Application of Siramesine in preparation of medicine for improving sperm function

Abstract

The invention discloses an application of Siramesine in preparation of a medicine for improving a sperm function. In-vitro experiments prove that the Siramecine can improve the sperm function; compared with a control group, Siramesine can induce a sperm intracellular calcium signal to be remarkably increased, enhance the ability of sperms to pass through a female genital tract and promote sperm chemotactic movement. The application of Siramesine can effectively improve the sperm function in the fertilization process, especially the movement ability of sperms, so as to improve the fertilization probability, optimize the assisted reproduction process and improve the fertility outcome of a patient by improving the sperm function.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and specifically relates to the application of Siramesine in preparing a medicine for improving human sperm function. Background Art

[0002] Male infertility, as a common health problem, has affected 10% to 15% of couples of childbearing age worldwide, among which male factors account for half of infertility cases. Sperm function reflects the ability of sperm to complete fertilization under physiological conditions. It is the core criterion for evaluating sperm quality and the key to ensuring the normal fertilization process. It mainly involves a series of complex and orderly behavioral changes, including activation of movement, capacitation, chemotaxis, acrosome reaction and sperm-egg fusion. Abnormalities in any of the above links may lead to abnormalities in the fertilization process. Therefore, these links are crucial for judging sperm function. Overall, sperm dysfunction is one of the main causes of male infertility, with a proportion between 60% and 70%. Sperm dysfunction can lead to adverse consequences such as failed conception, abnormal embryo development and early miscarriage in assisted reproductive cycles.

[0003] Mammalian sperm cells are very different from somatic cells in morphology and structure. The nucleus is highly condensed, most of the cytoplasmic components and organelles are lost, and they are specialized into special cells with flagella and motility. As transcriptionally and translationally inert cells, the realization of sperm functions does not rely mainly on gene expression regulation, but more on post-translational modification of proteins and the regulation of intracellular second messengers such as calcium signals and cAMP. Among these regulatory mechanisms, calcium signals play a vital role. They are the key signals for mature sperm to achieve a variety of physiological functions. The rapid mobilization and changes in intracellular calcium ion concentration can quickly regulate the activity of key enzymes and proteins, thereby triggering corresponding changes in cell function. A large number of studies have confirmed that sperm calcium signals are deeply involved in the regulation of various aspects of sperm function, from sperm maturation, motility, capacitation, superactivation, chemotaxis, to acrosome reaction and sperm-egg fusion, all of which are precisely regulated by calcium signals. Once the calcium ion regulation in sperm is unbalanced, it may lead to sperm dysfunction and then cause male infertility. In addition, calcium signaling, as a universal signaling pathway in cell behavior, also plays an indispensable role in egg activation after fertilization and early embryonic development. Therefore, regulating sperm calcium homeostasis or calcium signaling through drug means is expected to become an effective way to treat infertility caused by sperm dysfunction.

[0004] Low sperm motility is the most common disease in male infertility, clinically known as asthenozoospermia. Even if the egg can be fertilized by chance, poor sperm quality is not only not conducive to the eugenics of offspring, but may also increase the risk of miscarriage or premature birth. Although modern assisted reproductive technologies, such as intracytoplasmic sperm injection and preimplantation genetic testing, have made fertility possible, abnormal sperm function often poses hidden dangers during sperm-egg fusion, embryo transfer and development, affecting clinical outcomes. Therefore, for male infertility patients, the core goal of conventional treatment is to improve sperm motility through a variety of means, in order to achieve natural or artificial intrauterine injection conception, and reduce the potential health risks brought about by the use of other assisted reproductive technologies with excessive human intervention.

[0005] Siramesine is a high-affinity agonist of the sigma-2 receptor, which is 140 times more selective for the sigma-2 receptor than the sigma-1 receptor. Siramesine was originally developed as an anti-anxiety drug, and it was later found that it cleverly triggers the apoptosis program of target cells by interfering with mitochondrial stability rather than the lysosomal pathway. This unconventional cell death induction method has made Siramesine a hot topic in the exploration of anti-cancer research. Siramesine has shown strong killing power against a variety of cancer cell lines, such as HaCaT, Hsc-4, and HeLa. At the same time, its ability to induce apoptosis of refractory tumor cells such as the glioma cell line U-87MG has brought new hope for the treatment of complex diseases such as brain tumors. In addition, Siramesine also has anti-cancer activity in animals. For example, when treating mouse tumors, mice can be given 200 μL 20 mMSiramesine orally or 0.2 mL 20 mM Siramesine can be injected intraperitoneally; when treating rats, rats can be injected intraperitoneally with 1 mL 20 mM Siramesine. In addition, Siramesine has entered the second phase of clinical research and development. Therefore, existing studies have found that it has less toxic side effects on the human body and does not cause ethical problems. Literature and the inventor's research have found that sigma-2 receptors also exist on human sperm, suggesting that they may play an important role in regulating sperm function. Therefore, Siramesine, as an agonist ligand, may have great potential in improving sperm function and is worthy of further development and application. Summary of the invention

[0006] In view of the limited conventional treatment methods for improving sperm function in clinical practice, the present invention uses the agonist Siramesine to intervene in the expression of sigma-2 receptor protein located on human sperm, induce a significant increase in sperm intracellular calcium signals, enhance the ability of sperm to pass through the female reproductive tract, and promote chemotaxis. These functional changes help to increase the fertilization ability of sperm, so as to optimize the assisted reproductive process and improve the patient's reproductive outcomes by improving sperm function in vitro in clinical practice.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions: Application of Siramesine in the preparation of therapeutic drugs for improving sperm function.

[0008] Furthermore, the therapeutic drug can modulate sperm calcium signaling.

[0009] Further, the therapeutic drug can enhance the ability of sperm to pass through the female reproductive tract (climbing ability).

[0010] Furthermore, the therapeutic drug can promote sperm chemotaxis.

[0011] A therapeutic drug for improving sperm function, containing Siramesine.

[0012] Beneficial effects of the present invention: The present invention proves through in vitro experiments that Siramesine can improve sperm function; compared with the control group, Siramesine can induce a significant increase in sperm intracellular calcium signals, enhance the ability of sperm to pass through the female reproductive tract, and promote sperm chemotaxis. The application of Siramesine can effectively improve sperm function during in vitro fertilization, so as to optimize the assisted reproductive process and improve the patient's reproductive outcome by improving sperm function. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 The experimental results of Siramesine on sperm calcium signal activation in Example 1.

[0014] Figure 2 The experimental results of Example 2 show that Siramesine improves the climbing ability of sperm.

[0015] Figure 3 The experimental results of Siramesine promoting sperm chemotaxis in Example 3 are shown in FIG.

[0016] Figure 4 The experimental results in Example 3 verify that Siramesine promotes sperm chemotaxis. DETAILED DESCRIPTION

[0017] The preferred embodiments of the present invention will be described in detail below in conjunction with the examples. It should be understood that the following examples are provided only for the purpose of illustration and are not intended to limit the scope of the present invention. Those skilled in the art may make various modifications and substitutions to the present invention without departing from the purpose and spirit of the present invention.

[0018] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0019] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0020] In the present invention, the HS buffer formula used is: 135 mM NaCl, 5 mM KCl, 1 mM MgSO4, 2 mM CaCl2, 20 mM HEPES, 5 mM sucrose, 10 mM lactic acid, 1 mM sodium pyruvate, and the pH is adjusted to 7.4 using NaOH; the HTF energy acquisition solution formula is: 93.8 mM NaCl, 4.69 mM KCl, 0.2 mM MgSO4, 0.37 mM KH2PO4, 2.04 mM CaCl2, 21.4 mM lactic acid, 2.78 mM glucose, 21 mM HEPES, 4 mM NaHCO3, the pH is adjusted to 7.4, and 330 mM Na-Pyruvate (1000:1), HSA (25:1), and 21 mM NaHCO3 are added before use. Example 1

[0021] Detection of intracellular calcium signaling in sperm Sperm from volunteers with normal parameters were placed in a 37°C, 5% CO2 incubator for liquefaction. The semen was washed twice with 37°C preheated HS buffer to remove seminal plasma, and the sperm pellet was resuspended in HS buffer. 5 μM calcium ion fluorescent probe Fluo-4 AM and 0.05% (m / v) surfactant Pluronic F-127 were added to the resuspended sperm suspension to detect sperm intracellular calcium signals. Sperm was stained in a 37°C, 5% CO2 incubator in the dark for 30 min. Centrifuged at 1800 rpm for 6 min, resuspended in HS buffer, and this step was repeated twice. The sperm staining was observed under a fluorescence microscope. Flexstation3 was used to detect the changes in sperm intracellular calcium signals under the conditions of 488 nm excitation wavelength and 525 nm emission wavelength. HS buffer was used as negative control and progesterone P4 was used as positive control. 10 μL of Siramesine of different concentrations (1 μM, 10 μM, 25 μM, 50 μM, 100 μM) was added to 90 μL of fluorescently stained sperm through an automatic loading program.

[0022] like Figure 1 As shown in the figure, Siramesine can significantly induce an increase in sperm intracellular calcium signals. Siramesine can significantly increase the sperm intracellular calcium ion concentration at 10 μM, and within a concentration of 100 μM, the ability to activate calcium signals is concentration-dependent. At 50 μM, the induced first phase (secondary) calcium signal basically reaches saturation. Example 2

[0023] Climbing test to detect the ability of sperm to penetrate viscous media Sperm samples from volunteers with normal parameters were obtained after swimming to obtain sperm suspension with good vitality and no impurities. Centrifuge at 1800 rpm for 6 min, discard the supernatant, and resuspend in HTF capacitation solution. After capacitation in an incubator at 37℃ and 5% CO2 for 3 h, the sperm suspension was divided into 3 groups and incubated for 1 h after adding different drugs. Although Siramesine can significantly induce an increase in sperm intracellular calcium signal at 100 μM, its concentration can damage sperm cell vitality, so the Siramesine concentration adopted in the sperm function experiment is 50 μM. After the incubation, a capillary glass tube filled with 1% methylcellulose was inserted into the sperm suspension with different drugs added, and the sperm was allowed to climb in the incubator for 1 h. Finally, the number of sperm at 2 cm of the capillary glass tube was counted. Regarding the processing of the climbing ability results: the negative control HTF was used as the baseline value to calculate the climbing ability of sperm to the drug Siramesine and the positive control progesterone P4.

[0024] Figure 2It can be seen that Siramesine at a concentration of 50 μM significantly improves the climbing ability of sperm. Example 3

[0025] Chemotaxis assay to detect the ability of sperm to penetrate the medium along the concentration gradient Sperm samples from volunteers with normal parameters were obtained after swimming to obtain sperm suspension with good vitality and no impurities. Centrifuge at 1800 rpm for 6 min, discard the supernatant, and resuspend in HTF capacitation solution. After capacitation in an incubator at 37℃ and 5% CO2 for 2 h, the sperm suspension was divided into 2 groups, and capillary glass tubes filled with 10 μl HTF and 50 μM Siramesine prepared in advance were inserted into the sperm suspension, and the sperm were chemotactic in the incubator for 40 min. Finally, the number of sperm at 2 cm from the capillary glass tube was counted.

[0026] Figure 3 It shows that sperm tends to move towards 50 μM Siramesine. To further verify the promoting effect of Siramesine on sperm chemotaxis, another method was used to prove the preference of human sperm for Siramesine. Methods: Sperm samples with normal parameters from volunteers were obtained after swimming to obtain sperm suspension with good vitality and no impurities. Centrifuge at 1800 rpm for 6 minutes, discard the supernatant, and resuspend in HTF capacitation solution. After capacitation for 2 hours in a 37℃, 5% CO2 incubator, the sperm suspension was divided into 3 groups, 500 μL sperm was added to the bottom of the chemotaxis lower device, 60 μL of different drugs were added to the upper device, incubated for 40 minutes, and 10 μL sperm at the bottom of the upper device was taken to count under the sperm counting plate. Regarding the processing of chemotaxis results: Taking the negative control HTF as the benchmark value, the chemotactic effect of sperm on the drug Siramesine and the positive control Bourgeonal was calculated.

[0027] Figure 3 , Figure 4 The results together demonstrated that 50 μM Siramesine can promote sperm chemotaxis.

[0028] The above results show that Siramesine has the effect of significantly improving sperm climbing ability and promoting chemotaxis at 50 μM, and is expected to be used in the treatment of patients with sperm dysfunction in the future.

[0029] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention, which should all be included in the scope of the claims of the present invention.

Claims

1. Application of Siramesine in the preparation of therapeutic drugs for improving sperm function.

2. The use according to claim 1, characterized in that: The therapeutic agent can modulate sperm calcium signaling.

3. The use according to claim 1, characterized in that: The therapeutic drug can improve the sperm's ability to climb high.

4. The use according to claim 1, characterized in that: The therapeutic drug can enhance sperm chemotaxis.

5. A therapeutic drug for improving sperm function, characterized in that: Siramesine or a modified derivative drug thereof as an active ingredient is used as a main active ingredient for improving sperm function.

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