Application of salvianolic acid A in preparation of medicine for improving sperm function

Abstract

The invention discloses application of salvianolic acid A in preparation of a medicine for improving the sperm function, and belongs to the technical field of biological medicine. SAA is polyphenol acid formed by caffeic acid and tanshinol, namely salvianolic acid through an ester bond and an enol bond, is a soluble component generated by many salvia species, is mainly extracted from salvia miltiorrhiza, and has the advantages that the salvianolic acid A is prepared from salvianolic acid A, salvianolic acid B and salvianolic acid C; sAA is one of the most abundant salvianolic acid in a salvia miltiorrhiza extract, a large number of studies show that SAA has a remarkable antioxidant effect, SAA can improve the function of in-vitro mature sperms, compared with a control group, after SAA is added, sperm intracellular calcium signals and hyperactivity are remarkably improved, and the occurrence rate of sperm acrosome reaction is also remarkably increased; the application of SAA can effectively improve the sperm function in the in-vitro fertilization process and improve the sperm motility of weak sperm patients, so that infertility is intervened and treated through simple non-invasive means such as artificial fertilization, and the fertility outcome of the patients is improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to an application of salvianolic acid A in the preparation of a medicine for improving sperm function. Background Art

[0002] Male infertility, as a global health challenge, has affected many couples of childbearing age, with a high proportion of male factors. This disease not only causes physical and mental burdens on individuals, but also has a profound impact on the social demographic structure. Sperm function, as a core indicator of male fertility, directly determines the success rate of fertilization. Its integrity depends on the synergistic effect of a series of precise physiological processes, including passage through the female reproductive tract, response to environmental signals (such as pH gradients, chemokines), capacitation, hyperactivated movement, acrosome reaction, and sperm-egg membrane fusion. Studies have shown that many cases of male infertility are directly related to sperm dysfunction. Such abnormalities not only lead to difficulties in natural conception, but also significantly increase the risk of fertilization failure, embryonic development stagnation, and early miscarriage in assisted reproductive technology (ART) cycles. It is worth noting that even if fertilization is achieved through techniques such as intracytoplasmic sperm injection (ICSI), sperm function defects may still affect embryo quality and the long-term health of offspring through mechanisms such as abnormal epigenetic modifications.

[0003] As a terminally differentiated cell with extremely low transcription and translation activity, sperm functions are highly dependent on post-translational modification and second messenger system regulation rather than gene transcription activity. Calcium signaling, as a key regulatory hub, regulates the activity of effector molecules such as protein kinases and phosphatases through dynamic concentration gradient changes, thereby precisely controlling key physiological events such as sperm maturation, capacitation, chemotaxis, acrosomal exocytosis, and sperm-oocyte fusion. Existing studies have confirmed that an imbalance in calcium homeostasis in sperm can directly lead to abnormal hyperactivation patterns, impaired acrosomal enzyme release, and fusion protein dysfunction, ultimately leading to fertilization failure. In addition, the calcium oscillation signal transmitted by sperm has a transgenerational regulatory effect on oocyte activation and early embryonic development, further highlighting its biological importance.

[0004] Clinically, low sperm motility is one of the main phenotypes of male infertility. Even if such patients have offspring through human assisted reproductive technologies such as intracytoplasmic sperm injection and preimplantation genetic testing, abnormal sperm function often leads to challenges such as sperm-egg fertilization, embryo transfer and development failure, decreased embryo implantation rate, increased miscarriage rate and increased epigenetic risks for offspring. Although current mainstream assisted reproductive technologies can partially solve fertilization barriers, they are subject to problems such as large surgical trauma, high costs and unknown long-term health risks for offspring. Therefore, developing new intervention strategies based on the regulation of sperm function, which can improve the intrinsic quality of sperm to enable patients to conceive naturally or through low-invasive artificial insemination, has a dual significance for improving the health level of the population: on the one hand, it can reduce the clinical dependence on human assisted reproductive technologies, and on the other hand, it can help to protect the developmental potential of embryos from the source.

[0005] Salvianolic acid A (SAA) is a natural, water-soluble phenolic acid active ingredient extracted from the root of the traditional Chinese medicinal plant Salvia miltiorrhiza. SAA is one of the most potent antioxidant compounds among the active components of Salvia miltiorrhiza and exhibits a wide range of pharmacological actions, including cardiovascular protection, anti-inflammatory, antioxidant, antiviral, and anticancer properties. SAA can activate the Nrf2 / ARE pathway and the GSK3β / Nrf2 / HO-1 signaling pathway to exert antioxidant and anti-inflammatory effects. With its unique antioxidant-anti-inflammatory synergistic effects, SAA may exhibit advantages in sperm oxidative damage repair, inflammatory microenvironment regulation, and functional molecular target intervention. Therefore, SAA has great potential for improving sperm function. Summary of the Invention

[0006] Technical issues solved:

[0007] In response to the shortcomings of the existing technology, such as the limited conventional treatment methods for improving sperm function in clinical practice, this application provides an application of salvianolic acid A in the preparation of a drug for improving sperm function. The natural product SAA is used to intervene in sperm in vitro to improve sperm function, thereby intervening and treating infertile patients through simple non-invasive means such as artificial insemination, thereby improving the patients' reproductive outcomes.

[0008] Technical solution:

[0009] To achieve the above objectives, this application is implemented through the following technical solutions:

[0010] The invention discloses an application of salvianolic acid A in the preparation of a medicine for improving sperm function.

[0011] Furthermore, the drug for improving sperm function is a drug for non-disease diagnosis and treatment purposes that regulates sperm calcium signals.

[0012] Furthermore, the drug for improving sperm function is a drug for non-disease diagnosis and treatment purposes that enhances sperm superactivation ability.

[0013] Furthermore, the drug for improving sperm function is a drug for non-disease diagnosis and treatment purposes that improves the sperm acrosome reaction ability.

[0014] Furthermore, the drug for improving sperm function is a drug for non-disease diagnosis and treatment purposes, which is used to improve sperm motility in patients with asthenospermia.

[0015] Furthermore, the medicine includes salvianolic acid A and pharmaceutically acceptable excipients.

[0016] Furthermore, the auxiliary material is one or more of a diluent, an excipient, a filler, a binder, a wetting agent, a disintegrant, an absorption promoter, a surfactant, an adsorption carrier, and a lubricant.

[0017] Furthermore, the medicine is an oral or parenteral preparation.

[0018] Furthermore, the medicine is in the form of tablets, capsules, powders, pills, granules, solutions, suspensions, syrups, injections, suppositories, adsorbents or sprays.

[0019] Beneficial effects:

[0020] The present application provides a use of salvianolic acid A in the preparation of a medicament for improving sperm function, which has the following beneficial effects compared with the prior art:

[0021] 1. This invention utilizes SAA, a natural product, to intervene in sperm in vitro, improve sperm function, and enhance sperm motility in patients with asthenospermia. This approach aims to intervene and treat infertility through simple, non-invasive means such as artificial insemination, thereby improving patients' reproductive outcomes.

[0022] 2. This study demonstrates that SAA can activate the human sperm CatSper channel using patch clamp techniques. Compared to existing screening methods for drugs that improve sperm function, this study utilizes high-resolution patch clamp technology (whole-cell recording mode) for the first time, directly demonstrating at the single-cell level that a candidate molecule derived from a natural product can effectively and specifically activate the human sperm CatSper channel.

[0023] 3. Compared with existing synthetic compounds or natural extracts with unknown mechanisms of action, SAA in the present invention exhibits significant advantages in drug safety and target specificity, providing irreplaceable candidate molecules and research methods for the development of sperm function-improving drugs with clear mechanisms, high efficiency and safety. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The results of the present invention SAA regulation of sperm calcium signal, wherein a is different concentrations of SAA induced sperm [Ca 2 + ] i Add a schematic diagram, b is the sperm [Ca 2+ ] i Increased peak statistics, c is the EC of SAA 50 Fitting curves;

[0025] Figure 2The figure below shows the results of the application of SAA increasing sperm CatSper current. The left figure is a schematic diagram of SAA activating CatSper channels, and the right figure is a statistical analysis of the average CatSper current at -80mV and +80mV after adding SAA.

[0026] Figure 3 The figure below shows the results of the present application's SAA improving sperm superactivation ability, where HTF is the capacitation fluid. The left figure shows the sperm count at 2 cm from the capillary inlet, which is normalized to the sperm count of the control group. The right figure shows the sperm count at 3 cm from the capillary inlet, which is normalized to the sperm count of the control group.

[0027] Figure 4 This is a diagram showing the ability of the present SAA to enhance the occurrence of sperm acrosome reaction;

[0028] Figure 5 This is a graph showing the ability of SAA in this application to improve sperm motility in patients with asthenospermia. The left graph shows the effect of SAA on sperm motility in patients with asthenospermia, and the right graph shows the effect of SAA on the forward motility of sperm in patients with asthenospermia. DETAILED DESCRIPTION

[0029] The present invention will be further described in detail below in conjunction with the accompanying drawings and specific examples, but should not be construed as limiting the present invention. Without departing from the spirit and substance of the present invention, modifications or substitutions made to the inventive method, steps or conditions are intended to fall within the scope of the present invention. Experimental methods and reagents for which specific conditions are not specified in the examples are based on conventional conditions in the art.

[0030] The HS buffer used in the present invention has a formula of: 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.

[0031] The formula of HTF capacitation solution is: 93.8mM NaCl, 4.69mM KCl, 0.2mM MgSO4, 0.37mM KH2PO4, 2.04mM CaCl2, 21.4mM lactic acid, 2.78mM glucose, 21mM HEPES, 4mM NaHCO3, pH is adjusted to 7.4, and 330mM Na-Pyruvate (1000:1), HSA (25:1), and 21mM NaHCO3 are added before use.

[0032] Example 1

[0033] A use of salvianolic acid A in the preparation of a drug for improving sperm function, wherein the drug for improving sperm function is a drug for non-disease diagnosis and treatment purposes, which regulates sperm calcium signals. The specific steps for high-throughput detection of sperm intracellular calcium signals are as follows:

[0034] Step 1: Sperm from a volunteer bank with normal parameters is placed in a 37°C, 5% CO2 incubator to complete liquefaction. The semen is washed twice with 37°C preheated HS buffer to remove seminal plasma, and the sperm pellet is resuspended in HS buffer.

[0035] Step 2: Add 5 μM of the calcium ion fluorescent probe Fluo-4 AM and 0.05% of the surfactant Pluronic F-127 to the resuspended sperm suspension to detect the intracellular calcium signal of sperm;

[0036] Step 3: Sperm is stained in a 37°C, 5% CO2 incubator in the dark for half an hour;

[0037] Step 4: Centrifuge at 1800 rpm for 6 min, resuspend in HS buffer, repeat this step twice, and observe sperm staining under a fluorescence microscope;

[0038] Step 5: Use Flexstation3 to detect changes in sperm intracellular calcium signals under the conditions of 488nm excitation wavelength / 525nm emission wavelength, where HS buffer is used as a negative control and progesterone P4 is used as a positive control. 10μL of SAA at different concentrations is added to 90μL of fluorescently stained sperm through an automatic loading program.

[0039] Figure 1 The results of the present invention SAA regulation of sperm calcium signal, wherein a is different concentrations of SAA induced sperm [Ca 2 + ] i Add a schematic diagram, b is the sperm [Ca 2+ ] i The increased peak value statistics indicated that SAA induced sperm [Ca 2+ ] i Increase, c is the EC of SAA 50 Fit the curve to obtain the EC of SAA 50 =10.32μM; Figure 1 As shown in the results, SAA can significantly induce an increase in sperm intracellular calcium signals. SAA can significantly increase the sperm intracellular calcium concentration at 5 μM, and the effect increases with increasing concentration, reaching saturation at 40 μM.

[0040] Example 2

[0041] A use of salvianolic acid A in the preparation of a drug for improving sperm function, wherein the drug for improving sperm function is a drug for non-disease diagnosis and treatment purposes, which enhances sperm superactivation ability. The specific steps of detecting sperm CatSper current by patch clamp experiment are as follows:

[0042] Step 1: Take 0.5-1ml normal human sperm sample, add HS solution to 1.5ml, centrifuge twice at 1800rpm for 6min, remove the supernatant containing seminal plasma, and add 1ml HS to resuspend the sperm;

[0043] Step 2: Take 40μL of sperm sample and drop it into the dish. At the same time, add 400μL of HS and let it adhere to the wall for 10-20 minutes. After the adhesion is completed, add 1ml of HS to wash twice, and finally add 3ml of HS to start the experiment.

[0044] Step 3: Prepare the glass electrode and add an appropriate amount of electrode liquid. The electrode water resistance is about 20-30MΩ. Keep the electrode tip clean and gently absorb the droplet on the sperm neck or under the head to form a high-resistance seal with a resistance of about 5GΩ.

[0045] Step 4: After compensating the electrode capacitance, a voltage stimulus is applied to the sperm for 0.5 seconds, slowly increasing the voltage from 450mV to 650mV in 50mV increments. Simultaneously, a slight negative suction is applied to break the cell membrane, establishing whole-cell recording mode.

[0046] Step 5: Compensate for series resistance as much as possible and record CatSper currents induced by HS, DVF, and SAA by voltage clamp.

[0047] Step 6: All currents recorded by patch clamp were obtained from the HEKA amplifier; the effects of different drugs on sperm were achieved through a gravity perfusion system; and all recorded patch clamp data were exported using Clampfit 10.4 software.

[0048] Figure 2 The figure below shows the result of SAA increasing sperm CatSper current. The left figure is a schematic diagram of SAA activating CatSper channels, and the right figure is a statistical analysis of the average CatSper current at -80mV and +80mV after adding SAA. Figure 2 As shown in the results, SAA can significantly activate CatSper channels and induce an increase in CatSper current.

[0049] Example 3

[0050] A use of salvianolic acid A in the preparation of a drug for improving sperm function, wherein the drug for improving sperm function is a drug for non-disease diagnosis and treatment purposes, which enhances sperm hyperactivation ability. The specific steps of a climbing test for detecting the ability of sperm to penetrate a viscous medium are as follows:

[0051] Step 1: Sperm samples with normal parameters from the volunteer bank are passed through the upstream process to obtain sperm suspension with good motility and no impurities;

[0052] Step 2: Centrifuge at 1800 rpm for 6 min, discard the supernatant, and resuspend in HTF capacitation solution;

[0053] Step 3: After sperm capacitation in a 37°C, 5% CO2 incubator for 3 hours, the sperm suspension was divided into three groups and incubated for another 1 hour after adding P4 or SAA. Although SAA significantly induced an increase in sperm intracellular calcium signaling at 40 μM, its concentration far exceeded the normal blood concentration. Therefore, the SAA concentration adopted in this application for the sperm function experiment was 20 μM.

[0054] Step 4: After the incubation is completed, a capillary glass tube filled with 1% methylcellulose is inserted into the sperm suspension with different drugs added, and the sperm is allowed to climb in the incubator for 1 hour. Finally, the number of sperm at 2 and 3 cm in the capillary glass tube is counted.

[0055] Figure 3 The figure below shows the results of the present application SAA improving the super-activation ability of sperm, wherein the HTF capacitation solution, the left figure shows the number of sperm at 2 cm at the capillary inlet, and is normalized based on the number of sperm in the control group; the right figure shows the number of sperm at 3 cm at the capillary inlet, and is normalized based on the number of sperm in the control group; Figure 3 As shown, SAA at 20 μM can significantly enhance the superactivation ability of sperm at 2 cm and 3 cm, and P4 is a positive control.

[0056] Example 4

[0057] A use of salvianolic acid A in the preparation of a drug for improving sperm function, wherein the drug for improving sperm function is a drug for non-disease diagnosis and treatment purposes, wherein peanut agglutinin staining (PNA) is used to examine the effect of SAA on the acrosome reaction of human sperm, and the specific steps are:

[0058] Step 1: Take 1 mL of liquefied semen and perform swim-up purification. After 1 hour of swim-up, sperm with good motility are obtained.

[0059] Step 2: Centrifuge at 1800 rpm for 6 min, discard the supernatant, and resuspend in HTF capacitation solution;

[0060] Step 3: After the sperm were capacitated in a 37°C, 5% CO2 incubator for 3 hours, the sperm suspension was divided into 4 groups and incubated for 1 hour after adding P4, SAA, or both.

[0061] Step 4: Take 40 μL of resuspended sperm suspension, add 40 μL of 25 μg / ml PNA staining solution, stain for 30 minutes, and then add 10 μL of DNA fluorescent dye DAPI for fixation;

[0062] Step 5: Take photos and count them under an upright fluorescence microscope. At least 200 sperms should be counted in each experimental group to determine the acrosome reaction of the sperm.

[0063] Figure 4 This is a diagram showing the ability of the present invention's SAA to enhance the occurrence of sperm acrosome reaction. Figure 4 As shown in the results, SAA at 20 μM can increase the acrosome reaction rate of sperm, and the combined action of 20 μM SAA and 10 μM progesterone can also increase the acrosome reaction rate of sperm.

[0064] Example 5

[0065] A use of salvianolic acid A in the preparation of a drug for improving sperm function, wherein the drug for improving sperm function is a drug for non-disease diagnosis and treatment purposes, for improving sperm motility in patients with asthenospermia. The CASA system detects sperm motility and forward motility, and the specific steps are:

[0066] Step 1: Sperm samples with abnormal parameters from the volunteer pool, with sperm motility <40% and progressive motility <32%, are washed and separated to obtain a sperm suspension free of impurities;

[0067] Step 2: Centrifuge at 1800 rpm for 6 min, discard the supernatant, and resuspend in HTF capacitation solution;

[0068] Step 3: After the sperm were capacitated in a 37°C, 5% CO2 incubator for 3 hours, the sperm suspension was divided into two groups and incubated for another 1 hour after adding SAA;

[0069] Step 4: CASA detects sperm motility parameters, mainly recording Motility and Progressive motility.

[0070] Figure 5 This is the ability diagram of SAA in this application to improve sperm motility in patients with asthenozoospermia. The left figure is the effect of SAA on sperm motility in patients with asthenozoospermia, and the right figure is the effect of SAA on the forward motility of sperm in patients with asthenozoospermia. Figure 5 As shown in the results, SAA at 20 μM can increase the sperm motility and the percentage of progressively motile sperm in asthenospermic sperm.

[0071] The above results indicate that SAA at 20 μM has the effect of significantly improving the function of asthenozoospermia and is expected to be used in the treatment of patients with sperm dysfunction in the future.

[0072] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the present invention. 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. Use of salvianolic acid A in the preparation of a drug for improving sperm function.

2. The use of salvianolic acid A in the preparation of a medicament for improving sperm function according to claim 1, characterized in that: The drug for improving sperm function is a drug for non-disease diagnosis and treatment purposes, which regulates sperm calcium signals.

3. The use of salvianolic acid A in the preparation of a medicament for improving sperm function according to claim 1, characterized in that: The drug for improving sperm function is a drug for non-disease diagnosis and treatment purposes, which is used to enhance sperm superactivation ability.

4. The use of salvianolic acid A in the preparation of a medicament for improving sperm function according to claim 1, characterized in that: The drug for improving sperm function is a drug for non-disease diagnosis and treatment purposes, which improves the sperm acrosome reaction ability.

5. The use of salvianolic acid A in the preparation of a medicament for improving sperm function according to claim 1, characterized in that: The drug for improving sperm function is a drug for non-disease diagnosis and treatment purposes, which is used to improve sperm motility in patients with asthenospermia.

6. Use of salvianolic acid A according to any one of claims 1 to 5 in the preparation of a medicament for improving sperm function, characterized in that: The medicine comprises salvianolic acid A and pharmaceutically acceptable excipients.

7. The use of salvianolic acid A in the preparation of a medicament for improving sperm function according to claim 6, characterized in that: The auxiliary material is one or more of a diluent, an excipient, a filler, a binder, a wetting agent, a disintegrant, an absorption promoter, a surfactant, an adsorption carrier, and a lubricant.

8. The use of salvianolic acid A in the preparation of a medicament for improving sperm function according to claim 6, characterized in that: The medicine is an oral preparation or a parenteral preparation.

9. The use of salvianolic acid A in the preparation of a medicament for improving sperm function according to claim 6, characterized in that: The medicine is in the form of tablets, capsules, powders, pills, granules, solutions, suspensions, syrups, injections, suppositories, adsorbents or sprays.

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