Application of lycopene in preparation of medicine for treating hidden testis

Abstract

The invention discloses application of lycopene in preparation of medicines for treating hidden testis, and belongs to the technical field of biological medicines. As a naturally existing carotenoid, the lycopene can effectively improve spermatogenesis of a mouse suffering from cryptorrhea, and the research result provides a solid foundation for treatment of a patient suffering from cryptorrhea by the lycopene; the lycopene regulates and controls the spermatogenesis of the hidden testis through a hedgehog signal channel by reducing the level of active oxygen in tissues. The invention verifies the effects of lycopene on the aspects of reproductive system, influence on spermatogenesis, sperm motility protection and the like, and aims to verify factors influencing spermatogenesis and the action mechanism of lycopene on spermatogenesis of mammals. And a reliable theoretical basis is provided for the application of the gene in the aspects of excellent breeding of large animals, even male infertility and the like of human beings.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to the application of lycopene in the preparation of drugs for treating cryptorchidism. Background Art

[0003] In a large number of studies on male infertility factors, it has been found that the damage of excessive reactive oxygen species (ROS) in semen to sperm is the main cause of infertility in 30%-80% of infertile men. In the normal physiological fertilization process, there is usually a balance of oxidative stress (OS) between ROS family products and antioxidant-scavengers. An appropriate level of ROS is related to physiological functions such as highly active sperm motility, capacitation, acrosome reaction, and sperm-oocyte fusion. When sperm are attacked by excessive ROS, the fluidity and integrity of the sperm membrane are damaged, resulting in damage and breakage of the plasma membrane and even cell death, ultimately reducing sperm motility and fertilization ability.

[0004] Lycopene, also known as lycopin, has a molecular formula of C 40 H 56 , with 11 conjugated C=C bonds, is a non-cyclic planar conjugated polyunsaturated aliphatic hydrocarbon, and its structure is similar to that of carotenoids. Lycopene has cis and trans isomers, which can be interconverted during the processes of light, heat, processing, and cooking. Most of the lycopene present in natural plants is in the all-trans form. Lycopene is a fat-soluble unsaturated hydrocarbon, which is widely present in various red fruits and vegetables, such as the fruits of tomatoes, watermelons, carrots, grapes, strawberries, etc., and the content of lycopene in tomatoes is the highest.

[0005] Lycopene is a very strong antioxidant and has a strong ability to scavenge free radicals. Existing studies have shown that after the activation of the receptor for advanced glycation end products (RAGE) in human semen, it can induce cell reactions and lead to the production of ROS. Lycopene can reduce the level of the receptor for advanced glycation end products (RAGE) in human semen.

[0006] Most of the existing technologies disclose compound preparations. For example: a herbal compound preparation for preventing and treating male infertility and sterility. This herbal compound preparation is obtained by ethanol, methanol, acetone, and water extraction and concentration of the roots of Triangular Orchid, the seeds of Anders' Water Fern, the seeds of Mucuna pruriens, the seeds of Mimosa pudica, gum arabic, the roots of Astragalus membranaceus, the seed coats of Plantago asiatica, kapok gum, and the roots of Eurycoma longifolia. Verified by human trials, this herbal compound preparation can effectively improve the health level of male infertility patients, increase semen volume, sperm density, and sperm motility level, and significantly increase the levels of male testicular hormones and hormonal hormones. Therefore, this herbal compound preparation has potential value for development into drugs or related foods for treating male infertility and sterility.

[0007] However, there is no clear report on the use of lycopene to regulate cryptorchidism through the hedgehog signaling pathway. Summary of the Invention

[0008] The first object of the present invention is to provide an application of lycopene in the preparation of a drug for treating cryptorchidism. Lycopene, as a naturally occurring carotenoid, can effectively improve sperm production in cryptorchid mice. The research results provide a solid foundation for the treatment of cryptorchid patients with lycopene. Lycopene regulates cryptorchid sperm production by reducing the level of reactive oxygen species in tissues through the hedgehog signaling pathway.

[0009] The second object of the present invention is to provide an application of lycopene in the preparation of a spermatogenic reproductive drug for regulating cryptorchidism. The effects of lycopene on the reproductive system, spermatogenesis, and sperm motility protection are verified through experiments, aiming to verify the factors affecting spermatogenesis and the mechanism of action of lycopene on mammalian spermatogenesis, providing a reliable theoretical basis for its application in the excellent breeding of large animals and even in male infertility in humans.

[0010] The present invention is achieved through the following technical solutions:

[0011] Application of lycopene in the preparation of a drug for treating cryptorchidism, wherein the lycopene regulates and treats cryptorchidism through the hedgehog signaling pathway;

[0012] The concentration of the lycopene is 10 μg / g / d.

[0013] Preferably, the application of the drug in sperm count, testicular tissue, hormone level, and oxidative stress response.

[0014] Preferably, the sperm count is to increase the sperm count in cryptorchid mice.

[0015] Preferably, the testicular tissue is to relieve testicular tissue damage.

[0016] Preferably, the hormone level is to promote testosterone hormone production in cryptorchid mice.

[0017] Preferably, the oxidative stress response is to reduce the level of reactive oxygen species in the high-temperature tissue of cryptorchid mice.

[0018] Application of lycopene in the preparation of a spermatogenic reproductive drug for regulating cryptorchidism, wherein the concentration of the lycopene is 10 μg / g / d.

[0019] Preferably, the application of the drug in reproductive proteins.

[0020] Preferably, the expression level of the reproductive protein is to increase the protein expression level;

[0021] The protein includes glioma-associated carcinoprotein 1 and fusion inhibitory protein.

[0022] A drug for regulating and treating cryptorchidism based on the hedgehog signaling pathway, wherein the drug contains the above-mentioned lycopene as the main active ingredient.

[0023] Compared with the prior art, the present invention has at least the following technical effects:

[0024] The present invention provides an application of lycopene in the preparation of a drug for treating cryptorchidism. As a naturally occurring carotenoid, lycopene can effectively improve sperm production in cryptorchid mice. The research results provide a solid foundation for the treatment of cryptorchid patients with lycopene; lycopene regulates sperm production in cryptorchidism through the hedgehog signaling pathway by reducing the level of reactive oxygen species in tissues.

[0025] The application of the lycopene in the preparation of a drug for regulating spermatogenesis and reproduction in cryptorchidism verifies the effects of lycopene on the reproductive system, spermatogenesis, and sperm motility protection through experiments, aiming to verify the factors affecting spermatogenesis and the mechanism of action of lycopene on spermatogenesis in mammals, providing a reliable theoretical basis for its application in the excellent breeding of large animals and even in male infertility in humans. Description of the Drawings

[0026] Figure 1 Shows the effect of lycopene at different concentration groups on sperm production in cryptorchid mice;

[0027] Figure 2 Is a schematic diagram of the sperm count in different groups;

[0028] Figure 3 Is a statistical chart of lycopene promoting sperm production in cryptorchid mice;

[0029] Figure 4 Is a HE tissue section diagram of testicular tissues in different concentration groups, magnification 20×;

[0030] Figure 5 Is a schematic diagram of hormone determination in different concentration groups;

[0031] Figure 6 Is a diagram of the reactive oxygen species level and statistics of testicular tissues in each group;

[0032] Figure 7 Is for transcriptome sequencing experimental design and sample quality determination;

[0033] Figure 8 Is for Venn diagram and volcano plot analysis of differentially expressed genes;

[0034] Figure 9 For GO and KEGG pathway analysis of differential genes;

[0035] Figure 10 It is a schematic diagram of protein expression immunoblotting and gray scale statistics. Specific implementation manners

[0036] The following will describe the implementation scheme of the present invention in detail in combination with embodiments. However, those skilled in the art will understand that the following embodiments are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention. For the specific conditions not specified in the embodiments, they are carried out according to conventional conditions or the conditions recommended by the manufacturer. For the reagents or instruments not specified by the manufacturer, they are all conventional products that can be obtained by purchasing in the market.

[0037] Embodiment

[0038] Experimental design:

[0039] 1. Construction of cryptorchidism mouse model

[0040] According to the experimental requirements, a male mouse cryptorchid azoospermia model was established. In this experiment, 2-week-old male Kunming mice were selected for cryptorchid surgery. The bilateral testes were pulled into the abdominal cavity from the scrotum, and the traction bands on both sides of the testes were cut. Then, the testicular fat pads were fixed on the abdominal wall and the incision was sutured. After 1 week of conventional feeding after cryptorchid surgery, drugs were administered by gavage.

[0041] 2. Treatment with lycopene by gavage

[0042] The experiment was divided into four groups, namely normal control group (control), cryptorchid group (cryptorchid), low-dose lycopene treatment group (4 μg / g / d), medium-dose lycopene treatment group (10 μg / g / d), and high-dose lycopene treatment group (20 μg / g / d); the normal group and the cryptorchid group were directly gavaged with 0.2 mL of normal saline for 4 weeks.

[0043] 3. Tissue section

[0044] Take the testicular tissues of one side from four groups of mice and fix them in Bouin's fixative for 12 h; dehydrate them with gradient alcohol: first dehydrate with 70% alcohol (10 min) → 80% alcohol (10 min) → 95% alcohol (10 min) → 100% alcohol I (10 min) → 100% alcohol II (10 min); then perform gradient transparency treatment with alcohol and xylene; finally, embed them in paraffin; sectioning: cut them into 5-μm thin slices with a microtome, place them on the glass slides pre-coated with egg albumin glycerin, and air-dry them naturally. Dewax them with xylene; dehydrate them with alcohol; stain them with eosin and hematoxylin; decolorize them with hydrochloric acid-alcohol solution, and finally prepare the slides to observe the effect of lycopene on the testicular tissue sections of mice in each experimental group under a microscope. Detect the promoting effect of lycopene on mouse spermatogenesis from the histological aspect.

[0045] 4. Analysis of sperm production and motility

[0046] Take the epididymis, cut it into pieces and dissolve it in a physiological saline buffer at 37 °C, and detect the sperm motility and density of each group with a sperm analyzer. Use relevant analysis software to calculate the changes in sperm production and motility among groups.

[0047] 5. Detection of hormone levels

[0048] Take the serum samples from each group, and detect the changes in the hormone levels of LH, FSH, E2, and T in the serum of each group according to the operating steps of the enzyme-linked immunosorbent assay kit instructions, and collect and analyze the data with an enzyme-linked immunosorbent assay reader.

[0049] 6. Detection of ROS levels

[0050] Dewaxing of paraffin sections: put the sections into environmentally friendly dewaxing solution for 10 minutes-environmentally friendly dewaxing solution for 10 minutes-environmentally friendly dewaxing solution for 10 minutes-anhydrous ethanol I for 5 minutes-anhydrous ethanol II for 5 minutes-anhydrous ethanol III for 5 minutes-distilled water for washing; Antigen repair: Place the tissue sections in a repair box filled with EDTA antigen repair buffer (pH8.0) in a microwave oven for antigen repair, medium heat for 8 minutes, stop the fire for 8 minutes, and turn to medium-low heat for 7 minutes. After natural cooling, place the slides in PBS (pH7.4) and shake and wash on a decolorizing shaker for 3 times, each time for 5 minutes; Draw circles: Draw circles around the tissue with a tissue pen after the sections are slightly dried; Serum blocking: Add BSA in the circle and incubate for 30 minutes; Add primary antibody: Gently shake off the blocking solution, add PBS to the sections according to a certain ratio of primary antibodies, and lay the sections flat in a humidified box and incubate overnight at 4°C; Add secondary antibody: Place the slides in PBS (pH7.4) and shake and wash on a decolorizing shaker for 3 times, each time for 5 minutes. After the slices are slightly dried, add a secondary antibody labeled with the corresponding species of the primary antibody in a certain ratio to cover the tissue and incubate at room temperature for 50 minutes; DAPI counterstaining of cell nuclei: After the slices are slightly dried, add DAPI dye solution in the circle and incubate at room temperature for 10 minutes away from light; autofluorescence quenching: After the slices are slightly dried, add autofluorescence quencher in the circle for 5 minutes and rinse with running water for 10 minutes; sealing: Place the slides in PBS (pH7.4) and shake on a decolorizing shaker to wash 3 times, each time for 5 minutes. After the slices are slightly dried, seal them with anti-fluorescence quenching sealing agent.

[0051] 7. Transcriptome Sequencing

[0052] The testicular tissue of mice in each experimental group was taken, ground in a low-temperature environment, and then centrifuged and the supernatant was extracted with an RNA extraction kit to extract total RNA. Finally, the quality and concentration of the extracted RNA were detected by agarose gel electrophoresis and frozen in a -80℃ refrigerator for sequencing. The samples were sent to relevant companies for subsequent transcriptomics sequencing experiments to analyze the relevant mechanism of action, screen out the corresponding regulatory factors, and confirm the specific mechanism of action of lycopene in promoting spermatogenesis.

[0053] Experimental results:

[0054] 1. Lycopene promotes sperm production in cryptorchid mice

[0055] 1.1 Screening the optimal concentration of lycopene to promote spermatogenesis in cryptorchid mice

[0056] After establishing the cryptorchidism mouse model by surgery, the mice were divided into five groups: control group, cryptorchidism group, cryptorchidism + 4μg / g / d lycopene group, cryptorchidism + 10μg / g / d lycopene group, and cryptorchidism + 20μg / g / d lycopene group. The drug administration method was oral gavage. The experiment lasted for 35 days.

[0057] likeFigure 1 As shown in the figure, it is the effect of lycopene at different concentration groups on sperm production in cryptorchid mice. Among them, A: control (normal control group); B: cryptorchid (cryptorchid group); C: cryptorchid + 4 μg / g / d (cryptorchid + 4 μg / g / day lycopene); D: cryptorchid + 10 μg / g / d (cryptorchid + 10 μg / g / day lycopene); E: cryptorchid + 20 μg / g / d (cryptorchid + 20 μg / g / day lycopene). The result figure is the observation under a stereomicroscope, and the observation result is at 40 times magnification.

[0058] Combined with Figure 1 the experimental results show that lycopene at low, medium, and high concentrations can all effectively promote sperm production in cryptorchid mice, and show an upward trend with the increase in concentration. It shows that lycopene can effectively promote sperm production in cryptorchid mice.

[0059] The experimental results further show that 10 μg / g / d lycopene can more effectively promote sperm production in cryptorchid mice.

[0060] 1.2 On the basis of the above screening of lycopene concentration, lycopene at a medium concentration of 10 μg / g / d is selected for treatment, and the related indicators related to reproduction are detected:

[0061] (1) Sperm count; (2) Testis tissue section; (3) Change in hormone content; (4) Change in reactive oxygen species (ROS) in testis tissue; (6) Differentially expressed genes and related signaling pathways promoting sperm production in cryptorchid mice screened by transcriptomics: hedgehog signaling pathway; (6) Verify the change in the expression of related proteins in the hedgehog pathway.

[0062] Verify the sperm count and testicular tissue damage of lycopene in cryptorchid mice

[0063] As Figure 2 shown in the figure, it is a schematic diagram of sperm count in different groups. In the figure, A: control: normal control group; B: crytorchid: cryptorchid group; C: crytorchid + LYC: cryptorchid + 10 μg / g / d lycopene. Observation was carried out using a stereomicroscope at 40 times magnification.

[0064] The results combined with Figure 2 show that lycopene can effectively promote the sperm count in cryptorchid mice.

[0065] As Figure 3 shown in the figure, it is a statistical chart of lycopene promoting sperm production in cryptorchid mice.

[0066] The results combined with Figure 3It can be seen that compared with the cryptorchidism group, lycopene can effectively promote sperm production in cryptorchid mice.

[0067] As Figure 4 shown, it is a HE tissue section diagram of testicular tissues in different concentration groups, with a magnification of 20×. In the figure, A: normal control group; B: cryptorchidism group; C: cryptorchidism + lycopene group.

[0068] Figure 4 The results of HE staining of testicular tissues show that the seminiferous tubules in the testicular tissues of mice in the normal control group are arranged neatly, the spermatogenic cells at all levels are clearly stratified, the structure is tight, and sperm production can be seen in the tubules.

[0069] In the cryptorchidism group, the distance between seminiferous tubules in the testis of mice widened, the thickness of the tube wall was uneven, the spermatogenic cells were arranged disorderly, the stratification disappeared, the number decreased significantly, and a large blank area could be seen in the lumen.

[0070] Compared with the cryptorchidism group, in the cryptorchidism + lycopene group, the distance between seminiferous tubules decreased, the spermatogenic cells were arranged relatively neatly, the development level of spermatogenic cells could be seen, and the number of sperm in the tubules increased and approached normal.

[0071] The experimental results show that: 10 μg / g / d lycopene can effectively promote sperm production in cryptorchid mice and can effectively relieve testicular tissue damage.

[0072] 2. Verify the effect of lycopene on the production of testosterone hormone in cryptorchid mice

[0073] As Figure 5 shown, it is a schematic diagram of hormone determination in different concentration groups. In the figure, A: a diagram showing the change in testosterone content in each experimental group; B: a diagram showing the change in follicle-stimulating hormone content in each experimental group; C: a diagram showing the change in estradiol content in each experimental group; D: a diagram showing the change in luteinizing hormone content in each experimental group.

[0074] Figure 5 The experimental results show that compared with the cryptorchidism group, lycopene can effectively promote the production of testosterone and follicle-stimulating hormone in the cryptorchidism group.

[0075] 3. Verify the effect of lycopene on the level of reactive oxygen species (ROS) in testicular tissues of cryptorchid model mice

[0076] As Figure 6 shown, it is a diagram of the ROS level and statistics of testicular tissues in each group.

[0077] The results combined with Figure 6 The experimental results show that lycopene has an antioxidant effect and can significantly reduce the ROS level in testicular tissues of cryptorchid mice.

[0078] 4. Transcriptome sequencing of testicular tissues of cryptorchid model mice by lycopene

[0079] The transcriptome sequencing of the testis tissues of cryptorchidism model mice under the action of lycopene was preliminarily completed.

[0080] As Figure 7 shown, it is a schematic diagram of the transcriptome sequencing experiment design and sample quality determination. In the figure, A: Schematic diagram of the transcriptome sequencing process; B: Cluster analysis of the total expression of samples; C: Component analysis of the total expression of samples.

[0081] The results combined with Figure 7 A in it showed the process of transcriptome sequencing: Cryptorchidism surgery (UDT) was performed on 2-week-old mice. After normal feeding for one week, continuous intragastric administration of lycopene (LYC) was carried out for 4 weeks, and normal saline (Vehicle) was used in the control group. Total RNA was extracted from the testis tissues for high-throughput transcriptome sequencing, and each group was independently replicated 3 times.

[0082] The results combined with Figure 7 B and C in it showed the cluster analysis and principal component analysis of the total expression of samples.

[0083] As Figure 8 shown, it is the Venn diagram and volcano plot analysis of differentially expressed genes. In the figure, from left to right are the Venn diagram; volcano plot of the normal control group; volcano plot of the cryptorchidism + lycopene group.

[0084] The results combined with Figure 8 The Venn diagram in it showed that there was an obvious overlap between the differences between the control group and the cryptorchidism group and the differences between the cryptorchidism group and the cryptorchidism + lycopene group.

[0085] The results combined with Figure 8 The volcano plot in it showed that there were a large number of genes with significant expression differences in the cryptorchidism group compared with the control group; there were also great changes in gene expression in the lycopene administration group compared with the cryptorchidism group.

[0086] As Figure 9 shown, it is the Gene Ontology (GO) and KEGG pathway analysis of differentially expressed genes. In the figure, A is the GO analysis of differentially expressed genes between the cryptorchidism group and the cryptorchidism + lycopene group; B: KEGG analysis of differentially expressed genes between the cryptorchidism group and the cryptorchidism + lycopene group; the top 30 entries with the highest significance of differences are listed.

[0087] The results combined with Figure 9 , the Hedgehog signaling pathway was the most significantly different pathway, indicating that lycopene regulates spermatogenesis through this signaling pathway.

[0088] 5. Identification of the expression of proteins related to the Hedgehog signaling pathway screened

[0089] As Figure 10As shown, it is a schematic diagram of protein expression immunoblotting and gray scale statistics. In the figure, A: Detection of Gli1 protein expression levels in the normal group, cryptorchidism group, and cryptorchidism + lycopene group; B: Representing the gray scale values of each band in Figure A; C: Detection of sufu protein expression levels in the normal group, cryptorchidism group, and cryptorchidism + lycopene group; D: Representing the gray scale values of each band in Figure B.

[0090] The results combined Figure 10 show that the expression levels of Gli1 and SUFU proteins in the cryptorchidism group are both decreased, and the expression levels of Gli1 and SUFU proteins in the cryptorchidism + lycopene group are both increased compared with the cryptorchidism group, indicating that lycopene promotes sperm production by regulating the expression of Gli1 and SUFU proteins in the Hedgehog signaling pathway.

[0091] Finally, it should be noted that the above are only the preferred embodiments of the present invention and are not used to limit the protection scope of the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. Use of lycopene in the preparation of a drug for treating cryptorchidism, characterized in that, The lycopene regulates and treats cryptorchidism through the hedgehog signaling pathway; The concentration of the lycopene is 10 μg / g / d.

2. Use of lycopene in the preparation of a drug for treating cryptorchidism according to claim 1, wherein The application of the drug in sperm count, testicular tissue, hormone level, and oxidative stress response.

3. Use of lycopene in the preparation of a drug for treating cryptorchidism according to claim 2, characterized in that, The sperm count is to increase the oligospermia count in cryptorchidism.

4. Use of lycopene in the preparation of a drug for treating cryptorchidism according to claim 2, characterized in that, The testicular tissue is to relieve testicular tissue damage.

5. Use of lycopene in the preparation of a drug for treating cryptorchidism according to claim 2, characterized in that, The hormone level is to promote testosterone production in cryptorchid mice.

6. Use of lycopene in the preparation of a drug for treating cryptorchidism according to claim 2, characterized in that, The oxidative stress response is to reduce the reactive oxygen species level in the high-temperature tissue of cryptorchid mice.

7. Use of lycopene in the preparation of a spermatogenic and reproductive drug for regulating cryptorchidism, characterized in that, The concentration of the lycopene is 10 μg / g / d.

8. An application according to claim 7, wherein The application of the drug in reproductive proteins.

9. An application according to claim 8, wherein The reproductive protein expression level is to increase the protein expression level; The proteins include glioma-associated oncoprotein 1 and fusion inhibitory protein.

10. A drug for regulating and treating cryptorchidism based on the hedgehog signaling pathway, characterized in that, The drug contains the lycopene as described in claim 1 as the main active ingredient.

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