New application of testis metabolite 2-propyl thiazolidine-4-carboxylic acid

Abstract

The invention belongs to the technical field of reproductive health, and particularly relates to a new application of testis metabolite 2-propyl thiazolidine-4-carboxylic acid (2-n-propyylthiazolidine-4-carboxylic acid, PTCA), an application of the 2-propyl thiazolidine-4-carboxylic acid in preparation of a medicine for treating testis dyszoospermia induced by AFB1, the PTCA can promote testis interstitial cell testosterone secretion, and the medicine for treating testis dyszoospermia induced by AFB1 can be used for treating testis dyszoospermia induced by AFB1, so that the testis dyszoospermia induced by AFB1 can be used for treating testis dyszoospermia induced by AFB1. The PI3K / AKT / NRF2 antioxidant signal channel in the testis tissue is activated, the testis injury induced by AFB1 is relieved, and the ROS level in the testis tissue is reduced; according to the application, the important effect of PTCA as a novel testis metabolism marker and protective agent in the male reproductive system is disclosed for the first time, a new insight is provided for understanding the reproductive toxicity mechanism of AFB1, and an experimental foundation is laid for an intervention strategy of metabolite applied to male dyszoospermia.

Description

Technical Field

[0001] This invention belongs to the field of reproductive health technology, and in particular relates to a new application of 2-propylthiazoline-4-carboxylic acid, a testicular metabolite. Background Technology

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

[0003] Previous studies have confirmed that the viable cell counts of the three microorganisms—Saccharomyces cerevisiae, Lactobacillus casei, and Bacillus subtilis—are all 1.0 × 10⁻⁶. 5 A compound probiotic consisting of CFU / mL effectively alleviates AFB1-induced testicular Sertoli cell toxicity. This application aims to systematically explore the role of key metabolites in testicular tissue in combating AFB1 reproductive toxicity. Through non-targeted metabolomics analysis, we successfully identified four differentially expressed metabolites that significantly impaired spermatogenesis induced by AFB1, including indoleacetic acid, adenosine, 2-n-propylthiazolidine-4-carboxylicacid (PTCA), and L-cysteine. In in vitro testicular interstitial cell culture experiments, we found that PTCA can promote testosterone secretion from interstitial cells and alleviate AFB1-induced cytotoxicity. To further evaluate the function of these metabolites in spermatogenesis, we validated them one by one via intratestinal injection. The results showed that PTCA could almost completely reverse the AFB1-induced phenotypes of decreased sperm count, reduced sperm motility, and decreased testosterone levels, demonstrating excellent protective effects. In-depth mechanistic studies have shown that PTCA significantly enhances the body's antioxidant capacity and reduces ROS levels by activating the PI3K / AKT / NRF2 signaling pathway in the testes, thereby alleviating AFB1-induced oxidative stress damage. Summary of the Invention

[0004] The purpose of this invention is to provide a new application of the testicular metabolite 2-propylthiazolidin-4-carboxylic acid.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0006] A novel application of the testicular metabolite 2-propylthiazolidin-4-carboxylic acid (PTCA), wherein the 2-propylthiazolidin-4-carboxylic acid is used in the preparation of a drug for treating AFB1-induced testicular spermatogenesis disorders. PTCA relieves AFB1-induced testicular damage and reduces ROS levels in testicular tissue by promoting testosterone secretion from interstitial cells and activating the PI3K / AKT / NRF2 antioxidant signaling pathway in testicular tissue.

[0007] The advantages of this invention are: This application reveals for the first time the important role of PTCA as a novel testicular metabolic marker and protectant in the male reproductive system, which not only provides new insights into the reproductive toxicity mechanism of AFB1, but also lays an experimental foundation for the application of metabolites in intervention strategies for male spermatogenesis disorders. Attached Figure Description

[0008] Figure 1 This is an assessment of the comprehensive biological response index of different treatment groups in this invention; where AE represents the radar chart of reproductive toxicity assessment for the AFB1 group, MCP group, AFB1+LCP group, AFB1+MCP group, and AFB1+HCP group, respectively; F is a bar chart of IBRV2 values ​​for the five treatment groups; EPI: paired epididymal / body weight index, T: testosterone, SpC: sperm count, SpA: sperm abnormality rate, TSA: testicular cell apoptosis protein Bax / Bcl-2 ratio. Low (LCP), medium (MCP), and high (HCP) represent the number of live probiotics of 1.0 × 10⁻⁶, respectively. 5 1.0×10 6 and 1.0×10 7 CFU / mL

[0009] Figure 2 This is a volcano plot showing the differences in metabolites between the AFB1 group and the AFB1+MCP group. Note: log2FC: fold change in metabolite expression between the two groups; -log10(p_value): statistical test value for the difference in metabolite expression; VIP value: the influence of the inter-group difference in the corresponding metabolite on the classification of samples in the model; metabolites with VIP ≥ 1 are considered statistically significant (P < 0.05). MCP: The viable count of the compound probiotics is 1.0 × 10⁻⁶. 6 CFU / mL

[0010] Figure 3 This is a growth curve diagram of testicular interstitial cells under different density conditions in cell experiments.

[0011] Figure 4 These are microscopic images of testicular interstitial cells under different density conditions during cell experiments.

[0012] Figure 5 This is a histogram of Western blotting (WB) electrophoresis bands and grayscale values ​​in testicular 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 BPA + 200 μg / μL PTCA. On each bar chart, significant differences at the P < 0.05 level are indicated by different lowercase letters (a, b, and c), with the same letter indicating no significant difference. Each group contains 4 replicates.

[0013] Figure 6 These are immunofluorescence images of testicular tissue ROS analysis from different treatment groups and statistical data on the average optical density (scale bar 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 LPDCA.

[0014] Figure 7 HE staining and TUNEL analysis of different treatment groups. HE staining photomicrograph of testicular tissue (Figure A) (scale bar 100 μm); TUNEL staining photomicrograph of testicular tissue (Figure B) (scale bar 100 μm); Histogram of TUNEL-positive area of ​​testicular tissue (Figure C). 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 These are transmission electron micrographs of testicular tissue from group AFB1 (scale bars 5μm and 2μm). Figure A: Transmission electron micrograph of seminiferous tubules near the base; the two black bands in the middle of the left image represent the base of the seminiferous tubules, and the cells within the red box are spermatogonia. Figure B: Transmission electron micrograph of seminiferous tubules near the lumen; the cells within the red box in the left image are early spermatids. Figure C: Transmission electron micrograph of seminiferous tubules near the base; the cells within the red box in the left image are Sertoli cells. Figure D shows the testicular interstitium; the cells within the red box on the left are testicular interstitial cells. Red arrow: mitochondria; Orange arrow: rough endoplasmic reticulum; Green arrow: Golgi apparatus; Purple arrow: nucleus; Blue arrow: blood-testis barrier; Pink arrow: compact body; Yellow arrow: lysosome; Pink arrow: free lipid droplets. Detailed Implementation

[0016] Example

[0017] A novel application of a testicular metabolite, 2-propylthiazolidin-4-carboxylic acid, is characterized in that: the 2-propylthiazolidin-4-carboxylic acid is used in the preparation of a drug for treating AFB1-induced testicular spermatogenesis disorders. PTCA promotes the secretion of testosterone from 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.

[0018] Verification Experiment

[0019] 1. Materials and Methods

[0020] 1.1 Experimental Materials

[0021] Saccharomyces cerevisiae (CGMCC No. 2.3973), Lactobacillus casei (CGMCC No. 1.8727), and Bacillus subtilis (CGMCC No. 1.821) were purchased from the China General Microbiological Culture Collection Center (CGMCC), Chinese Academy of Sciences. L-cysteine, CAS: 52-90-4, molecular formula: C3H7NO2S, Beijing Solarbio Co., Ltd., product number: C0012. Indoleacetic acid, CAS: 87-51-4, molecular formula: C 10 H9NO2, Beijing Solarbio Co., Ltd., Product No.: I8020. Adenosine, CAS: 58-61-7, Molecular Formula: C 10 H 13 N5O4, Beijing Solarbio Co., Ltd., Product No.: A8240. 2-Propylthiazolidin-4-carboxylic acid (PTCA), CAS No.: 4165-34-8, Molecular formula: C7H 13 NO2S, Shanghai McLean Biochemical Technology Co., Ltd., Product No.: 785258.

[0022] 1.2 AFB1 and compound probiotics gavage experiment

[0023] Saccharomyces cerevisiae, Lactobacillus casei, and Bacillus subtilis were used. Lactobacillus casei was inoculated into sterilized MRS medium (g / L: peptone 10, yeast extract 10, glucose 20, Tween 80 1 mL, K₂HPO₄ 2, sodium acetate 5, sodium citrate 2, MgSO₄ 0.2, MnSO₄ 0.05, pH 6.20-6.60) at 121℃ for 30 min and incubated statically at 37℃ for 24 h. Bacillus subtilis was inoculated into LB medium (g / L: peptone 10, yeast extract 5, NaCl 10, pH 7.0) and incubated at 37℃ with shaking at 180 rpm for 24 h. Saccharomyces cerevisiae was inoculated into YPD medium (g / L: peptone 20, glucose 20, yeast extract 10) and incubated at 30℃ with shaking at 180 rpm for 24 h. After the three microorganisms were cultured, they were centrifuged at 4℃ and 10000r / min for 5min. 10%-50% skim milk powder, trehalose, monosodium glutamate, and surfactants were added to the bacterial pellet, followed by freeze-drying. The viable cell concentration (logCFU / mL) of each bacterial solution was determined by plate counting after freeze-drying.

[0024] Sixty 7-week-old male Kunming mice (purchased from Henan Huaxing Animal Experiment Center) were acclimatized for one week under the same environment (12 / 12-hour light / dark cycle). Subsequently, healthy mice of the same weight were randomly divided into 6 groups (n=10 / group), and their initial weights were recorded. Preliminary degradation experiments showed that the viable counts of the three types of probiotics were all 1.0 × 10⁻⁶. 5 The highest AFB1 degradation rate was observed at CFU / mL. Based on this, the viable bacteria concentrations for the low, medium, and high dose groups were set at 1.0 × 10⁻⁶ CFU / mL. 5 1.0×10 6 and 1.0×10 7 CFU / mL. Experimental groups and treatments are as follows:

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

[0026] Group B (AFB1): 150 μg / kg BWAFB1 (dissolved in 0.5 mL corn oil) was administered by gavage daily.

[0027] Group C (medium-dose probiotics, MCP): 0.5 mL of corn oil suspension containing 150 μg / kg BWAFB1 + MCP (live count 1.0 × 10⁻⁶) was administered daily by gavage. 6 (CFU / mL). The lyophilized powders of the three bacteria were resuspended in 0.5 mL of corn oil, and the final concentration of each microorganism was 3.0 × 10⁻⁶. 7 CFU / mL, dispersed by ultrasound for 15 seconds and then administered by gavage; prepare fresh before use.

[0028] Groups DE and F (AFB1 + probiotics): 0.5 mL of compound probiotics containing 150 μg / kg BWAFB1 and low (Group D, LCP), medium (Group E, MCP), and high (Group F, HCP) doses of probiotics were administered daily by gavage (final viable count of 1.0 × 10⁻⁶ cells). 5 1.0×10 6 and 1.0×10 7 A corn oil suspension (CFU / mL).

[0029] The experiment lasted 28 days, with weighing conducted weekly. After weighing on the 28th day, four mice from each group were randomly sacrificed (cervical dislocation after blood collection from the orbital pits). Tissues such as epididymis, testis, intestines, and liver were collected, weighed, and then classified and preserved for subsequent testing.

[0030] 1.3 Evaluation of the Integrated Bioresponse Ratio (IBRV2) of AFB1 and Compound Probiotics

[0031] Six indicators were selected for comprehensive biological response assessment (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).

[0032] 1.4 Metabolomics of Mouse Testicular Tissue

[0033] After the testes were removed from liquid nitrogen and ground, 1 mL of methanol / acetonitrile / water (2:2:1, V / V) cold extraction buffer was added to 80 mg of the sample, and the mixture was shaken thoroughly. The sample was homogenized twice using an MP Fastprep-245G homogenizer, followed by ultrasonic extraction at 4°C (30 min / time, twice). Finally, the supernatant was extracted by centrifugation at 14,000 g for 20 min and dried by vacuum centrifugation at 4°C. The dried sample was redissolved in acetonitrile / water (1:1, V / V) solution, and a 100 μL sample was taken for LC-MS.

[0034] LC-MS chromatographic conditions: ACQUITYUPLC T3 column (2.1 mm × 100 mm, 1.8 μm), column temperature 40 °C, 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 ionization source, positive ion mode, capillary voltage 3kV, ion source temperature 120℃, curtain gas flow rate 50L / h, desolvation gas flow rate 800L / h, gas flow temperature 400℃, cone voltage 40V, collision energy 20~40V, mass scan range m / z 50~1200; negative ion mode, capillary voltage 2.5kV, collision energy 25~45V, other parameters are the same as positive ion mode.

[0035] Each treatment group was repeated four times. The raw MS data (wiff.scan file) were converted to MzXML format using Proteo Wizard MSConvert and imported into the free software XCMS for analysis. Metabolite mass spectra were identified by matching with the NIST 14.0 mass spectrometry library. Differentially expressed metabolites were analyzed using MetaboAnalyst 5.0 (https: / / www.metaboanalyst.ca) software based on FC values ​​(FC>1.5) and P values ​​(P<0.05).

[0036] 1.5 Effects of four differentially metabolites on testosterone secretion from testicular interstitial cells in vitro

[0037] 1.5.1 Experimental Materials

[0038] TM3 mouse testicular interstitial cells (catalog number: Delf-10326) were provided by Hefei Wanwu Biotechnology Co., Ltd., and AFB1 was purchased from Sigma. DMEM / F12 basal medium, penicillin-streptomycin solution (10000U / mL penicillin G sodium salt, 10mg / mL streptomycin sulfate), trypsin, fetal bovine serum, and PBS buffer (1×) were all purchased from Wuhan Pronosai Life Science Co., Ltd.

[0039] 1.5.2 Cell Culture

[0040] Mesenchymal cells were removed from a -80°C freezer for cell resuscitation. They were placed in 5 mL of DMEM complete medium containing 10% fetal bovine serum and 1% penicillin-streptomycin solution and incubated at 37°C with 5% CO2 for 24–48 h. When cells reached 80%–90% confluence, they were digested with 800 μL of trypsin. After all cells detached from the culture vessel, digestion was stopped with 1 mL of complete medium. The cells were centrifuged at 3000 rpm for 5 min, the supernatant was discarded, and the cells were resuspended in 1 mL of complete medium for passage culture.

[0041] 1.5.3 Growth curves of mesenchymal cells under different seeding densities

[0042] Cells in the logarithmic growth phase and in good condition were selected for passage. Cells were digested with 800 μL of trypsin, centrifuged, and the supernatant was discarded. After resuspending the cells, 10–100 μL of the cell suspension was transferred to a cell counting plate and counted under a microscope. Cell suspensions of different densities were prepared and cultured in culture flasks. Cell viability was assessed using the MTT assay. 10 μL of 5 mg / mL MTT was added to each well, and the plate was incubated for 4 hours. The cell supernatant was then removed, and 150 μL of dimethyl sulfoxide (DMSO) was added to each well. The plate was then shaken at room temperature for 10 minutes. The absorbance was measured at 490 nm and 630 nm using a microplate reader. Relative cell viability (%) = (Experimental group A490 nm - A630 nm) / (Control group A490 nm - A630 nm) × 100%.

[0043] 1.5.4 Establishment of an aflatoxin B1 damage model

[0044] Mesenchymal cells in the logarithmic growth phase were digested, detached from the cell wall, centrifuged, and resuspended. 10–100 μL of each cell was placed in a cell counting chamber for observation and counting, with each well containing 1.0 × 10⁻⁶ cells. 5 Cells were seeded in 96-well plates and cultured for 24 h. Then, different concentrations of AFB1 (0, 2, 4, 6, 8, 10 μmol / L) were added, with 6 replicates per group. Cell viability was assessed using the MTT assay after 12 h, 24 h, and 48 h of further culture.

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

[0046] After digesting and detaching mesenchymal cells in the logarithmic growth phase, centrifuging and resuspending them, 10–100 μL was aspirated onto a cell counting plate. After observation under a microscope, the cells were counted at 1.0 × 10⁻⁶ cells per well. 5Cells were seeded in 96-well plates and cultured for 24 h. Then, 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, with six replicates per group. After culturing for 12 h, 24 h, and 48 h, cell viability was assessed using the MTT assay to screen for the optimal time and concentration of the four substances to alleviate cell damage, thus establishing a protective model.

[0047] 1.5.6 Effects of four metabolites on the protective effect against AFB1 damage and on testosterone secretion

[0048] Mesenchymal cells in the logarithmic growth phase were digested, decanted, centrifuged, and resuspended. 10–100 μL of each cell was placed on a cell counting plate and observed under a microscope. Cells were then counted at 1.0 × 10⁻⁶ cells per well. 5 Cells were seeded in 96-well plates with 6 replicates per group and cultured for 24 h. Four groups were established: control group (DMEM / F12 basal medium), AFB1 group (8 μmol / L LAFB1), IAA+AFB1 group (80 μmol / L IAA + 8 μmol / L LAFB1), and PTCA+AFB1 group (300 μmol / L PTCA + 8 μmol / L LAFB1), each group mixed 1:1. Cells were added to 96-well plates and cultured for 12 h, 24 h, and 48 h. Relative cell viability and testosterone levels were measured using the MTT assay. Testosterone levels were measured by centrifuging the cell incubation medium at 10000 rpm for 10 min at 4°C and then detecting the levels using a testosterone (T) ELISA kit (catalog number: CB10319-Mu, Shanghai Keabob Biotechnology Co., Ltd.).

[0049] 1.6 Verification of Differential Metabolites by Testicular Injection

[0050] 1.6.1 Preliminary test of testicular injection

[0051] To further verify the in vivo effects of four differentially metabolites—indoleacetic acid, adenosine 2-n-propylthiazolidine-4-carboxylic acid (PTCA), and L-cysteine—testicular injection experiments were conducted to establish a testicular injection model of AFB1-induced spermatogenesis disorder. Testicular injection procedure: Healthy male Kunming mice aged 8-10 weeks, weighing 22-25g, were used in an SPF-grade environment with standard diet and water, and under a 12 / 12 light / dark cycle. Testicular injection procedure: Mice were deeply anesthetized by isoflurane inhalation. The mice were then fixed, and the lower abdominal skin was prepared and disinfected. A small incision (approximately 0.5cm) was made in the scrotal skin, and the left testis was gently squeezed out to expose the field of vision. Using a microsyringe (equipped with a 33G fine needle), avoiding major blood vessels, 10μL of different treatment drug solutions were slowly injected. The needle was left in place for 10 seconds after injection to prevent fluid backflow. The testicle was gently returned to the scrotum, and the wound was sutured. In the sham surgery group, only the testicle was exposed before suturing. Postoperatively, physiological saline and analgesia (carbofen) were injected subcutaneously, and the patients were kept in single cages and observed until they recovered.

[0052] AFB1 spermatogenesis disorder model group. AFB1 solution (10 μL, 100 μg / μL dissolved in 5% DMSO solution) was injected into the left testis. Animals were sacrificed at weeks 1, 2, 3, 4, 5, and 7 post-injection, and epididymis was collected for sperm motility testing.

[0053] Exploring safe dosage groups of four substances: In each group, animals were injected into the left testis with different concentration gradients of a metabolite (100, 200, and 300 μg / μL dissolved in 5% DMSO solution, 10 μL). Animals were sacrificed three weeks post-injection (based on the peak time of the BPA effect), and sperm motility was assessed.

[0054] 1.6.2 Formal Experiment with Testicular Injection

[0055] Preliminary results showed that sperm motility significantly decreased on day 21 after AFB1 injection. The four substances had no significant effect on sperm motility within the concentration range of 100-300 μg / μL; therefore, 200 μg / μL was selected for the formal experiment. The pretreatment for the formal experiment was the same as the preliminary 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 μL, final concentration containing 100 μg / μL BPA + 200 μg / μL indoleacetic acid.

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

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

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

[0063] Ten mice were used in each group. On day 21 post-surgery, the mice were sacrificed and the left epididymis was harvested for sperm quality analysis, Western blot analysis, HE staining, TUNEL analysis, and transmission electron microscopy analysis.

[0064] 1.6.3 WB Analysis

[0065] Western blot analysis was performed on the left testes of groups A, B, C, and F. Testicular tissue was homogenized with liquid nitrogen, and total cellular protein was extracted using RIPA lysis buffer (Yamei Biotechnology Co., Ltd., Shanghai, China). The concentration of total cellular protein was detected using a BCA protein assay kit (Beijing Solarbio Biotechnology Co., Ltd., Shanghai, China). Equal volumes of protein were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene fluoride (PVDF) membrane. The membrane was blocked with TBST buffer containing 5% skim milk powder for 2 h, then incubated overnight with primary antibody at 4°C, followed by incubation with secondary antibody at room temperature for 2 h. Enhanced chemiluminescence immunoassay revealed immunopositive bands, and quantification was performed using ImageJ software.

[0066] 1.6.4 HE staining and TUNEL analysis

[0067] Left testes from groups A, B, C, and F were selected for HE staining and TUNEL analysis. Fixed tissues were washed, cleared, embedded in paraffin, and prepared into sections approximately 6 μm thick. These sections were stained with hematoxylin and eosin (HE), cleared with xylene, and finally covered with neutral resin at the edges before mounting and observation under an optical microscope. Testicular cell apoptosis was detected using a TUNEL assay kit (Nanjing Novizan Biotechnology Co., Ltd.), following the manufacturer's protocol. Cells were stained with DAPI for nuclear counterstaining and observed under an inverted microscope (Olympus, Tokyo, Japan). Images were randomly selected from three sections of each sample. TUNEL-positive areas were quantitatively analyzed using ImageJ software. The TUNEL-positive percentage was calculated as (TUNEL-positive area (green fluorescence image) / total nuclear area (blue fluorescence image)) × 100%. Mixed magnification (200x and 400x) was used to observe the location of tissue apoptosis.

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

[0069] To further clarify the extent of testicular cell damage, the AFB1 group was selected for electron microscopy analysis. Fresh testicular samples were fixed at 4°C with 2.5% phosphate-buffered glutaraldehyde for 6-8 hours, followed by neutralization and fixation with 1% osmium tetroxide for 1 hour, then rinsed with 0.1M phosphate buffer (pH 7.4), and subsequently dehydrated with different concentrations of ethanol. Finally, they were embedded in Epon 812 resin (SPI Supplies, West Chester, PA, USA). The samples were then rapidly frozen in isopentane at -80°C pre-cooled with liquid nitrogen. After equilibration at -20°C, the frozen tissue blocks were sectioned to a thickness of 5-8 μm and observed using a FEI Tecnai G212 transmission electron microscope (FEI Company, Hillsboro, OR, USA).

[0070] 1.6.6 Immunofluorescence assay 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 assessment indicators

[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 <![CDATA[AFB1]]> 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 <![CDATA[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 <![CDATA[AFB1+MCP]]> 0.79±0.11 0.29±0.08 1.08±0.09 3.31±0.25 61.29±2.12 2.31±0.12 <![CDATA[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 Differential metabolite analysis between the AFB1 group and the AFB1+MCP group in testicular tissue

[0080] Differential metabolites between the two groups were screened according to the criteria of FC>1.5 and P<0.05, and a volcano plot of differential metabolites was drawn as follows. Figure 2 As shown in Table 2, a total of 228 named differential metabolites were identified in the AFB1 and AFB1+MCP groups, of which 146 were identified in positive ion mode. Four typical differential metabolites were identified by FC value screening: indoleacetic acid, adenosine, 2-n-Propylthiazolidine-4-carboxylic acid, and L-cysteine ​​(P<0.05).

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

[0082]

[0083] 2.3 In vitro testicular interstitial cell experiment

[0084] 2.3.1 Growth curves and morphological observations of mesenchymal cells

[0085] Growth curves of mesenchymal cells under different densities are shown in the figure. Figure 3 Cell morphology is shown Figure 4 .Depend on Figure 3 As can be seen, the absorbance of mesenchymal cells increases with increasing density and culture time, with the absorbance at a cell density of 1.0 × 10⁻⁶ cells / year increasing. 6 and 5.0×10 5 After 60 hours of culture, the absorbance was greater than 1.0. Cell morphology showed irregular rod-shaped cells, and cell confluence reached 70% after 36–48 hours of culture. Based on absorbance and microscopic images, a cell density of 1.0 × 10⁶ cells was selected. 5 After 48 hours of culture, the cells were inoculated for use in subsequent models.

[0086] 2.3.2 Screening of AFB1 damage models

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

[0088] Table 3. Effect of AFB1 on cell viability (%)

[0089] Note: In the same column, different uppercase letters (A, B, C) indicate significant differences (P < 0.05), and the same uppercase letters indicate no significant differences (P > 0.05); in the same row, different lowercase letters (a, b, c, d) indicate significant differences (P < 0.05), and the same lowercase letters indicate no significant differences (P > 0.05), and so on.

[0090] 2.3.3 The alleviating effects of four substances on damaged cells

[0091] The results of the effect of indoleacetic acid (IAA) on cell viability are shown in Table 4. As can be seen from Table 4, when the IAA concentration is 80 μmol / L and the treatment time is 24 h, the cell viability reaches the highest level of 118.88%, which is significantly higher than other concentrations and times (P < 0.05). Therefore, this concentration and time were selected for subsequent protection experiments.

[0092] Table 4. Effect of IAA on cell viability (%)

[0093]

[0094] The results of PTCA on cell viability are shown in Table 5. As can be seen from Table 5, when the PTCA concentration is 300 μmol / L and the treatment time is 24 h, the cell viability reaches the highest level of 115.90%, which is significantly higher than other concentrations and times (P < 0.05). Therefore, this concentration and time were selected for subsequent protection experiments.

[0095] Table 5. Effect of PTCA on cell viability (%)

[0096]

[0097] The results of L-cysteine ​​on cell viability are shown in Table 6. As can be seen from Table 6, when L-cysteine ​​was used to treat cells for 24 hours, the cell viability was significantly lower than that of the control group with increasing concentration (P<0.05), and it had no effect on the proliferation of mesenchymal cells.

[0098] Table 6. Effect of L-cysteine ​​on cell viability (%)

[0099]

[0100] The results of adenosine on cell viability are shown in Table 7. As can be seen from Table 7, when adenosine was used to treat cells for 24 hours, the cell viability was significantly lower than that of the control group with increasing concentration (P<0.05), and it had no effect on the proliferation of mesenchymal cells.

[0101] Table 7. Effect of adenosine on cell viability (%)

[0102]

[0103] 2.3.4 Protective effect of IAA and PTCA against AFB1-induced cytotoxicity

[0104] Table 8 shows the effects of co-incubation of AFB1 with IAA and PTCA on cell viability. As shown in Table 8, cell viability and testosterone levels exhibited similar trends. PTCA significantly increased cell viability and testosterone levels (while AFB1 significantly decreased cell viability and testosterone levels, P < 0.05), while IAA had no significant effect on cell viability and testosterone levels (P > 0.05). When PTCA and IAA were added to the AFB1 group, cell viability and testosterone levels were upregulated to levels not significantly different from the control group (P < 0.05). Therefore, PTCA and IAA significantly alleviated AFB1-induced mesenchymal cell toxicity in vitro.

[0105] Table 8. Effects of co-incubation of IAA, PTCA, and AFB1 for 24 h on cell viability and testosterone levels.

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

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

[0108] 2.4.1 Differential metabolites alleviate AFB1-induced testicular spermatogenesis disorders

[0109] Data analysis results (Table 9) showed that sperm count, total sperm motility, and testosterone concentration in the AFB1 testicular injection group (Group C) were significantly lower than those in the control groups (Groups A and B) (P<0.05). Compared with the AFB1 group, the four differential metabolite protective treatments all showed varying degrees of protective effects. Among them, the PTCA protection group (Group F) showed the most significant protective effect, with sperm count, total motility, and testosterone levels recovering to levels not significantly different from the control group (P>0.05). In conclusion, PTCA exhibits near-complete protection against AFB1-induced reproductive toxicity.

[0110] Table 9. Effects of testicular injection of four differentially metabolites on weight and testosterone levels in Kunming mice.

[0111]

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

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

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

[0115] Figure 6 The ROS levels in the four treatment groups are shown. AFB1 (group B) significantly increased the ROS levels in mouse testicular tissue, significantly higher than the other three treatment groups. The PTCA+AFB1 group (group F) was significantly lower than group B, but higher than 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 HE staining and TUNEL analysis of the four treatment groups are shown. HE-stained images of testicular tissue show that group B (AFB1) exhibits typical seminiferous tubule atrophy and increased interstitial spaces (black arrows), while other groups show no significant pathological changes. TUNEL analysis results indicate that, compared to the control group, the AFB1 group significantly increased the proportion of TUNEL-positive areas in testicular tissue (P<0.05). After adding PTCA to the AFBI group (Group F), the proportion of TUNEL-positive area returned to a level not significantly different from the normal group (P>0.05).

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

[0119] AFB1-induced microscopic damage to testicular tissue is shown in [reference needed]. Figure 8The outer mitochondrial membrane partially dissolved, the mitochondrial matrix density decreased significantly in some areas, and the mitochondria exhibited focal areas of severe edema, with significant dissolution of the cristae. Figure 8 A, 8B, 8C, and 8D). The intervesicular spacing of the endoplasmic reticulum is widened, and the endoplasmic reticulum is moderately dilated. Figure 8 A and 8D). Golgi apparatus, slight dilation of the flattened sacs of the Golgi apparatus, partial dissolution of the membrane structure ( Figure 8 A). Significant dissolution of the nuclear membrane and slight expansion of the perinuclear space ( Figure 8 A and 8C), the blood-testis barrier, is distributed in a band-like pattern, with slight widening of the junctional gaps and slight dissolution of the membrane structure at the junctions (A and 8C). Figure 8 B).

[0120] 3. Conclusion

[0121] In summary, the study confirms that PTCA is a key metabolite exerting a protective effect on testicular tissue. In vitro testicular interstitial cell experiments showed that the addition of 300 μmol / L PTCA to the 8 μmol / LAFB1 group increased cell viability from 76.27% to 106.27%; testosterone levels increased from 315.89 pg / mL to 510.14 pg / mL, both without significant differences compared to the control group (P>0.05). Testicular injection validation experiments showed that PTCA (200 μg / μL) could almost completely reverse the spermatogenesis impairment caused by AFB1, reducing sperm count to 3.09 × 10⁻⁶. 4 The levels of spermatogenic cells / mL, total motility (43.94%), and testosterone (1.18 ng / mL) recovered to levels not significantly different from the normal control group (P>0.05), showing significantly better effects than other metabolites (such as indoleacetic acid and L-cysteine). PTCA antagonizes oxidative damage by activating the PI3K / AKT / NRF2 pathway: Western blot analysis showed that PTCA intervention significantly upregulated AFB1-inhibited PI3K and AKT phosphorylation and NRF2 protein expression (P<0.05), and enhanced the expression of downstream antioxidant genes, thereby alleviating 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

1. A novel application of a testicular metabolite, 2-propylthiazolidin-4-carboxylic acid (PTCA), characterized in that: The 2-propylthiazolidin-4-carboxylic acid is used in the preparation of a drug for treating AFB1-induced testicular spermatogenesis disorders. 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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