capacity of male germ cells. Background Technology

[0002] In recent years, extensive research has been conducted globally on male infertility, primarily driven by increased public awareness due to declining semen quality among healthy men worldwide (AGARWAL et al., 2020). Infertility is a significant public health and wellness issue with substantial social, psychological, and economic impacts (SUGANTHI et al., 2014). Advances in research on the molecular and cellular mechanisms of spermatogenesis have helped identify characteristics of many previously considered idiopathic conditions. Among the most important are hypogonadotropic hypogonadism, androgen receptor mutations, cystic fibrosis transmembrane conductance regulation gene mutations, genetic polymorphisms, and Y-linked infertility.

[0003] Selenium and zinc are both essential trace elements for the human body, playing a crucial role in scavenging reactive oxygen species. Compared to the normal semen group, the abnormal semen group showed a decrease of over 20% in serum and seminal plasma selenium concentrations. Furthermore, adding selenium to the routine treatment for oligospermia patients significantly increased sperm motility and the number of sperm with normal morphology compared to the conventional treatment group (Zhang Fengfeng et al., 2020). Compared to non-smokers, smokers showed a trend of decreasing zinc levels in their seminal plasma. Zinc levels in fertile and infertile men (smokers or non-smokers) are significantly correlated with sperm count and normal sperm morphology. These studies suggest that deficiencies in nutrients such as selenium and zinc may lead to poor sperm quality and idiopathic male infertility (COLAGAR et al., 2008). Icariin (ICA), a major flavonoid component derived from the traditional Chinese medicine epimedium (Epimedium brevicornum Maxim.), has a protective effect on male reproductive capacity. Studies have shown that Icariin promotes the proliferation of Sertoli cells in vitro by activating the ERK1 / 2 signaling pathway (NAN et al., 2014; Zhang et al., 2024). Taurine has a potential protective effect on male reproductive function. Taurine can delay the aging of testicular structure and function, maintain testicular homeostasis, and enhance sexual ability (LI et al., 2023). Studies have shown that coenzyme Q10, kallikrein, and pentoxifylline can all improve semen parameters (Peng, 2023; OMAR et al., 2019). Studies by Feng Jianhao et al. have shown that Dendrobium officinale extract can significantly enhance the proliferation of mouse TM3 cells (Feng Jianhao et al., 2024); studies by Lu Feng et al. have shown that retinol can promote the differentiation of spermatogenic cells in cryptorchidism by downregulating the expression level of the miR-210 gene (Lu Feng et al., 2022). Therefore, idiopathic infertility can be treated with hormonal drugs and nutritional supplements (N et al., 2021).

[0004] Urate-5' monophosphate is a precursor of uridine, a pyrimidine nucleoside that participates in RNA, biological membrane, and glycogen synthesis, playing a crucial role in cell growth and metabolism (Bai et al., 2023). Besides its indispensable role in RNA and DNA biosynthesis, uridine also participates in glycogen deposition and plays a vital regulatory role in protein and lipid glycosylation. Furthermore, uridine is involved in the biosynthesis of the extracellular matrix, which is essential for maintaining the stability of the extracellular environment; it also plays a key role in xenobiotic detoxification, helping to remove harmful substances from the body. Therefore, uridine plays multiple roles in organisms and is of great significance for maintaining normal life activities (DENG et al., 2017).

[0005] There are currently no reports on the use of uridine-5' monophosphate (Cas: 58-97-9) to enhance the proliferative capacity of male germ cells. Summary of the Invention

[0006] The purpose of this invention is to provide the application of uridine-5' monophosphate in the preparation of drugs that enhance the proliferative capacity of male germ cells.

[0007] Another object of the present invention is to provide the use of a compound containing uridine-5' monophosphate in the preparation of a medicament that enhances the proliferative capacity of male germ cells.

[0008] To achieve the above objectives, the present invention adopts the following technical measures:

[0009] The scope of protection of this invention includes:

[0010] Application of uridine-5' monophosphate in the preparation of drugs that enhance the proliferative capacity of male germ cells.

[0011] Application of compound containing uridine-5' monophosphate in the preparation of drugs that enhance the proliferative capacity of male germ cells.

[0012] In the above-described applications, the compound also contains xyloside lactone, zinc, selenium, and / or vitamin E as the main active ingredients. Preferably, in the above-described applications, the germ cells are testicular interstitial cells or testicular Sertoli cells.

[0013] In the above-described applications, preferably, the effective concentration of uridine-5' monophosphate is 0.01 μm to 1000 μm;

[0014] In the above-described applications, preferably, the effective concentration of uridine-5' monophosphate is 0.1 μm to 100 μm;

[0015] In the above-described applications, preferably, the effective concentration of uridine-5' monophosphate is 1 μm to 10 μm.

[0016] The dosage form of the drug described above is a pharmaceutically acceptable dosage form, including tablets, capsules, granules, injections, powders, or drops.

[0017] Compared with the prior art, the present invention has the following advantages:

[0018] This application is the first to discover that uridine-5' monophosphate has the ability to promote the proliferation of male germ cells. The proliferation effect is optimal when the concentration of uridine-5' monophosphate is 10 μM. Compared with the control, the proliferation effect on mouse TM3 cells (mouse testicular interstitial cells) is increased by 52.87%, and the proliferation effect on mouse TM4 cells (normal mouse testicular Sertoli cells) is increased by 53.57%. Furthermore, when combined with xylosidone, it also has a synergistic effect, further enhancing the proliferation effect on TM3 cells, laying the foundation for the treatment of male reproductive system diseases in the future. Attached Figure Description

[0019] Figure 1 The effect of different concentrations of uridine-5' monophosphate on the proliferation of mouse testicular cells.

[0020] Figure 2 The effects of uridine-5' monophosphate, uridine-5' monophosphate and their complexes on mouse testicular cell proliferation. Detailed Implementation

[0021] Unless otherwise specified, the technical solutions described in this invention are all conventional solutions in the field; the reagents or materials described, unless otherwise specified, are all from commercial sources.

[0022] The materials and conventional methods involved in this invention are as follows:

[0023] All xylosidones mentioned in this invention are D-xylosidones (Cas: 18423-66-0).

[0024] The mouse TM3 (mouse testicular interstitial cells) and mouse TM4 (normal mouse testicular Sertoli cells) cells were obtained from the Oil Crops Research Institute of the Chinese Academy of Agricultural Sciences.

[0025] Accurately weigh an appropriate amount of uridine-5' monophosphate powder and prepare a 10 mmol / L stock solution using ultrapure water. Filter the solution through a 0.22 μm sterile filter membrane, label with the preparation date, drug name, and concentration, and store in a sealed container at -80℃. Dilute to the required concentration with DEME / F12 serum-free medium before use.

[0026] Xyloside lactone solution: Accurately weigh an appropriate amount of xyloside lactone solution, prepare a 10 mmol / L stock solution using ultrapure water, filter through a 0.22 μm sterile filter membrane, label with the preparation date, drug name and concentration, store in a sealed container at -80℃, and dilute to the required concentration with DEME / F12 serum-free medium before use.

[0027] This invention uses Prism 9.0 to analyze cell experiment results, which are expressed as mean ± standard deviation (±s). One-way ANOVA was used to analyze the means of multiple groups, and independent samples t-tests were used to compare two groups. P < 0.05 was considered statistically significant.

[0028] Effects of different concentrations of uridine-5' monophosphate on mouse testicular cell proliferation:

[0029] Cell incubation experiment:

[0030] (1) Experimental group: Cells (TM3 or TM4) were seeded into cell culture plates (6-well culture plates, 2.5 × 10⁶ cells / wells). 5 cells / well; 1×10⁶ 96-well culture plate 4 Cells were cultured in wells, and 100 μL of uridine-5' monophosphate solution of different concentrations (1000 μM, 100 μM, 10 μM, 1 μM, 0.1 μM, 0.01 μM) was added according to experimental requirements. After culturing for 24 hours, cell viability was detected using CCK-8 assay.

[0031] (2) Control group: Cells (TM3 or TM4) were seeded into cell culture plates (6-well plates, 2.5 × 10⁶ cells / wells). 5 Cells / well;) were added, and 100 μL of DEME / F12 serum-free medium was added, and the cells were cultured together with the experimental group for another 24 hours. The viability and number of cells were also detected using the CCK-8 reagent.

[0032] (3) Blank group: To obtain experimental blank values, six wells were set up on the same 96-well plate without seeding any cells. 100 μL of PBS buffer was added to these wells. After the other two groups (experimental group and control group) were co-cultured for 24 hours, 100 μL of serum-free medium was added to these blank wells, and the plates were cultured for another 24 hours. These blank wells will be used for subsequent detection to obtain experimental blank values.

[0033] CCK-8 assay for cell viability: After the treatment time, remove the culture plate, discard the original culture medium or drug solution, and add 100 μL of freshly prepared culture medium containing 10% CCK-8. Return the plate to the incubator and continue culturing for 30 min. Remove the culture plate and measure the absorbance at wavelengths of 450 nm and 650 nm using a microplate reader.

[0034] The results are as follows Figure 1 As shown, the proliferation effect was best when the concentration of uridine-5' monophosphate was 10 μM. Compared with the control, the proliferation effect on mouse TM3 cells increased by 52.87% and the proliferation effect on mouse TM4 cells increased by 53.57%.

[0035] Example 2:

[0036] Application of uridine-5' monophosphate and xylosidone complex in the preparation of drugs that enhance the proliferative capacity of male germ cells:

[0037] In this embodiment, the synergistic effect between xylosidone and uridine-5' monophosphate, two active ingredients, was explored.

[0038] 100 μM xylosidone and 10 μM uridine-5' monophosphate were mixed at a volume ratio of 1:1 to form a compound, and the proliferation of mouse testicular cells was detected by cell incubation experiment.

[0039] Cell incubation experiment:

[0040] (1) Experimental group: Cells (TM3 or TM4) were seeded into cell culture plates (6-well culture plates, 2.5 × 10⁶ cells / wells). 5 cells / well; 1×10⁶ 96-well culture plate 4 Cells / well were collected and 100 μL of the test reagent was added according to experimental requirements. After culturing for another 24 hours, cell viability was detected using the CCK-8 reagent.

[0041] (2) Control group: Cells (TM3 or TM4) were seeded into cell culture plates (6-well plates, 2.5 × 10⁶ cells / wells). 5 Cells / well;) were added, and 100 μL of DEME / F12 serum-free medium was added, and the cells were cultured together with the experimental group for another 24 hours. The viability and number of cells were also detected using the CCK-8 reagent.

[0042] (3) Blank group: To obtain experimental blank values, six wells were set up on the same 96-well plate without seeding any cells. 100 μL of PBS buffer was added to these wells. After the other two groups (experimental group and control group) were co-cultured for 24 hours, 100 μL of serum-free medium was added to these blank wells, and the plates were cultured for another 24 hours. These blank wells will be used for subsequent detection to obtain experimental blank values.

[0043] CCK-8 assay for cell viability: After the treatment time, remove the culture plate, discard the original culture medium or drug solution, and add 100 μL of freshly prepared culture medium containing 10% CCK-8. Return the plate to the incubator and continue culturing for 30 min. Remove the culture plate and measure the absorbance at wavelengths of 450 nm and 650 nm using a microplate reader.

[0044] The results are as follows Figure 2As shown, the complex has a proliferative effect on both mouse TM3 and mouse TM4 cells. Compared with the control, the proliferation effect on mouse TM3 cells increased by 64.80%, and the proliferation effect on mouse TM4 cells increased by 57.20%.

[0045] There was no significant difference in the proliferation of mouse TM3 cells between the xylosidone treatment group and the uridine-5' monophosphate treatment group (p = 0.618); there was no significant difference in the proliferation of mouse TM3 cells between the xylosidone treatment group and the combined treatment group (p = 0.079); there was a significant difference in the proliferation of mouse TM3 cells between the uridine-5' monophosphate treatment group and the combined treatment group (p = 0.041).

[0046] There was no significant difference in the proliferation of mouse TM4 cells between the xylosidone treatment group and the uridine-5' monophosphate treatment group (p = 0.218); there was no significant difference in the proliferation of mouse TM4 cells between the xylosidone treatment group and the combined treatment group (p = 0.289); there was no significant difference in the proliferation of mouse TM4 cells between the uridine-5' monophosphate treatment group and the combined treatment group (p = 0.769).

[0047] The above results indicate that xyloside lactone, when combined with uridine-5' monophosphate, can promote the efficacy of uridine-5' monophosphate. After combination, it significantly improves the proliferation of mouse TM3 cells, demonstrating a synergistic effect.

[0048] Example 3:

[0049] Effects of uridine-5' monophosphate and its compounds on the cell cycle of mouse testicular cells:

[0050] In this embodiment, flow cytometry was used to identify the state of cells after incubation in Example 1. The steps are as follows:

[0051] (1) After the treatment time has elapsed, carefully remove the original culture medium or drug solution and gently rinse the cells twice with PBS buffer to remove any residue. Then, add 500 μL of 0.25% trypsin to digest the cells. After the cells have digested, quickly add 1 mL of TM4 medium to stop the digestion process, and carefully pipette any cells that have not completely detached to ensure they are completely separated from the cell wall. Collect the cells into a 15 mL centrifuge tube and centrifuge at 300 x g for 5 min. After centrifugation, discard the culture medium in the centrifuge tube and resuspend the cells with 1 mL of PBS buffer. Then, centrifuge again and discard the supernatant. Finally, add 0.3 mL of PBS buffer to resuspend the cells, then add 1.2 mL of -20°C anhydrous ethanol, mix thoroughly, and fix the cell suspension in a -20°C freezer for 1 hour or overnight.

[0052] (2) After fixation, the centrifuge tubes were placed back into the centrifuge and centrifuged at 300xg for 5 min. Then, the supernatant was discarded, 1 mL of PBS buffer was added to resuspend the cells, and the mixture was allowed to stand at room temperature for 15 min.

[0053] (3) Place the centrifuge tube back into the centrifuge and centrifuge at 300xg for 5 minutes. After centrifugation, discard the supernatant and add 100μL of RNase A reagent to fully suspend the cells. Then incubate the cells in a 37°C water bath for 30 minutes.

[0054] (4) Add 400 μL of PI reagent (concentration of 50 μg / mL) and mix thoroughly. Then, incubate the cell suspension in a dark environment at 2-8°C for 30 minutes to allow the PI reagent to bind to the cell DNA.

[0055] (5) Immediately conduct the test and record the signal at the excitation wavelength of 488nm.

[0056] (6) Use Flowjo software to analyze and obtain cell cycle distribution.

[0057] The results showed that for TM3 cells, compared with the control group, the proportion of cells in G1 phase decreased by 11.43% in the uridine-5' monophosphate treatment group, which was highly significant (p = 0.003), and the proportion of cells in S phase increased by 7.733%, which was highly significant (p = 0.0016). Compared with the control group, the proportion of cells in G1 phase decreased by 13.93% in the drug combination treatment group, which was highly significant (p = 0.0001), and the proportion of cells in S phase increased by 8.50%, which was highly significant (p = 0.0008).

[0058] For TM4 cells, compared with the control group, the proportion of cells in G1 phase decreased by 11.40% in the uridine-5' monophosphate treatment group, which was highly significant (p = 0.00004), and the proportion of cells in S phase increased by 5.380%, which was highly significant (p = 0.0024). Compared with the control group, the proportion of cells in G1 phase decreased by 10.91% in the drug combination treatment group, which was highly significant (p = 0.00007), and the proportion of cells in S phase increased by 8.367%, which was highly significant (p = 0.0023).

[0059] The above results indicate that uridine-5' monophosphate and its complexes promote the proliferation of TM3 and TM4 cells by facilitating the S phase of TM3 and TM4 cells.

[0060] Previous studies have shown that zinc, selenium, and vitamin E can effectively improve male reproductive capacity. Therefore, these substances can also be combined with uridine-5' monophosphate or its complex in this invention to enhance the proliferation capacity of male germ cells.

Claims

1. The application of a compound of uridine-5' monophosphate and xylanolone in the preparation of a drug that enhances the proliferation of testicular interstitial cells and testicular Sertoli cells, wherein the concentration of uridine-5' monophosphate is 10 μM and the concentration of xylanolone is 100 μM.

2. In the application according to claim 1, the dosage form of the drug is a pharmaceutically acceptable dosage form.

3. The application according to claim 2, wherein the dosage form is tablet, capsule, granule, injection, powder or drops.

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