CN117695258A - Application of resveratrol in preparation of medicament for improving sperm motility - Google Patents
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- CN117695258A CN117695258A CN202311574733.1A CN202311574733A CN117695258A CN 117695258 A CN117695258 A CN 117695258A CN 202311574733 A CN202311574733 A CN 202311574733A CN 117695258 A CN117695258 A CN 117695258A
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- Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)
Abstract
The invention discloses application of resveratrol in preparing a medicament for improving sperm motility. The invention carries out differential analysis on protein modification groups in seminal plasma of group 2 (healthy people NZS and weak seminal disorder people AZS), screens to obtain a key protein posttranslational modification difference, namely lysine succinylation modification, then finds out the most important dissuccinylase SIRT5 in sperms by screening relevant factors, adopts a specific sperm in-vitro culture solution to co-culture human sperms and the SIRT5 activator resveratrol with different concentrations, and detects sperm motility, thus the result also proves that the compound can improve sperm motility. The method successfully utilizes protein modification histology analysis and combines sperm cells and a modification enzyme activator co-culture experiment to screen out a compound capable of improving sperm motility. The method provides a chemical substance with prospect for improving sperm motility, and can bring new hope for treating asthenozoospermia.
Description
Technical Field
The invention belongs to the field of biological medicine, and in particular relates to application of resveratrol in improving sperm motility.
Background
At present, infertility affects about 15% -20% of couples worldwide, and infertility has become the third biggest disease besides cancers and cardiovascular and cerebrovascular diseases, wherein male factor-induced infertility accounts for about half. Sperm motility (term mobility) refers to the ability of Sperm to move forward. According to WHO standard, the ratio of forward motile sperm in semen is greater than or equal to 32% of normal sperm motility, and less than this standard is hypomotility or oligospermia (AZS). Asthenozoospermia is a common cause of male infertility. 80% of male infertility is reported to be associated with sperm motility disorders, and 20% of male infertility is reported to be directly associated with low sperm motility. The large sample data in China show that the incidence rate of asthenozoospermia in all men subjected to semen analysis is up to more than 50%.
Lysine succinylation (Lysine succinylation, ksuc) is a novel posttranslational modification of proteins discovered in recent years, and Ksuc is likely to be involved in sperm function regulation as one of the important ways of regulating cellular energy metabolism. The modification is the incorporation of a two negatively charged four-carbon succinyl group on the lysine residue, resulting in a change in the charge of the lysine group from +1 to-1. This change in charge properties is similar to the effect of protein phosphorylation, and can alter the structure of proteins to regulate their function. SIRT5, the major dissuccinylase, is located in the mitochondrial matrix and is involved in regulating many metabolic processes.
Resveratrol (RES) is a natural non-flavonoid polyphenol compound with cis-form and trans-form, is widely distributed in medicinal plants such as grape, peanut, giant knotweed, mulberry and the like, and has the effects of resisting apoptosis, inflammation, oxidization, aging and the like. RES has been reported to activate SIRT5. Thus, REV may promote its dissuccinylation by enhancing SIRT5 activity, regulating energy metabolism. RES is expected to be a novel pharmaceutical preparation for regulating sperm motility.
Disclosure of Invention
The invention aims to provide application of resveratrol in preparing a medicament for improving sperm motility.
According to the invention, 4D succinylation modification analysis is carried out on sperm proteins in healthy people (NZS) and weak sperm disease people (AZS) semen, an agonist-Resveratrol (Resveratrol) for regulating and controlling succinylation modification key enzyme is obtained through screening, then, a specific sperm in-vitro culture solution is adopted to co-culture human sperm cells with the agonist, and sperm activity is detected, so that the result also proves that the small molecule preparation can improve sperm activity.
Accordingly, the present invention provides the use of resveratrol, or a derivative thereof, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for enhancing sperm motility.
Preferably, the sperm motility enhancing drug is a drug for treating asthenospermia.
Preferably, the medicine contains resveratrol or its derivative or its pharmaceutically acceptable salt and pharmaceutically acceptable supplementary material in effective amount.
Preferably, the preparation can be an oral preparation, an injection and other pharmaceutically common dosage forms.
Preferably, the oral preparation is various dosage forms such as oral liquid, electuary, pill, tablet, granule and the like.
Preferably, the sperm is human sperm.
A second object of the present invention is to provide a medicament for improving sperm motility, which contains resveratrol as an active ingredient in an effective amount.
It is a third object of the present invention to provide a method of improving sperm motility by adding resveratrol to a sperm-containing system.
A fourth object of the present invention is to provide the use of a formulation for detecting the level of lysine succinylation modification in a sperm protein in the preparation of a formulation for diagnosing asthenospermia.
The method successfully utilizes 4D succinylation modification histology detection and analysis, and combines the sperm cell and the agonist co-culture experiment of the modification enzyme, thereby screening out a small molecule preparation capable of improving the sperm motility. The method provides a good chemical substance for improving the sperm motility, and can bring new hope for treating the asthenozoospermia.
Description of the drawings:
FIG. 1 is a smear test chart of semen samples from both healthy men and asthenozoospermia populations.
FIG. 2 shows the analysis of the difference and enrichment of succinylation modification of sperm proteins in both healthy men and asthenozoospermia populations.
FIG. 3 is a flow chart of screening candidate metabolites for sperm motility related activity.
FIG. 4 is a diagram showing sperm smear detection after co-culturing sperm cells with resveratrol.
FIG. 5 is a graph showing sperm motility assays after co-culturing sperm cells with resveratrol. (a) sperm cell forward motility; (B) total sperm motility; (C) Linear Velocity (VSL); (D) curve rate (Curvilinear velocity, VCL); (E) Average path rate (Average path velocity, VAP); (F) forward (Straightness coefficient, STR); (G) Sperm head lateral swing amplitude (Amplitude of lateral head displacement, ALH); (H) whipping frequency (Beat cross frequency, BCF).
Detailed Description
The following examples are further illustrative of the invention and are not intended to be limiting thereof.
Example 1:
1. collection of semen samples from two groups of people
Semen sampling is carried out on the patients who come from hospital and visit, clinical detection is carried out, the sampling standard achieves aseptic sampling, and interference conditions of experiments are reduced. The sampling standard procedure is as follows: after 3-7 days of abstinence, the patient can go to the hospital to finish sampling; before formal sampling, the hands are washed 2-3 times with soapy water, the penis, particularly the glans and the coronary sulcus, are washed with warm soapy water, then are scrubbed 2-3 times with 75% alcohol, and finally the semen is directly injected into a sterile container through masturbation, taking care to avoid contacting the sterile wall of the container.
The freshly collected semen is detected by using a conventional semen clinical detection means. And taking a part of samples of each semen sample, and detecting indexes of the samples by using a semen automatic analyzer, wherein the detection contents comprise pH value, semen liquefaction time, semen volume, semen count and motile semen percentage. The remaining semen samples were transferred to sterile microcentrifuge tubes, incubated at 37℃for 30min, and stored at-80℃for 2 hours after collection.
In the 22-45 year old male population, collection of patient-related information was accomplished while respecting patient privacy, and volunteers were recruited, with the volunteer criteria as follows: history of heart, liver, kidney, digestive tract, nervous system, mental abnormalities, metabolic abnormalities, and the like; male subjects aged 22-45 years (including 22 years and 45 years); body Mass Index (BMI) =body weight (kg)/height 2 (M 2 ) The BMI is 19.0-26.0kg/m 2 Within a range (including a critical value); all test procedures were signed with informed consent prior to the test, willing to follow and capable of being completed. The 60-person cohort was divided into 2 groups, including healthy groups (sperm motility>32%) with weak sperm disease population (sperm motility rate)<30%) and the smear observations for both groups are shown in figure 1. And then, separating semen according to the standard step of sperm gradient separation to finish the collection of sperm samples.
2. Sperm protein 4D succinylation modification histology detection and analysis
The semen sample is subjected to steps such as sperm purification, protein extraction, pancreatin enzymolysis, antibody enrichment, liquid chromatography-mass spectrometry and the like to carry out 4D-succinylation modification group analysis, 2765 sites in 677 proteins are identified to generate lysine succinylation (Ksuc), 1983 sites in 441 proteins are quantified, and difference multiples of >1.5 and <0.66 are respectively determined to be up-regulated and down-regulated. It was found that 208 of the protein succinylation levels were up-regulated in the asthenozoospermia population and 51 of the protein succinylation modification levels were down-regulated in the asthenospermia population; the succinylation modification level of lysine at 521 sites was up-regulated in the asthenospermia population, while only 73 sites were down-regulated (fig. 2A).
To further compare the major occurrence and course of altered lysine succinylation levels in the asthenospermia group with healthy group sperm proteins. Differentially modified succinylated proteins were found, 46% mitochondrial protein, 29% cytoplasmic protein, 10% extracellular protein (fig. 2B). GO analysis showed that differentially modified proteins were present in multiple cellular components and involved in multiple biological processes and metabolic pathways (fig. 2C).
3. Co-culture of sperm cells and resveratrol and sperm motility detection
Since the reduced level of succinylation modification of lysine occurs mainly in mitochondria and cytoplasm of sperm of the asthenozoospermia population, we screened the related factors regulating succinylation of protein, found that SIRT5 is the most predominant protein desuccinilase, and this enzyme is also mainly localized in mitochondria and cytoplasm. At the same time, we have found that SIRT5 expression is also reduced in sperm of asthenozoospermia patients. Thus, we attempted to increase sperm motility by activating the dissuccinylase SIRT5 to reduce the succinylation level of sperm proteins in the asthenospermia population. The resveratrol can be used as an SIRT5 activator, has the functions of resisting oxidization, resisting apoptosis and the like, and can protect sperm motility.
The process comprises the following four steps: 1) Semen analysis. Before semen samples are collected, determining information such as age of a patient, last semen discharging time and the like; and then, after the collection of the sample is completed, the indexes such as semen volume, PH, viscosity and the like are analyzed, after 30 minutes of semen is completely liquefied, the concentration and the activity of sperms in the semen are analyzed by a sperm analyzer, and for sperms which cannot be identified by a computer, the activity and the concentration data of the sperms are counted after the manual correction is completed. 2) And collecting sperm samples. Classifying semen samples according to a fifth WHO edition, selecting samples with sperm motility less than 30% as weak sperm disease groups, obtaining sperm cells by adopting a density gradient centrifugation method for the samples, and adjusting sperm concentration. 3) Culturing sperms in vitro: adding sperm cells with adjusted concentration into sperm in vitro culture solution (namely fertilized oviduct liquid culture solution, HTF, produced by Gossdadadada) respectively (0 uM, 0.8uM, 1.5uM, 3uM, 6uM, 12uM and 20uM resveratrol are added into culture solution, wherein resveratrol is dissolved by using proper amount of absolute ethanol) to make sperm final concentration 5×10 in each repetition (n=3) 6 Each/ml was placed in an incubator at 37℃for cultivation. 4) And (5) analyzing results. After sperm cells are cultured for 1h in vitro, the motility of the sperm cells is detectedAs shown in FIG. 5, four groups of 3uM, 6uM, 12uM and 20uM resveratrol improved sperm motility compared to the 0uM group, wherein 12uM is the optimal action concentration of resveratrol (FIGS. 4 and 5).
The method of the invention obtains a medicament for improving the sperm motility, and can improve the sperm motility in the culture process of a proper condition culture medium.
Claims (9)
1. Use of resveratrol, or a derivative or pharmaceutically acceptable salt thereof in the manufacture of a medicament for improving sperm motility.
2. The use according to claim 1, wherein the agent that increases sperm motility is an agent that treats asthenozoospermia.
3. The use according to claim 1, wherein the medicament comprises an effective amount of resveratrol or a derivative thereof, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable adjuvant.
4. The use according to claim 3, wherein the preparation is an oral preparation or an injection.
5. The use according to claim 4, wherein the oral preparation is an oral liquid, granule, pill, tablet, or granule.
6. The use of claim 1, wherein the sperm is human sperm.
7. A medicament for improving sperm motility, which comprises resveratrol or a derivative thereof or a pharmaceutically acceptable salt thereof as an active ingredient in an effective amount.
8. A method for improving sperm motility, comprising adding resveratrol, a derivative thereof, or a pharmaceutically acceptable salt thereof to a system comprising sperm.
9. The application of succinylation modified level preparation for detecting lysine in sperm protein in preparing preparation for diagnosing asthenozoospermia.
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| CN202311574733.1A CN117695258A (en) | 2023-11-23 | 2023-11-23 | Application of resveratrol in preparation of medicament for improving sperm motility |
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| CN202311574733.1A CN117695258A (en) | 2023-11-23 | 2023-11-23 | Application of resveratrol in preparation of medicament for improving sperm motility |
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