CN114306306B

CN114306306B - Application of 2-bromopalmitic acid in preparation of medicine for treating diseases related to spermatogenic dysfunction

Info

Publication number: CN114306306B
Application number: CN202210160692.0A
Authority: CN
Inventors: 戈榭; 姚兵; 马汝钧; 靖俊; 赵薇
Original assignee: Eastern Theater General Hospital of PLA
Current assignee: Eastern Theater General Hospital of PLA
Priority date: 2022-02-22
Filing date: 2022-02-22
Publication date: 2023-08-22
Anticipated expiration: 2042-02-22
Also published as: CN114306306A

Abstract

The invention discloses application of 2-bromopalmitic acid in preparing a medicament for treating diseases related to spermatogenic dysfunction. The invention makes clear for the first time that 2-bromopalmitic acid can improve barrier injury of testis support cells under the stimulation of saturated fatty acid; by constructing a high saturated fatty acid mouse spermatogenic dysfunction model, it is clear that 2-bromopalmitic acid can effectively improve spermatogenic dysfunction caused by high saturated fatty acid in vivo, and restore the barrier function of testis support cells; the in vivo experimental data foundation for treating the spermatogenic dysfunction caused by the lipid metabolism disorder by the 2-bromopalmitic acid is also provided, and a novel method is provided for treating the disease.

Description

Application of 2-bromopalmitic acid in preparation of medicine for treating diseases related to spermatogenic dysfunction

Technical Field

The invention belongs to the technical field of biomedicine, and in particular relates to application of 2-bromopalmitic acid in preparing a medicament for treating diseases related to spermatogenic dysfunction.

Background

Infertility is an important medical problem which cannot be ignored in modern society, and male factors account for 30-50% of the infertility couples. Seminiferous dysfunction is one of the leading causes of male infertility. In recent years, the rise in obesity rate has made lipid metabolism disorders an important cause of spermatogenic dysfunction. The abnormal increase of saturated fatty acids is a major feature of lipid metabolism disorders and is also a key factor in the initiation of spermatogenic dysfunction. Therefore, the treatment of spermatogenic dysfunction due to lipid metabolism disorder and abnormal increase of saturated fatty acids has become a medical problem to be solved in recent years.

The spermatogenesis process is carried out in the seminiferous tubules of the testes and is precisely regulated and controlled by various cells in the microenvironment of the testes. In the spermatogenic microenvironment, testis support cells provide an immune-free microenvironment for spermatogenesis by forming a blood-testosterone barrier and protect the spermatogenic cells from the threat of external harmful substances. In addition, testicular interstitial cells, peritubular myoid cells and testicular support cells together regulate the spermatogenic process by paracrine action to ensure the normal progression of spermatogenesis. However, the major cell types and specific mechanisms of action that lipid metabolism disorders affect in the spermatogenic microenvironment are not yet defined, which limits the medical scope of diseases associated with spermatogenic dysfunction.

Previous studies have shown that resveratrol, phenylbutyrate sodium salt, curcumin, asiatic acid and the like can partially restore the spermatogenic function of fat animal models caused by high-fat diet. In addition, melatonin has been shown to improve spermatogenic function by alleviating apoptosis, endoplasmic reticulum stress, oxidative stress, and the like of spermatogenic stem cells caused by saturated fatty acids. However, in the case of dysspermia due to lipid metabolism disorder, it is still not known how to restore the function of spermatogenesis by improving the cell function in the microenvironment of spermatogenesis. In the testicular microenvironment of spermatogenesis, testicular support cells are the only type of somatic cells in direct contact with spermatogenic cells, which not only form a blood-testicular barrier to protect the spermatogenic cells from injury, but also provide support for spermatogenesis through nutrition transmission and paracrine functions. Thus, studies directed to functional protection of testicular support cells would provide a new strategy for treating disorders associated with spermatogenic dysfunction.

2-bromopalmitic acid is a known inhibitor of protein palmitoylation, which is reduced in protein palmitoylation by inhibition of palmitoyltransferase. According to the prior studies, saturated fatty acids can significantly increase the palmitoylation level of proteins in cells. Abnormal palmitoylation can alter the structure and water solubility of proteins, further affecting the state and function of cells, thereby causing a variety of diseases including metabolic syndrome, cancer, and the like. As palmitoylation inhibitors, 2-bromopalmitic acid has been demonstrated to relieve inflammatory pain, relieve bone cancer pain, prevent and treat bone loss-related diseases, and the like. However, there is no report on the use of 2-bromopalmitic acid to improve the spermatogenic function.

Disclosure of Invention

The invention aims to: the application of the 2-bromopalmitic acid in preparing the medicine for treating the diseases related to the spermatogenic dysfunction is provided, a new method for treating the diseases related to the spermatogenic dysfunction can be developed, and a new target point and thought are provided for improving the spermatogenic function.

The technical scheme is as follows: the invention provides an application of 2-bromopalmitic acid in preparing a medicament for treating diseases related to spermatogenic dysfunction.

As a further limiting aspect of the application of the present invention, the disorders related to spermatogenic dysfunction include, but are not limited to, spermatogenic dysfunction caused by lipid metabolism disorder.

As a further limiting scheme of the application of the invention, the medicine for treating diseases related to spermatogenic dysfunction comprises 2-bromopalmitic acid.

As a further limiting scheme of the application of the invention, the medicament for treating the diseases related to spermatogenic dysfunction is a medicament for improving the cell barrier function of testis support, a medicament for improving characteristic genes in the cell barrier forming process or a medicament for inhibiting the endoplasmic reticulum stress caused by saturated fatty acid.

As a further limiting aspect of the invention's application, the signature genes include one or more of ZO-1, occludin, claudin-11 and Claudin-5.

As a further limiting scheme of the application of the invention, when the medicine for treating the diseases related to the spermatogenic dysfunction is used, the administration dosage of the 2-bromopalmitic acid is 2-40mg/kg.

As a further limiting scheme of the application of the invention, the dosage form of the medicament for treating the diseases related to the spermatogenic dysfunction is injection, capsule, oral preparation or microcapsule preparation.

As a further limiting aspect of the application of the present invention, the mode of administration of the medicament for treating a disease associated with spermatogenic dysfunction is at least one of oral administration, parenteral administration, inhalation spray administration, topical administration, rectal administration, nasal administration, buccal administration, testicular injection administration or administration via an implanted reservoir.

As a further limiting aspect of the use of the present invention, parenteral administration includes at least one of subcutaneous, intradermal, intravenous, intramuscular, intra-articular, intra-arterial, intra-synovial, intra-sternal, lesion site or intracranial administration.

The invention also provides a medicament for treating diseases related to spermatogenic dysfunction, wherein the diseases related to spermatogenic dysfunction comprise but are not limited to spermatogenic dysfunction caused by lipid metabolism disorder, and the medicament comprises 2-bromopalmitic acid.

Compared with the prior art, the invention has the beneficial effects that: by utilizing various experimental means such as transmembrane resistance detection, transmembrane fluorescence leakage detection, western immunoblotting and the like, it is clear that 2-bromopalmitic acid can improve barrier injury of testis supporting cells in vitro under the stimulation of saturated fatty acid; by constructing a mouse highly saturated fatty acid spermatogenic disorder model, it is clear that 2-bromopalmitic acid also has the function of restoring the integrity of blood-testosterone barriers in vivo, and can obviously improve the spermatogenic disorder caused by lipid metabolism disorder; provides the basis of in vivo experimental data of treating the spermatogenic dysfunction caused by the lipid metabolism disorder by the 2-bromopalmitic acid, defines the protective effect of the 2-bromopalmitic acid on the blood-testosterone barrier, and provides a new treatment strategy for the diseases related to the spermatogenic dysfunction.

Drawings

FIG. 1 shows the improvement effect of 2-bromopalmitic acid (2-BP) on the damage of testis support cell barrier caused by saturated fatty acid Palmitic Acid (PA) in a transmembrane resistance experiment;

FIG. 2 shows the improvement effect of 2-BP on the damage of testis support cell barrier caused by saturated fatty acid PA by a transmembrane macromolecule fluorescence leakage experiment;

FIG. 3 shows the recovery of saturated fatty acid PA by Western immunoblotting to detect 2-BP resulting in decreased expression of claudin;

FIG. 4 shows the recovery effect of Western immunoblotting detection of 2-BP on the increase of endoplasmic reticulum stress marker protein expression caused by saturated fatty acid PA;

FIG. 5 shows that excessive palmitoylation of total testis protein by saturated fatty acid PA is significantly inhibited after lavage of 2-BP in mice;

FIG. 6 shows that 2-BP restores saturated fatty acid PA-induced decline in epididymal tail sperm count in mice;

FIG. 7 shows that 2-BP restores saturated fatty acid PA-induced decline in epididymal tail sperm motility in mice;

FIG. 8 shows in vivo FITC-I fluorescence leakage assay detection of 2-BP restored saturated fatty acid PA-induced blood-testosterone barrier injury in mice;

figure 9 shows that electron microscopy of 2-BP restored saturated fatty acid PA-induced blood-testosterone barrier damage in mice.

Detailed Description

The present invention will be further illustrated with reference to the following drawings and specific examples, which are intended to be illustrative of the invention and not to be limiting of the scope of the invention, and modifications or substitutions made to the methods, steps or conditions of the invention without departing from the spirit and nature of the invention are to be understood to be within the scope of the invention.

The technical means used in the examples are conventional means well known to those skilled in the art unless otherwise indicated; the reagents used in the examples were commercially available.

Example 1:

examples of barrier damage of 2-bromopalmitic acid in vitro to improve testis support cells upon stimulation with saturated fatty acids are as follows.

(1) Isolation and culture of primary testis support cells in mice:

taking a 20-day-old ICR male mouse, killing the neck, soaking the male mouse in 75% sterile alcohol for 2-5 minutes, cutting the lower abdominal skin in a super clean bench, taking out testes, cutting off seminal vesicles from the tail of the epididymis, taking out testes on two sides, placing the testes into a culture dish of PBS (phosphate buffered saline) at 37 ℃, tearing off adipose tissues, testis, tunica albuginea and blood vessels by using ophthalmic forceps, exposing a seminiferous tubule, and placing the seminiferous tubule into another culture dish with PBS. Cutting the tissue to a size of less than 1mm ³ Is placed in 5ml of 0.25% (m/V) trypsin and digested with shaking at 37℃for 4-6 minutes to disperse the seminiferous tubules. PBS 25-30ml was washed once and the supernatant removed. Digestion with 5ml of type 1 collagenase at 0.1% (m/V) was performed at 37℃for 6-8 minutes with shaking, using DMEM/F12 (V/V, 1:1) with 10% FBS as stop solution. The cell suspension was filtered through a 100 mesh screen. PBS 5-10ml wash pass, 4 degree centrifuge, 1200 rpm, centrifugal 6 minutes, remove supernatant, PBS 30ml wash pass, remove supernatant. The cells were seeded into petri dishes and incubated at 34℃in an incubator (5% CO) ₂ ) After culturing for 4 hours, the spermatogenic cells were removed by washing three times. The next day to change liquid for removal againNon-adherent spermatogenic cells. Culturing was continued until the cells grew to a density of 80% or more.

(2) Culture of TM4 mouse testis support cell line:

TM4 mouse testis support cell line was purchased from Saiborin (Shanghai) Biotechnology Co., ltd (iCell Bioscience Inc) and used DMEM/F12 containing 5% horse serum and 2.5% fetal bovine serum as medium at 37℃and 5% CO ₂ Culturing under the condition.

(3) Digestion and enumeration of cells:

discarding culture medium, washing cells twice with PBS, performing pancreatin digestion for 1 min, observing cell rounding under microscope, adding complete culture medium containing serum, stopping digestion, re-suspending cells, centrifuging at 1500rpm for 5 min at room temperature, discarding supernatant, adding appropriate amount of complete culture medium, blowing, mixing, dripping 20 μl into a blood cell counting plate, observing under microscope, counting the number of cells in 5 medium cells at upper left, lower left, upper right, lower right and middle of the counting plate, and counting 5 medium cell number×5×10 per ml cell number according to "5 medium cell number" ⁴ The x dilution factor "was calculated.

(4) Cell barrier integrity was tested by transmembrane resistance experiments:

primary testis support cells of mice were prepared according to 0.5X10 ⁶ /cm ² Is seeded into Millicell suspension cell chambers (PET material, 0.4 μm pore size, merck Millipore Co.) when the diary is Day1, and then cultured for 3 days to form cell barriers. After barrier formation, cells were treated in three groups: PA group was added with 0.4mM PA,2-BP+PA group was added with 10. Mu.M 2-BP and 0.4mM PA, and Control group (Control) was added with the same amount of dimethyl sulfoxide solvent as 2-BP+PA group. Culturing for 3 days after the drug adding treatment. Transmembrane resistance was measured daily for the duration of the experiment using a Millicell resistance measurement system (Merck Millipore Co.). A decrease in resistance indicates an impaired cell barrier integrity.

(5) Macromolecular fluorescence leakage assay to detect cell barrier integrity:

primary testis support cells of mice were prepared according to 0.5X10 ⁶ /cm ² Is inoculated into Millicell suspension cell chamber (PET material, pore size of 0.4 μm, merck Millipore Co., ltd.)Driver). After cell barrier formation, cells were treated for 24 hours with three groups of drugs: PA group was added with 0.4mM PA,2-BP+PA group was added with 10. Mu.M 2-BP and 0.4mM PA, and Control group (Control) was added with the same amount of dimethyl sulfoxide solvent as 2-BP+PA group. For fluorescence leakage detection, 200. Mu.l of FITC-dextran 1mg/ml in phenol red free DMEM/F12 was added to the upper chamber, and 1.2ml of phenol red free DMEM/F12 was added to the lower chamber. The incubator was further incubated for 4 hours, 200. Mu.l of the solution was placed in a 96-well plate in the lower chamber, and fluorescence measurement was performed using an enzyme-labeled instrument (excitation light 490nm, emission light 520 nm). An increase in fluorescence indicates an impaired cellular barrier integrity.

(6) Protein sample processing and extraction:

TM4 cells were seeded in 6-well plates (30X 10) ⁴ Well), divided into a Control group, a PA group and a 2-BP+PA group, wherein 0.4mM PA was added to the PA group, 10. Mu.M 2-BP and 0.4mM PA were added to the 2-BP+PA group, and the Control group (Control) was added with the same amount of dimethyl sulfoxide solvent as that of the 2-BP+PA group. After 24 hours of drug addition, cells were collected, RIPA cell lysate containing protease inhibitor was added, lysed on ice for 30 minutes, the cell lysate was transferred to a 1.5ml centrifuge tube, centrifuged at 13000rpm for 15 minutes at 4℃and the supernatant was collected to a new 1.5ml centrifuge tube, protein concentration was determined by BCA method, 1/4 volume of 5 Xprotein loading buffer was added, after 5 minutes of boiling at 98℃it was stored in a refrigerator at-80 ℃.

(7) Protein immunoblotting detection of protein expression level:

the prepared protein samples were loaded onto a PAGE gel at 20. Mu.g per sample, and transferred to PVDF membrane after electrophoresis. The primary antibody was incubated overnight at 4℃and washed three times with TBST buffer per shaker for 10 minutes. The secondary antibody was incubated at room temperature for 1 hour and washed three times with TBST buffer per shaking table for 10 minutes. The bands were developed using a horseradish peroxidase chromogenic kit and photographed using a chemiluminescent imaging system. The gray scale detection of the bands was performed with ImageJ software to achieve quantification of protein expression.

The result shows that: both the transmembrane resistance test (shown in figure 1) and the macromolecular fluorescence leakage test (shown in figure 2) show that 2-bromopalmitic acid (2-BP) can significantly improve the damage of testis supporting cell barrier caused by PA. Protein immunofluorescence detection shows that 2-BP can not only increase the expression level of the tight junction proteins ZO-1, occludin (OCLN), claudin-11 (CLDN 11) and Claudin-5 (CLDN 5) (as shown in FIG. 3), but also decrease the expression levels of endoplasmic reticulum stress marker proteins CHOP, GRP78, ATF4 and ATF6 (as shown in FIG. 4).

Example 2:

examples of the 2-bromopalmitic acid which can significantly improve the dysfunction of spermatogenesis and the damage of blood-testosterone barrier caused by lipid metabolism disorder in vivo are as follows.

(1) The experimental materials are selected from:

animals are selected from ICR male mice of 4 weeks old; the 2-BP and PA were selected from Sigma, america. The 2-BP is prepared into a storage solution of 100mg/ml by using ethanol, and is prepared into a working solution of 5mg/ml by using physiological saline for injection during experiments. The PA was chelated with BSA, and a stock solution (i.e., working solution) of 20mM (about 5.13 mg/ml) was prepared using physiological saline as a solvent.

(2) Mouse feeding conditions:

the mice are bred in the environment with the temperature of 23-27 ℃ and the relative humidity of 40-70%, and are free to drink water and forge; the illumination period is 12 hours of illumination-12 hours of darkness. The experiment was started after 1 week of adaptive feeding of the mice.

(3) Mouse grouping and model setup:

mice were randomly divided into three groups: the group 2-BP+PA is injected with 200mg/kg PA per abdominal cavity every day, and the stomach is irrigated every other day with 40mg/kg 2-BP; the PA group is injected with 200mg/kg of PA per abdominal cavity every day, and the same amount of physiological saline containing ethanol as the 2-BP+PA group is infused every other day; the Control group (Control) was intraperitoneally injected with an equal volume of BSA solution (dissolved in physiological saline) every other day and was perfused with the same amount of ethanol-containing physiological saline as the 2-BP+PA group. The experimental time lasted 30 days.

(4) Animal material selection:

after the mice are anesthetized by chloral hydrate, eyeballs are removed to obtain blood, and testes and epididymis are obtained after neck breakage and sacrifice.

(5) Detection of total testis protein palmitoylation level:

after removal of the mouse testis, 1/4 of the testis was cut out for total protein palmitoylation detection by liquid nitrogen quick freezing. The assay uses an acyl-biotin displacement method. The specific operation method comprises the following steps: tissues were immersed in RIPA lysate containing 50mM N-ethylmaleimide, and after shearing the tissues, they were further sonicated. Centrifugation was carried out at 13000rpm for 15 minutes at 4℃and the supernatant was collected into a new 1.5ml centrifuge tube and spun at 4℃overnight. Protein was precipitated with glacial acetone, resuspended in 4% sds solution (containing 4% sds, 50mM Tris and 5mM edta, ph=7.4), and divided into 2 parts per sample on average: one portion was added with an equal volume of 1.5M Hydroxylamine (HA) solution and the other portion was added with an equal volume of 0.1M Tris-HCl solution (ph=7.4). After 2 hours of spin mixing at room temperature, the protein was precipitated with ice acetone, resuspended in 4% SDS solution (containing 4% SDS, 50mM Tris and 5mM EDTA, pH=6.2), and 4 volumes of BMCC-Biotin solution (containing 6.25. Mu.M BMCC-Biotin, 150mM NaCl, 50mM Tris, 5mM EDTA and 0.2% Triton X-100) were added and spin mixed at room temperature for 2 hours. The protein was reprecipitated with ice acetone, the solubilized protein was resuspended in a 2% SDS solution (containing 2% SDS, 50mM Tris and 5mM EDTA, pH=7.4, protease inhibitor added prior to use), the protein concentration was determined by BCA method, 1/4 volume of 5 Xprotein loading buffer was added, and after 5 minutes of boiling at 98℃the protein was stored in a refrigerator at-80 ℃. In electrophoresis, each sample was loaded onto a PAGE gel in equal volume, transferred to a PVDF membrane after electrophoresis, blocked with 3% BSA at room temperature for 2 hours, incubated with horseradish peroxidase-conjugated streptavidin (HRP-Strep) at room temperature for 1 hour, washed for half an hour with TBST, subjected to band development using a horseradish peroxidase chromogenic kit, and photographed using a chemiluminescent imaging system. The gray scale detection of the bands was performed with ImageJ software to achieve quantification of protein expression.

(6) Sperm suspension preparation and sperm concentration calculation:

epididymis was removed from mice, epididymal tails were placed in 400 μl HTF solution, and gently transferred with syringe needles to allow sperm to swim out of epididymal tails. Sperm were collected, incubated in a 37 ℃ water bath for 10 minutes, and after mixing by blowing, the concentration of sperm was measured by CASA.

(7) Sperm motility analysis:

sucking the sperm suspension prepared in the step (6), dripping the sperm suspension on a glass slide, covering the glass slide, and detecting the sperm motility by CASA.

(8) In vivo FITC-I fluorescence leakage assay to detect blood-testosterone barrier integrity:

2 hours prior to mice sacrifice, 5mg/ml FITC-I solution (in PBS) was freshly prepared and 200. Mu.l each mouse tail was intravenously injected. When dissecting mice, frozen sections were prepared from unilateral testes. The fluorescence microscope was used to observe the leakage of FITC-I fluorescence into seminiferous tubules in testes. Fluorescence in the seminiferous tubules indicates impaired blood-testosterone barrier integrity.

(9) And (3) observing tight connection by using an electron microscope:

the testis tissue of the mouse is taken out and cut out to 1mm ³ Tissue pieces of the size were fixed by immersing them in 0.1M phosphate buffer containing 2.5% glutaraldehyde and stained with 1% osmium tetroxide, 2% uranyl acetate and lead citrate in that order. After slicing, observation was performed using a transmission electron microscope. The normal tight connection structure is tight and continuous, and the structure becomes loose and breaks after the tight connection is damaged.

The result shows that: 2-bromopalmitic acid (2-BP) was effective in reducing the abnormal elevation of protein palmitoylation in the testes of mice caused by PA injection (as shown in FIG. 5). For PA-induced dysspermia, 2-BP has significant therapeutic effects, not only increasing sperm concentration (as shown in figure 6), but also increasing sperm motility (as shown in figure 7). The in vivo FITC-I fluorescence leakage assay (shown in FIG. 8) and electron microscopy (shown in FIG. 9) also showed that 2-BP has the function of restoring blood-testosterone barrier integrity.

As described above, although the present invention has been shown and described with reference to certain preferred embodiments, it is not to be construed as limiting the invention itself. Various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims.

Claims

The application of 2-bromopalmitic acid in preparing medicine for treating diseases related to spermatogenic dysfunction is characterized in that: the diseases related to spermatogenic dysfunction are spermatogenic dysfunction caused by lipid metabolism disorder.
2. The use according to claim 1, characterized in that: the medicine for treating diseases related to spermatogenic dysfunction comprises 2-bromopalmitic acid.
3. The use according to claim 1, characterized in that: the medicine for treating diseases related to spermatogenic dysfunction is a medicine for improving the function of testis supporting cell barrier, a medicine for improving characteristic genes in the process of forming cell barrier or a medicine for inhibiting endoplasmic reticulum stress caused by saturated fatty acid.
4. A use according to claim 3, characterized in that: the characteristic genes comprise one or more of ZO-1, occludin, claudin-11 and Claudin-5.
5. A use according to claim 3, characterized in that: when the medicine for treating the diseases related to spermatogenic dysfunction is used, the administration dosage of the 2-bromopalmitic acid is 2-40mg/kg.
6. The use according to claim 1, characterized in that: the medicament for treating the diseases related to the spermatogenic dysfunction is in the form of injection, oral preparation or microcapsule preparation.
7. The use according to claim 1, characterized in that: the medicine for treating the diseases related to spermatogenic dysfunction is at least one of oral administration, parenteral administration, inhalation spray administration or local administration.
8. The use according to claim 1, characterized in that: the medicine for treating the diseases related to the spermatogenic dysfunction is at least one of rectal administration, nasal administration, buccal administration, testicular injection administration or administration through an implanted medicine storage device.
9. The use according to claim 7, characterized in that: parenteral administration includes at least one of subcutaneous, intradermal, intravenous, intramuscular, intra-articular, intra-arterial, intra-synovial, intrasternal, lesion or intracranial administration.