Application of sodium 4-phenylbutyrate in product for improving dyszoospermia of obese males

Abstract

The invention discloses application of sodium 4-phenylbutyrate in a product for improving dyszoospermia of obese males, and belongs to the technical field of biological medicines. High-fat feed is adopted to construct a rat obesity model, the protection effect of 4-PBA on the reproductive function of obese rats is studied, and experimental results show that after the 4-PBA intervenes the obese rats, the testosterone content can be obviously increased, the estradiol level can be reduced, the sperm motility can be improved, the sperm malformation rate can be reduced, the spermatogenic dysfunction can be obviously relieved, and the anti-obesity effect on testis tissue morphology, testis tissue morphology, testis tissue morphology, testis tissue morphology and testis tissue morphology can be improved. The functions of supporting cell mitochondria, endoplasmic reticulum and MAM structures are obviously improved, and a new treatment strategy and a new drug target are provided for intervening and controlling obesity induced testis dyszoospermia and improving fertility of obese males.

Description

TECHNICAL FIELD

[0001] The application relates to the field of biological medicine, in particular to application of sodium 4-phenylbutyrate in improving spermatogenic disorder of obese males. BACKGROUND

[0002] Obesity is a chronic metabolic disease caused by the interaction of multiple factors such as genetics, lifestyle and environment, and is characterized by fat tissue accumulation and systemic inflammation. Obesity-induced diseases such as diabetes, cardiovascular disease and mental abnormalities seriously threaten human health. Among them, the different degrees of sperm quantity reduction, semen quality decline and testosterone synthesis disorder caused by obesity in men of childbearing age are important causes of male infertility, which seriously affect the reproductive health of men of childbearing age. Therefore, in the current environment of the decreasing male fertility, it is a current research hotspot to clarify the molecular mechanism of obesity-induced spermatogenic disorder and to explore new drugs for preventing and treating obesity and its complications.

[0003] The mechanism of obesity-induced male reproductive dysfunction is complex and is still unclear. High-fat diet (HFD) is one of the main causes of obesity, and the correlation between obesity induced by high-fat diet and spermatogenic disorder has not been fully addressed. At present, there is a lack of specific treatment drugs in the clinic, and most of them focus on lifestyle adjustment or hormone therapy, which lack direct targeting drugs, resulting in limited efficacy or obvious side effects. Endoplasmic reticulum stress inhibitor sodium 4-phenylbutyrate has been proved to have protective effect in various disease models such as obesity, but its application in obesity-related spermatogenic disorder has not been reported. SUMMARY

[0004] In view of the above problems, the present application provides a new application of 4-PBA in improving spermatogenic disorder of obese rats, which provides a new treatment strategy and drug target for intervention and control of obesity-induced testicular spermatogenic disorder and improvement of male fertility in obese males.

[0005] To achieve the above purpose, the technical scheme adopted by the present application is as follows:

[0006] The application of sodium 4-phenylbutyrate in improving spermatogenic disorder of obese males is the application in preventing, improving and / or treating male infertility disorder in obese males.

[0007] Further, the spermatogenic disorder in the application is caused by lipid metabolism disorder.

[0008] Further, the improvement in the application is improved by at least one of the following ways:

[0009] (a) protecting spermatogenic cells and / or supporting cells;

[0010] (b) reducing the rate of abnormal sperm;

[0011] (c) improving sperm motility;

[0012] (d) increasing testosterone content;

[0013] (e) reducing estradiol level;

[0014] (f) protecting mitochondrial-endoplasmic reticulum structure coupling function in testicular Sertoli cells;

[0015] (g) up-regulating expression of MAMs marker proteins VDAC1 / IP3R1 and / or key tethering protein Mfn2;

[0016] (h) down-regulating endoplasmic reticulum stress core marker protein GRP78 and / or inhibiting products of PERK pathway activation.

[0017] Further, the product in the application comprises a drug.

[0018] Another object of the present application is to provide a pharmaceutical composition for preventing or treating obesity-related male spermatogenic dysfunction, comprising sodium 4-phenylbutyrate or a pharmaceutically acceptable salt thereof.

[0019] Further, the pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers.

[0020] Further, the dosage form of the pharmaceutical composition is a tablet, a capsule, a granule, a powder or an oral suspension.

[0021] According to the above technical solution, the present application discloses the application of sodium 4-phenylbutyrate in products for preventing obesity-related male spermatogenic dysfunction. The present application uses high-fat feed to construct a rat obesity model, studies the protective effect of 4-PBA on the reproductive function of obese rats, and the experimental results show that after 4-PBA intervention in obese rats, the testosterone content can be significantly increased, the estradiol level can be reduced, the sperm motility can be increased, the sperm deformity rate can be reduced, the spermatogenic dysfunction can be significantly relieved, and the testicular tissue morphology, Sertoli cell mitochondria, endoplasmic reticulum and MAM structure function can be obviously improved. The present application provides a new treatment strategy and drug target for intervention and control of obesity-induced testicular spermatogenic dysfunction and improvement of fertility in obese men. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 : Testicular morphological observation chart of each group of rats (×200);

[0023] Figure 2 : Testicular Sertoli cell ultrastructure observation chart of each group of rats (TEM, ×4000);

[0024] Figure 3 : Testicular seminiferous tubule VDAC1 and IP3R1 co-expression result chart of each group of rats (×400);

[0025] Figure 4 Fig. 4 is a graph showing the expression of Mfn2 in the testicular seminiferous tubules of rats in each group (x400);

[0026] Figure 5 Fig. 5 is a graph showing the expression of GRP78 in the testicular seminiferous tubules of rats in each group (x400);

[0027] Figure 6 Fig. 6 is a graph showing the expression of PERK in the testicular seminiferous tubules of rats in each group (x400). DETAILED DESCRIPTION

[0028] The technical solutions in the embodiments of the present application will be clearly and completely described below. Obviously, the described embodiments are only some of the embodiments of the present application, but not all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0029] The reagents involved in the embodiments of the present application are all purchased from the market channel, and the methods not mentioned are conventional experimental methods, which will not be described one by one here.

[0030] Rat E2 ELISA Kit (Item No.: MM-0575R2), Rat T ELISA Kit (Item No.: MM-0577R2), Rat P ELISA Kit (Item No.: MM-0551R2), Rat FSH ELISA Kit (Item No.: MM-70867R2), Rat LH ELISA Kit (Item No.: MM-0624R2), and Rat PRL ELISA Kit (Item No.: MM-0563R2) are all purchased from Jiangsu Enzyme Immune Industry Co., Ltd.

[0031] Example 1

[0032] I. Experimental materials

[0033] 1. Experimental animals

[0034] A total of 30 4-5-week-old male SD rats (purchased from Beijing Huafukang Biosciences Co., Ltd.) were selected. Before formal modeling, the rats were fed in cages (5 rats per cage), and adaptive feeding was performed for 3 days. The experimental environment temperature was 22℃, the relative humidity was 40%, the day and night time distribution was uniform, and the self-feeding and drinking water were 12h.

[0035] 2. Feed

[0036] Basic feed: purchased from Beijing Keao Cooperation Feed Co., Ltd.

[0037] High-fat feed: 70% basic feed + 15% lard + 15% sucrose, purchased from Beijing Keao Cooperation Feed Co., Ltd.

[0038] 3. Related solutions and drug preparation

[0039] 4-phenylbutyric acid sodium aqueous solution: 1 g of 4-phenylbutyric acid sodium solid powder (purchased from MedChemExpress LLC, purity 99.86%) was weighed into 10 mL of purified water, ultrasonic assisted dissolution (power: 5 W to 15 W, temperature: 15-20 °C, time: 1-3 minutes), prepared into a 4-phenylbutyric acid sodium aqueous solution with a concentration of 100 mg / mL, and filtered and sterilized with a 0.22 μm PVDF material needle filter before use, and prepared as needed.

[0040] II. Construction and intervention of obese rat model

[0041] (1) 4-5 week old male SD rats were selected, and after 3 days of adaptive feeding, they were randomly divided into 10 control group and 20 model group. The control group was given basic feed, and the model group was given high-fat feed, and both groups were free to drink water and eat, and after 20 weeks of continuous feeding, the body weight of the rats was detected. When the average body weight of the rats in the model group was 10%-25% higher than that of the control group, it was determined that the obese rat model was successfully constructed.

[0042] (2) The SD rats with successfully constructed models were evenly distributed according to the body weight interval into 10 model group and 10 4-PBA group. The 4-PBA group was given 4-phenylbutyric acid sodium aqueous solution (concentration 100 mg / mL) to the rats at a dose of 350 mg / kg by gavage, and the control group and the model group were given the same volume of normal saline by gavage, once a day, for 4 weeks of continuous intervention, and the diet conditions of the animals in each group remained unchanged during the period.

[0043] III. Sample collection

[0044] After the intervention, the SD rats in each group were fasted for 24 h, and the body weight of the rats was weighed and labeled with an electronic balance. The rats were given anesthesia (50 (15 mg / kg) + xylazine (5 mg / kg) combined intraperitoneal injection), fixed in a supine position, and blood was collected from the abdominal aorta. The testis tissue was taken, and the left testis of each rat was placed in Davidson's fixative (the volume ratio of fixative to tissue was not less than 50:1), and after 24 hours of fixation, the tissue was cut into pieces, and conventional paraffin sections were prepared with a thickness of 3 μm. The right testis fresh tissue was taken 1 mm x 1 mm x 1 mm with 2.5% glutaraldehyde fixative (Solarbio, P1126, Beijing Solarbio Technology Co., Ltd.) at 4°C, and another part was directly frozen at -80°C. The tail of the epididymis tissue was taken for sperm function detection.

[0045] Four, 4-phenyl sodium butyrate multi-dimensional intervention effect evaluation

[0046] 1. Determination of serum lipid metabolism indicators

[0047] Whole blood was taken from 6 rats in each group, centrifuged at 3000 rpm and 4°C for 10 min, and 0.5 mL of supernatant serum was taken. The levels of triglyceride (TG) and cholesterol (CHO) in the serum were detected by using a full-automatic biochemical analyzer. The experimental data was represented by SPSS19.0 statistical software was used, and the significant difference test between groups was performed by one-way ANOVA and pairwise comparison. P<0.05 was considered statistically significant, and the results are shown in Table 1.

[0048]

[0049] As shown in Table 1, compared with the control group, the serum triglyceride level and cholesterol of the model group rats were increased, especially the triglyceride level was significantly increased (P<0.01); compared with the model group, the triglyceride and cholesterol levels of the 4-PBA group were decreased, especially the triglyceride level was significantly decreased (P<0.05).

[0050] 2. Determination of serum sex hormone indicators

[0051] The serum of all rats in each group was taken, and the commercially available biochemical kit of Jiangsu Enzyme Immune Industry Co., Ltd. was used to detect the sex hormone indicators of rats, such as testosterone, follicle stimulating hormone, estradiol, luteinizing hormone, prolactin and progesterone, according to the manufacturer's instructions. The experimental data was represented by SPSS19.0 statistical software was used, and the significant difference test between groups was performed by one-way ANOVA and pairwise comparison. P<0.05 was considered statistically significant, and the results are shown in Table 2.

[0052]

[0053] As shown in Table 2, compared with the control group, the serum estradiol level of the model group rats was significantly increased (P<0.05), and the testosterone level was significantly decreased (P<0.01); compared with the model group, the estradiol level of the 4-PBA group was decreased (P<0.05), and the testosterone level was significantly increased (P<0.05).

[0054] 3. Sperm function detection

[0055] Take the epididymal tail tissue of all rats in each group, remove the adipose tissue, and then place in 37℃ preheated 0.9% NaCl solution (NaCl solution and tissue volume ratio is not less than 10:1), use ophthalmic surgical scissors to break, then culture in 37℃, 5% CO2 incubator for 15 min, so that the sperm can fully swim out, and then take the supernatant to 1.5 mL Ep tube, mix thoroughly, and then take 10 μL to preheated detection plate, place on the sperm analyzer (Mai Lang computer aided sperm analyzer, ML-608JZ III), and collect 5 field images in sequence according to the upper, middle, lower, left and right of the slide, and take the average value as the detection result. After the image collection is completed, adjust the gray value, and complete the detection of sperm density, motility and deformity rate through automatic tracking.

[0056] The experimental data are represented by SPSS19.0 statistical software, and the significant difference test between groups uses one-way ANOVA and pairwise comparison, and P<0.05 is considered to have statistical significance, and the results are shown in Table 3.

[0057]

[0058] As shown in Table 3, compared with the control group, the sperm motility of the model group was significantly reduced (P<0.05), the sperm deformity rate was significantly increased (P<0.01), and the sperm density, linear velocity and curved velocity were decreased, but the difference was not significant. Compared with the model group, the sperm motility of the 4-PBA group was increased (P<0.05), and the sperm deformity rate was decreased (P<0.05).

[0059] 4. Hematoxylin-eosin staining to observe the morphological and structural changes of testis of rats in each group

[0060] The paraffin sections of each rat were sequentially immersed in xylene I for 30 min, xylene II for 30 min, anhydrous ethanol for 5 min, 95% ethanol for 5 min, 85% ethanol for 5 min, 75% ethanol for 5 min, and distilled water for 5 min; stained with hematoxylin dye for 4 min, washed with tap water; differentiated with 1% hydrochloric acid alcohol differentiation solution for 30 s, washed with tap water, and returned to blue with dilute ammonia water (1%); stained with eosin dye for 1 min, washed with tap water, and then sequentially immersed in 75% ethanol for 30 s, 85% ethanol for 30 s, 95% ethanol for 30 s, anhydrous ethanol I for 30 s, and anhydrous ethanol II for 1 min, air-dried, and then transparent in xylene I and II for 5 min each time, and finally added with neutral balsam for mounting. Observed under an optical microscope (×200), and the results are shown in Figure 1 .

[0061] From Figure 1It was found that in the control group, the seminiferous tubules of the testis were intact, the cells were arranged tightly and orderly, and there were no obvious changes in the seminiferous epithelium. In the model group, the spermatogenic cells of the testis were sparsely arranged, the number of cell layers was reduced, the supporting cells were degenerated and showed a "vacuolar" change, and the intercellular spaces between adjacent cells were widened, indicating that obesity caused damage to the testis tissue, especially affecting the spermatogenic cells and supporting cells. In the 4-PBA group, the cells in the seminiferous tubules of the testis were arranged more tightly and orderly, and the number of cell layers was increased.

[0062] 5. Changes in the endoplasmic reticulum, mitochondria, and mitochondrial-associated endoplasmic reticulum membrane of rat testicular supporting cells in each group: Fresh testicular tissue (1mm × 1mm × 1mm) from each rat was fixed at 4℃ for 6 hours with pre-cooled 2.5% glutaraldehyde fixative (Solarbio, P1126, Beijing Solarbio Technology Co., Ltd.). After fixing, the fixative was discarded, and the tissue blocks were rinsed with pre-cooled 0.1M PBS (pH 7.4) (rinsing 3 times, 15 min each time). Fixation was then performed for 2 hours in the dark with 1% osmium tetroxide (prepared in 0.1M PBS, pH 7.2-7.4). Gradient ethanol dehydration was performed: 50% ethanol (4℃), 70% ethanol (4℃), 80% ethanol (4℃), 90% ethanol (room temperature), 95% ethanol (room temperature), and 100% ethanol (room temperature), 15 min each. After ethanol dehydration, the tissue was permeated with epoxy resin for 4 hours and then transferred to pure resin overnight (12-24 hours) to ensure complete resin penetration into the tissue. The fully infiltrated tissue blocks were placed in embedding plates filled with fresh resin and heated in a 60°C oven for 24 hours to allow the resin to fully polymerize and harden. After polymerization, the embedded blocks were trimmed and sectioned at 50-70 nm for ultrathin sectioning and staining. A transmission electron microscope (JEOL JEM1200EX, Nippon Electron Ltd.) was used with an accelerating voltage of 80 kV, and a small-to-medium aperture condenser was selected. 100 μm or 50 The objective aperture was 30 μm. Observations and imaging of mitochondria, endoplasmic reticulum, and MAM at different magnifications are shown in the figure. Figure 2 .

[0063] Depend on Figure 2 It was observed that in the control group, the mitochondria (Mi) within the supporting cells were round, with intact inner and outer membrane structures, dense mitochondrial cristae, and neat, clear arrangement. The endoplasmic reticulum (ER) in the control group had normal structure, arranged in flattened sacs, with tight mitochondrial-associated reticulum membrane (MAM) connections and frequent contact. In the model group, mitochondria were significantly swollen, with broken or missing cristae, appearing vacuolated. The endoplasmic reticulum was dilated and swollen, with partial membrane rupture, loose MAMs, and fewer coupling structures. In the 4-PBA group, mitochondrial and endoplasmic reticulum damage was less severe than in the model group, with tighter MAM connections and more coupling structures.

[0064] 6. Immunofluorescence staining to trace seminiferous tubule MAMs marker proteins, binding protein Mfn2, endoplasmic reticulum stress, and the PERK pathway.

[0065] The paraffin sections of each rat were sequentially immersed in xylene I for 30 min, xylene II for 30 min, anhydrous ethanol for 5 min, 95% ethanol for 5 min, 85% ethanol for 5 min, 75% ethanol for 5 min, and distilled water for 5 min; 3% hydrogen peroxide was added dropwise, and incubated at room temperature for 10 min to block endogenous peroxidase; the antigen was repaired by microwave heating with citric acid buffer, and blocked with 50 g / L bovine serum albumin for 20 min; the diluted primary antibody working solution (see Table 4 for specific information) was added dropwise, and incubated at 4°C overnight; after washing with PBS, the IP3R1 and VDAC1 double staining group was treated according to the double-labeling three-color fluorescence detection kit (RS0036, Immunoway), and the rest of the groups were added with Alexa Fluor 488 fluorescently labeled secondary antibody, and incubated at room temperature for 1 h in the dark; after washing with PBS, the sections were mounted with anti-fluorescence quenching mounting medium containing DAPI (S2110, Beijing Solaybao Technology Co., Ltd.), and observed under a fluorescence microscope in a dark room. The results of VDAC1 and IP3R1 co-expression, Mfn2 expression, GRP78 expression, and PERK expression in the testis seminiferous tubules of the rats in each group are shown in Figs. Figure 3 、 4 、5、6.

[0066]

[0067] The expression of mitochondrial and endoplasmic reticulum specific marker proteins VDAC1 and IP3R1, and MAM binding protein Mfn2 was reduced in the model group, and the fluorescence intensity was significantly weakened, and the expression was increased in the 4-PBA group compared with the model group. Figures 3-4 The expression of endoplasmic reticulum stress marker proteins GRP78 and PERK was increased in the model group, and the fluorescence intensity was significantly increased, and the expression was significantly reduced in the 4-PBA group compared with the model group. Figures 5-6

[0068] In summary, obesity can cause endoplasmic reticulum stress in the testis of rats, leading to changes in the structure and function of MAM. Therefore, it can be inferred that the spermatogenic dysfunction induced by high-fat diet in obese rats may be caused by changes in the structure and function of mitochondria, endoplasmic reticulum, and mitochondria-related endoplasmic reticulum membrane in Sertoli cells due to endoplasmic reticulum stress. As an endoplasmic reticulum stress inhibitor, sodium 4-phenylbutyrate can significantly increase the content of testosterone, reduce the level of estradiol, increase sperm motility, reduce the rate of sperm abnormality, and significantly alleviate spermatogenic dysfunction. It also has a significant improvement effect on the structure and function of testicular tissue, Sertoli cell mitochondria, endoplasmic reticulum, and MAM. The results show that sodium 4-phenylbutyrate can reduce endoplasmic reticulum stress in the testis of obese rats, improve the structure and function of Sertoli cell MAM, and improve spermatogenic dysfunction.

[0069] ​The various embodiments described in this specification are intended to be illustrative only and in no way limit the scope of the application. Changes and modifications can be made by those skilled in the art, which employ the principles of the application, without departing from the scope of the application. Accordingly, the application is not limited to the embodiments described herein, but instead has scope to encompass any choice whatsoever that is dependent on, or can be substituted in, the principal, new and inventive features that are described and claimed herein.

[0070] The above description of disclosed embodiments is intended to be illustrative only and not limiting of the application. Numerous modifications to these embodiments can be made by those skilled in the art without departing from the spirit or scope of the application. The scope of the application is not limited to the embodiments described herein, but rather extends to any that are dependent on, or can be substituted in, the principal, new and inventive features that are described and claimed herein.

Claims

1. Use of sodium 4-phenylbutyrate in improving spermatogenic impairment in obese men, characterized in that, The application is the application of the product in preventing, improving and / or treating obesity male infertility disorder.

2. The use of sodium 4-phenylbutyrate according to claim 1 for improving spermatogenic disorders in obese men, characterized in that, The spermatogenic disorder is caused by lipid metabolism disorder. 3.The use of sodium 4-phenylbutyrate according to claim 1 in a product for improving spermatogenic disorder in obese men, characterized in that, The improvement is achieved by at least one of the following ways: (a) protecting spermatogenic cells and / or Sertoli cells; (b) reducing the rate of abnormal sperm; (c) improving sperm motility; (d) increasing the content of testosterone; (e) reducing the level of estradiol; (f) protecting the mitochondrial-endoplasmic reticulum structure coupling function in Sertoli cells; (g) up-regulating the expression of MAMs marker protein VDAC1 / IP3R1 and / or key tethering protein Mfn2; (h) down-regulating the endoplasmic reticulum stress core marker protein GRP78 and / or inhibiting the product of PERK pathway activation.

4. Use according to any one of claims 1 to 3, characterized in that, The product comprises a drug.

5. A pharmaceutical composition for preventing or treating obesity-related male spermatogenic dysfunction, characterized by, The drug composition further comprises one or more pharmaceutically acceptable carriers.

6. The pharmaceutical composition for preventing or treating obesity-induced male spermatogenic dysfunction according to claim 5, wherein the pharmaceutical composition is administered to a patient in need of the treatment. The dosage form of the drug composition is a tablet, a capsule, a granule, a powder or an oral suspension.

7. The pharmaceutical composition for preventing or treating obesity-induced male spermatogenic dysfunction according to any one of claims 5-6, wherein the composition is administered to a patient in need of the treatment in an amount of 0.1-10 mg / kg per day. ​

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