CN116459242B - Application of 3-hydroxyphenylacetic acid in preparation of medicines for preventing and/or treating reproductive dysfunction of aged men - Google Patents
Application of 3-hydroxyphenylacetic acid in preparation of medicines for preventing and/or treating reproductive dysfunction of aged men Download PDFInfo
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- CN116459242B CN116459242B CN202310728831.XA CN202310728831A CN116459242B CN 116459242 B CN116459242 B CN 116459242B CN 202310728831 A CN202310728831 A CN 202310728831A CN 116459242 B CN116459242 B CN 116459242B
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Abstract
The invention provides an application of 3-hydroxyphenylacetic acid in preparing medicaments for preventing and/or treating reproductive dysfunction of aged men. According to the invention, the effect of 3-hydroxyphenylacetic acid in testis seminiferous dysfunction of an aging mouse is discussed through an aging mouse model, and the result shows that the 3-hydroxyphenylacetic acid can improve the sperm concentration of the aging mouse, and the 3-hydroxyphenylacetic acid can enable the arrangement of testis endogenous sperm cells of the aging mouse to be more orderly through the HE staining of testis tissues, so that the immunofluorescence of the testis shows that after the treatment of the 3-hydroxyphenylacetic acid, the sperm cells at all levels are obviously increased. The invention also discovers that the 3-hydroxyphenylacetic acid can reduce the oxidative stress level of the testis of the aging mouse.
Description
Technical Field
The invention relates to an application of 3-hydroxyphenylacetic acid in preparing a medicament for preventing and/or treating reproductive dysfunction of aged men, belonging to the field of medicaments.
Background
In recent years, life expectancy has increased significantly due to advances in medical and public health policies. However, as men age, testis function gradually declines and results in its fertility, overall health and quality of life being affected. Therefore, it is of great importance to explore new methods for improving male sexual dysfunction due to aging.
At present, medicines for clinically improving the sperm quality of men mainly comprise vitamin E, coenzyme Q10, lycopene and the like, and the main functions of the medicines are antioxidation stress, but no definite report exists for finding therapeutic targets of the medicines at present.
The current literature reports that 3-hydroxyphenylacetic acid improves ventricular pathological remodeling in mouse models of myocardial infarction (microbiome. 2022 May 31;10 (1): 82. Doi: 10.1186/s 40168-022-01271-6.) and also reports that 3-hydroxyphenylacetic acid has a blood pressure lowering (nutrients. 2022 Jan 13;14 (2): 328. Doi: 10.3390/nu 14020328.) and an effect of preventing alcoholic liver injury (Int J Mol Sci. 2022 Feb 3;23 (3): 1762. Doi: 10.3390/ijms 23031762.), but no relationship between 3-hydroxyphenylacetic acid and male reproductive function has been found.
Disclosure of Invention
The invention aims to provide a novel pharmaceutical application of 3-hydroxyphenylacetic acid.
The novel application of the 3-hydroxyphenylacetic acid provided by the invention is the application of the 3-hydroxyphenylacetic acid in preparing medicaments for preventing and/or treating male reproductive dysfunction.
The male reproductive dysfunction may specifically be male reproductive dysfunction due to aging.
The male reproductive dysfunction specifically refers to testis seminiferous dysfunction.
It is another object of the present invention to provide the use of 3-hydroxyphenylacetic acid for the manufacture of a medicament having at least one of the following functions 1) -4):
1) The sperm concentration is improved;
2) The arrangement level of the testicle endophytic sperm cells is increased and the arrangement is tidy;
3) The number of sperm cells at each level is obviously increased;
4) Reducing the level of oxidative stress in the testes.
The reduction of the level of oxidative stress in the testes means at least one of the following: 1) Increasing total glutathione peroxidase in testis; 2) The ratio of reduced glutathione to reduced glutathione/oxidized glutathione in testis is increased; 3) The content of malondialdehyde is reduced.
According to the invention, the effect of 3-hydroxyphenylacetic acid in testis seminiferous dysfunction of an aging mouse is discussed through an aging mouse model, and the result shows that the 3-hydroxyphenylacetic acid can improve the sperm concentration of the aging mouse, and further through HE dyeing of testis tissues, the 3-hydroxyphenylacetic acid can enable the arrangement of testis endogenous sperm cells of the aging mouse to be more orderly, and testis immunofluorescence shows that after the 3-hydroxyphenylacetic acid is treated, each grade of spermatogenic sperm cells are obviously increased. Finally, the invention also finds that 3-hydroxyphenylacetic acid can reduce the oxidative stress level of the testis of the aged mice.
The 3-hydroxyphenylacetic acid has a clear chemical formula, is a human body source metabolite, and is mainly used for treating the reduction of reproductive capacity of old men and is different from the existing medicines for improving the sperm quality of men. The invention solves the problem of serious deficiency of medicines for clinically preventing or treating male reproductive dysfunction caused by aging, and discovers that 3-hydroxyphenylacetic acid obviously improves the spermatogenic function of aged mice for the first time, thereby providing a new prevention and treatment option for the male reproductive dysfunction of the aged.
Drawings
The accompanying drawings are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate the invention and together with the embodiments of the invention, serve to explain the invention. In the drawings:
FIG. 1 shows the results of sperm density and sperm motility (A+B-stage sperm ratio, A+B+C-stage sperm ratio) of mice in example 1 of the present invention, wherein the mice aged for 18 to 20 months were used as a control group, and 3-hydroxyphenylacetic acid was administered to the mice aged for gastric lavage (the same applies hereinafter). * ,P< 0.05; a is the sperm density of the mice, B is the proportion of A+B-level sperm, and C is the proportion of A+B+C-level sperm.
FIG. 2 shows the results of the intrinsic parameters of sperm motility in mice in example 1 of the present invention; in fig. 2, a is a linear velocity, B is a curved velocity, C is an average path velocity, D is a whipping frequency, E is a sperm head lateral swing amplitude, and F is a handedness.
FIG. 3 shows the results of HE staining of mouse testis tissue in example 1 of the present invention. A in fig. 3 is a representative graph of testes HE of the aged mice group, and B is a 2-fold enlarged representative graph of a; c is a representative graph of testis HE of a lavage group given 3-hydroxyphenylacetic acid to aged mice, and D is a 2-fold enlarged representative graph of C.
FIG. 4 shows immunofluorescence of sperm cells of each grade of mouse testis tissue in example 1 of the present invention. DAZL is a marker of spermatogenic cells, SYCP3 is a marker of spermatogenic cells, PGK2 is a marker of round sperm, TNP1 is a marker of elongated sperm, WT1 is a marker of supporting cells.
FIG. 5 shows the results of examination of antioxidant substances in testis in example 1 of the present invention. A in fig. 5 is the result of total antioxidant capacity, B is the result of total glutathione peroxidase content, C is the result of superoxide dismutase content, wherein,P＜0.01。
FIG. 6 shows the results of measuring glutathione content in testis in example 1 of the present invention. A in fig. 6 is the result of the reduced glutathione content, B is the ratio of reduced glutathione to oxidized glutathione, where,P＜0.05，**，P＜0.01。
FIG. 7 shows the results of the measurement of malondialdehyde content in testes in example 1 of the present invention. Wherein, the first and second parts are used for the treatment of the first and second diseases,P＜0.05。
Detailed Description
The preferred embodiments of the present invention will be described below with reference to the accompanying drawings, it being understood that the preferred embodiments described herein are for illustration and explanation of the present invention only, and are not intended to limit the present invention.
The terms used in the present invention generally have meanings commonly understood by those of ordinary skill in the art unless otherwise indicated.
The specific techniques or conditions are not identified in the examples and are described in the literature in this field or are carried out in accordance with the product specifications. The reagents or equipment used were conventional products available for purchase through regular channels, with no manufacturer noted.
The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the examples described below, unless otherwise specified, are all commercially available products.
Example 1
1. Experimental animals:
aged mice 18-20 months, C57BL/6 strain, male, grade SPF, offered by the department of animals of the university of Beijing, medical department. After 1 week of adaptive feeding, the control group (lavage saline), model group (lavage 25mg/kg of 3-hydroxyphenylacetic acid (10 mg/ml concentration using saline)) were fed once daily for 6 weeks. After the experiment is finished, the mice are sacrificed, epididymis and testis are taken out, and the testis is weighed and subjected to corresponding treatment.
Animal experiments strictly follow the rules of animal experiment management of a third hospital of Beijing university and are audited by the ethical committee of animal experiments.
(1) Tissue leaving: mice were weighed and testis tissue was weighed. Fresh epididymis was left. 4% paraformaldehyde fixation, conventional alcohol dehydration and paraffin embedding slicing of testicular tissue on one side; the testis tissue on the other side is directly extracted with protein or frozen in liquid nitrogen.
(2) The effect on the morphology of the seminiferous tubules of testicular tissue was examined: paraffin sections of testis tissue were subjected to conventional histomorphometric staining (hematoxylin-eosin staining) to observe the morphology changes of seminiferous tubules of testis tissue of each group of mice, such as arrangement of seminiferous tubules, shape of lumen, arrangement and number of seminiferous cells of each stage, etc.
(3) Determining the effect on semen quality: collecting epididymal sperms by a diffusion method, and detecting the sperm density, forward movement proportion and total sperm movement proportion of the mice by a computer-aided semen analysis system (Beijing ear plus software Co., ltd.); linear velocity, curve velocity, average path velocity, whipping frequency, sperm head lateral amplitude, and handedness.
(4) Testicle immunofluorescence pathology detection: paraffin sections of testis tissue were routinely deparaffinized, antigen repaired in EDTA at 95 ℃ for 30 min, blocked with 10% donkey serum for 1 h, marker antibodies of each grade of spermatogenic cells were incubated overnight at 4 ℃, secondary incubation and DAPI staining, and photographed under a zeiss LSM880 confocal microscope.
(5) The testicle oxidation stress state is detected according to the description of the detection kit of total antioxidant capacity, total glutathione peroxidase, superoxide dismutase, reduced glutathione/oxidized glutathione and malondialdehyde provided by Biyundian corporation.
(6) All data represent at least three independent experimentsData are expressed as mean ± Standard Error (SEM). Use of GraphPad Prism9 for carrying outtThe test is carried out and the analysis is carried out,Pa value < 0.05 considers the difference statistically significant, meaningP< 0.05: <P< 0.01: <P＜0.001。
2. Experimental results:
2.1 Index of sperm density and sperm motility of mice
First, we examined semen quality at the end of treatment in both control and 3-hydroxyphenylacetic acid mice by means of computer-aided semen analysis, and found (see fig. 1): compared with the mice in the control group, the sperm density of the 3-hydroxyphenylacetic acid mice is obviously increased,Pand < 0.05, while the forward movement proportion and the total movement proportion of the sperms of the 3-hydroxyphenylacetic acid mice are not significantly changed compared with the control group.
2.2 intrinsic parameters of sperm motility in mice
The intrinsic parameters of sperm movement of two groups of mice are detected by a method of computer-aided semen analysis, and as shown in fig. 2, the intrinsic movement indexes of sperm of the STZ mice comprise linear velocity (A), curve velocity (B), average path velocity (C), whipping frequency (D), sperm head sideslip amplitude (E) and forward direction (F), and no obvious difference is found between the two groups.
2.3 HE staining results of mouse testis tissue
Hematoxylin-eosin (HE) staining of testis tissue was performed on both groups of mice, and as a result, it was found (see fig. 3): the arrangement level of each grade of spermatogenic cells in the spermatogenic tubule of the aging group mice is reduced, and the sperms in the lumen are sparse; and the arrangement level of each grade of spermatogenic cells in the seminiferous tubules of the 3-hydroxyphenylacetic acid mice is increased, the arrangement is tidy, and the increased mature sperms are visible in the lumen.
2.4 results of immunofluorescence of mouse testis tissue
Immunofluorescence results of testis tissues of mice (see fig. 4) show that the expression of the marker PGK2 of round sperms and the marker TNP1 of long sperms is obviously higher in the 3-HPAA group than in the control group, and it can be seen that the treatment of 3-hydroxyphenylacetic acid can improve the sperm concentration of the mice with aging.
2.5 Results of expression of mouse testis oxidative stress level
The total antioxidant capacity, total glutathione peroxidase, superoxide dismutase, reduced glutathione/oxidized glutathione and malondialdehyde levels of two groups of mice were tested by means of a kit, and as a result, it was found (see fig. 5, 6 and 7): the total glutathione peroxidase (GPx) in the testes of the mice of the 3-hydroxyphenylacetic acid group is remarkably increased,P< 0.01; meanwhile, after the treatment of the 3-hydroxyphenylacetic acid, the ratio of the reduced glutathione to the reduced glutathione/oxidized glutathione in the testes of the mice is obviously improved,P< 0.05; malondialdehyde reflecting lipid oxidation levels were significantly reduced in the 3-hydroxyphenylacetic acid group,P＜0.05。
according to the embodiment, after the treatment of the 3-hydroxyphenylacetic acid on the basis of the aged mice, the 3-hydroxyphenylacetic acid can be found to improve the sperm concentration of the aged mice, improve the testis structure of the aged mice, increase sperm cells and reduce the oxidation stress level of testes of the aged mice through a computer-aided semen analysis system, testis histology dyeing, immunofluorescence and testes oxidation stress level detection. Accordingly, the 3-hydroxyphenylacetic acid can be applied to the preparation of the therapeutic drugs for improving the reproductive dysfunction of the aged men.
It will be apparent to those skilled in the art that various modifications and variations can be made to the present invention without departing from the spirit or scope of the invention. Thus, it is intended that the present invention also include such modifications and alterations insofar as they come within the scope of the appended claims or the equivalents thereof.
Claims (3)
- Use of 3-hydroxyphenylacetic acid in the manufacture of a medicament for the prevention and/or treatment of male reproductive dysfunction; the male reproductive dysfunction is male reproductive dysfunction caused by aging;the male reproductive dysfunction refers to testis seminiferous dysfunction.
- Use of 3-hydroxyphenylacetic acid for the manufacture of a medicament having at least one of the following functions 1) -4):1) The sperm concentration is improved;2) The arrangement level of the testicle endophytic sperm cells is increased and the arrangement is tidy;3) The number of sperm cells at each level is obviously increased;4) Reducing the level of oxidative stress in the testes.
- 3. The use of claim 2, wherein said reducing the level of oxidative stress in the testes is at least one of: 1) Increasing total glutathione peroxidase in testis; 2) The ratio of reduced glutathione to reduced glutathione/oxidized glutathione in testis is increased; 3) The content of malondialdehyde is reduced.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| TW234724B (en) * | 1992-06-20 | 1994-11-21 | Basf Ag | |
| US5639643A (en) * | 1992-06-20 | 1997-06-17 | Basf Aktiengesellschaft | Preparation of 3-hydroxyphenylacetic acid |
| CN111514124A (en) * | 2020-04-13 | 2020-08-11 | 上海大学 | Application of 3-hydroxyphenylacetic acid in preparation of medicine for improving pathologic ventricular remodeling and/or heart failure after myocardial infarction |
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- 2023-06-20 CN CN202310728831.XA patent/CN116459242B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| TW234724B (en) * | 1992-06-20 | 1994-11-21 | Basf Ag | |
| US5639643A (en) * | 1992-06-20 | 1997-06-17 | Basf Aktiengesellschaft | Preparation of 3-hydroxyphenylacetic acid |
| CN111514124A (en) * | 2020-04-13 | 2020-08-11 | 上海大学 | Application of 3-hydroxyphenylacetic acid in preparation of medicine for improving pathologic ventricular remodeling and/or heart failure after myocardial infarction |
Non-Patent Citations (1)
| Title |
|---|
| 男性不育相关代谢组学研究进展;吴华等;《中华生殖与避孕杂志》;第40卷(第10期);864-870 * |
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