Application of N-carbamoylglutamate in the prevention and treatment of bovine follicular cysts: related models and screening methods

Abstract

This invention provides the application of N-carbamoylglutamate (NCG) in the prevention and treatment of bovine follicular cysts, along with related models and screening methods. Specifically, it relates to the application of NCG in the preparation of products for the prevention and / or treatment of bovine follicular cysts, and preventive and therapeutic compositions containing NCG. This invention also establishes an in vitro cell model of bovine follicular cysts using 10... ‑7 Treatment of bovine ovarian granulosa cells with M-adrenocorticotropic hormone (ACTH) for 48 hours stably induces a cyst-like state of hormonal imbalance. Based on this model, an in vitro screening method using NCG as a positive control is provided to evaluate the preventive and therapeutic potential of candidate substances. In vivo experiments have confirmed that adding NCG to the diet of dairy cows can effectively treat and prevent follicular cysts. This invention provides a safe and efficient new non-hormonal strategy for the prevention and treatment of bovine follicular cysts, and is accompanied by reliable research and screening tools.

Description

Technical Field

[0001] This invention belongs to the fields of veterinary medicine, animal reproduction, and feed additives, specifically relating to the novel use of N-carbamoylglutamate (NCG) in the prevention and / or treatment of bovine follicular cysts (also known as cystic ovary disease). This invention also relates to methods for establishing in vitro cell models of bovine follicular cysts, drug screening methods based on these models, preventive and therapeutic compositions containing NCG, related feeding methods, and kits or platforms for screening. Background Technology

[0002] Bovine follicular cysts are one of the most common reproductive disorders in dairy cows, characterized by the persistent presence of abnormally enlarged anovulatory cystic structures on the ovary. This disease leads to estrous cycle disorders, decreased conception rates, and prolonged non-pregnant periods, resulting in significant economic losses. Its pathogenesis is complex, but it is generally believed to be closely related to hypothalamic-pituitary-gonadal (HPG) axis dysfunction, endocrine imbalances, oxidative stress, and abnormalities in the local metabolic microenvironment of the ovary.

[0003] Currently, the clinical prevention and control of bovine follicular cysts mainly relies on hormone therapy, such as the use of gonadotropin-releasing hormone (GnRH), human chorionic gonadotropin (hCG), or prostaglandins. However, hormone therapy has problems such as large individual variability, unstable efficacy, potential interference with normal endocrine cycles, and the risk of drug residues. Therefore, developing safe, effective, and non-hormonal prevention and treatment strategies has become a research hotspot and urgent need in this field.

[0004] N-Carbamoylglutamate (NCG) is a stable structural analog of N-acetylglutamate (NAG) and a key activator in the synthesis of endogenous arginine. Previous studies have shown that NCG plays a role in improving reproductive performance, stress resistance, and antioxidant properties in animals (such as pigs and sheep). However, its preventive and therapeutic effects, specific targets, and molecular mechanisms in bovine follicular cysts, a specific reproductive disorder, have not been systematically elucidated, and it has not yet been identified as a clearly defined active ingredient for the preparation of specialized products for the prevention and treatment of this disease.

[0005] Furthermore, the lack of stable, reliable in vitro cell models that can simulate the key pathological features of bovine follicular cysts hinders in-depth research and efficient drug screening when studying the pathogenesis of bovine follicular cysts and screening for prevention and treatment drugs. Summary of the Invention

[0006] The first object of the present invention is to provide a novel use of N-carbamoylglutamic acid (NCG) or a pharmaceutically acceptable salt thereof in the preparation of products for the prevention and / or treatment of bovine follicular cysts.

[0007] A second objective of this invention is to provide a composition for the prevention and treatment of bovine follicular cysts.

[0008] The third objective of this invention is to provide a method for establishing an in vitro cell model that can simulate the core pathological features of bovine follicular cysts.

[0009] A fourth objective of this invention is to provide a screening method based on the above-mentioned in vitro cell model for evaluating the potential of candidate substances in preventing and treating bovine follicular cysts.

[0010] The fifth objective of this invention is to provide a feeding method for preventing or treating bovine follicular cysts.

[0011] The sixth objective of this invention is to provide a kit or platform for screening drugs for the prevention and treatment of bovine follicular cysts.

[0012] To achieve the above objectives, the present invention adopts the following technical solution:

[0013] This invention first provides a novel use of N-carbamoylglutamic acid (NCG) or a pharmaceutically acceptable salt thereof in the preparation of products for the prevention and / or treatment of bovine follicular cysts (also known as cystic ovary disease).

[0014] Further, the product is a feed additive, veterinary preparation, or cell culture medium additive. When used as a feed additive or veterinary preparation, the dosage is such that the daily intake of cattle is 5-40 g / head. Preferably, the daily intake is 15-25 g / head. More preferably, the daily intake is 20 g / head, fed continuously for 30-60 days, for example, 40 days, to obtain significant preventive or therapeutic effects.

[0015] The present invention also provides a composition for the prevention and / or treatment of bovine follicular cysts, comprising a therapeutically effective amount of N-carbamoylglutamic acid (NCG) or a pharmaceutically acceptable salt thereof as an active ingredient, and at least one pharmaceutically acceptable or feed-acceptable carrier.

[0016] Furthermore, the composition may also contain one or more additional active ingredients selected from antioxidants (such as vitamin E, selenium, etc.), vitamins, trace elements, or probiotics to synergistically enhance the effect or provide comprehensive nutritional support.

[0017] This invention also provides a method for establishing an in vitro cell model of bovine follicular cysts, which can stably simulate the core pathological features of granulosa cells in a cystic state. The method includes the following steps:

[0018] a) Provide bovine ovarian granulosa cells;

[0019] b) Treat the granulosa cells with adrenocorticotropic hormone (ACTH);

[0020] c) Treat for a sufficient time to induce a hormonal secretion disorder phenotype in cells, including significant suppression of estradiol (E2) secretion and / or abnormally elevated progesterone (P4) secretion.

[0021] Preferably, in step b), the concentration range of ACTH is 10. -10 M to 10 -6 M.

[0022] More preferably, the concentration of ACTH is 10. -7 M.

[0023] Preferably, in step c), the processing time is at least 24 hours.

[0024] More preferably, the processing time is 48 hours. Using 10 -7 The 48-hour MACCH treatment regimen can stably and efficiently induce typical hormonal imbalances, most closely resembling the pathological state of cysts in vivo.

[0025] Based on the above-described in vitro cell model, this invention also provides an in vitro screening method for evaluating the potential of candidate substances to prevent or treat bovine follicular cysts. The method includes the following steps:

[0026] a) Establish an in vitro cell model of bovine follicular cysts using the methods described above;

[0027] b) The candidate substance to be evaluated is applied to the cell culture system during model establishment (co-processing), before model establishment (pre-processing), or after model establishment (post-processing);

[0028] c) Detect and analyze one or more biological indicators of the cells, the indicators being selected from: estradiol (E2) secretion level, progesterone (P4) secretion level, E2 / P4 ratio, cell viability (e.g., measured by MTT assay), and mRNA expression levels of key genes in the steroid hormone synthesis pathway (e.g., STAR, CYP17A1, FSHR, P450arom).

[0029] d) Compare and analyze the detection results of the candidate substance treatment group with the results of the untreated model control group and the positive control group containing the known effective substance N-carbamoylglutamic acid (NCG);

[0030] If the candidate substance can significantly reverse or alleviate ACTH-induced E2 secretion inhibition and / or P4 secretion elevation, and / or restore the expression levels of abnormally expressed steroid synthesis-related genes (such as upregulation of STAR and CYP17A1 and downregulation of FSHR and P450arom) to normal physiological state, it indicates that the candidate substance has potential activity in preventing or treating bovine follicular cysts.

[0031] Furthermore, during the screening process, NCG can be used as a standard positive control to verify the effectiveness of the model and evaluate the relative potency of candidate substances.

[0032] The present invention also provides a feeding method for preventing or treating bovine follicular cysts. The method includes adding and continuously feeding an effective amount of N-carbamoylglutamate (NCG) or a pharmaceutically acceptable salt thereof to the diet of cattle that are at risk of developing follicular cysts (such as during the peripartum period, early postpartum period, or heat stress period) or have been diagnosed with follicular cysts for a period of time.

[0033] Furthermore, the effective amount is 5-40 g NCG per cow per day.

[0034] Preferably, the effective amount is 20 g NCG per cow per day.

[0035] Furthermore, the continuous feeding period is at least 20 days.

[0036] Preferably, the duration is 30 to 60 days, for example, 40 days.

[0037] This invention also provides a kit or integrated screening platform for screening drugs for the prevention and treatment of bovine follicular cysts. The kit or platform comprises:

[0038] a) Adrenocorticotropic hormone (ACTH), as a modeling agent;

[0039] b) N-Carbamoylglutamic acid (NCG) was used as a positive control to evaluate the effectiveness of the model and the activity of candidate substances.

[0040] c) Detection reagents for detecting hormone secretion levels (such as ELISA or RIA reagents for E2 and P4) or gene expression levels (such as qRT-PCR reagents) in bovine ovarian granulosa cells.

[0041] Furthermore, the kit or platform may also contain bovine ovarian granulosa cells (e.g., primary cells or cell lines), or a complete set of reagents (e.g., digestive enzymes, culture media, serum, etc.) for isolating and culturing bovine ovarian granulosa cells, to provide a ready-to-use research or screening system.

[0042] This invention also provides the application of N-carbamoylglutamate (NCG) as a positive control in establishing or validating drug screening models for bovine reproductive disorders (especially bovine follicular cysts).

[0043] The present invention has the following beneficial effects:

[0044] This invention reveals for the first time that NCG does not work through a simple antioxidant pathway, but rather by activating the arginine-NO metabolic axis and regulating downstream key signaling pathways such as PI3K / Akt / mTOR. Specifically, it upregulates the expression of follicle-stimulating hormone receptor (FSHR) and aromatase (P450arom), while downregulating the expression of acute regulatory steroid production protein (STAR) and 17α-hydroxylase (CYP17A1). This precise "reprogramming" of the gene network directly reverses the ACTH stress-induced adrenalization shift in granulosa cell function, restoring their normal estrogen synthesis capacity and pre-ovulatory functional state at the molecular level, thus achieving targeted intervention at the core of the disease.

[0045] This invention translates a well-defined cellular and molecular mechanism into a verifiable holistic animal prevention and treatment program. Controlled trials demonstrated that adding NCG (20 g / head / day, 40 days) to the diet resulted in a cure rate of 91.67% (11 / 12) for existing follicular cysts, significantly higher than the melatonin treatment group (8.33%, 1 / 12) and the blank control group (0%, 0 / 12) (P < 0.01). In the prevention trial, the new cyst incidence rate in the NCG group was 0%, also significantly lower than the blank control group (15%) and the melatonin group (20%).

[0046] Traditional hormone therapy suffers from problems such as significant individual variability in response, disruption of normal reproductive cycles, and potential residue risks. The NCG feed additive solution provided by this invention, as a nutritional regulation method, avoids these risks and offers high safety. Its application is simple; it can be directly mixed into feed without altering existing farm management processes. It is highly applicable and easily promoted for large-scale dairy farming, demonstrating promising industrialization prospects and economic benefits.

[0047] Furthermore, based on NCG's well-defined cellular mechanism of action and excellent overall preventive and therapeutic effects, this invention establishes a standardized in vitro screening system using an ACTH-induced granulocyte dysfunction model and NCG as a systemic suitability control and efficacy evaluation benchmark. This model provides a reliable tool for efficiently screening and evaluating novel anti-cystic compounds and has significant methodological value. Attached Figure Description

[0048] Figure 1 Effects of different concentrations of ACTH treatment for 48 h on the secretion of estradiol (A) and progesterone (B) by bovine granulosa cells.

[0049] Figure 2 Effects of simultaneous ACTH + NCG treatment (24 h) on estradiol (A) and progesterone (B) secretion from granulosa cells.

[0050] Figure 3Effects of simultaneous ACTH + NCG treatment (48 h) on estradiol (A) and progesterone secretion (B) in granulosa cells.

[0051] Figure 4 Effects of ACTH treatment for 24 h followed by NCG treatment for 24 h on the secretion of estradiol (A) and progesterone (B) in bovine granular cells.

[0052] Figure 5 Effects of NCG pretreatment for 24 h combined with ACTH treatment for 24 h on the secretion of estradiol (A) and progesterone (B) in bovine granular cells.

[0053] Figure 6 : Detection of key genes for steroid synthesis in bovine granulocytes treated with ACTH by NCG using qRT-PCR ( STAR, CYP17A1, FSHR, P450arom The effect of relative mRNA expression levels. Detailed Implementation

[0054] The present invention will be described in detail below with reference to embodiments and accompanying drawings. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless specific conditions are specified, are generally performed under conventional conditions in the art or as recommended by the manufacturer.

[0055] Example 1: Establishment of an ACTH-induced model of bovine ovarian granulosa cell dysfunction

[0056] 1.1 Main reagents and instruments:

[0057] Ovarian source: Ovaries were collected from healthy adult Holstein cows within 30 minutes of slaughter.

[0058] Cell culture medium: DMEM / F12 basal medium, supplemented with 10% (v / v) fetal bovine serum and 1% (v / v) penicillin-streptomycin solution.

[0059] Digestive fluid: 0.25% (w / v) trypsin solution containing 0.02% EDTA.

[0060] Main reagent: Adrenocorticotropic hormone (ACTH), purity ≥95%, prepared with sterile phosphate buffer to a concentration of 10... - 4 M stock solution, aliquoted and stored at -20℃.

[0061] Main instruments: Clean bench, CO2 incubator (37℃, 5% CO2), inverted phase contrast microscope, low-speed centrifuge, fully automated cell counter.

[0062] 1.2 Isolation and primary culture of bovine ovarian granulosa cells:

[0063] The collected ovaries were rinsed three times with preheated sterile saline (containing 1% penicillin and oxytocin). Follicular fluid was extracted from follicles with a diameter of 3-8 mm on the surface of the ovary using a 10 mL sterile syringe (with an 18G needle) and collected in a 15 mL sterile centrifuge tube.

[0064] Centrifuge the collected follicular fluid at 1000 rpm for 5 minutes and discard the supernatant. Gently resuspend the cell pellet in pre-warmed PBS buffer and wash three times under the same conditions. Filter the resulting cell suspension through a 200-mesh filtration to remove cell clumps and centrifuge at 1000 rpm for 5 minutes. Wash twice with PBS and centrifuge at 1000 rpm for 5 minutes. Wash once more with cell culture medium and discard the supernatant. Resuspend the cells in 1 mL of cell culture medium, agitate thoroughly, and count the cells. Adjust the cell concentration to 1 × 10⁶ cells / mL. 5 Cells / mL were seeded in 25T culture flasks and incubated statically in a 37℃, 5% CO2 incubator. After 12 hours, granular cells adhered to the culture medium and could be observed; at this point, the culture medium was replaced.

[0065] 1.3 ACTH treatment to establish a functional disorder model:

[0066] Once the granulosa cells have grown to 80-90% confluence, proceed with the experimental treatment. Discard the original culture medium and gently wash the cells twice with sterile PBS.

[0067] Replace with serum-free DMEM / F12 medium and subject to serum starvation for 1 hour to synchronize the cell cycle and reduce basal disturbances.

[0068] After starvation, the culture medium was discarded. The experimental groups were then treated with different concentrations of ACTH (final concentrations were 10...). -10 M, 10 -9 M, 10 -8 M, 10 -7 M, 10 -6 M) Serum-free culture medium. The control group was given an equal volume of serum-free culture medium without ACTH.

[0069] The cells were returned to the incubator to continue culturing. The culture was terminated after 24 hours and 48 hours of treatment, respectively.

[0070] Sample collection: Carefully collect the cell culture supernatant from each well / flask, centrifuge at 4°C and 2000 rpm for 10 minutes to remove suspended cells and debris, aliquot the supernatant and store at -80°C for hormone detection. Simultaneously, wash cells twice with pre-chilled PBS, add an appropriate amount of TRIzol reagent to lyse the cells, and store at -80°C for subsequent RNA extraction.

[0071] 1.4 Hormone level detection (radioimmunoassay, RIA):

[0072] Hormone concentration detection: This invention uses radioimmunoassay to analyze the concentrations of progesterone and estrogen in the culture medium. The inter-group and intra-group variability are both less than 10%. The specific operation steps are as follows:

[0073] (1) Preparation of standard curve: Prepare 11 centrifuge tubes and label them 1-11; add 500 μL of RIA buffer to each tube; add 500 μL of hormone standard sample solution to tube 1 and vortex to mix; add 500 μL from tube 1 to tube 2 and vortex to mix; add 500 μL from tube 2 to tube 3, and so on, until tube 11. Take another centrifuge tube and label it starting from 10. Add 100 μL of the diluent from tubes 1-11 to each tube. The tube number correspondence is as follows: the diluent from tube 1 is distributed to tubes 10-12, the diluent from tube 2 is distributed to tubes 13-15, and so on.

[0074] (2) Diluting the antibody and labeled antigen: 100 μL of antibody was diluted with 11.5 mL of RIA buffer; 5 μL of labeled antigen was diluted with 30 mL of RIA buffer.

[0075] (3) Set up negative controls: TC group represents all labeled antigens, with only 100 μL of labeled antigen added, three tubes in total, labeled 1-3; NSB group represents the separation ability of activated carbon, with 100 μL of labeled antigen and 200 μL of activated carbon diluent added, labeled 4-6; B0 group represents the binding ability of antibody and labeled antigen, with 100 μL of antibody, 100 μL of labeled antigen and 200 μL of activated carbon diluent added, labeled 7-9.

[0076] (4) Sample preparation: After centrifuging the culture medium, take the supernatant, dilute it 10 times with RIA buffer, take 100 μL and add it to the centrifuge tube. Make two replicates for each sample, starting from 43 and 44.

[0077] (5) Antigen-antibody binding: Add 100 μL of antibody, 100 μL of labeled antigen, and 200 μL of activated charcoal dilution to the centrifuge tubes of the standard curve and samples, and label them. Balance each tube with RIA buffer, so that the total volume of each tube is 700 μL. After vortexing and mixing, incubate overnight at 4°C.

[0078] (6) Add activated carbon: Add 200 μL of activated carbon dilution to the standard curve tube and sample tube, shake to mix, and let stand at 4°C for 15 minutes.

[0079] (7) Centrifuge at 4℃ for 15 minutes at a speed of 2500 rpm.

[0080] (8) Pour the supernatant into the bottle, add 4 mL of scintillation liquid to each bottle, close the cap and shake vigorously to mix.

[0081] 1.5 Results and Analysis:

[0082] like Figure 1 and 2 As shown, ACTH treatment had a time- and concentration-dependent effect on hormone secretion in bovine granulocytes. After 24 hours of treatment, all concentrations of ACTH significantly inhibited E2 secretion (p < 0.05 compared to the control group), with 10% of the concentrations significantly lower. - 7 M and 10 -6 The inhibitory effect on M was most significant (p < 0.01), but it had no significant effect on P4 secretion. After 48 hours of treatment, the inhibitory effect of E2 persisted and intensified; simultaneously, 10 -7 M and higher concentrations of ACTH significantly promoted P4 secretion (p < 0.05). This successfully mimicked the typical hormonal dysregulation features of granulosa cells in follicular cysts: insufficient estrogen synthesis accompanied by abnormal progesterone levels. Therefore, 10... -7 48 hours of M ACTH treatment was used as the standard modeling condition for subsequent experiments.

[0083] Example 2: The intervention effect of NCG on ACTH-induced cellular dysfunction

[0084] 2.1 Experimental grouping and treatment:

[0085] Based on the primary granular cells established in Example 1, the following treatment groups were set up, with 6 replicates per group:

[0086] Control group: serum-free culture medium, culture time is the same as the corresponding experimental group.

[0087] ACTH model group: containing 10 -7 M ACTH used serum-free medium and cultured for the same time as the corresponding experimental group.

[0088] NCG intervention group I (co-treatment): containing 10 -7 M ACTH and 10 -7 M NCG in serum-free medium, cultured for 24 / 48 hours.

[0089] NCG intervention group II (pretreatment): first used a solution containing 10 -7 M NCG was pretreated with serum-free medium for 24 hours, then the medium was discarded and replaced with medium containing 10 -7 Continue culturing in serum-free medium for 24 hours.

[0090] NCG intervention group III (post-treatment): first use a drug containing 10-7 M ACTH was treated with serum-free medium for 24 hours, then the medium was discarded and replaced with medium containing 10... -7 Continue culturing the M NCG in serum-free medium for 24 hours.

[0091] (Note: NCG was directly dissolved in serum-free medium and prepared to a concentration of 10...) -2 M stock solution, after filtration and sterilization, is stored at -20℃. Dilute to the working concentration before use.

[0092] 2.2 Processing and Sample Collection:

[0093] Cell seeding and starvation procedures were the same as in Example 1.3. Culture media corresponding to the treatment conditions were added according to the above groupings. Except for the 24-hour co-treatment group and the corresponding control, the total time for all other treatments was 48 hours (the total time for the "pre-treatment" and "post-treatment" modes was also 48 hours). After treatment, cell culture supernatants were collected and E2 and P4 concentrations were measured using the methods in Examples 1.3 and 1.4.

[0094] 2.3 Results and Analysis:

[0095] like Figure 3-5 As shown, compared with the control group, E2 secretion was significantly suppressed and P4 secretion was significantly increased in the ACTH model group. All three intervention modalities of NCG showed varying degrees of antagonism. Specifically:

[0096] The NCG co-treatment group significantly reversed the inhibitory effect of ACTH on E2 (p < 0.01, vs ACTH group) and significantly reduced the abnormally elevated P4 level (p < 0.05).

[0097] The NCG pretreatment group showed the strongest protective effect, with no significant difference in E2 and P4 levels compared to the Control group (p > 0.05), almost completely blocking the damaging effects of ACTH.

[0098] The NCG posttreatment group also showed significant recovery of E2 secretion and reduction of P4 levels, demonstrating therapeutic potential.

[0099] The results showed that NCG has a clear preventive (pretreatment) and therapeutic (co-treatment and post-treatment) effect on ACTH-induced granulocyte cytokine secretion disorder.

[0100] Example 3: Effects of NCG on the expression of key genes in steroid synthesis

[0101] 3.1 Cell processing and RNA extraction:

[0102] Cells from the Control group, ACTH model group, and NCG co-treatment group in Example 2 were collected, the culture medium was discarded, and the cells were washed once with PBS. 1 mL of TRIzol lysis buffer was added directly to each well, mixed thoroughly by pipetting, and transferred to RNase-free centrifuge tubes. The cells were incubated at room temperature for 5 minutes. Subsequent RNA extraction was performed strictly according to the kit instructions.

[0103] 1. Add 200 μL of chloroform, shake vigorously for 15 seconds, and let stand at room temperature for 3 minutes.

[0104] 2. Centrifuge at 12000 g for 15 minutes at 4℃. Carefully transfer approximately 400 μL of the colorless aqueous phase from the top layer to a new tube.

[0105] 3. Add an equal volume of pre-cooled 70% ethanol (prepared with DEPC water) and mix well.

[0106] 4. Transfer the mixture to an adsorption column, centrifuge at 12000 g for 30 seconds at 4°C, and discard the waste liquid.

[0107] 5. Add 500 μL of protein removal solution RW1 to the adsorption column, centrifuge for 30 seconds, and discard the waste liquid.

[0108] 6. Add 500 μL of rinse buffer RW (diluted with anhydrous ethanol), centrifuge for 30 seconds, and discard the waste liquid. Repeat this step once.

[0109] 7. Centrifuge the empty column for 2 minutes to completely remove residual ethanol. Place the adsorption column in a new centrifuge tube and let it air dry for 2 minutes.

[0110] 8. Add 30-50 μL of RNase-Free ddH2O dropwise to the center of the adsorption membrane, let stand at room temperature for 2 minutes, centrifuge at 12000 g for 2 minutes at 4℃, and collect the RNA solution. Determine the RNA concentration and purity (A260 / A280 between 1.8 and 2.0) using an ultra-micro spectrophotometer, and store at -80℃.

[0111] 3.2 Reverse transcription to synthesize cDNA:

[0112] Using the Tiangen Biotech FastKing gDNA Dispelling RT SuperMix kit, prepare a 20 μL reaction mixture on ice:

[0113] Total RNA: 1 μg

[0114] 5×FastKing-RT SuperMix: 4 μL

[0115] RNase-Free ddH2O: Add to a final volume of 20 μL

[0116] After gently mixing, run the following program on a PCR instrument: 42℃ for 15 minutes (reverse transcription); 95℃ for 3 minutes (enzyme inactivation). Dilute the resulting cDNA 5-fold with RNase-free ddH2O and store at -20℃ for later use.

[0117] 3.3 Real-time quantitative PCR:

[0118] The SuperReal PreMix Plus (SYBR Green) kit from Tiangen Biotech was used. Primer sequences are shown in Table 1 and were synthesized by Sangon Biotech Co., Ltd.

[0119] Table 1. qRT-PCR primer sequences

[0120] Prepare a 20 μL reaction mixture in a 96-well plate:

[0121] 2×SuperReal PreMix Plus: 10 μL

[0122] Upstream primer (10 μM): 0.6 μL

[0123] Downstream primer (10 μM): 0.6 μL

[0124] cDNA template: 2 μL (approximately 50 ng)

[0125] RNase-Free ddH2O: 6.8 μL

[0126] Three technical replicates were set up for each sample. The following program was run on a Bio-Rad CFX96 Touch real-time quantitative PCR instrument: 95°C pre-denaturation for 15 min; 95°C denaturation for 10 s, 60°C annealing / extension for 30 s, 40 cycles; finally, melting curves were plotted (read every 0.5°C from 65°C to 95°C). Two... -ΔΔCt The method calculates the target gene relative to the internal reference gene ( GAPDH The relative expression level of ).

[0127] 3.4 Results and Analysis:

[0128] like Figure 6 As shown, compared to the Control group, the ACTH model group STAR and CYP17A1 Gene expression was significantly upregulated (p < 0.01), while FSHR and P450arom Gene expression levels were significantly downregulated (p < 0.01). This abnormal gene expression profile was significantly reversed in the NCG co-treatment group. STAR and CYP17A1 The expression level was significantly lower than that of the ACTH model group (p < 0.05), while FSHR and P450arom The expression levels of NCG significantly rebounded (p < 0.05), all trending towards the levels in the Control group. This reveals the core molecular mechanism by which NCG corrects steroid synthesis in granulosa cells at the transcriptional level.

[0129] Example 4: In vivo efficacy test of NCG in preventing bovine follicular cysts (compared to melatonin)

[0130] 4.1 Laboratory Animals and Their Management:

[0131] Two hundred and forty healthy Holstein lactating cows with similar parities (2-3 parities), milk yield (30-35 kg / d), and body condition scores (2.5-3.5) were selected. All cows were housed in the same standardized barn, with free access to a total nutritional supplement (TMR) diet and water. The basal diet was formulated according to the NRC (2001) nutritional requirements for dairy cows. A two-week pre-feeding period was provided to allow the cows to acclimatize to the environment.

[0132] 4.2 Experimental Design and Grouping:

[0133] After the pre-feeding period, experienced veterinarians performed rectal examinations combined with B-mode ultrasound (7.5 MHz linear array probe) on all experimental cattle to determine ovarian status. Based on the examination results, the cattle were grouped as follows:

[0134] The confirmed cyst group consisted of 36 follicles with a diameter ≥25 mm, thin walls, and no luteinized structure as observed on ultrasound, and their presence was confirmed to be persistent in two consecutive examinations (7 days apart). These follicles were randomly divided into 3 treatment trial groups, with 12 follicles in each group.

[0135] NCG treatment group: basic TMR + NCG (20 g / head / day).

[0136] Melatonin treatment group: basal TMR + melatonin (46.4 mg / head / day).

[0137] Blank control group: Basic TMR.

[0138] Healthy control group: 60 animals, with normal ovarian and follicular development as determined by ultrasound. They were randomly divided into 3 prevention experimental groups, with 20 animals in each group.

[0139] NCG prevention group: basic TMR + NCG (20 g / head / day).

[0140] Melatonin prevention group: Basal TMR + melatonin (46.4mg / head / day).

[0141] Blank control group: Basic TMR.

[0142] NCG (purity ≥97%) was weighed daily and manually premixed with approximately 1 kg of concentrate supplement before being sprinkled on the surface of the TMR diet, ensuring that each cow consumed it. Melatonin was administered via injection, which is the currently accepted method for ensuring effective melatonin action in cattle. The trial lasted 40 days.

[0143] 4.3 Observation indicators and detection methods:

[0144] Follicular cyst examination: Ultrasound examinations were performed by the same reproductive specialist (who was unaware of the group assignments) on the start day (D0) and end day (D40) of the trial. The number, size, and morphology of all follicles ≥10 mm in diameter on both ovaries were recorded. Cure / effectiveness criteria: The cystic follicles disappeared or shrank in diameter to <20 mm, and normally developing follicles or corpora lutea appeared simultaneously. New occurrence criteria: The presence of cystic follicles ≥25 mm in diameter that persisted until the examination day.

[0145] Production performance record: Record the milk production of each group of dairy cows daily.

[0146] Blood parameters monitoring: Blood was collected from the tail vein before morning feeding on days 0, 20, and 40. Serum was separated and stored at -80°C. The concentrations of glucose (GLU), non-esterified fatty acids (NEFA), and β-hydroxybutyrate (BHB) in serum were measured using a fully automated biochemical analyzer. The levels of estradiol (E2), progesterone (P4), and cortisol (COR) were measured using an ELISA kit.

[0147] 4.4 Data Processing and Statistical Analysis:

[0148] The experimental data were statistically analyzed using SPSS 26.0 software. Categorical data such as cyst incidence and cure rate were compared using the chi-square test. Quantitative data such as milk production and hormone levels were expressed as mean ± standard deviation. Normality and homogeneity of variance tests were performed first, and those meeting the criteria were analyzed using one-way ANOVA. Multiple comparisons between groups were performed using Duncan's new multiple range test. A p-value < 0.05 was considered statistically significant, and a p-value < 0.01 was considered highly statistically significant.

[0149] 4.5 Results and Analysis:

[0150] 4.5.1 Treatment efficacy for follicular cysts (Table 2)

[0151] At the end of the trial, the cyst cure rate in the NCG treatment group was as high as 91.67% (11 / 12), significantly higher than that in the melatonin treatment group (8.33%, 1 / 12) and the blank control group (0%, 0 / 12) (P < 0.01). There was no significant difference between the latter two groups (P > 0.05). Ultrasound imaging showed that most of the cystic structures on the ovaries of the cows in the NCG treatment group disappeared, replaced by normal-sized follicles or newly formed corpora lutea.

[0152] 4.5.2 Preventive effect on follicular cysts (Table 2)

[0153] During the 40-day trial period, no new cysts were observed in the NCG prevention group, with a new incidence rate of 0%, which was significantly lower than that in the melatonin prevention group (20%, 4 / 20) and the blank control group (15%, 3 / 20) (P < 0.01). There was no significant difference in the new incidence rate between the melatonin prevention group and the blank control group.

[0154] Table 2. Statistical analysis of the effects of in vivo feeding trials

[0155] Note: Different lowercase letters in the same column indicate significant differences (P < 0.05), and different uppercase letters indicate extremely significant differences (P < 0.01).

[0156] 4.5.3 Effects on serum markers

[0157] In the later stages of the experiment (D20, D40), the serum E2 levels of dairy cows in the NCG treatment and prevention groups were significantly higher than those in their corresponding melatonin groups and the blank control group (P < 0.05), while the P4 and COR levels were significantly lower (P < 0.05). The serum NEFA and BHB levels in the NCG group also showed a decreasing trend, indicating improved metabolic status. The melatonin group showed no significant improvement in the above blood indicators compared to the blank control group.

[0158] Conclusion: This controlled trial confirms that dietary NCG supplementation is effective in treating and preventing bovine follicular cysts, with significantly better results than melatonin supplementation under the same experimental conditions. Melatonin failed to show a significant preventive or therapeutic effect in this trial, indicating that the effect of NCG is unique and specific.

[0159] Example 5: Preparation and application of NCG-containing cell culture medium additives

[0160] 5.1 Preparation of NCG culture medium additives:

[0161] Weigh 190.2 mg of N-carbamoylglutamic acid powder (molecular weight 190.16) and dissolve it in 10 mL of sterile, pyrogen-free phosphate-buffered saline (PBS, pH 7.4) to prepare a 100 mM stock solution. Filter the solution through a 0.22 μm sterile syringe filter for sterilization, aliquot into 1.5 mL sterile centrifuge tubes, and store at -20°C protected from light. Shelf life is 6 months.

[0162] 5.2 Application in granulocyte stress protection:

[0163] Bovine ovarian granulosa cells were isolated and cultured according to the method in Example 1 and seeded into 96-well plates.

[0164] The experiment was divided into four groups: normal control group, ACTH stress group, and ACTH+NCG protection group.

[0165] Pretreatment of the protection group: Two hours before adding ACTH, NCG stock solution was added to the culture medium of the ACTH+NCG protection group to bring the final concentration to 10. -7 M. The normal control group and the ACTH stress group were given an equal volume of PBS or ethanol solvent.

[0166] Stress treatment: Two hours later, ACTH stock solution was added to the ACTH stress group and the ACTH+NCG protection group, with a final concentration of 10. -7 M. An equal volume of PBS was added to the normal control group.

[0167] Cell viability assay (MTT method): After 48 hours of treatment, add 20 μL of MTT solution (5 mg / mL) to each well and continue culturing for 4 hours. Carefully aspirate the culture medium from the wells, add 150 μL of dimethyl sulfoxide (DMSO) to each well, and shake gently for 10 minutes to fully dissolve the crystals. Measure the absorbance (OD) of each well at 490 nm using a microplate reader. 490 Cell viability (%) = (OD value of experimental group - OD value of blank well) / (OD value of control group - OD value of blank well) × 100%.

[0168] Results: Cell viability in the ACTH stress group decreased significantly to approximately 65%. Cell viability in the ACTH+NCG protection group recovered to approximately 92%, which was significantly higher than that in the ACTH stress group (P < 0.01) and showed no significant difference from the normal control group.

[0169] Example 6: Application of an in vitro screening model using NCG as a positive control

[0170] 6.1 Establishment and validation of the standardized screening model:

[0171] Following the standard procedure of Example 1, the same batch of isolated bovine granular cells was used each time to perform ACTH (10) assays in 96-well plates. -7 M) Modeling treatment, with blank control wells and NCG positive control wells set up (10 -7 M ACTH + 10 -7 M NCG).

[0172] Criteria for judging model validity: A batch of cell models is considered qualified and can be used for screening only if all three of the following conditions are met simultaneously: a) E2 secretion in the ACTH model group is reduced by more than 50% compared with the blank control group. b) P4 secretion in the ACTH model group is increased by more than 100% compared with the blank control group. c) E2 secretion in the NCG positive control group is increased by more than 80% compared with the ACTH model group, and P4 secretion is reduced by more than 30%.

[0173] This validation board must be run before each screening experiment. Only if the validation passes will the subsequent screening data be considered valid.

[0174] 6.2 Activity screening of candidate compounds:

[0175] On a validated cell model plate, the following wells were set up: blank control group, ACTH model group, and NCG positive control group (10⁻⁶ wells). -7 M), and groups of test compounds at different concentrations (e.g., compound A, let's say 10). -8 M, 10 -7 M, 10 -6 M has three concentrations, and each concentration has 6 replicates.

[0176] After 48 hours of processing, the supernatant was collected and the E2 and P4 concentrations were detected using ELISA (faster than RIA and suitable for high-throughput). Simultaneously, RNA could be extracted from selected wells and detected by qRT-PCR. FSHR and P450arom The expression is used as a secondary validation metric.

[0177] Activity evaluation:

[0178] Initial screening: Calculate the E2 / P4 ratio for each treatment group. If treatment with a certain concentration of compound can restore this ratio to more than 70% of that in the NCG positive control group, the compound is considered to have preliminary activity and proceeds to secondary screening.

[0179] Rescreening and dose-effect: For compounds that initially tested positive, the concentration gradient was expanded for testing, and their half-maximal effective concentration (EC50) was calculated. 50 This refers to the compound concentration at which the E2 / P4 ratio is restored to the median value between the model group and the positive control group. It is also compared with the EC of NCG. 50 Compare and evaluate their relative effectiveness.

[0180] The above description is merely a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. The use of NCG or a pharmaceutically acceptable salt thereof in the preparation of pharmaceutical compositions for the prevention and / or treatment of bovine follicular cysts.

2. The application according to claim 1, characterized in that, In the pharmaceutical composition, the amount of NCG added is such that the daily intake of cattle is 5-40 g / head.

3. The application according to claim 2, characterized in that, The amount of NCG added is to ensure that the daily intake of cattle is 15-25g / head.

4. The application according to claim 1, characterized in that, The pharmaceutical composition comprises a therapeutically effective amount of NCG or a pharmaceutically acceptable salt thereof as an active ingredient, and at least one pharmaceutically acceptable carrier, and further comprises one or more additional active ingredients selected from antioxidants, vitamins, trace elements or probiotics.

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