New applications of a testicular metabolite, 2-propylthiazolidine-4-carboxylic acid

Abstract

The application belongs to the technical field of reproductive health, and particularly relates to a new application of a testicular metabolite 2-n-propylthiazolidine-4-carboxylic acid (PTCA), and the 2-n-propylthiazolidine-4-carboxylic acid is used for preparing a drug for treating testicular spermatogenic disorder induced by AFB1. The PTCA can promote the secretion of testosterone of testicular interstitial cells, activate the PI3K / AKT / NRF2 antioxidant signaling pathway in testicular tissue, relieve testicular injury induced by AFB1, and reduce the ROS level in testicular tissue. The application discloses, for the first time, the important role of the PTCA as a new testicular metabolic marker and protective agent in the male reproductive system, provides a new insight into the reproductive toxicity mechanism of AFB1, and lays an experimental foundation for the application of metabolites to the intervention strategy of male spermatogenic disorder.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of reproductive health, and particularly relates to a new application of a testicular metabolite 2-n-propylthiazolidine-4-carboxylic acid. BACKGROUND

[0002] Aflatoxin B1 (AFB1) is a secondary metabolite produced by Aspergillus flavus, which widely contaminates food crops and animal feed. It is a major challenge to global food safety and agricultural economy. AFB1 not only has strong hepatotoxicity and carcinogenicity, but also can accumulate in the human and animal body through the food chain, causing serious damage to the reproductive system. Studies have shown that AFB1 exposure can induce oxidative stress in testicular tissue, damage the blood-testis barrier, interfere with hormone synthesis, and induce sperm cell apoptosis, ultimately leading to decreased sperm quality and male fertility decline. In recent years, the development of metabolomics technology has provided a new perspective for discovering key endogenous small molecules that regulate the process of spermatogenesis. These metabolites are expected to become new strategies for antagonizing exogenous toxins and improving spermatogenic function.

[0003] In our previous study, we have confirmed that the complex probiotics composed of 1.0 x 10 5 CFU / mL of Saccharomyces cerevisiae, Lactobacillus casei and Bacillus subtilis can effectively alleviate the testicular Sertoli cell toxicity induced by AFB1. The present application aims to systematically explore the role of key metabolites in the testicular tissue in resisting the reproductive toxicity of AFB1. Through non-targeted metabolomics analysis, we successfully identified four differential metabolites that showed significant changes during the process of AFB1-induced spermatogenic disorder, including indoleacetic acid, adenosine, 2-n-propylthiazolidine-4-carboxylic acid (PTCA) and L-cysteine. In the in vitro testicular Leydig cell culture experiment, it was found that PTCA can promote the secretion of testosterone by Leydig cells and alleviate the cell toxicity induced by AFB1. To further evaluate the function of these metabolites in spermatogenesis, we verified them one by one by intratesticular injection. The results showed that PTCA can almost completely reverse the phenotypes of decreased sperm count, weakened sperm motility and decreased testosterone level caused by AFB1, showing excellent protective effect. In-depth mechanism research showed that PTCA can significantly enhance the body's antioxidant capacity and reduce the level of ROS by activating the PI3K / AKT / NRF2 signaling pathway in the testis, thereby alleviating the oxidative stress damage caused by AFB1. SUMMARY

[0004] The purpose of the present application is to provide a new application of a testicular metabolite 2-n-propylthiazolidine-4-carboxylic acid.

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

[0006] A new application of a testicular metabolite 2-propylthiazolidine-4-carboxylic acid (PTCA) for preparing a drug for treating testicular spermatogenic disorders induced by AFB1, wherein PTCA relieves testicular damage induced by AFB1 by promoting the secretion of testosterone in Leydig cells, activates the PI3K / AKT / NRF2 antioxidant signaling pathway in testicular tissue, and reduces the ROS level in testicular tissue.

[0007] The present application has the advantages that: the present application discloses for the first time the important role of PTCA as a new testicular metabolic marker and protective agent in the male reproductive system, not only provides new insights into the reproductive toxicity mechanism of AFB1, but also lays an experimental foundation for the application of metabolites in the intervention strategy for male spermatogenic disorders. BRIEF DESCRIPTION OF DRAWINGS

[0008] Figure 1 is the comprehensive biological response index evaluation of different treatment groups in the present application; wherein A-E are radar charts for reproductive toxicity evaluation of AFB1 group, MCP group, AFB1+LCP group, AFB1+MCP group and AFB1+HCP group, respectively; F is a columnar statistical chart of IBRV2 values of the five treatment groups; EPI: paired epididymal / body weight index, T: testosterone, SpC: sperm count, SpA: sperm abnormality rate, TSA: ratio of testicular cell apoptosis proteins Bax / Bcl-2. Low (LCP), medium (MCP) and high (HCP) respectively represent that the viable cell count of the complex probiotics is 1.0x10 5 , 1.0x10 6 and 1.0x10 7 CFU / mL

[0009] Figure 2 is a volcano plot of differential metabolites between the AFB1 group and the AFB1+MCP group. Note: log2FC: fold change of metabolite expression difference between the two groups; -log10(p_value): statistical test value of metabolite expression change difference; VIP value: influence intensity of corresponding metabolite difference in sample classification discrimination in the model, metabolites with VIP≥1 are significantly different (P<0.05). MCP: the viable cell count of the complex probiotics is 1.0x10 6 CFU / mL

[0010] Figure 3 is a growth curve graph of testicular Leydig cells under different density conditions in the cell experiment.

[0011] Figure 4 is a cell morphology microscopic observation graph of testicular Leydig cells under different density conditions in the cell experiment.

[0012] Figure 5 Figure 6 is the WB electrophoresis band and gray value statistical histogram of testis tissue. Group A: control group, no treatment; Group B: solvent control group, injection volume 10 μL, 5% DMSO solution; Group C: BPA group, 10 μL, 100 μg / μL dissolved in 5% DMSO solution; Group F: PTCA protection group, 10 μL, final concentration containing 100 μg / μL AFB1+200 μg / μL PDCA. On each histogram, significant differences at the level of P<0.05 are indicated by different lowercase letters (a, b and c), and the same letter indicates no significant difference. Each group contains 4 repeated samples.

[0013] Figure 6 Figure 7 is the immunofluorescence picture and average optical density statistics of ROS analysis of testis tissue of different treatment groups (scale 100 μm). Group A: control group, no treatment; Group B: solvent control group, injection volume 10 μL, 5% DMSO solution; Group C: BPA group, 10 μL, 100 μg / μL dissolved in 5% DMSO solution; Group F: PTCA protection group, 10 μL, final concentration containing 100 μg / μL AFB1+200 μg / μL PDCA.

[0014] Figure 7 Figure 8 is the HE staining and TUNEL analysis of different treatment groups. Testis tissue (Figure A) HE staining micrograph (scale 100 μm); testis tissue (Figure B) TUNEL staining micrograph (scale 100 μm); testis tissue (Figure C) TUNEL positive area statistical histogram. Group A: control group, no treatment; Group B: solvent control group, injection volume 10 μL, 5% DMSO solution; Group C: BPA group, 10 μL, 100 μg / μL dissolved in 5% DMSO solution; Group F: PTCA protection group, 10 μL, final concentration containing 100 μg / μL AFB1+200 μg / μL PDCA.

[0015] Figure 8 Figure 9 is the transmission electron micrograph of testis tissue of AFB1 group (scale 5 μm and 2 μm). Figure A: transmission electron micrograph of seminiferous tubule near the basement, the two black bands in the middle of the left picture are the basement of the seminiferous tubule, and the cells in the red box are spermatogonia; Figure B: transmission electron micrograph of seminiferous tubule near the lumen, the red box in the left picture is early spermatid; Figure C: transmission electron micrograph of seminiferous tubule near the basement, the cell in the red box in the left picture is Sertoli cell; Figure D is testicular interstitium, and the red box in the left picture is testicular interstitial cell. Red arrow: mitochondria; orange arrow: rough endoplasmic reticulum; green arrow: Golgi body; purple arrow: nucleus; blue arrow: blood-testis barrier; pink arrow: dense body; yellow arrow: lysosome; pink arrow: free lipid droplet. DETAILED DESCRIPTION

[0016] Embodiment

[0017] The application of a testicular metabolite, 2-propylthiazolidine-4-carboxylic acid, is characterized in that the 2-propylthiazolidine-4-carboxylic acid is used for preparing a drug for treating testicular spermatogenic disorder induced by AFB1, and the PTCA can promote the secretion of interstitial cell testosterone, activate the PI3K / AKT / NRF2 antioxidant signaling pathway in testicular tissue, relieve testicular injury induced by AFB1, and reduce the ROS level in testicular tissue.

[0018] Verification experiment

[0019] 1 Materials and methods

[0020] 1.1 Experimental materials

[0021] S. cerevisiae (CGMCC No. 2.3973), L. casei CGMCC No. 1.8727 and B. Subtilis CGMCC No. 1.821 were purchased from the China General Microbiological Culture Collection Center (CGMCC). L-cysteine, CAS: 52-90-4, molecular formula: C3H7NO2S, Beijing Solabio Co., Ltd., Catalog No.: C0012. Indoleacetic acid, CAS: 87-51-4, molecular formula: C 10 H9NO2, Beijing Solabio Co., Ltd., Catalog No.: I8020. Adenosine, CAS: 58-61-7, molecular formula: C 10 H 13 N5O4, Beijing Solabio Co., Ltd., Catalog No.: A8240. 2-propylthiazolidine-4-carboxylic acid (PTCA), CAS No.: 4165-34-8, molecular formula: C7H 13 NO2S, Shanghai Maikelin Biochemical Technology Co., Ltd., Catalog No.: 785258.

[0022] 1.2 AFB1 and compound probiotic gavage experiment

[0023] S. cerevisiae, L. casei and B. subtilis. L. casei was inoculated in MRS medium (g / L: peptone 10, yeast extract 10, glucose 20, Tween 80 1 mL, K2HPO42, sodium acetate 5, sodium citrate 2, MgSO40.2, MnSO40.05, pH 6.20-6.60) after sterilization at 121℃ for 30 min, and incubated at 37℃ for 24 h. B. subtilis was inoculated in LB medium (g / L: peptone 10, yeast extract 5, NaCl 10, pH 7.0), and incubated at 37℃ with 180 rpm for 24 h. S. cerevisiae was inoculated in YPD medium (g / L: peptone 20, glucose 20, yeast extract 10), and incubated at 30℃ with 180 rpm for 24 h. After the culture of the three microorganisms, the bacterial precipitate was centrifuged at 10000 r / min at 4℃ for 5 min, and then freeze-dried by adding 10%-50% skim milk powder, trehalose, sodium glutamate and surfactin, etc. The viable bacterial concentration (log CFU / mL) of each bacterial solution was determined by plate counting method after freeze-drying.

[0024] 60 7-week-old male Kunming mice (purchased from Henan Huaxing Animal Experiment Center) were adapted for one week under the same environment (12 / 12 h light-dark cycle). Then, healthy mice with the same body weight were randomly divided into 6 groups (n=10 / group), and the initial body weight was recorded. The previous body degradation experiment showed that the three kinds of viable bacteria of the compound probiotics were 1.0×10 5 CFU / mL, which had the highest AFB1 degradation rate. Therefore, the viable bacterial concentrations of the low, medium and high dose groups were set to 1.0×10 5 , 1.0×10 6 and 1.0×10 7 CFU / mL, respectively. The experimental grouping and treatment were as follows:

[0025] Group A (control): 0.5 mL of corn oil was administered intragastrically daily.

[0026] Group B (AFB1): 150 μg / kg BW of AFB1 (dissolved in 0.5 mL of corn oil) was administered intragastrically daily.

[0027] Group C (medium dose probiotics, MCP): 0.5 mL of corn oil suspension containing 150 μg / kg BW of AFB1+MCP (viable bacteria were all 1.0×10 6 CFU / mL) was administered intragastrically daily. The freeze-dried powder of the three bacterial bodies was resuspended with 0.5 mL of corn oil, and the final concentration of the three microorganisms was 3.0×10 7 CFU / mL. After ultrasonic dispersion for 15 s, it was administered intragastrically, and it was prepared on the spot).

[0028] D.E and F groups (AFB1 + probiotics): 0.5 mL of corn oil suspension containing 150 pg / kg BW AFB1 and low (D group, LCP), medium (E group, MCP) and high (F group, HCP) doses of complex probiotic bacteria (final concentration of viable bacteria was 1.0 x 10 5 , 1.0 x 10 6 and 1.0 x 10 7 CFU / mL, respectively) were administered daily by gavage.

[0029] The experimental period was 28 days, and the mice were weighed weekly. After weighing on day 28, 4 mice from each group were randomly sacrificed (cervical dislocation after orbital blood collection), and epididymis, testis, intestine, liver and other tissues were collected. After weighing, the samples were stored for subsequent testing.

[0030] 1.3 AFB1 and complex probiotic comprehensive biological response index evaluation (IBRV2)

[0031] TSI: paired testis / body weight index, EPI: paired epididymis / body weight index, T: testosterone, SpC: sperm count, SpA: sperm abnormality rate, TSA: testicular cell apoptosis protein Bax / Bcl-2 ratio, 6 indexes were selected for comprehensive biological response index evaluation (IBRV2).

[0032] 1.4 Metabolomics of mouse testis tissue

[0033] After the testis was taken out in liquid nitrogen, 80 milligrams of sample was added to 1 milliliter of methanol / acetonitrile / water (2:2:1, V / V) cold extraction solution and shaken thoroughly. First, use the homogenizer (MP Fastprep-245G) to homogenize twice, then ultrasonic extraction (30 minutes / time, twice) at 4°C, finally centrifuge the supernatant at 14,000g for 20 min and vacuum centrifuge the extraction solution at 4°C. The dried sample was redissolved with acetonitrile / water (1:1, V / V) solution, and the sample amount was 100 microliters for LC-MS.

[0034] LC-MS chromatographic conditions: ACQUITY UPLC T3 column (2.1 mm x 100 mm, 1.8 μm), column temperature 40 ℃, injection volume 2 μL, mobile phase 0.1% formic acid aqueous solution (A) - acetonitrile (B), flow rate 0.35 mL / min. Gradient elution (0-0.2 min, 5% B; 0.2-3 min, 5%-20% B; 3-6 min, 20%-45% B; 6-8 min, 45%-55% B; 8-14 min, 55%-65% B; 14-16 min, 65%-75% B; 16-20 min, 75%-95% B; 20-23 min, 95%-5% B). Mass spectrometry conditions: electrospray ion source, positive ion mode, capillary voltage 3 kV, ion source temperature 120 ℃, gas curtain flow rate 50 L / h, desolvation gas flow rate 800 L / h, gas flow temperature 400 ℃, cone hole voltage 40 V, collision energy 20-40 V, mass scan range m / z 50-1200; negative ion mode, capillary voltage 2.5 kV, collision energy 25-45 V, other parameters same as positive ion mode.

[0035] Each treatment group was repeated 4 times. The original MS data (wiff.scan file) was converted to MzXML format using Proteo Wizard MSConvert and imported into free software XCMS for analysis. Metabolite mass spectra were identified by matching with NIST 14.0 mass spectral library. Using MetaboAnalyst 5.0 (https: / / www.metaboanalyst.ca) software, differential expression metabolite analysis was performed according to FC value (FC>1.5) and P value (P<0.05).

[0036] 1.5 Effects of four differential metabolites on testosterone secretion of in vitro testicular Leydig cells

[0037] 1.5.1 Experimental materials

[0038] TM3 mouse Leydig cells (Cat. No. Delf-10326) were provided by HeFei WanWu Biological Technology Co., Ltd., AFB1 was purchased from Sigma Company; DMEM / F12 basic culture medium, penicillin-streptomycin solution (10000 U / mL penicillin G sodium salt, 10 mg / mL streptomycin sulfate), trypsin, fetal bovine serum, PBS buffer (1x) were purchased from Wuhan Punsen Life Science Co., Ltd.

[0039] 1.5.2 Cell culture

[0040] The interstitial cells were taken out from the freezer at -80°C for cell recovery. Placed in 5 mL of DMEM complete medium containing 10% fetal bovine serum, 1% penicillin-streptomycin solution, placed in a 37°C, 5% CO2 incubator for 24-48 h, when the cells grow to 80%-90% adherent, use 800 μL of trypsin for digestion, when the cells are all off the wall, stop digestion with 1 mL of complete medium. Centrifuge at 3000 r / min for 5 min, discard the supernatant, resuspend with 1 mL of complete medium and subculture.

[0041] 1.5.3 Growth curve of interstitial cells under different inoculation density conditions

[0042] The cells in the logarithmic growth phase and in good condition were selected for subculture. The cells were digested with 800 μL of trypsin, centrifuged, resuspended, and 10-100 μL of the cell suspension was taken to the cell counting plate under a microscope for counting, and different density cell suspensions were prepared and cultured in culture bottles. The cell viability was detected by MTT method. 10 μL of MTT with a concentration of 5 mg / mL was added to each well, and acted for 4 h. The cell supernatant was removed, 150 μL of dimethyl sulfoxide (DMSO) was added to each well, and then the plate was shaken at room temperature for 10 min. The absorbance values were measured at 490 nm and 630 nm wavelengths by a microplate reader. Relative cell viability (%) = (experimental group A490 nm-A630 nm) / (control group A490 nm-A630 nm) x 100%.

[0043] 1.5.4 Establishment of aflatoxin B1 injury model

[0044] The interstitial cells in the logarithmic growth phase were digested, centrifuged, resuspended, and 10-100 μL was taken to the cell counting plate for observation and calculation. 1.0 x 10 5 The cells were inoculated in a 96-well plate at a density of 1.0 x 10

[0045] 1.5.5 Establishment of IAA, PTCA, L-cysteine, and adenosine protection models

[0046] The interstitial cells in the logarithmic growth phase were digested, centrifuged, resuspended, and 10-100 μL was taken to the cell counting plate for observation and calculation. 1.0 x 10 5Cells were seeded in 96-well plates, and after 24 h of culture, different concentrations of IAA (0, 20, 40, 60, 80, 100 μmol / L), PTCA (0, 100, 200, 300, 400, 500 μmol / L), L-cysteine (0, 200, 400, 600, 800, 1000 μmol / L), and adenosine (0, 0.25, 0.5, 1 μmol / L) were added, respectively, with 6 replicates in each group. After 12 h, 24 h, and 48 h of continuous culture, the cell viability was detected by MTT method to screen the optimal relief time and concentration of the four substances for cell damage, and a protection model was established.

[0047] 1.5.6 Effects of the four metabolites on AFB1 damage protection and testosterone secretion

[0048] The interstitial cells in the logarithmic growth phase were digested, detached, centrifuged, and suspended, and 10-100 μL thereof was taken to a cell counting plate, which was observed under a microscope, and 1.0 x 10 5 Cells were seeded in 96-well plates, and after 24 h of culture, different concentrations of IAA (0, 20, 40, 60, 80, 100 μmol / L), PTCA (0, 100, 200, 300, 400, 500 μmol / L), L-cysteine (0, 200, 400, 600, 800, 1000 μmol / L), and adenosine (0, 0.25, 0.5, 1 μmol / L) were added, respectively, with 6 replicates in each group. After 12 h, 24 h, and 48 h of continuous culture, the cell viability was detected by MTT method to screen the optimal relief time and concentration of the four substances for cell damage, and a protection model was established.

[0049] 1.6 Verification of differential metabolites by testicular injection

[0050] 1.6.1 Pre-test of testicular injection

[0051] To further verify the in vivo application effect of the four different metabolites: indoleacetic acid, adenosine 2-n-propylthiazolidine-4-carboxylic acid (PTCA), L-cysteine, a test of testicular injection of the four substances was carried out, and an AFB1 spermatogenic disorder testicular injection model was established. Testicular injection operation: 8-10 week old healthy male Kunming mice, body weight 22-25 g, SPF level environment, standard diet and water, 12 / 12 light and dark cycle. Testicular injection operation: the mice were anesthetized with isoflurane inhalation, and were deeply anesthetized. Fix it, prepare the skin and disinfect the lower abdomen. Make a small incision (about 0.5 cm) on the scrotal skin, gently squeeze out the left testis, and expose it to the visual field. Avoid major blood vessels with a microsyringe (with a 33G fine needle), and slowly inject 10 μL of different treatment drug solution. Leave the needle for 10 seconds after injection to prevent liquid backflow. Gently put the testis back into the scrotum and suture the wound. The sham operation group was only sutured after the testis was exposed. Subcutaneous injection of saline and analgesic drugs (carprofen) after operation, single-cage feeding observation until recovery.

[0052] AFB1 spermatogenic disorder model group. The left testis was injected with AFB1 solution (10 μL, 100 μg / μL dissolved in 5% DMSO solution). One batch of animals was sacrificed at 1, 2, 3, 4, 5, and 7 weeks after injection, and the epididymis was removed for sperm motility test.

[0053] 4 substance safety dose group exploration: each group of animals injected with different concentrations of one metabolite (100, 200 and 300 μg / μL dissolved in 5% DMSO solution, 10 μL) into the left testis. Killed at 3 weeks after injection (according to the peak time of BPA effect) and test sperm motility.

[0054] 1.6.2 Testicular injection formal experiment

[0055] The results of the pre-experiment showed that the sperm motility decreased significantly on the 21st day after AFB1 injection, and the four substances had no significant effect on sperm motility in the concentration range of 100-300 μg / μL, so 200 μg / μL was selected for the formal experiment. The pre-treatment was the same as the pre-experiment, and the experimental design and grouping were as follows:

[0056] Group A: control group, no treatment

[0057] Group B: solvent control group, injection volume 10 μL, 5% DMSO solution

[0058] Group C: BPA group, 10 μL, 100 μg / μL dissolved in 5% DMSO solution

[0059] Group D: Indoleacetic acid protection group, 10 μΐ, final concentration containing 100 μg / μΐ BPA + 200 μg / μΐ indoleacetic acid

[0060] Group E: Adenosine protection group, 10 μΐ, final concentration containing 100 μg / μΐ BPA + 200 μg / μΐ adenosine

[0061] Group F: PTCA protection group, 10 μΐ, final concentration containing 100 μg / μΐ BPA + 200 μg / μΐ PDCA

[0062] Group G: L-cysteine protection group, 10 μΐ, final concentration containing 100 μg / μΐ BPA + 200 μg / μΐ L-cysteine.

[0063] 10 mice per group, on the 21st day after operation, after killing, the left epididymis was taken for sperm quality analysis, WB analysis, HE staining, TUNEL analysis and transmission electron microscopy analysis, respectively.

[0064] 1.6.3 WB analysis

[0065] The left testis of group A, B, C and F was selected for WB analysis. The testis tissue was ground with liquid nitrogen, and the total cell protein was extracted with RIPA lysis buffer (Yaenzyme Biotechnology Co., Ltd., Shanghai, China), and the total cell protein concentration was detected with BCA protein detection kit (Beijing Solaybao Biotechnology Co., Ltd., Shanghai, China). Equal amounts of protein were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membrane. The membrane was blocked with 5% skim milk in TBST buffer for 2 h, then incubated with primary antibody at 4°C overnight, then incubated with secondary antibody at room temperature for 2 h. Enhanced chemiluminescence was used to show the immunopositive band, and Image J software was used for quantitative analysis.

[0066] 1.6.4 HE staining and TUNEL analysis

[0067] HE staining and TUNEL analysis were performed on the left testis of groups A, B, C, and F. After fixation, the tissues were washed with water, dehydrated, and embedded in wax. Sections of approximately 6 pm in thickness were prepared, stained with hematoxylin-eosin (HE), and dehydrated with xylene. Finally, the sections were mounted with neutral resin on the edge, and observed under an optical microscope. Testicular cell apoptosis was detected using a TUNEL detection kit (Nanjing KeyGen Biotech Co., Ltd.). The detection process was performed according to the manufacturer’s protocol. The cells were counterstained with DAPI, and observed under an inverted microscope (Olympus, Tokyo, Japan). Images were randomly selected from three sections of each sample. The TUNEL-positive area was quantitatively analyzed using ImageJ software. The proportion of TUNEL-positive cells = (TUNEL-positive area (green fluorescence image) / total nuclear area (blue fluorescence image)) x 100%. Mixed images (200x and 400x) were used to observe the location of tissue apoptosis.

[0068] 1.6.5 Electron microscopy analysis of testicular tissue in the AFB1 group

[0069] To further clarify the damage to testicular cells, electron microscopy analysis was performed on the AFB1 group. Fresh testicular samples were fixed at 4°C for 6-8 h with 2.5% glutaraldehyde in phosphate buffer, followed by 1% osmium tetroxide for 1 h. The samples were then washed with 0.1 M phosphate buffer (pH 7.4), dehydrated with different concentrations of ethanol, and embedded in Epon 812 resin (SPI Supplies, West Chester, PA, USA). Rapid freezing was performed in liquid nitrogen-precooled isopentane at -80°C. After equilibration at -20°C, 5-8 pm thick sections were prepared, and observed using a FEI Tecnai G212 transmission electron microscope (FEI Company, Hillsboro, OR, USA).

[0070] 1.6.6 Immunofluorescence determination of ROS levels in testicular tissue

[0071] ROS levels in the left testes of groups A, B, C, and F were measured using immunofluorescence. Fresh testicular samples were fixed at 4°C in pre-cooled 4% paraformaldehyde for 6-8 hours and thoroughly washed with PBS. A sucrose gradient dehydration process was then performed to protect tissue morphology. After embedding with OCT compounds, the samples were rapidly frozen in isopentane pre-cooled by liquid nitrogen at -80°C. After equilibration at -20°C, the frozen tissue blocks were sectioned to a thickness of 5-8 μm, mounted on glass slides, and air-dried at room temperature for 30 minutes for subsequent staining. The sections were first incubated with 10 μmol / L LDHE solution for 30 minutes, followed by incubation in the dark for 10 minutes for nuclear staining with 4′,6-diamino-2-phenylindole (DAPI) staining solution. Finally, the sections were washed with PBS and photographed using fluorescence imaging. Cyt c and NRF2 protein expression levels were measured: cells were incubated with primary antibodies against NRF2 and Cyt c, respectively, at 4°C for 12 hours, followed by three washes with PBS. Next, cells were incubated with Cy3-linked anti-IgG secondary antibody at 25°C for 1 h, and the cell nuclei were stained with 4',6-diamidinyl-2-phenylindole (DAPI) (G1012; Wuhan Sangon Biotech Co., Ltd.). After standing in the dark at 25°C for 2 min, fluorescence imaging was performed. The fluorescence intensity of DHE and Cy3 in each group was observed and compared using a DM4000B inverted fluorescence microscope. The average fluorescence intensity was calculated by dividing the total fluorescence intensity of the recognition region by the area of ​​the recognition region.

[0072] 1.7 Data Processing

[0073] Experimental data are expressed as mean ± standard deviation. Analysis of variance (ANOVA) was performed using SPSS 20.0. The Duncan multivariate test was used to compare differences in means, and p < 0.05 was used to indicate a significant difference.

[0074] 2 Results Analysis

[0075] 2.1 Evaluation of comprehensive biological response indicators in the gavage experiment of AFB1 and compound probiotics

[0076] Six ratios were selected for comprehensive biological response index (IBRV2): TSI (paired testis / body weight index), EPI (paired epididymis / body weight index), T (testosterone), SpC (sperm count), SpA (sperm abnormality rate), and TSA (testicular cell apoptosis protein Bax / Bcl-2 ratio). Specific data are shown in Table 1. IBRV2 values ​​for the five treatment groups are shown in Table 1. Figure 1 ,Depend on Figure 1 F shows that the IBRV2 of the AFB1 group was 3.52, which was significantly higher than that of the other treatment groups (P < 0.05); the IBRV2 of the AFB1+MCP group was 0.48, which was not significantly different from that of the MCP group (P > 0.05), but was significantly lower than that of the AFB1 group (P < 0.05).

[0077] Table 1 Comprehensive biological response index evaluation index

[0078] TSI % EPI % Tng / mL SPC SPA % TSA Control group 0.66±0.05 0.28±0.02 1.12±0.11 3.12±0.23 57.65±2.12 2.12±0.11 ​ 0.58±0.06 0.26±0.07 0.72±0.09 2.56±0.13 70.82±2.12 7.56±0.23 MCP 0.65±0.08 0.23±0.07 1.07±0.08 3.35±0.11 56.24±2.12 2.35±0.09 [["afb1 + lcp"]] ​ 0.75±0.2 0.25±0.04 0.91±0.07 2.82±0.16 68.83±2.12 3.82±0.15 ​ 0.79±0.11 0.29±0.08 1.08±0.09 3.31±0.25 61.29±2.12 2.31±0.12 [["afb1+hcp"]] ​ 0.69±0.09 0.27±0.11 1.11±0.13 3.32±0.19 57.15±2.12 3.32±0.17

[0079] 2.2 Analysis of differential metabolites between AFB1 group and AFB1+MCP group in testicular tissue

[0080] According to the standard of FC>1.5, P<0.05, the screening of differential metabolites between the two groups was carried out, and the volcano plot of differential metabolites was drawn as shown in Figure 2 . A total of 228 named differential metabolites were identified between AFB1 group and AFB1+MCP group, of which 146 were identified in positive ion mode, and the statistics of typical differential metabolites are shown in Table 2. Four typical differential metabolites (P<0.05) were screened by FC value, including indoleacetic acid, adenosine, 2-n-propylthiazolidine-4-carboxylic acid and L-cysteine.

[0081] Table 2 Details of typical differential metabolites between AFB1 group and AFB1+MCP group in positive ion mode

[0082]

[0083] 2.3 In vitro Leydig cell experiment

[0084] 2.3.1 Growth curve and morphological observation of Leydig cells

[0085] The growth curve of Leydig cells under different cell density conditions is shown in Figure 3 , and the cell morphology is shown in Figure 4 . As can be seen from Figure 3 , the absorbance value of Leydig cells increases with the increase of cell density and culture time, and the absorbance value of Leydig cells cultured for 60h under cell density of 1.0×10 6 and 5.0×10 5 is greater than 1.0. The cell morphology observation shows that the cells are irregular rods, and the cell fusion degree reaches 70% after 36-48h of culture. According to the absorbance value and microscopic observation, the cell density of 1.0×10 5 was selected, and the cells were inoculated after 48h of culture, which were used for subsequent model.

[0086] 2.3.2 Screening of AFB1 damage model

[0087] The results of AFB1 on cell survival rate are shown in Table 3. As shown in Table 3, the cell viability decreased to 77.89% when AFB1 was used at a concentration of 8 μmol / L for 24 h, which was significantly higher than other concentrations and time (P<0.05). Therefore, 8 μmol / L of AFB1 was selected as the toxin damage model for subsequent studies.

[0088] Table 3 Effects of AFB1 on cell survival rate (%)

[0089] Note: In the same column, different capital letters (A, B, C) represent significant differences (P<0.05), and the same capital letter represents no significant difference (P>0.05); in the same row, different small letters (a, b, c, d) represent significant differences (P<0.05), and the same small letter represents no significant difference (P>0.05), and so on.

[0090] 2.3.3 Mitigating effects of four substances on damaged cells

[0091] The results of IAA on cell survival rate are shown in Table 4. As shown in Table 4, the cell viability reached a maximum of 118.88% when IAA was used at a concentration of 80 μmol / L for 24 h, which was significantly higher than other concentrations and time (P<0.05). Therefore, this concentration and time were selected for subsequent protection experiments.

[0092] Table 4 Effects of IAA on cell survival rate (%)

[0093]

[0094] The results of PTCA on cell survival rate are shown in Table 5. As shown in Table 5, the cell viability reached a maximum of 115.90% when PTCA was used at a concentration of 300 μmol / L for 24 h, which was significantly higher than other concentrations and time (P<0.05). Therefore, this concentration and time were selected for subsequent protection experiments.

[0095] Table 5 Effects of PTCA on cell survival rate (%)

[0096]

[0097] The results of L-cysteine on cell survival rate are shown in Table 6. As shown in Table 6, the cell viability was significantly lower than the control group (P<0.05) as the concentration of L-cysteine increased when it was used for 24 h. L-cysteine had no effect on the proliferation of interstitial cells.

[0098] Table 6 Effects of L-cysteine on cell survival rate (%)

[0099]

[0100] The results of adenosine on cell survival rate are shown in Table 7. As shown in Table 7, when adenosine acts on cells for 24 hours, the cell viability is significantly lower than that of the control group (P<0.05) with the increase of the concentration, and has no effect on the proliferation of interstitial cells.

[0101] Table 7 Effect of adenosine on cell survival rate (%)

[0102]

[0103] 2.3.4 IAA, PTCA protective effect on AFB1 induced cytotoxicity

[0104] The results of IAA and PTCA respectively incubated with AFB1 on cell survival rate are shown in Table 8. As shown in Table 8, the change trend of cell viability and testosterone is the same. PTCA can significantly improve the cell viability and testosterone level (AFB1 group significantly reduces the cell viability and testosterone level P<0.05), and IAA has no significant effect on the cell viability and testosterone level (P>0.05). When PTCA and IAA are added to the AFB1 group respectively, the cell viability and testosterone level are up-regulated to have no significant difference with the control group (P<0.05), so PTCA and IAA have significant alleviating effect on AFB1 induced interstitial cell toxicity in vitro.

[0105] Table 8 Effect of IAA and PTCA incubated with AFB1 for 24 hours on cell viability and testosterone

[0106] Control group IAA group PTCA group ​ IAA + AFB1 group PTCA + AFB1 group Cell viability (%) 100.0 ± 7.21 b ]] 100.13 ± 3.11 b ]] 110.03 ± 2.17 a ]] 76.27 ± 4.11 c ]] 101.50 ± 7.21 b ]] 106.27 ± 4.11 ab ]] Testosterone (pg / mL) 532.13 ± 16.21 b ]]> 519.21 ± 17.19 b ]] 586.65 ± 24.21 a ]] 315.89 ± 25.32 c ]] 515.54 ± 31.69 b ]] 510.14 ± 21.29 b ]]

[0107] 2.4 Verification of testicular injection effect of differential metabolites

[0108] 2.4.1 Differential metabolites alleviate AFB1 induced spermatogenic disorder in testis

[0109] The data analysis results (Table 9) show that the sperm count, total sperm motility and testosterone concentration of AFB1 testicular injection (group C) are significantly lower than those of the control group (groups A and B) (P<0.05). Compared with the AFB1 group, the four kinds of differential metabolite protection treatments all show different degrees of protection effect. Among them, the protection effect of PTCA protection group (group F) is the most significant, and the sperm count, total motility and testosterone level are restored to the level without significant difference with the control group (P>0.05). In summary, PTCA shows almost complete protection in antagonizing the reproductive toxicity caused by AFB1.

[0110] Table 9 Effect of testicular injection of four kinds of differential metabolites on the weight and testosterone of Kunming mice

[0111]

[0112] 2.4.2 PTCA alleviates AFB1-induced testicular spermatogenic disorder by activating NRF2 antioxidant pathway

[0113] Figure 5 The results showed that AFB1 (group B) significantly down-regulated the expression levels of PI3k, AKT and NRF2 antioxidant pathway related proteins (P<0.05). The expression levels of the above proteins were increased to no significant difference with the control group after AFB1 and PTCA co-injection into testis (group F). Consistent with the change trend of PI3K protein expression, the phosphorylation level of PI3K p110 catalytic subunit was significantly reduced under the induction of AFB1 (P<0.05); and the phosphorylation level after co-injection of AFB1 and PTCA into testis was not significantly different from the control group (P>0.05). The phosphorylation level of NRF2 was significantly higher in PTCA (group C) and PTCA+AFB1 (group F) than in the control and AFB1 groups, and there was no significant difference between the latter two groups (P>0.05). The expression level of P62 protein was the highest in group C, followed by group F, both of which were significantly higher than those in the control and AFB1 groups (P<0.05).

[0114] 2.4.3 PTCA reduces ROS level in AFB1 testicular tissue

[0115] Figure 6 The results showed that AFB1 (group B) significantly increased the ROS level in testicular tissue, which was significantly higher than that in the other three groups. The ROS level in PTCA+AFB1 group (group F) was significantly lower than that in group B and higher than that in the control group (P<0.05).

[0116] 2.4.5 PTCA reduces testicular tissue damage and DNA damage (HE staining and TUNEL analysis)

[0117] Figure 7 The results of HE staining and TUNEL analysis of the four groups are shown. From the HE staining photos of testicular tissue, it can be seen that group B (AFB1) showed typical atrophy of seminiferous tubules with increased interstitial space (black arrows), and the other groups showed no significant pathological changes. The statistical results of TUNEL analysis showed that the proportion of TUNEL positive area in testicular tissue was significantly increased in AFB1 group compared with the control group (P<0.05), and the proportion of TUNEL positive area in AFB1 group with PTCA (group F) was significantly lower than that in the control group (P>0.05).

[0118] 2.4.6 PTCA reduces AFB1-induced damage to testicular cell structure (transmission electron microscopy analysis)

[0119] The microscopic damage of testicular tissue induced by AFB1 is shown Figure 8The outer membrane of mitochondria was partially dissolved, the density of mitochondrial matrix decreased significantly, mitochondria showed focal high edema, and the number of cristae was reduced Figure 8 The endoplasmic reticulum cisternae were expanded moderately (Fig. 8A and 8D). The Golgi apparatus was slightly expanded, and the flattened cisternae were partially dissolved (Fig. 8A and 8D). The nuclear membrane was partially dissolved, and the perinuclear space was slightly expanded (Fig. 8A and 8C). The blood-testis barrier was distributed in bands, and the intercellular space was slightly widened (Fig. 8B). Figure 8 Figure 8 Figure 8 Figure 8

[0120] 3 Conclusion

[0121] In summary, the study confirmed that PTCA is a key metabolite that plays a protective role in testicular tissue. In vitro testicular Leydig cell experiments showed that the cell viability increased from 76.27% to 106.27% and the testosterone level increased from 315.89 pg / mL to 510.14 pg / mL in the 8 μmol / L AFB1 group with the addition of 300 μmol / L PTCA, which showed no significant difference from the control group (P > 0.05). The testicular injection verification experiment showed that PTCA (200 μg / μL) can almost completely reverse the spermatogenic impairment caused by AFB1, making the sperm count (3.09 × 10 4 The total viability (43.94%), and testosterone level (1.18 ng / mL) returned to no significant difference from the normal control group (P > 0.05), which was significantly better than other metabolites (such as indole acetic acid and L-cysteine, etc.). PTCA antagonizes oxidative damage by activating the PI3K / AKT / NRF2 pathway: Western Blot analysis showed that PTCA intervention significantly up-regulated the phosphorylation of PI3K, AKT, and NRF2 protein expression inhibited by AFB1 (P < 0.05), and enhanced the expression of downstream antioxidant genes, thereby reducing oxidative stress and apoptosis in testicular tissue and improving spermatogenesis. This study provides a solid theoretical basis and experimental support for PTCA as an intervention strategy for AFB1 reproductive toxicity.​​​​

Claims

The use of 1,2-propylthiazolidin-4-carboxylic acid (PTCA) in the preparation of a medicament for treating AFB1-induced testicular spermatogenesis disorders, characterized in that: PTCA promotes testosterone secretion from testicular interstitial cells, activates the PI3K / AKT / NRF2 antioxidant signaling pathway in testicular tissue, alleviates AFB1-induced testicular damage, and reduces ROS levels in testicular tissue.

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