Application of butyric acid or derivative thereof in preparation of zearalenone reproductive toxicity resisting preparation

Abstract

The invention discloses an application of butyric acid or a derivative thereof in preparation of a zearalenone reproductive toxicity resisting preparation. Also disclosed is an antitoxic feed composition comprising a specific base ration and a butyric acid derivative. In-vivo and in-vitro tests and molecular mechanism researches prove that butyric acid and derivatives thereof (such as tributyrin and sodium butyrate) can effectively relieve zearalenone (ZEN)-induced animal ovary injury. The action mechanism of the traditional Chinese medicine composition is that follicular atresia is reduced, the balance of reproductive hormones (such as GnRH, E2, AMH and P4) is recovered, and clinical symptoms caused by ZEN poisoning such as vulva swelling and the like are relieved by activating a BMPs / SMAD signal channel, especially up-regulating SMAD4 protein expression and inhibiting oxidative stress (ROS) and Caspase-3 mediated granular cell apoptosis.

Description

Application of butyric acid or its derivatives in the preparation of anti-zearalenone reproductive toxicity agents Technical Field

[0001] This invention belongs to the field of biological feed and veterinary drug technology, specifically relating to the application of butyric acid or its derivatives (especially tricresyl glycerol and sodium butyrate) in the preparation of a product for alleviating or preventing zearalenone-induced ovarian damage and granulosa cell apoptosis. Background Technology

[0002] Zearalenone (ZEN) is a non-steroidal mycotoxin produced by Fusarium fungi, commonly found in feed ingredients such as corn and wheat, as well as their processing byproducts. ZEN has estrogen-like activity, competitively binding to estrogen receptors, leading to severe reproductive dysfunction in animals (especially sows), such as vulvar swelling, ovarian atrophy, abortion, and stillbirth, causing huge economic losses to the livestock industry.

[0003] Existing research indicates that the reproductive toxicity of ZEN is closely related to its induction of apoptosis in ovarian granulosa cells (GCs). Apoptosis of GCs disrupts the microenvironment for follicular development, leading to follicular atresia. Although physical adsorbents or biodegrading enzymes are available for removing toxins from feed, effective nutritional interventions or repair strategies are still lacking for target organ damage (such as ovarian) caused by ZEN that has already been ingested.

[0004] Butyric acid (BA) is an important short-chain fatty acid produced by fermentation by intestinal microorganisms, possessing energy-providing and anti-inflammatory properties. However, current technologies have not systematically elucidated whether butyric acid and its derivatives can antagonize the reproductive toxicity of ZEN, especially regarding the mechanism by which it regulates ovarian function through the BMPs / SMAD signaling pathway, which remains a blank. Summary of the Invention

[0005] The purpose of this invention is to provide a new application of butyric acid or its derivatives, namely, its application in the preparation of products for preventing or alleviating reproductive system damage caused by zearalenone poisoning in animals.

[0006] The technical solution adopted by this invention to solve its technical problem is:

[0007] The application of butyric acid or its derivatives in the preparation of products for preventing or alleviating reproductive system damage caused by zearalenone poisoning in animals. This invention demonstrates that butyric acid derivatives (such as tributyrate (TB) and sodium butyrate (NaB)) can significantly alleviate ZEN-induced growth performance decline, vulvar swelling, and hormonal imbalances in pigs, and effectively inhibit oxidative stress and apoptosis in ovarian granulosa cells. Mechanistic studies show that ZEN exposure inhibits the BMPs / SMAD signaling pathway (SMAD4 protein expression is downregulated, BMP6 protein expression is upregulated), while butyric acid derivatives can reverse the downregulation of SMAD4 expression and activate the BMPs / SMAD pathway, thereby exerting an ovarian protective effect. When SMAD4 expression is interfered with, the anti-apoptotic protective effect of butyric acid derivatives disappears, confirming the key role of this pathway.

[0008] Preferably, the butyric acid derivative is selected from at least one of butyrate and butyrate ester; preferably, it is glyceryl tributyrate or sodium butyrate.

[0009] Preferably, the product is animal feed; the butyric acid or its derivative is added to the animal feed at an amount of 0.1%-0.5%, based on the total weight of the animal feed as 100%; preferably 0.3%.

[0010] Preferably, the reproductive system damage manifests as ovarian granulosa cell apoptosis, follicular atresia, oxidative stress damage to ovarian tissue, or reproductive hormone secretion disorder.

[0011] Preferably, the animal is a pig; the product is used to upregulate the expression of SMAD4 protein in ovarian tissue.

[0012] A feed additive for alleviating zearalenone poisoning in animals, comprising an effective amount of butyric acid or its derivatives as an active ingredient.

[0013] An animal feed with resistance to zearalenone reproductive toxicity, comprising a basal feed and an antitoxin additive; the antitoxin additive is butyric acid or its derivative, and its content in the animal feed is 0.1%-0.5% (by weight); the basal feed comprises the following raw materials in parts by weight: 35-45 parts corn, 8-12 parts puffed corn, 12-16 parts broken rice, 1.5-2.5 parts soybean oil, 18-22 parts soybean meal, 5-7 parts puffed soybeans, 1.5-3 parts fermented soybean meal, 1.5-2.5 parts fish meal, 1-1.5 parts whey powder, 1.3-1.7 parts dicalcium phosphate, 0.4-0.6 parts limestone powder, 0.3-0.5 parts sodium chloride, 0.3-0.5 parts amino acid additive, and 0.5-0.8 parts premix.

[0014] Preferably, the antitoxin additive is glyceryl tribanilate.

[0015] Preferably, the basic feed comprises the following raw materials in parts by weight: 39.35 parts corn, 10.00 parts puffed corn, 14.00 parts broken rice, 2.00 parts soybean oil, 19.50 parts soybean meal, 6.49 parts puffed soybean, 2.09 parts fermented soybean meal, 1.78 parts fish meal, 1.16 parts whey powder, 1.54 parts dicalcium phosphate, 0.57 parts limestone powder, 0.44 parts sodium chloride, 0.40 parts amino acid additive, and 0.68 parts premix.

[0016] Preferably, the amino acid additive is selected from at least one of methionine, lysine, threonine, and tryptophan.

[0017] The beneficial effects of this invention are:

[0018] Compared with the prior art, the present invention has the following significant advantages:

[0019] 1. The tributyric acid glyceride (TB) provided by this invention, as a feed additive, can effectively alleviate growth inhibition and reproductive organ lesions caused by zearalenone (ZEN). Experimental data show that feeding ZEN (2 mg / kg) significantly increases the vulvar area and total weight gain in pigs, resulting in typical estrogen poisoning symptoms; while adding 0.3% TB effectively alleviated reproductive tract congestion and edema by reducing vulvar area on day 35 and the total increase in vulvar area from day 1 to day 35 (P < 0.05). Although ZEN significantly reduced the average daily gain (ADG) of pigs, the addition of TB improved the health status of pigs to some extent and reduced the systemic depletion caused by poisoning.

[0020] 2. This invention confirms that butyrate derivatives can reverse the hormonal characteristics of "false estrus" induced by ZEN and restore endocrine homeostasis. ZEN exposure led to abnormally high levels of gonadotropin-releasing hormone (GnRH), prolactin (PRL), and estradiol (E2) in serum (P < 0.05); the TB treatment group significantly reduced serum GnRH and E2 levels on days 21 and 35 (P < 0.05), bringing them back to levels close to the control group. ZEN exposure led to a significant decrease in key pregnancy-preserving / follicle-developing hormones such as anti-Müllerian hormone (AMH) and progesterone (P4) (P < 0.05); the TB treatment group significantly increased serum AMH and P4 levels on day 21 (P < 0.05) and AMH levels on day 35 (P < 0.05), indicating that it has a protective effect on ovarian reserve and corpus luteum function.

[0021] 3. This invention quantifies the protective efficacy of butyrate derivatives against ovarian granulosa cells (GCs) through dual in vitro and in vivo verification. In an in vitro model, treatment with 0.25 mM sodium butyrate (NaB) for 24 hours significantly reversed the decrease in AVG-16 cell (a type of porcine ovarian granulosa cell) viability induced by 20 μM ZEN (P < 0.05), demonstrating the optimal protective concentration effect. ZEN stimulation led to a significant surge in intracellular reactive oxygen species (ROS) levels (P < 0.05); the ROS levels in the NaB-treated group were significantly reduced (P < 0.05), effectively blocking the oxidative damage pathway. Flow cytometry analysis showed a significantly increased apoptosis rate in the ZEN group and a significantly decreased apoptosis rate in the NaB-treated group (P < 0.05), while the expression of the apoptosis-executing protein c-Caspase3 was also significantly inhibited (P < 0.05). In vivo TUNEL staining further confirmed that TB treatment significantly reduced the number of apoptotic cells in the ovarian granulosa layer.

[0022] 4. Immunohistochemical and Western blot quantitative analyses showed that ZEN poisoning significantly decreased SMAD4 protein expression and significantly increased BMP6 expression in ovarian tissues and cells; while butyrate derivative treatment significantly upregulated SMAD4 protein expression (P < 0.05), thereby activating the BMPs / SMAD signaling pathway. After downregulating SMAD4 gene expression by approximately 30% using RNA interference technology, the inhibitory effect of sodium butyrate on ZEN-induced apoptosis and c-Caspase3 expression disappeared (no significant difference), quantitatively confirming that SMAD4 is a key target for butyrate to exert its anti-ovarian damage effect.

[0023] Compared to the direct use of butyric acid, the tributyric acid glyceride (TB) preferred in this invention is odorless, non-volatile, and has good gastric stability, allowing for the targeted release of butyric acid in the intestine, resulting in higher bioavailability. Sodium butyrate (NaB), on the other hand, has good water solubility and is suitable for administration via drinking water or in vitro studies. Both can be conveniently applied to livestock production as feed additives or veterinary drugs, possessing significant economic value. Attached Figure Description

[0024] Figure 1. Effect of TB on serum hormone levels in pigs fed a ZEN-contaminated diet (21 days).

[0025] Figure 2 Effect of TB on serum hormone levels in pigs fed a ZEN-contaminated diet (35 days).

[0026] Figure 3. Effects of TB on the morphology of ovarian tissue in pigs fed a ZEN-contaminated diet. From left to right: normal follicle morphology and structure, normal follicle morphology and structure, red arrow: atretic follicle, red arrow: atretic follicle.

[0027] Figure 4. Effects of TB on the ultrastructure of pig ovaries fed a ZEN-contaminated diet (8000 ×), where thin red arrows indicate mitochondrial vacuolation and thick red arrows indicate nuclear condensation.

[0028] Figure 5. Effect of TB on apoptosis of ovarian cells in pigs fed a ZEN-contaminated diet (400 ×), where thin red arrows indicate GC apoptosis;

[0029] Figure 6. Effect of TB on BMP2 protein expression in pig ovaries fed a ZEN-contaminated diet (800 ×), where thin red arrows represent oocytes; thick red arrows represent GCs layer; the same applies below.

[0030] Figure 7 Effect of TB on BMP5 protein expression in pig ovaries fed a ZEN-contaminated diet (800 ×).

[0031] Figure 8 Effect of TB on BMP6 protein expression in pig ovaries fed a ZEN-contaminated diet (800 ×).

[0032] Figure 9 Effect of TB on SMAD4 protein expression in pig ovaries fed a ZEN-contaminated diet (800 ×).

[0033] Figure 10. Morphological changes of porcine AVG-16 cells after treatment with different concentrations of NaB for 12 h (200 ×).

[0034] Figure 11. Morphological changes of porcine AVG-16 cells after treatment with different concentrations of NaB for 24 h (200 ×).

[0035] Figure 12. Morphological changes of porcine AVG-16 cells after treatment with different concentrations of NaB for 36 h (200 ×).

[0036] Figure 13. Effects of different concentrations of NaB on the viability of AVG-16 cells;

[0037] Figure 14. Effects of different concentrations of NaB on the morphology of ZEN-stimulated AVG-16 cells (200 ×).

[0038] Figure 15. Effects of different concentrations of NaB treatment on the viability of ZEN-stimulated AVG-16 cells;

[0039] Figure 16. Effect of NaB on ROS production in ZEN-stimulated AVG-16 cells;

[0040] Figure 17 Effect of NaB on the apoptosis rate of ZEN-stimulated AVG-16 cells;

[0041] Figure 18 Effect of NaB on c-Caspase3 protein expression in ZEN-stimulated AVG-16 cells;

[0042] Figure 19. Effect of NaB on the expression levels of proteins related to the BMPs / SMAD signaling pathway in ZEN-stimulated AVG-16 cells;

[0043] Figure 20 Verification of SMAD4 interference efficiency;

[0044] Figure 21. Effect of NaB on ROS in ZEN-stimulated AVG-16 cells after SMAD4 interference;

[0045] Figure 22 Effect of NaB on apoptosis rate of ZEN-stimulated AVG-16 cells after SMAD4 interference;

[0046] Figure 23 Effect of NaB on c-Caspase3 protein expression in ZEN-stimulated AVG-16 cells after SMAD4 interference;

[0047] Figure 24. Effect of NaB on the expression levels of proteins related to the BMPs / SMAD signaling pathway in ZEN-stimulated AVG-16 cells after SMAD4 interference. Detailed Implementation

[0048] The technical solution of the present invention will be further described in detail below through specific embodiments. It should be understood that the implementation of the present invention is not limited to the following embodiments, and any modifications and / or alterations made to the present invention will fall within the protection scope of the present invention.

[0049] In this invention, unless otherwise specified, all parts and percentages are by weight, and the equipment and raw materials used are commercially available or commonly used in the art. Unless otherwise specified, the methods in the following embodiments are conventional methods in the art.

[0050] Unless otherwise specified, the reagents used in the following examples can be purchased from a regular biochemical reagent store.

[0051] Table 1. Description of primary and secondary immunohistochemical antibodies

[0052]

[0053] Table 2. Description of primary and secondary antibodies used in Western blot analysis.

[0054]

[0055] Example 1: In vivo protective effect of tributyrate (TB) against ZEN-induced porcine ovarian injury.

[0056] 1. Experimental Animals and Experimental Design

[0057] The ZEN added to the diet had a purity of 98.22% and was purchased from MCE (product number HY-103447); the TB had a purity of 60% and was purchased from Jialiduo Biotechnology Co., Ltd.

[0058] Thirty-two healthy, 33-day-old Duroc × Landrace × Large White crossbred female piglets (purchased from Hubei Aodeng Agricultural and Animal Husbandry Technology Co., Ltd.) with similar parity and body weight (BW) were selected. Their initial BW was 9.5 ± 0.2 kg. Based on similar BW, they were randomly divided into four treatment groups (N = 8 / group): control group (CON group, fed a basal diet), TB group (fed a diet supplemented with 0.3% TB), ZEN group (fed a diet supplemented with 2 mg / kg ZEN), and ZEN + TB group (fed a diet supplemented with 2 mg / kg ZEN and 0.3% TB). The basal diet used in the experiment was formulated according to the NRC (2012) nutritional requirements for piglets (formula shown in Table 3). The experimental period was 35 days. At the end of the experiment, six piglets from each group were randomly selected, anesthetized, and slaughtered. Ovarian tissue was collected (see Sample Collection for details).

[0059] Table 3. Dietary composition and nutrient content (air-dried basis, %)

[0060]

[0061] Samples were collected from the anterior vena cava on days 21 and 35 of the experimental period. Blood samples were collected in non-anticoagulated vacuum blood collection tubes and allowed to stand at room temperature for at least 30 minutes before being transferred to the laboratory for centrifugation (3500 rpm, 4℃, 15 min). Serum samples were then aliquoted and stored at -80℃ for later analysis. After blood collection on day 35, female piglets were anesthetized, slaughtered, and dissected. Ovarian tissue was placed on ice, and 0.5 cm of the tissue was collected. 3 Ovaries were fixed in 4% paraformaldehyde solution for subsequent hematoxylin-eosin (HE) staining analysis. A 1.5 mm sample was taken. 3 The ovaries were fixed in 2.5% glutaraldehyde solution for subsequent transmission electron microscopy analysis. The remaining ovarian tissue was minced to an appropriate size, flash-frozen in liquid nitrogen, and then stored at -80°C for later analysis.

[0062] 2. Animal test indicators and methods

[0063] Growth performance was measured by weighing on days 1, 7, 14, 21, 28 and 35 of the experiment, and the average daily weight gain (ADG) of female piglets was calculated using the following formula: ADG (g / d) = (final BW - initial BW) / number of days of the experiment.

[0064] The vulvar area was measured using vernier calipers on days 1, 7, 14, 21, 28, and 35 of the experiment. The vulvar length and width were calculated using the rhombus area formula: Vulvar area (mm²) 2 = (vaginal length × vulvar width) / 2.

[0065] Ovarian tissue morphology analysis

[0066] (1) Morphological analysis: Ovarian tissue was fixed with 4% paraformaldehyde, dehydrated and embedded, and then made into 5 μm paraffin sections. After HE staining and resin mounting, the morphology of ovarian tissue was observed under an Olympus optical microscope.

[0067] (2) Ultrastructure analysis: 1.5 mm 3 Ovarian tissue samples of various sizes were fixed with 2.5% glutaraldehyde, then post-fixed with osmium tetroxide, dehydrated with graded acetone, and embedded in epoxy resin. Sample processing and observation by transmission electron microscopy (HT7700) were performed by Wuhan Saiweier Biotechnology Co., Ltd.

[0068] Serum hormone levels were determined using radioimmunoassay to measure the levels of gonadotropin-releasing hormone (GnRH), follicle-stimulating hormone (FSH), luteinizing hormone (LH), AMH, prolactin (PRL), P4, testosterone (T), and E2 in serum. The kits were purchased from Lunchangshuo Biotechnology Co., Ltd., and the procedure was strictly performed according to the instructions.

[0069] To determine ovarian cell apoptosis, paraffin sections of ovarian tissue were dewaxed and hydrated, and then processed according to the instructions of the TUNEL reagent kit from Wuhan Fabre Biotechnology Co., Ltd. Antigens were repaired with proteinase K (20 μg / mL), and apoptotic cells were labeled with TUNEL reaction solution (37℃, 1 h). Cell nuclei were counterstained with DAPI, and observation and image acquisition were performed under a fluorescence microscope. The percentage of apoptotic cells was calculated using ImageJ software (number of TUNEL-positive cells / total number of cells × 100%).

[0070] The expression levels of proteins related to the BMPs / SMAD signaling pathway in ovarian tissue were determined using immunohistochemical staining to measure the distribution and relative expression of BMP2, BMP5, BMP6, and SMAD4 proteins. Sections fixed in 4% paraformaldehyde and embedded in paraffin were dewaxed, rehydrated, and treated with H2O2 for 10 min. After washing with PBS, sections were mounted with 5% BSA for 10 min, and then 10 μL of BMP2, BMP5, BMP6, and SMAD4 antibodies (1:50) were added to each section and incubated overnight at 4°C. After washing with phosphate-buffered saline (PBS), sections were incubated with biotinylated secondary antibody at room temperature for 10 min. After washing, catalase-labeled streptavidin was added, followed by staining with 3,3'-diaminobenzidine. Sections were observed and images were acquired under a fluorescence microscope. Descriptions of the primary and secondary immunohistochemical antibodies are shown in Table 1.

[0071] Experimental data were analyzed using SPSS 26.0 statistical software in a two-way ANOVA. The main effects of the model included TB and ZEN, and their interaction. Results are expressed as mean ± standard error (SEM). When interaction effects were present, Duncan's multiple comparisons were performed. Statistical results were plotted using GraphPad Prism 8.0. P < 0.05 was considered statistically significant, and 0.05 ≤ P < 0.1 was considered to indicate a significant trend.

[0072] 3. Experimental Results

[0073] 3.1 Effects of tributyrate on growth performance of pigs fed diets contaminated with zearalenone

[0074] Table 4 shows that dietary ZEN supplementation tended to reduce pig body size (BW) on day 35 (0.05 ≤ P < 0.1), and significantly reduced abortion rate (ADG) at 8-21 days, 22-35 days, and 1-35 days (P < 0.05). TB had no significant effect on BW or ADG regardless of ZEN supplementation.

[0075] 3.2 Effect of tributyrate on the vulva area of ​​pigs fed a diet contaminated with zearalenone

[0076] Table 5 shows that pigs fed a ZEN-contaminated diet exhibited significantly increased vulvar area on days 7, 14, 21, 28, and 35, as well as a significantly increased total vulvar area increase from day 1 to day 35 (P < 0.05). TB and ZEN had a significant interaction effect on vulvar area on day 35 and the total increase in vulvar area from day 1 to day 35 (P < 0.05). For pigs fed a ZEN-contaminated diet, TB significantly reduced vulvar area on day 35 and the total increase in vulvar area from day 1 to day 35 (P < 0.05). For pigs not fed a ZEN-contaminated diet, TB had no significant effect on vulvar area on day 35 or the total increase in vulvar area from day 1 to day 35.

[0077] Table 4. Effects of TB on growth performance of pigs fed ZEN-contaminated diets.

[0078]

[0079] Note: Contains different letters abc The numbers indicate significant differences between treatment groups (P < 0.05), while the numbers containing the same letter or without a letter indicate no significant differences (P > 0.05), and so on.

[0080] Table 5. Effects of TB on the vulva area of ​​pigs fed ZEN-contaminated diets.

[0081]

[0082] 3.3 Effects of tributyrate on serum hormone levels in pigs fed a diet contaminated with zearalenone

[0083] As shown in Figure 1, on day 21, the serum levels of GnRH, PRL, and E2 in pigs fed a ZEN-contaminated diet were significantly increased (P < 0.05), while the levels of FSH, LH, AMH, P4, and T were significantly decreased (P < 0.05). TB and ZEN had a significant interaction effect on the serum levels of GnRH, AMH, P4, and E2 on day 21 (P < 0.05). In pigs fed a ZEN-contaminated diet, TB significantly decreased the serum levels of GnRH and E2 on day 21 (P < 0.05) and significantly increased the serum levels of AMH and P4 on day 21 (P < 0.05). In pigs not fed a ZEN-contaminated diet, TB had no significant effect on the serum levels of GnRH, AMH, P4, and E2 on day 21.

[0084] As shown in Figure 2, on day 35, the serum levels of GnRH, PRL, and E2 in pigs fed a ZEN-contaminated diet were significantly increased (P < 0.05), while the levels of FSH, LH, AMH, P4, and T were significantly decreased (P < 0.05). TB and ZEN had a significant interaction effect on the serum AMH and E2 levels on day 35 (P < 0.05). In pigs fed a ZEN-contaminated diet, TB significantly decreased the serum E2 level on day 35 (P < 0.05) and significantly increased the serum AMH level on day 35 (P < 0.05). In pigs not fed a ZEN-contaminated diet, TB had no significant effect on the serum AMH and E2 levels on day 35.

[0085] 3.4 Effects of tributyrate on ovarian morphology in pigs fed a diet contaminated with zearalenone

[0086] 3.4.1 Effects of tributyrate on morphological changes in ovarian tissue of offspring fed a diet contaminated with zearalenone

[0087] As shown in Figure 3, the ovarian follicles in the CON and TB groups had normal morphology and structure with no obvious pathological changes; a large number of follicles in the ZEN group were atretic, causing ovarian damage; only a small number of follicles were atretic in the ZEN + TB group, and the ovarian tissue damage was relatively mild.

[0088] 3.4.2 Effects of tributyrate on the ultrastructure of pig ovaries fed a diet contaminated with zearalenone

[0089] As shown in Figure 4, the morphology and structure of mitochondria, endoplasmic reticulum and nucleus of GCs in the CON group and TB group were normal; the degree of mitochondrial edema and severe vacuolization of GCs in the ZEN group (thin red arrows), endoplasmic reticulum expansion and severe nuclear pyknosis (thick red arrows) were observed; only a small number of mitochondria in GCs in the ZEN + TB group showed vacuolization and mild nuclear pyknosis.

[0090] 3.5 Effect of tributyrate on apoptosis of ovarian cells in pigs fed a diet contaminated with zearalenone

[0091] As shown in Figure 5, dietary ZEN supplementation significantly increased the ovarian cell apoptosis rate (P < 0.05), with apoptosis mainly occurring in the granulosa layer of follicles (red arrows). TB and ZEN showed a significant interaction effect on the ovarian cell apoptosis rate (P < 0.05). In pigs fed a ZEN-contaminated diet, TB significantly reduced the ovarian cell apoptosis rate (P < 0.05). In pigs not fed a ZEN-contaminated diet, TB had no significant effect on the ovarian cell apoptosis rate.

[0092] 3.6 Effect of tributyrate on the expression levels of proteins related to the BMPs / SMAD signaling pathway in pigs fed a diet contaminated with zearalenone

[0093] Figures 6, 7, 8, and 9 show that most BMP2-positive cells were found in oocytes (thin red arrows), with a smaller portion in the GC layer (thick red arrows). Conversely, most BMP5, BMP6, and SMAD4-positive cells were found in the GC layer (thick red arrows), with a smaller portion in oocytes (thin red arrows). Pigs fed a ZEN-contaminated diet showed significantly increased BMP5 and BMP6 protein expression levels (P < 0.01) and significantly decreased SMAD4 protein expression levels (P < 0.05). In pigs fed a ZEN-contaminated diet, TB significantly increased SMAD4 protein expression levels (P < 0.05). In pigs not fed a ZEN-contaminated diet, TB had no significant effect on SMAD4 protein expression levels.

[0094] Experimental results:

[0095] ZEN exposure significantly increased vulvar area and decreased growth performance in pigs; TB treatment significantly reduced vulvar area and total increase on day 35, alleviating ZEN-induced edema. ZEN caused abnormally high levels of serum GnRH, PRL, and E2, and significantly decreased levels of FSH, LH, AMH, and P4. TB treatment significantly restored these hormone levels to normal. Histological and ultrastructural observations showed that ZEN caused extensive follicular atresia, granulosa cell layer disorder, mitochondrial vacuolization, and nuclear pyknosis. TB intervention significantly reduced ovarian structural damage and decreased atretic follicles. TUNEL assays showed that ZEN significantly increased ovarian cell apoptosis, while TB significantly inhibited this process. Immunohistochemistry showed that ZEN inhibited SMAD4 protein expression, while TB significantly restored SMAD4 expression levels, suggesting that TB may exert its anti-ZEN reproductive toxicity effect by regulating the BMPs / SMAD signaling pathway.

[0096] Example 2: In vitro protective effect of sodium butyrate (NaB) against ZEN-induced porcine ovarian granulosa cells (AVG-16).

[0097] 1. Materials and Methods

[0098] AVG-16 cells required for the experiment were purchased from Qingqi (Shanghai) Biotechnology Development Co., Ltd. (sourced from the European Certified Cell Culture Collection Center, ECACC). ZEN: purity ≥ 98.22% (MCE, catalog number HY-103447); NaB: purity ≥ 98% (Solarbio, catalog number ISO190).

[0099] 2. Test Methods

[0100] Complete cell culture medium was prepared by adding 50 mL fetal bovine serum (FBS), 5 mL penicillin-streptomycin mixture (PSF) (100 U / mL penicillin, 100 U / mL streptomycin), 500 μL insulin-transferrin-selenium (ITS-X), and 250 μL epidermal growth factor (EGF) to a final volume of 500 mL. After thorough mixing, the mixture was filtered and sterilized, then aliquoted into 50 mL centrifuge tubes and stored at 4°C. Preheating to 37°C before use was recommended.

[0101] Cell culture

[0102] (1) Cell resuscitation

[0103] AVG-16 cells removed from liquid nitrogen were rapidly thawed in a 37°C water bath until the cell suspension thawed. The cell suspension was then transferred to a 15 mL sterile centrifuge tube, 1 mL of complete culture medium was added and mixed well. After centrifugation at 1000 rpm for 5 min, the supernatant was discarded, and the cells were resuspended in 2 mL of complete culture medium. The cells were then seeded into 25 cm² culture media containing 4 mL of complete culture medium. 2 Gently shake the culture flask to mix well, and place it in a cell culture incubator at 37℃, 5% CO2, and saturated humidity to culture the cells.

[0104] (2) Cell passage

[0105] When the cells reach 80%-90% confluence, wash with PBS, then digest with trypsin for 4 min (37℃), add 2 mL of complete culture medium to stop digestion, centrifuge at 1000 rpm for 5 min, discard the supernatant, resuspend the cells in 2 mL of complete culture medium, and then seed in fresh 75 cm⁻¹ cells. 2 In the culture flask.

[0106] 3 Experimental Design

[0107] AVG-16 cells were selected as the research object in this experiment. Based on the previous research of our group, the appropriate treatment conditions for ZEN were determined to be: AVG-16 cells treated with 20 μM ZEN for 24 h (Zuo Cuige, 2024). Cells were treated with different concentrations of NaB (0.125, 0.25, 0.5, 1, and 2 mM) for 12, 24, and 36 h, respectively. Then, using a 2 × 6 factorial design, AVG-16 cells were pretreated for 12 h with culture media containing different concentrations of NaB (0, 0.125, 0.25, 0.5, and 1 mM), followed by co-treatment with different concentrations of NaB and ZEN (0 and 20 μM) for 24 h to screen for the optimal NaB concentration and treatment time. Using a 2 × 2 factorial design, AVG-16 cells were divided into four treatment groups: CON group, NaB group (0.25 mM), ZEN group (20 μM), and ZEN + NaB group (20 μM ZEN + 0.25 mM NaB). Cell samples were collected to measure relevant indicators.

[0108] 4. Test indicators and methods

[0109] Determination of cell morphology and viability

[0110] (1) Determination of AVG-16 cell morphology: AVG-16 cells were seeded in 96-well plates (70%-80% confluence). After the experimental treatment, the cells were washed 1-2 times with PBS, replaced with fresh culture medium, and quickly placed under an inverted microscope (200 ×) to observe and record the cell morphology.

[0111] (2) Cell viability determination: AVG-16 cells resuspended after trypsin digestion were seeded into 96-well plates. After the experimental treatment, medium containing CCK-8 was added (CCK-8 and complete medium were prepared at a ratio of 1:9), and after incubation for 1.5-2 h, the absorbance (OD value) at 450 nm was measured using an ELISA reader.

[0112] To determine the reactive oxygen species (ROS) in cells, AVG-16 cells resuspended after trypsin digestion were seeded in 6-well plates. After the experimental treatment, following the steps of the Solarbio ROS detection kit (catalog number CA1410), the positive control group was first given medium containing Rosup (Rosup and serum-free medium were prepared at a ratio of 1:1000), and the cells were incubated at 37°C in the dark for 20-30 min. After that, the medium was replaced, and the cells were washed 1-2 times with serum-free medium. Then, the treatment groups (excluding the blank control group) were given medium containing 2,7-dichlorofluorescein diacetate (DCFH-DA) (DCFH-DA and serum-free medium were prepared at a ratio of 1:1000), and the cells were incubated at 37°C in the dark for 20 min. After that, the medium was replaced, and the cells were washed 3 times with serum-free medium to thoroughly remove any DCFH-DA that had not entered the cells. After washing, replace each well with 1 mL of fresh serum-free culture medium and detect the fluorescence intensity of ROS using a fluorescence microplate reader (488 nm excitation wavelength, 525 nm emission wavelength).

[0113] Assay for sodium butyrate-induced apoptosis in AVG-16 cells: AVG-16 cells, resuspended after trypsin digestion, were seeded in 6-well plates. Following the grouping and treatment described in section 2.3, cells were washed 1-2 times with PBS according to the Solarbio ANNEXIN V-FITC / PI apoptosis detection kit (catalog number CA1020). Cells were then digested with trypsin without EDTA, and 1 mL of complete culture medium was added to terminate the digestion and collect the cell sample. The cell suspension was then transferred to a 1.5 mL sterile centrifuge tube, centrifuged at 1000 rpm for 5 min, the supernatant was discarded, 1 mL of pre-chilled PBS (4°C) was added, the cells were resuspended, and the cell pellet was centrifuged again, discarding the supernatant. 100 μL of the cell suspension was transferred to a 5 mL flow cytometry tube, 5 μL of Annexin V / FITC was added, and the mixture was incubated at room temperature in the dark for 5 min. Then, 5 μL of propidium iodide (PI) solution was added, followed by 400 μL of PBS. The mixture was then analyzed by flow cytometry.

[0114] Determination of BMPs / SMAD signaling pathway-related protein expression levels in AVG-16 cells stimulated by sodium butyrate: AVG-16 cells, after trypsin digestion and resuspending, were seeded in 6-well plates. Following the grouping and treatment described in section 2.3, 100 μL of Lysis Buffer Mix was added to each well. The lysis buffer was spread evenly with a cell scraper to ensure complete cell lysis. Cell samples were scraped and transferred to 1.5 mL sterile EP tubes. After centrifugation, the supernatant was collected for protein content determination, and sample protein was prepared. The expression levels of BMPs / SMAD signaling pathway-related proteins were determined using Western blot, with β-actin as an internal control. Information on the primary and secondary antibodies used is shown in Table 2.

[0115] Experimental data were analyzed using SPSS 26.0 statistical software for two-way ANOVA and one-way ANOVA. The main effects of the model included NaB and ZEN, and their interaction. Results are expressed as mean ± SEM. When interaction effects were present, Duncan's multiple comparisons were performed. Statistical results were plotted using GraphPad Prism 8.0. P < 0.05 was considered statistically significant, and 0.05 ≤ P < 0.1 was considered to indicate a significant trend.

[0116] 5. Experimental Results

[0117] 5.1 Effects of different concentrations of sodium butyrate on the morphology and viability of AVG-16 cells

[0118] 5.1.1 Effects of different concentrations of sodium butyrate on the morphology of AVG-16

[0119] As shown in Figure 10, after AVG-16 cells were treated with NaB for 12 h, the number of adherent cells increased significantly with the increase of NaB concentration (0-2 mM), and the cell morphology changed significantly: from the initial round shape to the willow leaf shape, the number of cell protrusions increased, and the cells showed a clearer spindle-shaped feature.

[0120] Figure 11 shows that after 24 h of NaB treatment, the number of adherent AVG-16 cells initially increased and then decreased. The highest cell density was observed in the 0.25 mM NaB treatment group, while higher concentrations (>0.5 mM) of NaB led to a decrease in cell number. Notably, cells in all treatment groups exhibited significant morphological changes, including a willow-leaf shape transformation and increased protrusions, with a more pronounced spindle-shaped feature.

[0121] As shown in Figure 12, after AVG-16 cells were treated with NaB for 36 h, the number of adherent cells decreased from NaB concentrations of 0.5 mM and above, but the morphological changes were more obvious: the cell processes became significantly longer and the spindle-shaped features were further enhanced.

[0122] 5.1.2 Effects of different concentrations of sodium butyrate on the viability of AVG-16 cells

[0123] As shown in Figure 13, NaB treatment exhibited a significant time- and concentration-dependent effect on AVG-16 cell viability: after 12 h of treatment, cell viability was significantly increased compared to the control group at 0.125–2 mM NaB treatment (P < 0.05). After 24 h of treatment, cell viability was significantly enhanced at 0.25–0.5 mM NaB treatment (P < 0.05), while 2 mM NaB significantly decreased cell viability (P < 0.05). After 36 h of treatment, cell viability decreased in a dose-dependent manner at 0.125–2 mM NaB treatment, while ≥ 0.25 mM NaB significantly inhibited cell viability (P < 0.05).

[0124] 5.2 Effects of sodium butyrate on the morphology and viability of AVG-16 cells stimulated by zearalenone

[0125] 5.2.1 Effects of sodium butyrate on the morphology of AVG-16 cells stimulated by zearalenone

[0126] As shown in Figure 14, the effects of different concentrations of NaB on the morphology of ZEN-stimulated AVG-16 cells are as follows: Compared with the control group, cells treated with 20 μM ZEN showed shorter processes and fewer adherent cells. Based on ZEN treatment, cells treated with different concentrations of NaB (0.125, 0.25, 0.5, and 1 mM) showed longer processes, and the typical spindle-shaped morphology of AVG-16 cells became increasingly clear.

[0127] 5.2.2 Effect of sodium butyrate on the viability of AVG-16 cells stimulated by zearalenone

[0128] As shown in Figure 15, compared with the control group, ZEN treatment for 24 h resulted in a significant decrease in cell viability (P < 0.05). In addition to ZEN treatment, 0.125, 0.25, 0.5, and 1 mM NaB all significantly increased cell viability (P < 0.05).

[0129] 5.3 Effect of sodium butyrate on reactive oxygen species in AVG-16 cells stimulated by zearalenone

[0130] As shown in Figure 16, the effects of NaB on ROS of ZEN-stimulated AVG-16 cells were as follows: compared with the control group, the ROS of ZEN-stimulated AVG-16 cells was significantly increased (P < 0.05); compared with the ZEN group, the ROS of the ZEN + NaB group was significantly decreased (P < 0.05).

[0131] 5.4 Effect of sodium butyrate on apoptosis induced by zearalenone in AVG-16 cells

[0132] As shown in Figure 17, compared with the control group, the apoptosis rate of AVG-16 cells in the ZEN group was significantly increased (P < 0.05); compared with the ZEN group, the apoptosis rate of cells in the ZEN + NaB group was significantly decreased (P < 0.05). Figure 18 shows that compared with the control group, the expression level of c-Caspase3 protein, which is related to apoptosis, in ZEN-stimulated AVG-16 cells was significantly increased (P < 0.05), and compared with the ZEN group, the expression level of c-Caspase3 protein in the ZEN + NaB group was significantly decreased (P < 0.05).

[0133] 5.5 Effect of sodium butyrate on the expression levels of proteins related to the BMPs / SMAD signaling pathway in AVG-16 cells stimulated by zearalenone

[0134] As shown in Figure 19, compared with the control group, ZEN stimulation significantly reduced the protein expression levels of SMAD4 and BMP6 in AVG-16 cells (P < 0.05), and tended to increase the protein expression level of BMP5 (0.05 ≤ P < 0.1). NaB and ZEN showed an interactive effect on the protein expression level of SMAD4 (0.05 ≤ P < 0.1). In ZEN-treated cells, NaB tended to increase the protein expression level of SMAD4 (0.05 ≤ P < 0.1). Neither NaB nor ZEN had a significant effect on the protein expression level of BMP2.

[0135] Experimental results:

[0136] In this study, an in vitro injury model was established using the AVG-16 cell line and treated with 20 μM ZEN for 24 h. The optimal protective concentration of NaB was determined to be 0.25 mM. NaB pretreatment significantly improved cell viability and morphology under ZEN stimulation (promoting protrusion growth and maintaining spindle-shaped structure). ZEN induced a surge in intracellular ROS levels, increased apoptosis rate, and elevated c-Caspase3 expression. 0.25 mM NaB significantly reduced ROS levels and inhibited c-Caspase3 expression, thereby reducing apoptosis. Western blot analysis showed that NaB could reverse the ZEN-induced downregulation of SMAD4 protein.

[0137] In summary, NaB can activate the BMPs / SMAD signaling pathway, effectively inhibit ZEN-induced apoptosis of porcine ovarian granulosa cells (pGCs), and thus alleviate the damage caused by ZEN to pGCs.

[0138] Example 3: Verification of the molecular mechanism based on SMAD4 interference

[0139] 1. Experimental Materials

[0140] The cells required for the experiment are the same as in Example 2.

[0141] The standard negative control interfering RNA oligomers (NC-siRNA oligo) and SMAD4-siRNA oligo were designed and synthesized by Shanghai Jima Pharmaceutical Technology Co., Ltd.; GP-transfect Mate transfection reagent was purchased from Shanghai Jima Pharmaceutical Technology Co., Ltd. The remaining reagents were the same as in Example 2.

[0142] 2. Test Methods

[0143] The preparation of complete cell culture medium and cell culture are the same as in Example 2.

[0144] The synthesis and transfection of SMAD4 interfering RNA were performed according to the SMAD4 gene ID (ID: 397142) found on NCBI. The most effective NC-siRNA and SMAD4-siRNA sequences were designed targeting this gene (see Table 6 for details). The dissolution of the siRNA oligo powder, the preparation of the transfection complex, and the cell transfection procedure were all performed according to the RNAi product manual of Shanghai Gemma Biotechnology Co., Ltd. and the instructions for the GP-transfect Mate transfection reagent. After transfection, the culture medium was replaced with complete medium, and protein was extracted after 36 hours for interference efficiency analysis or subsequent experiments.

[0145] Table 6 Gene Interference RNA Sequences

[0146]

[0147] 3 Experimental Design

[0148] AVG-16 cells were selected as the research object. Based on Example 2, the cells were divided into the following four treatment groups: ZEN + NC-siRNA group, ZEN + SMAD4-siRNA group, ZEN + NaB + NC-siRNA group, and ZEN + NaB + SMAD4-siRNA group. Cell samples were collected to measure relevant indicators.

[0149] 4. Test indicators and methods

[0150] To verify the efficiency of SMAD4 interference, AVG-16 cells, resuspended after trypsin digestion, were seeded in 6-well plates. The experiment consisted of two treatment groups, with three replicates per group. After the adherent cells reached 30-40% confluence, siRNA oligo transfection was performed. Thirty-six hours after transfection, the cells were washed 1-2 times with PBS, and 100 μL of Lysis Buffer Mix was added to each well to collect cell samples for protein extraction. Western blot was used to verify the SMAD4 interference efficiency, following the same experimental procedures as in Example 2. Information on the primary and secondary antibodies is shown in Table 2.

[0151] Determination of the effect of sodium butyrate on reactive oxygen species induced by zearalenone in AVG-16 cells after SMAD4 interference

[0152] AVG-16 cells, resuspended after trypsin digestion, were seeded in 6-well plates. A 2 × 2 factorial design was used, with four treatment groups and ten replicates per group. Once the adherent cells reached 30-40% confluence, siRNA oligo transfection was performed. After 12 h of transfection, cells were pretreated with 0.25 mM NaB for 12 h, followed by co-treatment with different concentrations (0, 0.25 mM) of NaB and ZEN (0, 20 μM) for 24 h. After treatment, the culture medium was changed, and ROS fluorescence intensity was detected as in step 4 of Example 2.

[0153] Determination of the effect of sodium butyrate on zearalenone-induced apoptosis in AVG-16 cells after SMAD4 interference

[0154] AVG-16 cells, digested with trypsin and resuspended, were seeded into 6-well plates. After grouping and treatment as in 2.4.2, the experimental procedure was the same as step 4 of Example 2, using flow cytometry to detect and analyze apoptosis.

[0155] Determination of the effect of sodium butyrate on the expression levels of BMPs / SMAD signaling pathway-related proteins in AVG-16 cells induced by SMAD4 interference

[0156] AVG-16 cells, resuspended after trypsin digestion, were seeded into 6-well plates. After grouping and treatment as described in 2.4.2, cells were washed 1-2 times with PBS, and 100 μL of Lysis Buffer Mix was added to each well. Cell samples were collected, and proteins were extracted. Western blot was used to determine the expression levels of proteins related to the BMPs / SMAD signaling pathway, following the same experimental procedure as step 4 in Example 2. Information on primary and secondary antibodies is shown in Table 2.

[0157] Data on SMAD4 interference efficiency validation were analyzed using t-tests with SPSS 26.0 statistical software. Other data were analyzed using one-way ANOVA combined with Duncan's multiple comparison method. Statistical results were plotted using GraphPadPrism 8.0. Results are expressed as mean ± SEM. P < 0.05 was considered statistically significant, and 0.05 ≤ P < 0.1 was considered to indicate a significant trend.

[0158] 5. Experimental Results

[0159] 5.1 Verification of SMAD4 Interference Efficiency

[0160] As shown in Figure 20, compared with the NC-siRNA group, the protein expression of SMAD4 in the SMAD4-siRNA group was significantly downregulated (P < 0.05), and its expression level was downregulated by 30% compared with the NC group.

[0161] 5.2 Effects of sodium butyrate on reactive oxygen species induced by zearalenone in AVG-16 cells after SMAD4 interference

[0162] As shown in Figure 21, the effects of NaB on the ROS of AVG-16 cells after SMAD4 interference were as follows: Compared with the ZEN + NC-siRNA group, the ROS of NaB-treated cells was significantly reduced (P < 0.05); compared with the ZEN + SMAD4-siRNA group, the ROS of the ZEN + NaB + SMAD4-siRNA group was significantly reduced (P < 0.05).

[0163] 5.3 Effect of sodium butyrate on zearalenone-induced apoptosis in AVG-16 cells after SMAD4 interference

[0164] As shown in Figure 22, compared with the ZEN + NC-siRNA group, NaB treatment significantly reduced the apoptosis rate of AVG-16 cells (P < 0.05); compared with the ZEN + SMAD4-siRNA group, NaB treatment had no significant effect on the apoptosis rate. Figure 23 shows that compared with the ZEN + NC-siRNA group, NaB treatment significantly reduced the expression level of the apoptosis-related protein c-Caspase3 (P < 0.05); compared with the ZEN + SMAD4-siRNA group, NaB treatment had no significant effect on the expression level of c-Caspase3.

[0165] 5.4 Effects of sodium butyrate on the expression levels of proteins related to the BMPs / SMAD signaling pathway in AVG-16 cells induced by zearalenone after SMAD4 interference

[0166] As shown in Figure 24, the effects of NaB on the expression levels of BMPs / SMAD signaling pathway-related proteins in AVG-16 cells after SMAD4 interference were as follows: Compared with the ZEN + NC-siRNA treatment group, NaB-treated cells showed a significant decrease in BMP6 protein expression (P < 0.05) and a trend towards increasing SMAD4 protein expression (0.05 ≤ P < 0.1). Compared with the ZEN + SMAD4-siRNA group, the NaB + ZEN + SMAD4-siRNA group showed no significant effect on the protein expression levels of SMAD4 and BMP6. Neither NaB nor SMAD4-siRNA had a significant effect on BMP5 protein expression.

[0167] Experimental results:

[0168] This embodiment utilizes siRNA technology to interfere with SMAD4 gene expression in AVG-16 cells, setting up ZEN+NC-siRNA, ZEN+SMAD4-siRNA, ZEN+NaB+NC-siRNA, and ZEN+NaB+SMAD4-siRNA groups. Experiments showed that after interfering with SMAD4, although NaB still reduced ROS levels (suggesting that antioxidant activity may involve other pathways), the inhibitory effect of NaB on apoptosis rate and c-Caspase3 expression disappeared (the difference was not statistically significant). This indicates that NaB's inhibition of ZEN-induced granulosa cell apoptosis depends on the intact BMPs / SMAD signaling pathway (especially SMAD4 protein mediation).

[0169] In summary, this invention demonstrates through in vitro and in vivo experiments that butyric acid and its derivatives effectively antagonize ZEN-induced oxidative stress and granulosa cell apoptosis by activating the BMPs / SMAD signaling pathway, particularly by upregulating the expression of SMAD4 protein, thereby protecting animal ovarian function and showing clear application prospects for preventing or alleviating ZEN reproductive toxicity.

Claims

1. The use of butyric acid or a derivative thereof in the preparation of products for preventing or alleviating reproductive system damage caused by zearalenone poisoning in animals.

2. The application as described in claim 1, characterized in that, The butyric acid derivative is selected from at least one of butyrate and butyrate ester.

3. The application as described in claim 1, characterized in that, The product is animal feed; the butyric acid or its derivative is added to the animal feed at a rate of 0.1%-0.5%, based on the total weight of the animal feed as 100%.

4. The application as described in claim 1, characterized in that, The damage to the reproductive system manifests as ovarian granulosa cell apoptosis, follicular atresia, oxidative stress damage to ovarian tissue, or reproductive hormone secretion disorders.

5. The application as described in claim 1, characterized in that, The animal is a pig; the product is used to upregulate the expression of SMAD4 protein in ovarian tissue.

6. An animal feed with resistance to zearalenone reproductive toxicity, characterized in that, The animal feed includes a base feed and an antitoxin additive; the antitoxin additive is butyric acid or its derivative, and its content in the animal feed is 0.1%-0.5% (by weight); the base feed includes the following raw materials in parts by weight: 35-45 parts corn, 8-12 parts puffed corn, 12-16 parts broken rice, 1.5-2.5 parts soybean oil, 18-22 parts soybean meal, 5-7 parts puffed soybeans, 1.5-3 parts fermented soybean meal, 1.5-2.5 parts fishmeal, 1-1.5 parts whey powder, 1.3-1.7 parts dicalcium phosphate, 0.4-0.6 parts limestone powder, 0.3-0.5 parts sodium chloride, 0.3-0.5 parts amino acid additive, and 0.5-0.8 parts premix.

7. The animal feed according to claim 6, characterized in that, The antitoxin additive is glyceryl tartrate.

8. The animal feed according to claim 6, characterized in that, The basic feed comprises the following raw materials in parts by weight: 39.35 parts corn, 10.00 parts puffed corn, 14.00 parts broken rice, 2.00 parts soybean oil, 19.50 parts soybean meal, 6.49 parts puffed soybeans, 2.09 parts fermented soybean meal, 1.78 parts fish meal, 1.16 parts whey powder, 1.54 parts dicalcium phosphate, 0.57 parts limestone powder, 0.44 parts sodium chloride, 0.40 parts amino acid additives, and 0.68 parts premix.

9. The animal feed according to claim 6, characterized in that, The amino acid additive is selected from at least one of methionine, lysine, threonine, and tryptophan.

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