Uridine-5 '-monophosphate and application of compound of uridine-5'-monophosphate in preparation of medicine for enhancing proliferation capacity of male germ cells

Abstract

The invention belongs to the technical field of medicines, and discloses uridine-5 '-monophosphate and application of a compound of uridine-5'-monophosphate in preparation of a medicine for enhancing the proliferation capacity of male germ cells. It is found for the first time that uridine-5 '-monophosphate has multiplication capacity on male germ cells, when the concentration of uridine-5'-monophosphate is 10 [mu] M, the multiplication effect is optimal, and compared with a control group, the multiplication effect on mouse TM3 cells is increased by 52.87%, and the multiplication effect on mouse TM4 cells is increased by 53.57%; and after being compounded with xylonic acid lactone, the compound also has a synergistic capability, so that the proliferation effect on TM3 cells is further improved, and a foundation is laid for treating male reproductive system diseases in the future.

Description

Technical Field

[0001] The invention belongs to the field of medical technology, and particularly relates to the use of uridine-5' monophosphate and its compound in the preparation of a drug for enhancing the proliferation ability of male germ cells. Background Art

[0002] In recent years, a great deal of research has been conducted globally on male infertility, mainly due to the increase in public awareness caused by the decline in semen quality in healthy men worldwide (AGARWAL et al., 2020). Infertility is a major public health issue and health problem with enormous social, psychological and economic impacts (SUGANTHI et al., 2014). Today, advances in the study of the molecular and cellular mechanisms of spermatogenesis have helped to define the characteristics of many diseases that were previously considered idiopathic. The most important of these are hypogonadotropic hypogonadism, androgen receptor mutations, cystic fibrosis transmembrane conductance regulator gene mutations, genetic polymorphisms, and Y chromosome-linked infertility.

[0003] Selenium and zinc are both essential trace elements for the human body and play an important role in removing reactive oxygen species. Compared with the normal semen group, the selenium concentration in the serum and seminal plasma of the abnormal semen group decreased by more than 20%, and the addition of selenium as a treatment to the conventional treatment of oligospermia patients significantly increased sperm motility and the number of sperm with normal morphology compared with the conventional treatment group (Zhang Fengfeng et al., 2020); compared with non-smokers, the zinc level in the seminal plasma of smokers tended to decrease. Zinc in fertile and infertile (smokers or non-smokers) men is significantly correlated with sperm count and normal sperm morphology. These studies have shown that the lack of nutrients such as selenium and zinc may lead to poor sperm quality and the occurrence of idiopathic male infertility (COLAGAR et al., 2008). Icariin (ICA) is the main component of flavonoids from the traditional Chinese medicine Epimedium brevicornum Maxim. It has a protective effect on male reproductive ability. Studies have shown that icariin promotes the proliferation of supporting cells in vitro by activating the ERK1 / 2 signaling pathway (NAN et al., 2014; Zhang Jingyi et al., 2024). Taurine has a potential protective effect on the reproductive function of male animals. Taurine can delay the aging of testicular structure and function, maintain the homeostasis of the testicular environment, and enhance sexual ability (LI et al., 2023). Studies have shown that coenzyme Q10, kallikrein and pentoxifylline can improve semen parameters (Peng Jing, 2023; OMAR et al., 2019). Feng Jianhao et al.'s study showed that Dendrobium officinale extract can significantly increase the proliferation activity of mouse TM3 cells (Feng Jianhao et al., 2024); Lu Feng et al.'s study showed that retinol can promote the differentiation of cryptorchid spermatogenic cells by downregulating the expression level of the miR-210 gene (Lu Feng et al., 2022). Therefore, idiopathic infertility can be treated through hormone drugs and nutritional supplements (N et al., 2021).

[0004] Uridine-5' monophosphate is the precursor of uridine, a pyrimidine nucleoside that participates in the synthesis of RNA, biological membranes and glycogen, and plays an important role in cell growth and metabolism (Bai Xueyi et al., 2023). In addition to playing an indispensable role in the biosynthesis of RNA and DNA, uridine is also involved in the deposition of glycogen and plays an important regulatory role in the glycosylation of proteins and lipids. In addition, uridine is also involved in the biosynthesis of the extracellular matrix, which is essential for maintaining the stability of the extracellular environment; uridine also plays a key role in the detoxification of xenobiotics, helping to remove harmful substances from the body. Therefore, uridine plays multiple roles in the body and is of great significance for maintaining the normal progress of life activities (DENG et al., 2017).

[0005] There is no report on the use of uridine-5' monophosphate (Cas: 58-97-9) to enhance the proliferation capacity of male germ cells. Summary of the invention

[0006] The purpose of the present invention is to provide the use of uridine-5' monophosphate in the preparation of a drug for enhancing the proliferation ability 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 drug for enhancing the proliferation ability of male germ cells.

[0008] In order to achieve the above object, the present invention adopts the following technical measures:

[0009] The protection scope of the present invention includes:

[0010] Application of uridine-5' monophosphate in the preparation of drugs for enhancing the proliferation ability of male germ cells.

[0011] Application of a compound containing uridine-5' monophosphate in the preparation of a drug for enhancing the proliferation ability of male germ cells.

[0012] In the above application, the compound further contains xylonolactone, zinc, selenium and / or vitamin E as main active ingredients. In the above application, preferably, the germ cells are testicular interstitial cells or testicular Sertoli cells;

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

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

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

[0016] In the above-mentioned application, the dosage form of the drug 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] The present application discovered for the first time that uridine-5' monophosphate has the ability to proliferate male germ cells. The proliferation effect is best 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) increased by 52.87%, and the proliferation effect on mouse TM4 cells (normal mouse testicular Sertoli cells) increased by 53.57%; and after it is compounded with xylonolactone, it also has synergistic ability, which further enhances the proliferation effect on TM3 cells, laying a foundation for the future treatment of male reproductive system diseases. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0020] Figure 2 The present invention relates to the effects of uridine-5' monophosphate, uridine-5' monophosphate and their complexes on the proliferation of mouse testicular cells. DETAILED DESCRIPTION

[0021] The technical solutions described in the present invention, unless otherwise specified, are all conventional solutions in the art; the reagents or materials described, unless otherwise specified, are all from commercial channels.

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

[0023] The xylonic acid lactones described in the present invention are all D-xylonic acid lactone (Cas: 18423-66-0).

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

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

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

[0027] The present invention uses Prism 9.0 to perform data analysis on the cell experiment results, and expresses them as mean ± standard deviation (±s). The means of multiple groups are analyzed by one-way analysis of variance, and the comparison between two groups is performed by independent sample t test, and P < 0.05 indicates that the difference is 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 inoculated into cell culture plates (2.5×10 5 cells / well; 96-well culture plate 1×10 4 cells / well), and add 100 μL of uridine-5' monophosphate solution of different concentrations (1000 μM, 100 μm, 10 μm, 1 μm, 0.1 μm, 0.01 μm) according to the experimental requirements. After culturing for 24 hours, CCK-8 reagent was used to detect the cell survival rate.

[0031] (2) Control group: cells (TM3 or TM4) were inoculated into cell culture plates (2.5×10 5 cells / well;), and added 100 μL of DEME / F12 serum-free medium, and continued to culture for 24 hours together with the experimental group. CCK-8 reagent was also used to detect cell viability and quantity.

[0032] (3) Blank group: In order to obtain the blank value of the experiment, 6 wells were set up on the same 96-well plate without inoculating 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 culture medium was added to these blank wells and cultured for another 24 hours. These blank wells will be used for subsequent tests to obtain the blank value of the experiment.

[0033] CCK-8 method to detect cell viability: After the treatment time is reached, take out the culture plate, discard the original culture medium or drug solution, add 100 μL of newly prepared culture solution containing 10% CCK-8, and return to the incubator to continue culturing for 30 minutes. Take out the culture plate and detect the absorbance value at 450nm and 650nm on the 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] Embodiment 2:

[0036] Application of a compound of uridine-5' monophosphate and xylonolactone in the preparation of a drug for enhancing the proliferation ability of male germ cells:

[0037] In this example, the synergistic effect between the two active ingredients, xylonolactone and uridine-5' monophosphate, was investigated.

[0038] 100 μM xylonolactone and 10 μM uridine-5' monophosphate were mixed at a volume ratio of 1:1 as 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 inoculated into cell culture plates (2.5×10 5 cells / well; 96-well culture plate 1×10 4 cells / well), and add 100 μL of the test agent according to the experimental requirements. After culturing for 24 hours, the CCK-8 reagent was used to detect the cell survival rate.

[0041] (2) Control group: cells (TM3 or TM4) were inoculated into cell culture plates (2.5×10 5 cells / well;), and added 100 μL of DEME / F12 serum-free medium, and continued to culture for 24 hours together with the experimental group. CCK-8 reagent was also used to detect cell viability and quantity.

[0042] (3) Blank group: In order to obtain the blank value of the experiment, 6 wells were set up on the same 96-well plate without inoculating 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 culture medium was added to these blank wells and cultured for another 24 hours. These blank wells will be used for subsequent tests to obtain the blank value of the experiment.

[0043] CCK-8 method to detect cell viability: After the treatment time is reached, take out the culture plate, discard the original culture medium or drug solution, add 100 μL of newly prepared culture solution containing 10% CCK-8, and return to the incubator to continue culturing for 30 minutes. Take out the culture plate and detect the absorbance value at 450nm and 650nm on the microplate reader.

[0044] The results are as follows Figure 2As shown: the complex has a proliferation effect on mouse TM3 cells 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 xylonolactone 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 xylonolactone 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 xylonolactone 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 xylonolactone 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 the combination of xylonolactone and uridine-5' monophosphate can promote the efficacy of uridine-5' monophosphate. After the combination, it significantly improves the proliferation effect of mouse TM3 cells and has a synergistic effect.

[0048] Embodiment 3:

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

[0050] In this example, flow cytometry was used to identify the cell status after incubation in Example 1, and the steps were as follows:

[0051] (1) When the treatment time is reached, carefully remove the original culture medium or drug solution, and gently rinse the cells twice with PBS buffer to remove residues. Then, add 500 μL of 0.25% trypsin to digest the cells. After the cell digestion is completed, quickly add 1 mL of TM4 medium to terminate the digestion process, and carefully blow those cells that have not completely fallen off to ensure that they are completely away from the wall. Collect the cells into a 15 mL centrifuge tube and place it in a centrifuge. Centrifuge at 300xg for 5 minutes. After centrifugation, discard the culture medium in the centrifuge tube and resuspend the cells with 1 mL of PBS buffer. After that, 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℃ anhydrous ethanol, mix thoroughly, and place the cell suspension in a -20℃ refrigerator to fix for 1 hour or overnight;

[0052] (2) After fixation, place the centrifuge tube back into the centrifuge and centrifuge at 300 x g for 5 min. Then, discard the supernatant, add 1 mL of PBS buffer to resuspend the cells, and let stand at room temperature for 15 min.

[0053] (3) Place the centrifuge tube in the centrifuge again and centrifuge at 300 x g for 5 min. After centrifugation, discard the supernatant and add 100 μL of RNase A reagent to fully suspend the cells, then incubate them in a 37°C water bath for 30 min.

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

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

[0056] (6) Analyze using Flowjo software to obtain cell cycle distribution.

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

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

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

[0060] Previous studies have shown that zinc, selenium, and VE can effectively improve male reproductive capacity, so these substances can also be combined with the uridine-5' monophosphate or its complex of the present invention for administration to improve the proliferation capacity of male germ cells.

Claims

1. Application of uridine-5' monophosphate in the preparation of drugs for enhancing the proliferation ability of male germ cells.

2. Use of a compound containing uridine-5' monophosphate in the preparation of a drug for enhancing the proliferation ability of male germ cells.

3. The use according to claim 2, wherein the compound further contains xylonolactone, zinc, selenium and / or vitamin E as main active ingredients.

4. The use according to claim 1 or 2, wherein the germ cells are testicular Leydig cells or testicular Sertoli cells.

5. The use according to claim 1 or 2, wherein the effective concentration of uridine-5' monophosphate is 0.01 μM to 1000 μM.

6. The use according to claim 1 or 2, wherein the effective concentration of uridine-5' monophosphate is 0.1 μM to 100 μM.

7. The use according to claim 1 or 2, wherein the effective concentration of uridine-5' monophosphate is 1 μM to 10 μM.

8. The use according to claim 1 or 2, wherein the dosage form of the drug is a pharmaceutically acceptable dosage form.

9. The use according to claim 8, wherein the dosage form is tablets, capsules, granules, injections, powders or drops.

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