Application of sodium butyrate in prevention and treatment of cow mastitis

Abstract

The invention discloses application of sodium butyrate to prevention and treatment of dairy cow mastitis, and belongs to the technical field of biology. The invention can provide an application of sodium butyrate in preventing and treating cow mastitis, in particular an application in preparing a preparation for preventing and treating cow mastitis induced by lipopolysaccharide (LPS) in escherichia coli. According to the application of the sodium butyrate in the aspect of preventing and treating the dairy cow mastitis, expression of key factors such as mammary gland tight junction protein is up-regulated under stimulation of lipopolysaccharide (LPS), and the sodium butyrate can effectively inhibit apoptosis and enhance the barrier function of mammary gland acinus, so that the effect of preventing and treating the dairy cow mastitis is achieved.

Description

Technical Field

[0001] The invention belongs to the technical field of biology, and particularly relates to application of sodium butyrate in preventing and treating cow mastitis. Background Art

[0002] Mastitis in dairy cows is an inflammatory disease of the mammary gland caused by pathogenic microbial infection, mechanical damage, or environmental stress. It seriously threatens the health of dairy cows, reduces milk production and quality, and causes significant economic losses to the dairy industry. The peripartum period (three weeks before and three weeks after parturition) is a peak period for mastitis in dairy cows. During this period, cows must cope with the physiological stress of childbirth while also stimulating lactation. This period typically places them in a negative energy metabolic balance, leading to decreased immune function and increased vulnerability to pathogenic microbial invasion.

[0003] Escherichia coli is one of the main pathogens causing mastitis in dairy cows. Lipopolysaccharide (LPS) in its cell wall has significant immune-activating properties, inducing a strong inflammatory response, thereby exacerbating mammary tissue inflammation and damage. Currently, clinical treatment for mastitis in dairy cows relies primarily on antibiotics. However, the increasing prevalence of antibiotic resistance and the potential threat posed by residual antibiotics to food safety and public health are becoming increasingly prominent. There is an urgent need to explore new, non-antibiotic or antimicrobial-reducing prevention and treatment options for mastitis in dairy cows, in order to achieve a green, safe, and sustainable dairy farming model.

[0004] To reduce reliance on antibiotics, researchers are exploring a variety of alternative treatment strategies, including: natural plant extracts (such as flavonoids and polyphenols) with antibacterial, anti-inflammatory, and immunomodulatory properties; probiotics and their metabolites that can inhibit pathogens and enhance immunity by regulating the structure of intestinal and mammary microbiota; antimicrobial peptides (AMPs) with broad-spectrum antimicrobial activity and less prone to developing drug resistance; immunomodulators (such as β-glucan) and specific vaccines that can enhance dairy cows' resistance to pathogens; nanomaterials and targeted technologies that can help improve the therapeutic efficiency of antimicrobial ingredients; and Chinese herbal compound preparations that can prevent and assist in the treatment of mastitis by clearing away heat and toxins, reducing swelling, and relieving pain. These "non-antibiotic" or "antibiotic-reducing" approaches offer a new, green, and safe path for the prevention and control of mastitis in dairy cows.

[0005] Therefore, the development of "non-antibiotic" or "antibiotic-reduced" alternative drugs has become an important research direction for the prevention and treatment of dairy cow mastitis, which is of great significance for promoting the high-quality development of animal husbandry and ensuring food safety. Summary of the Invention

[0006] In view of this, the present invention provides an application of sodium butyrate in preventing and treating cow mastitis. By upregulating the expression of key factors such as mammary tight junction proteins under lipopolysaccharide (LPS) stimulation, sodium butyrate can effectively inhibit cell apoptosis and enhance the barrier function of mammary alveoli, thereby achieving the effect of preventing and treating cow mastitis.

[0007] To achieve the above object, the present invention provides the following technical solutions:

[0008] One of the purposes of the present invention is to provide an application of sodium butyrate in preventing and treating mastitis in dairy cows.

[0009] A second object of the present invention is to provide a preparation for preventing and treating cow mastitis, which comprises sodium butyrate.

[0010] Furthermore, the dosage of sodium butyrate is 1% of daily feed intake (kg).

[0011] A third object of the present invention is to provide an application of sodium butyrate in the preparation of a preparation for preventing and treating cow mastitis.

[0012] Furthermore, the sodium butyrate is used in the preparation of a preparation for preventing and treating cow mastitis induced by Escherichia coli infection.

[0013] Furthermore, the sodium butyrate is used in the preparation of a preparation for preventing and treating cow mastitis induced by lipopolysaccharide (LPS) in Escherichia coli.

[0014] Compared with the prior art, the present invention has the following beneficial effects:

[0015] (1) Perinatal oral administration of sodium butyrate can promote inflammatory cell infiltration and the expression of proinflammatory cytokines in the early stages of mammary gland infection, thereby enhancing the body's immune response and contributing to the recovery of mammary gland infection.

[0016] (2) Perinatal oral administration of sodium butyrate can enhance the expression of mammary tight junction proteins and other factors under LPS stimulation, inhibit cell apoptosis, and thus enhance the barrier function of mammary alveoli. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Grouping and model building process for the present invention;

[0018] Figure 2 The present invention is about the effect of oral administration of sodium butyrate on LPS-induced mammary gland pathological damage in mice;

[0019] Figure 3 This is the effect of oral administration of sodium butyrate on the MPO activity of mouse mammary gland tissue induced by LPS;

[0020] Figure 4The present invention is to investigate the effect of sodium butyrate administered orally on polymorphonuclear neutrophils (PMN) in LPS-induced mouse mammary tissue;

[0021] Figure 5 This is the effect of oral administration of sodium butyrate on CD68 and CD177 in mouse mammary tissue induced by LPS;

[0022] Figure 6 The effect of sodium butyrate of the present invention on the distribution of tight junction proteins in mouse mammary gland induced by LPS;

[0023] Figure 7 The effect of sodium butyrate on LPS-induced tight junction protein and gene expression in mouse mammary glands;

[0024] Figure 8 The effect of sodium butyrate on LPS-induced apoptosis in mouse mammary glands is shown in the present invention.

[0025] Figure 9 This is the effect of sodium butyrate on the expression of natural immunity-related genes of MAC-T cells induced by LPS. DETAILED DESCRIPTION

[0026] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without creative work are within the scope of protection of the present invention.

[0027] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0028] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0029] The present invention will be further described below with reference to the accompanying drawings and specific embodiments:

[0030] Example 1

[0031] 1. Experimental Animals

[0032] This example uses SPF-grade Kunming mice (Liaoning Changsheng Biotechnology Co., Ltd.) about 8 weeks old and weighing 25±5g. Before the experiment, male and female mice were caged separately and adaptively raised for one week, with free access to food. The feeding system is a one-way airflow feeding system with a temperature of 20±2°C and a humidity of 50±5%. Mice with normal mental and appetite were selected for subsequent animal experiments. All animal operations in this experiment were approved by the Experimental Animal Ethics Committee of Henan Agricultural University (HNND20211921901).

[0033] 2. The main reagents used in the test are shown in Table 1 below.

[0034] Table 1 Main reagents

[0035]

[0036] 3. Preparation of main reagents

[0037] (1) Preparation of LPS solution: Use a pipette to draw 10 mL of ddH2O and add it to a 10 mg LPS dry powder bottle. Ultrasonicate for 10 min to fully dissolve it to prepare a 1 mg / mL LPS stock solution. Filter through a 0.22 μm filter membrane and then divide it into smaller portions. Store at -20°C in the dark until use. When modeling, dilute the LPS stock solution with ddH2O to a concentration of 0.2 mg / mL.

[0038] (2) Preparation of NaB solution: Weigh 2 mg of NaB and dissolve it in 10 mL of normal saline. Vortex thoroughly to mix. The solution concentration is 0.2 mg / mL and stored at 4°C.

[0039] 4. Experimental Grouping and Construction of Mouse Mastitis Model

[0040] The mice were co-housed at a male:female ratio of 1:2. After 12 hours of co-housing, vaginal plugs were examined to determine pregnancy. After pregnancy was confirmed, the female mice were housed individually. Seven days before delivery, the female mice were randomly divided into six groups, each consisting of six mice: control group (Ctrl group), sodium butyrate intervention group (NaB group), 24-hour LPS stimulation group (LPS-24h group), sodium butyrate intervention plus 24-hour LPS stimulation group (NaBL-24h group), 36-hour LPS stimulation group (LPS-36h group), and sodium butyrate intervention plus 36-hour LPS stimulation group (NaBL-36h group). The specific treatments were as follows:

[0041] ① Ctrl group: The mice were continuously gavaged with normal saline from 7 days before the expected date of delivery to 7 days after delivery, and 50 μL of normal saline was infused into the fourth right mammary gland on the 8th day after delivery.

[0042] ②NaB group: The rats were continuously gavaged with 1% of the dietary intake of sodium butyrate solution from 7 days before the expected date of delivery to 7 days after delivery, and 50 μL of normal saline was injected into the fourth pair of right mammary glands on the 8th day after delivery.

[0043] ③LPS-24h group: The rats were continuously gavaged with normal saline from 7 days before the expected date of delivery to 7 days after delivery, and 50 μL LPS (0.2 mg / mL) was infused into the fourth right mammary gland on the 8th day after delivery.

[0044] ④NaBL-24h group: The mice were continuously gavaged with 1% of the dietary intake of sodium butyrate solution from 7 days before the expected date of delivery to 7 days after delivery. On the 8th day after delivery, 50 μL of LPS (0.2 mg / mL) was infused into the fourth right mammary gland.

[0045] ⑤LPS-36h group: The rats were continuously gavaged with normal saline from 7 days before the expected date of delivery to 7 days after delivery, and 50 μL LPS (0.2 mg / mL) was infused into the fourth right mammary gland on the 8th day after delivery.

[0046] ⑥NaBL-36h group: The mice were continuously gavaged with 1% of the dietary intake of sodium butyrate solution from 7 days before the expected date of delivery to 7 days after delivery. On the 8th day after delivery, 50 μL of LPS (0.2 mg / mL) was infused into the fourth right mammary gland.

[0047] All the above groups were modeled on the 8th day after birth. The pups were removed 3 hours before modeling, the female mice were restrained, and the fourth pair of right mammary glands were disinfected. The tip of the nipple was removed by ophthalmic scissors about 1mm to fully expose the milk duct, and the corresponding reagent was aseptically perfused along the milk duct using a microsyringe. Among them, mammary tissue samples were collected from the Ctrl group, NaB group, LPS-24h group, and NaBL-24h group 24 hours after modeling; samples were collected from the LPS-36h group and NaBL-36h group 36 hours after modeling; the detailed grouping and model construction process are as follows. Figure 1 shown.

[0048] 5. Breast tissue collection

[0049] The mammary gland tissue collection process for each group of mice was as follows: After the mice were anesthetized and sacrificed by cervical dislocation, the fourth pair of mammary gland tissue from the right side was collected. First, the collected mammary gland tissue was quickly rinsed in physiological saline to ensure that there was no milk residue on the surface; then, the moisture on the surface of the tissue was blotted dry with absorbent paper. A portion of the mammary gland tissue was fixed in 4% formaldehyde fixative and used for histopathology, immunohistochemistry, and immunofluorescence analysis in the following examples; the remaining mammary gland tissue was quickly frozen in liquid nitrogen and stored at -80°C for use in molecular-level studies such as RNA or protein extraction in the following examples.

[0050] Statistical analysis was performed using IBM SPSS Statistics 27.0 (SPSS Inc, USA). All experimental data are presented as mean ± standard error of the mean (SEM). One-way ANOVA was used to compare differences between groups, and the LSD method was used for multiple comparisons when homogeneity of variance was met. The differences between the two groups were analyzed using the independent sample t-test. All statistical results were considered statistically significant at p < 0.05. Graphs were drawn using GraphPad Prism 8.0.2.263 software.

[0051] Example 2

[0052] This example is to perform pathological analysis on the mammary gland tissues of mice in each test group (Ctrl group, NaB group, LPS-24h group, NaBL-24h group, LPS-36h group and NaBL-36h group) in Example 1. The specific operation process is as follows:

[0053] (1) Fixation: The collected breast tissue is fixed in tissue fixative for 1-2 days. If turbidity occurs in the middle, the fixative can be replaced according to the situation.

[0054] (2) Dehydration: The fixed tissue was placed in 70%, 90%, 95% ethanol and anhydrous ethanol for 1 hour each for gradient dehydration.

[0055] (3) Transparency: The dehydrated tissue was placed in xylene I and xylene II for 30 min each to make the tissue transparent and facilitate subsequent wax immersion.

[0056] (4) Wax immersion: Soak the tissue in melted paraffin for 1 hour, changing the wax solution three times during this period to ensure that the tissue is fully permeated with paraffin.

[0057] (5) Embedding: Place the tissue that has been completely soaked in wax in a paraffin embedding frame, pour in the newly molten wax solution, and let it cool at -20°C until the wax block is completely solidified and ready for use.

[0058] (6) Sectioning: Use a pathology slicer to cut the wax block into continuous sections with a thickness of about 4 μm. Place it in warm water to flatten it, pick it up with a glass slide, and dry it at 60°C to ensure that the sections are firmly attached to the glass slide.

[0059] (7) Staining: Immerse the sections in hematoxylin solution for 5 minutes, turn blue in tap water for 10 minutes, and then dehydrate in gradient ethanol solutions for 5 minutes; then stain with eosin solution for 5 minutes, and finally soak in xylene I and xylene II for 10 minutes each to remove alcohol and make the tissue sections transparent, which is convenient for sealing and microscopic observation.

[0060] (8) Sealing: Aspirate the residual liquid on the slice, air-dry it, and then add a drop of neutral resin sealing medium to the center of the slice. After drying at room temperature, observe it under a microscope and perform breast tissue pathological histological evaluation.

[0061] Figure 2 Effects of oral administration of sodium butyrate on LPS-induced mammary gland pathological damage in mice. Figure 2 (A) is a representative epitope of mouse mammary gland tissue; Figure 2 (B) Representative H&E staining images of mouse mammary gland tissue. The upper scale is 50 μm and the lower scale is 20 μm. The blue arrow indicates inflammatory cell infiltration; the red arrow indicates incomplete alveolar structure; and the black arrow indicates milk stasis in the alveoli.

[0062] like Figure 2 As shown in (A), macroscopic observation and histopathological comparison of the right fourth mammary gland (test mammary region) of mice in each group revealed no significant differences between the right and left mammary glands of mice in the Ctrl and NaB groups. The mammary gland tissue showed a smooth surface and intact structure, with no abnormalities such as congestion, swelling, or damage, indicating a normal physiological state. Twenty-four hours after LPS stimulation, the right mammary glands of mice in both the LPS-24h and NaBL-24h groups exhibited significant inflammatory responses, manifested by marked swelling, congestion, and vasodilation, accompanied by numerous hemorrhagic spots and even localized tissue necrosis, indicating successful establishment of an acute mastitis model. Thirty-six hours after LPS stimulation, the inflammatory response in the right mammary glands of mice in the LPS-36h group was further exacerbated, with more severe tissue redness and vasodilation. However, pathological changes such as redness, congestion, and vasodilation in the right mammary glands of mice in the NaBL-36h group were significantly reduced, and the tissue appearance returned to normal. There was no significant difference in tissue morphology compared to the left control group.

[0063] like Figure 2(B) Representative H&E-stained images of the fourth pair of mammary glands (tested mammary regions) from the right side of each group of mice were analyzed. Results showed that in the Ctrl group, the glandular duct structure was clear, the epithelial cells were tightly arranged, the alveolar structure was intact, and no inflammatory cell infiltration was observed. No significant changes were observed in the NaB group compared with the Ctrl group, indicating that NaB has no significant effect on mammary gland tissue under normal circumstances. Twenty-four hours after LPS stimulation, the alveolar walls in the LPS-24h group thickened, with numerous inflammatory cells infiltrating within the alveoli (indicated by blue arrows), and the alveolar structure was incomplete (indicated by red arrows), suggesting that the inflammatory response leads to tissue damage. Compared with the LPS-24h group, the inflammatory cell infiltration within the mammary alveoli of mice in the NaBL-24h group was further enhanced, indicating that NaB may temporarily enhance the local immune response during the early stages of LPS stimulation but does not immediately alleviate inflammatory symptoms. After 36 hours of LPS stimulation, the inflammatory response in the LPS-36h group was further aggravated, with severe alveolar necrosis and atrophy and milk stasis (indicated by black arrows). Compared with the LPS-36h group, the inflammatory infiltration, alveolar necrosis and atrophy, and milk stasis in the mammary gland acinus of mice in the NaBL-36h group were significantly improved, indicating that a longer period of NaB pretreatment can effectively promote the recovery of mammary inflammation and reduce tissue damage.

[0064] In summary, oral administration of sodium butyrate can not only enhance the local immune response induced by LPS, but also help accelerate the recovery of mammary gland inflammation in perinatal mice and reduce the degree of tissue damage after a long period of time.

[0065] Example 3

[0066] This example is to detect the myeloperoxidase (MPO) activity of the mouse mammary tissues of each test group (Ctrl group, NaB group, LPS-24h group, NaBL-24h group, LPS-36h group and NaBL-36h group) in Example 1. The specific operation process is as follows:

[0067] Follow the instructions for the myeloperoxidase (MPO) assay kit. Accurately weigh 50 mg of frozen mouse mammary tissue and add 950 μL of pre-chilled homogenization medium to prepare a 5% (w / v) tissue homogenate. After thorough mixing, transfer 180 μL of the homogenate to a 2 mL centrifuge tube. Set aside as a control tube and assay tube.

[0068] According to the kit instructions, add the corresponding volume of reaction reagent to each tube in sequence and allow the reaction to proceed at room temperature in the dark for the specified time. Upon completion of the reaction, immediately measure the absorbance of each tube using a spectrophotometer at a wavelength of 460 nm in a 1 cm pathlength cuvette, adjusting the absorbance to zero with double-distilled water (ddH2O). Calculate the MPO activity in each sample using the standard curve and the formula provided in the kit. Results are expressed as enzyme activity per unit mass of tissue (U / g tissue). Comparison of MPO levels between experimental groups will assess the effects of different treatments on the inflammatory response in breast tissue.

[0069] Figure 3 To investigate the effect of oral administration of sodium butyrate on LPS-induced MPO activity in mouse mammary gland. Myeloperoxidase (MPO) activity is an important marker of neutrophil activation and can directly reflect the degree of neutrophil infiltration in mammary gland tissue.

[0070] from Figure 3 As can be seen, there was no significant difference in MPO activity between the Ctrl and NaB groups, indicating that NaB treatment alone had no significant effect on MPO activity in mouse mammary gland tissue. Compared with the Ctrl group, MPO activity in the LPS-24h group was significantly increased (p<0.05), indicating that neutrophil infiltration in mammary gland tissue increased 24 hours after LPS induction. Furthermore, MPO activity in the NaBL-24h group was significantly higher than that in the LPS-24h group (p<0.05). After 36 hours of LPS stimulation, MPO activity in the NaBL-36h group was significantly lower than that in the LPS-36h group (p<0.05). This suggests that sodium butyrate treatment significantly reduced MPO activity 36 hours after LPS induction, demonstrating a certain anti-inflammatory effect and reducing neutrophil infiltration in mammary gland tissue.

[0071] Example 4

[0072] This example is to perform polymorphonuclear neutrophil (PMN) immunohistochemical detection on the mammary gland tissue of mice in each test group (Ctrl group, NaB group, LPS-24h group, NaBL-24h group, LPS-36h group and NaBL-36h group) in Example 1. The specific operation process is as follows:

[0073] (1) Dewaxing of sections: The sections prepared in Example 2 were sequentially placed in dewaxing solution I, dewaxing solution II, and dewaxing solution III and soaked for 10 min each to ensure complete removal of paraffin.

[0074] (2) Dehydration: Dehydrate the dewaxed sections in anhydrous ethanol three times for 5 minutes each time.

[0075] (3) Antigen repair: Place the slices in citric acid buffer (pH 6.0), heat in a microwave oven at medium heat for 8 minutes, let it stand for 8 minutes, and then heat at medium-low heat for 7 minutes; after natural cooling, place the slices in PBS (pH 7.4) and wash for 5 minutes each time, repeat 3 times.

[0076] (4) Blocking endogenous peroxidase: The sections were placed in 3% hydrogen peroxide in the dark, incubated at room temperature for 25 minutes, and then washed in PBS (pH 7.4) for 5 minutes.

[0077] (5) Serum blocking: 3% BSA (bovine serum albumin) was added to the sections and blocked at room temperature for 30 minutes to reduce nonspecific binding.

[0078] (6) Add primary antibody: Gently shake off the blocking solution, add appropriately diluted primary antibody to the slice, place it flat in the incubation box, and incubate overnight at 4°C.

[0079] (7) Adding secondary antibody: On the second day, wash the slides in PBS three times, 5 minutes each time; after the sections are slightly dried, add appropriate secondary antibody and incubate at room temperature for 50 minutes.

[0080] (8) DAB color development: After the slides were washed again in PBS for 3 times, DAB color development solution was added dropwise. When the color changed to the ideal level, the slides were immediately rinsed with tap water to terminate the color development reaction.

[0081] (9) Re-staining of cell nuclei: Re-stain sections with hematoxylin for 3 min, wash off the sections with differentiation solution for a few seconds, rinse with tap water, and treat with bluing solution to make the cell nuclei appear clear blue.

[0082] (10) Dehydration and mounting: The sections were placed in anhydrous ethanol and xylene in turn for dehydration and transparency; the sections were mounted with mounting glue and observed under a white light microscope, and the images were analyzed using Fiji Image J software.

[0083] Figure 4 This study aimed to investigate the effects of sodium butyrate administered orally on polymorphonuclear neutrophils (PMNs) in LPS-induced mouse mammary glands. Immune cells, particularly PMNs, serve as the body's first line of defense against pathogenic microorganisms and play a crucial role in combating infection. The number and functional status of PMNs are important indicators of mammary gland health.

[0084] Figure 4 (A) Representative immunohistochemical staining images of PMNs. The red arrows show the accumulation of PMNs in mammary alveoli. Figure 4(B) Relative expression levels of PMNs. As can be seen from the figure: almost no PMN infiltration was observed in the mammary tissues of the Ctrl and NaB groups; this indicates that under normal circumstances, the number of PMNs in mammary tissues is maintained at a low level, and sodium butyrate alone does not cause significant changes. After 24 hours of LPS stimulation: Microscopic images of the LPS-24h group show ( Figure 4 A), a large number of PMN infiltrations appeared in the breast tissue, especially in the periacinar area. The statistical results showed that ( Figure 4 B), compared with the Ctrl group, the number of PMNs increased significantly 24 hours after LPS stimulation (p<0.05), indicating that LPS can effectively induce inflammatory response in mammary tissue, leading to the accumulation of a large number of immune cells; the microscopic images of the NaBL-24h group showed ( Figure 4 A), the PMN infiltration degree was more obvious than that of LPS-24h group. The statistical results ( Figure 4 This was also confirmed by Figure B, where the number of PMNs in the NaBL-24h group was significantly higher than that in the LPS-24h group, suggesting that sodium butyrate may further enhance the immune response and promote PMN recruitment in the early stages of LPS stimulation. 36 hours after LPS stimulation, PMN infiltration remained significant in the LPS-36h group, but the number of PMNs in the NaBL-36h group was significantly reduced. This suggests that sodium butyrate may exert an anti-inflammatory effect in the later stages of LPS stimulation, helping to mitigate excessive immune responses and thereby protect mammary tissue from damage.

[0085] These results suggest that, in the early phase (24 hours) of LPS stimulation, sodium butyrate may enhance the immune response and promote resistance to pathogenic microorganisms, as evidenced by a further increase in the number of polymorphonuclear neutrophils (PMNs). Furthermore, in the later phase (36 hours) of LPS stimulation, as inflammation progresses, sodium butyrate exhibits significant anti-inflammatory properties, significantly reducing excessive PMN infiltration and thereby alleviating tissue damage caused by proteases and reactive oxygen species released by neutrophils. Thus, perinatal oral administration of sodium butyrate modulates PMN infiltration in LPS-induced mouse mammary glands in a time-dependent manner, potentially playing a bidirectional regulatory role in the development and progression of inflammation. Sodium butyrate not only helps enhance host defenses during the initial phase of infection but also prevents immune responses from becoming uncontrolled during the persistent phase of inflammation, thereby maintaining mammary tissue homeostasis. This dual mechanism of action suggests that sodium butyrate has promising potential for the prevention and treatment of inflammatory diseases such as perinatal mastitis. Furthermore, these results further support the application of sodium butyrate as an immunomodulator.

[0086] Example 5

[0087] This example is to perform immunofluorescence detection of CD68 (total macrophage marker) and CD177 (neutrophil marker) positive cells in the mammary gland tissue of mice in each test group (Ctrl group, NaB group, LPS-24h group, NaBL-24h group, LPS-36h group and NaBL-36h group) in Example 1. The specific operation process is as follows:

[0088] (1) According to the experimental steps in Example 2 and Example 4, the sample was dewaxed to water, antigen repaired, endogenous peroxidase blocked, and the section was blocked.

[0089] (2) Add CD177 primary antibody to the slices, place in a humidified chamber, and incubate overnight at 4°C in the dark.

[0090] (3) Wash the slides with PBS and add the corresponding secondary antibody, then incubate at room temperature in the dark for 50 minutes.

[0091] (4) After washing the slides, add TSA fluorescent colorimetric solution and incubate at room temperature for 10 min in the dark.

[0092] (5) Immerse the sections in citric acid repair solution and microwave-heat until boiling for 10 minutes to remove the primary and secondary antibodies bound to the tissue and complete the quenching of the first round of staining.

[0093] (6) Repeat the blocking process in step (1), then add CD68 primary antibody and incubate overnight; the next day, perform secondary antibody incubation and TSA color development according to steps (3) and (4), and microwave treatment again to ensure complete removal of residual signals.

[0094] (7) After the slices were slightly dried, DAPI staining solution was added and incubated at room temperature for 10 min in the dark to mark the cell nuclei. The slices were then washed with PBS.

[0095] (8) To reduce the interference of tissue autofluorescence, add an appropriate amount of autofluorescence quencher and let it act at room temperature for 5 minutes, then rinse with running water for 10 minutes.

[0096] (9) After sealing with anti-fluorescence quenching sealing agent, the images were observed and collected under a fluorescence microscope, and the images were quantitatively analyzed using FijiImage J software.

[0097] Figure 5To further elucidate the effect of sodium butyrate on LPS-induced mammary gland CD68 and CD177 in mice, we used immunofluorescence staining to analyze the expression of macrophages (marked by CD68) and neutrophils (marked by CD177) in mammary gland tissues of different treatment groups in Example 1. The results are as follows:

[0098] Figure 5 (A) is the immunofluorescence image of CD68 and CD177 (scale bar 20 μm), Figure 5 (B) is the relative fluorescence intensity of CD68, Figure 5 (C) Relative fluorescence intensity of CD177. Figure 5 (A) Figure 5 (B) and Figure 5 As can be seen in (C), the expression of CD68 and CD177 was low in the Ctrl group, indicating that the number of macrophages and neutrophils in breast tissue is low under normal circumstances. After 24 hours of LPS stimulation, the expression of CD68 and CD177 in the LPS-24h group was significantly increased compared with the Ctrl group (p<0.05), indicating that LPS successfully induced the infiltration of macrophages and neutrophils in breast tissue, reflecting a strong inflammatory response. Compared with the LPS-24h group, the NaBL-24h group further promoted the LPS-induced expression of CD68 and CD177, suggesting that sodium butyrate may enhance the local immune response at the early stage (24 hours), leading to the accumulation of more macrophages and neutrophils. After 36 hours of LPS stimulation: the expression of CD68 and CD177 continued to increase in the LPS-36h group, indicating that the inflammatory response was still intensifying and macrophages and neutrophils continued to infiltrate the mammary tissue in large numbers; compared with the LPS-36h group, the expression of CD68 and CD177 was significantly reduced in the NaBL-36h group, and there was no significant difference compared with the Ctrl group, indicating that at a later time point (36 hours), sodium butyrate exerted an anti-inflammatory effect, reduced the excessive infiltration of macrophages and neutrophils, and helped to restore the homeostasis of mammary tissue.

[0099] In summary, sodium butyrate exerts a time-dependent, bidirectional regulatory effect on LPS-induced innate immune responses in the mouse mammary gland: in the early stages of LPS stimulation, sodium butyrate enhances local immune responses and promotes the recruitment of macrophages and neutrophils. Later in LPS stimulation, sodium butyrate exhibits significant anti-inflammatory properties, reducing the excessive infiltration of these immune cells and thereby alleviating tissue damage caused by their release of proteases and reactive oxygen species. These findings further support the potential role of sodium butyrate as a potential immunomodulator for the prevention and treatment of inflammatory diseases such as peripartum mastitis.

[0100] Example 6

[0101] This example is to detect the mRNA of tight junction-related genes and apoptosis-related genes in the mouse mammary tissue of each test group in Example 1 (Ctrl group, NaB group, LPS-24h group, NaBL-24h group, LPS-36h group and NaBL-36h group).

[0102] (1) Tissue RNA extraction

[0103] This experiment was performed according to the instructions of the "RNA isolater total RNA extraction reagent" reagent. The specific steps are as follows:

[0104] ① Take about 50 mg of the breast tissue sample stored at -80°C in Example 1 above and place it in a 1.5 mL enzyme-free tube (pre-added with 1 mL of Trizol and grinding beads and kept on ice).

[0105] ② Pre-cool the base of the tissue grinder, place the sample tube into the instrument for homogenization, grind at 70 Hz for 20 seconds, and repeat 3 to 4 times until there are no obvious tissue particles in the lysate.

[0106] ③ After homogenization, the sample was allowed to stand at 4°C for 5 min, centrifuged at 12000 rpm at 4°C for 5 min, and the supernatant was transferred to a new 1.5 mL enzyme-free EP tube.

[0107] ④ Add 200 μL of pre-cooled chloroform to the supernatant, shake vigorously to mix until it becomes emulsified, place it in a 4°C centrifuge for 5 minutes, and then centrifuge it at 12,000 rpm at 4°C for 15 minutes to promote phase separation.

[0108] ⑤ After centrifugation, carefully aspirate about 400 μL of the upper transparent aqueous phase (avoid touching the middle protein layer) and transfer it to a new 1.5 mL enzyme-free EP tube.

[0109] ⑥ Add an equal volume (400 μL) of pre-cooled isopropanol, mix thoroughly by inverting, and place in a 4°C centrifuge for 10 minutes. Centrifuge at 12,000 rpm for 10 minutes to obtain RNA precipitation.

[0110] ⑦ Carefully discard the supernatant, centrifuge briefly, and then use a yellow and white combination pipette tip to completely remove the remaining liquid, retaining the white RNA precipitate at the bottom.

[0111] ⑧ Add 1 mL of pre-cooled 75% ethanol (prepared by RNase-free ddH2O and anhydrous ethanol in a volume ratio of 3:1), gently flick the bottom of the tube to suspend the white precipitate, invert it several times to mix, let it stand at 4°C for 5 minutes, and then centrifuge it at 12000 rpm at 4°C for 5 minutes to wash.

[0112] ⑨ Repeat steps ⑦ and ⑧ to further remove impurities and improve RNA purity.

[0113] ⑩ After washing, the RNA precipitate was air-dried in a clean bench at room temperature until it became a gel (approximately 5 minutes). 30 μL RNase-free ddH2O was added and the bottom of the tube was gently flicked. After it was fully dissolved, it was stored in a -80°C refrigerator for later use.

[0114] (2) RNA concentration and purity determination

[0115] The concentration and purity of the extracted RNA samples were detected by spectrophotometer. 260 / OD 280 RNA samples with a ratio close to 2.0 indicate high purity and minimal protein contamination. RNA integrity was assessed by electrophoresis on a 1.2% denaturing agarose gel, revealing three distinct bands. The 28S rRNA band was approximately twice as bright as the 18S rRNA band, indicating no significant RNA degradation. RNA samples meeting quality standards were uniformly diluted or concentrated to 500 ng / μL for subsequent experiments.

[0116] (3) cDNA synthesis

[0117] cDNA synthesis was performed using a reverse transcription kit. The specific steps are as follows:

[0118] ① Genomic DNA removal reaction: Add the following components in a 200 μL enzyme-free PCR tube in sequence: 10 μL RNase-free ddH2O, 4 μL 4×gDNA wiper mix, and 2 μL RNA template (concentration of 500 ng / μL). Mix by pipetting and incubate in a 42°C water bath for 2 minutes.

[0119] ② Reverse transcription reaction: Add 4 μL of 5×HiScript II qRT SuperMix II to the above reaction system, mix thoroughly and centrifuge, then place the reaction tube in a gene thermal cycler for reverse transcription reaction. The program is set as follows: 50°C, 15 minutes (reverse transcription); 85°C, 5 seconds (terminate reaction).

[0120] ③cDNA dilution and storage: After reverse transcription is completed, the obtained cDNA product is diluted 10-fold with RNase-free ddH2O, aliquoted and stored in a -80℃ refrigerator for long-term storage for subsequent qRT-PCR detection.

[0121] (4) qRT-PCR detection

[0122] Based on the mRNA sequence information of the target gene in the NCBI database, specific qPCR primers were designed online and Shanghai Sangon Biotechnology Co., Ltd. was commissioned to synthesize the primers. The specific primer sequence information is shown in Table 2 (where: F represents the upstream primer and R represents the downstream primer). The qRT-PCR reaction system was prepared according to the instructions of the relevant reagents. The detailed components are shown in Table 3. The reaction procedure is as follows: pre-denaturation (95°C 30s); cycle reaction (95°C 10s → 60°C 30s, 40 cycles); melting curve (95°C 15s → 60°C 60s → 95°C 15s). Use 2 -ΔΔCt The data were statistically analyzed and processed by the method, and each sample was tested three times to ensure the accuracy and repeatability of the results.

[0123] Table 2 qPCR primer information

[0124]

[0125] Table 3 qPCR reaction system

[0126]

[0127] Figure 6 The effect of sodium butyrate on the distribution of tight junction proteins in mouse mammary gland induced by LPS. Figure 6(AC) are representative immunohistochemical staining images of Claudin-3, Occludin and ZO-1 in mouse mammary gland tissue. It can be seen from the figures that: in the Ctrl group, the expression of tight junction proteins was normal and the tissue structure was clear; in the NaB group, the expression of ZO-1 (p<0.05) was significantly increased, and the expression of other proteins did not change significantly; in the LPS-24h group, the expression of the three proteins was significantly decreased (p<0.05), indicating that LPS stimulation had an inhibitory effect on tight junction proteins; in the NaBL-24h group, under the intervention of sodium butyrate, the expression of Claudin-3, Occludin and ZO-1 was significantly increased (p<0.05), indicating that sodium butyrate can alleviate the reduction of tight junction proteins caused by LPS. Figure 6 (DF) are statistical graphs of the relative expression levels of Claudin-3, Occludin and ZO-1. It can be seen from the figures that compared with the Ctrl group, the expression of ZO-1 in the NaB group was significantly increased (p<0.05); after 24 hours of LPS stimulation, the protein expression levels of Claudin-3, Occludin and ZO-1 in the LPS-24h group were significantly decreased (p<0.05), while the expression levels of the above three tight junction proteins in the NaBL-24h group were significantly increased, and the differences were significant compared with the LPS-24h group (p<0.05).

[0128] Conclusions: LPS induction significantly reduces the expression of tight junction proteins (Claudin-3, Occludin, and ZO-1) in mouse mammary tissue, impairing barrier function. Sodium butyrate intervention significantly alleviates LPS-induced reductions in tight junction proteins, potentially demonstrating a protective effect. Furthermore, ZO-1 expression was significantly elevated in the NaB group, suggesting that sodium butyrate may directly regulate ZO-1 expression.

[0129] Figure 7 Effects of sodium butyrate on LPS-induced tight junction protein and gene expression in mouse mammary gland. Figure 7 (AC) are the mRNA levels of Claudin-3, Occludin, and ZO-1 genes; Figure 7 (D, I) Representative protein grayscale images of Claudin-1, Claudin-3, Occludin, and ZO-1; Figure 7(EH, JM) Quantitative protein analysis results for Claudin-1, Claudin-3, Occludin, and ZO-1. The figures show that compared with the Ctrl group, the expression levels of tight junction-related genes and proteins were significantly downregulated after 24 and 36 hours of LPS stimulation (p < 0.05). However, in the sodium butyrate-treated groups (NaBL-24h and NaBL-36h), the expression levels of these proteins were significantly increased (p < 0.05), a trend consistent with the immunohistochemical results.

[0130] Example 7

[0131] To investigate whether the barrier function of the mammary gland tissue of mice in each experimental group (Ctrl group, NaB group, LPS-24h group, NaBL-24h group, LPS-36h group, and NaBL-36h group) in Example 1 is associated with cell apoptosis, TUNEL staining analysis was performed. The specific steps are as follows:

[0132] ① Dewaxing to water: The sections prepared in Example 2 were placed in a dewaxing solution for dewaxing, and then placed in anhydrous ethanol for dehydration, with the treatment time being 10 min.

[0133] ② Proteinase K repair: add an appropriate amount of proteinase K solution, incubate at 37°C for 20 minutes, and then wash with PBS buffer three times, each time for 5 minutes.

[0134] ③ Room temperature equilibrium: Add buffer solution and incubate the slices at room temperature for 10 minutes to reach equilibrium.

[0135] ④ Add reaction solution: add reaction solution (TDT enzyme, dUTP, buffer mixed in a ratio of 1:5:50) dropwise and incubate at 37°C for 1 h.

[0136] ⑤ DAPI counterstaining of cell nuclei: After washing the sections with PBS, add DAPI staining solution and incubate at room temperature in the dark for 10 minutes to mark all cell nuclei.

[0137] ⑥ Sealing: After washing with PBS, use anti-fluorescence quenching mounting medium to seal the sample.

[0138] ⑦ Microscopic examination and photography: Observe and collect images under a fluorescence microscope. The ultraviolet excitation wavelength used for DAPI is 330-380 nm, and the excitation wavelength used for 488 fluorescein labeling is 465-495 nm.

[0139] ⑧ Interpretation of results: DAPI-labeled nuclei appear blue, while 488-fluorescein-labeled apoptotic nuclei appear green. By comparing the amount and intensity of green fluorescence signals between different groups, the degree of apoptosis in each sample can be assessed.

[0140] Figure 8 The effect of sodium butyrate on LPS-induced mouse mammary apoptosis phase was studied. The results showed that compared with the Ctrl group and the NaB group, the number of apoptosis-positive cells in the LPS-24h group was significantly increased. As the stimulation time was extended to 36h, the degree of apoptosis in the LPS-36h group was further aggravated. In the NaBL-24h group and the NaBL-36h group, the LPS-induced apoptosis was significantly alleviated under the intervention of sodium butyrate.

[0141] Example 8

[0142] This example investigates the effects of serum after in vivo intervention with sodium butyrate (NaB) on the LPS-induced inflammatory response and innate immune function of bovine mammary epithelial cells.

[0143] 1. Materials and Methods

[0144] (1) Cell culture

[0145] MAC-T cells (a bovine mammary epithelial cell line) were routinely cultured in RPMI 1640 complete medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin solution (100 U / mL penicillin, 100 μg / mL streptomycin). The cells were incubated in a 37°C, 5% CO2 incubator with the culture medium replaced every 24-48 h. Cells were used for subsequent experiments when the cell confluence reached 80%-90%.

[0146] (2) Preparation of serum from animals gavaged with NaB

[0147] Healthy adult Wistar rats were used for the experiment. Sodium butyrate was administered orally by gavage at a dose of 1% of their daily feed intake (kg) once daily for 7 consecutive days. A control group received 1× PBS by gavage. Blood was collected after the final gavage: the animals were lightly anesthetized with sodium pentobarbital (50 mg / kg), and blood was collected via the abdominal aorta. The collected blood samples were allowed to stand at room temperature for 30 minutes. After natural coagulation, the blood was centrifuged at 3000 rpm for 10 minutes at 4°C, and the supernatant serum was collected to prepare sodium butyrate gavage serum (NaBs). The resulting serum was immediately inactivated by water bath at 56°C for 30 minutes to inactivate complement components. The serum was then aliquoted and stored at -80°C until further use.

[0148] (3) Experimental group design

[0149] Set up the following four groups of processing:

[0150] Ctrl group (control group): MAC-T cells were cultured normally in complete medium containing 10% regular FBS for 24 h without any intervention.

[0151] LPS group: MAC-T cells were pretreated with PBS for 18 h; then, MAC-T cells were stimulated with 1 μg / mL lipopolysaccharide (LPS) for 6 h.

[0152] NaB serum group (NaBs): MAC-T cells were pretreated with a medium containing 10% of the above-mentioned NaBs for 18 h; then the culture medium was replaced with a complete medium containing 10% conventional FBS and cultured for another 6 h.

[0153] NaB+LPS group: MAC-T cells were pretreated with a medium containing 10% of the above-mentioned NaBs for 18 h, and then stimulated with a medium containing 1 μg / mL LPS and 10% regular FBS for 6 h.

[0154] To evaluate the regulatory effect of serum on inflammatory response after NaB intervention in vivo.

[0155] 2. MAC-T cell RNA extraction, cDNA synthesis, and related gene expression detection

[0156] (1) Total RNA extraction

[0157] The MAC-T cells after treatment in each group were collected, and total RNA was extracted using RNA isolater Total RNA Extraction Reagent strictly according to the instructions. The main steps are as follows:

[0158] ① Sterile preparation: All centrifuge tubes, pipette tips, etc. use RNase-free consumables;

[0159] ②Cell lysis: discard the culture medium, wash with PBS and add lysis buffer to fully lyse the cells;

[0160] ③ Phase separation: Add chloroform and shake to mix, centrifuge at 12,000 × g for 15 min at 4°C, and collect the upper aqueous phase;

[0161] ④ RNA precipitation and washing: add isopropanol to precipitate RNA, wash with 75% ethanol, air dry and dissolve in RNase-free ddH2O;

[0162] ⑤Quality detection: Use NanoDrop to measure RNA concentration and A 260 / A 280 The ratio of 28S and 18S rRNA was determined (qualified range: 1.8–2.0). 1% agarose gel electrophoresis was performed to examine the clarity of 28S and 18S rRNA bands and confirm that there was no significant degradation. Qualified RNA samples were stored at -80°C until use.

[0163] (2) cDNA synthesis (reverse transcription)

[0164] use IIQ RT SuperMix for qPCR (Vazyme) was used for reverse transcription reaction. The system is shown in Table 4 below.

[0165] Table 4 Reverse transcription system

[0166]

[0167] Procedure: Add gDNA Wiper Mix to remove genomic DNA contamination and incubate at 42°C for 2 minutes. Add HiScript II qRT SuperMix and perform reverse transcription: 25°C for 5 minutes (primer annealing), 50°C for 30 minutes (reverse transcription), and 85°C for 5 minutes (enzyme inactivation). The resulting cDNA can be used for qPCR amplification or stored short-term at -20°C.

[0168] (3) Primer design and sequence information

[0169] Based on the experimental requirements, the mRNA sequences (Accession numbers) of the target genes in dairy cows (Bos taurus) were retrieved from the NCBI database. RT-qPCR primers were designed and synthesized online through the website of Shanghai Sangon Biotechnology Co., Ltd. The primer sequences for inflammatory factors and innate immunity-related genes required for this experiment are shown in Table 5 (where F represents the upstream primer and R represents the downstream primer). The qRT-PCR reaction system was prepared according to the relevant reagent instructions. The detailed components are shown in Table 3.

[0170] Table 5 Target genes and primer sequences required for fluorescence quantitative PCR analysis

[0171]

[0172] qPCR amplification was performed using the SYBR Green method, and the reaction system is shown in Table 6 below:

[0173] Table 6 Reaction system

[0174]

[0175] The reaction procedure was as follows: pre-denaturation (95°C 30s), two-step amplification (95°C 10s → 60°C 30s, 40 cycles), melting curve (95°C 15s → 60°C 60s → 95°C 15s). -ΔΔCt The data were statistically analyzed and processed by the method, and each sample was tested three times to ensure the accuracy and repeatability of the results.

[0176] Figure 9 The effect of sodium butyrate on the expression of innate immunity-related genes of MAC-T cells induced by LPS can be seen from the figure:

[0177] (i) Compared with the Ctrl group, the LPS group significantly induced the expression of CXCL8, IL-1β, and IL-6 proinflammatory cytokines (P<0.05), indicating that the LPS group successfully established an in vitro mastitis inflammation model.

[0178] (ii) Compared with the Ctrl group, NaB alone had little effect on proinflammatory factors such as CXCL8, IL-1β, and IL-6, and even had a slight inhibitory effect (such as IL-6), which indicates that NaB itself does not trigger the body's immune response.

[0179] (iii) Compared with the LPS group, the addition of NaB in the NaB+LPS group on the basis of LPS stimulation further increased the expression of pro-inflammatory factors such as CXCL8 and IL-1β (P<0.05), indicating that NaB may enhance the body's immune response to LPS. In addition, it can be seen that although LPS alone can significantly increase the expression of DEFB5 (P<0.05), the addition of NaB inhibits this effect, which may be a negative feedback regulatory mechanism to prevent excessive inflammatory response from causing tissue damage.

[0180] (iv) Compared with the Ctrl group, although the expression of proinflammatory factors (CXCL8, IL-1β, and IL-6) in the NaB+LPS group was higher than that in the Ctrl group, this increase was controllable and accompanied by other beneficial changes, indicating that NaB can balance the inflammatory response caused by LPS to a certain extent;.

[0181] These results indicate that NaB promotes the expression of key inflammatory mediators (CXCL8, IL-1β, and IL-6), helping to rapidly recruit immune cells to the site of infection. Furthermore, NaB inhibits LPS-induced overexpression of DEFB5, preventing tissue damage caused by excessive inflammatory responses. The above analysis also demonstrates that NaB modulates LPS-induced expression of innate immunity-related factors in bovine mammary epithelial cells by regulating the expression of CXCL8 and IL-1β and inhibiting the expression of the acute phase response protein-related gene SAA2.

[0182] In summary, NaB can not only activate the innate immune response, but also adjust the degree of inflammatory response, thereby effectively alleviating LPS-induced cow mammary inflammation (especially cow mastitis induced by lipopolysaccharide (LPS) in Escherichia coli), providing a theoretical basis for the development of new treatment strategies.

[0183] The above describes in detail the application of sodium butyrate disclosed in the present invention for preventing and treating mastitis in dairy cows. Specific examples are used herein to illustrate the principles and implementation methods of the present invention. The description of the above examples is intended only to help understand the method and core concept of the present invention. It should be noted that, for those skilled in the art, various improvements and modifications may be made to the present invention without departing from the principles of the present invention, and such improvements and modifications also fall within the scope of protection of the claims of the present invention.

Claims

1. Application of sodium butyrate in preventing and treating mastitis in dairy cows.

2. A preparation for preventing and treating cow mastitis, characterized in that: Contains sodium butyrate.

3. The preparation for preventing and treating cow mastitis according to claim 2, characterized in that: The dosage of sodium butyrate is 1% of daily feed intake (kg).

4. Use of sodium butyrate in the preparation of a preparation for preventing and treating mastitis in dairy cows.

5. The use according to claim 4, characterized in that The sodium butyrate is used in preparing a preparation for preventing and treating cow mastitis induced by Escherichia coli infection.

6. The use according to claim 5, characterized in that The sodium butyrate is used in preparing a preparation for preventing and treating cow mastitis induced by lipopolysaccharide (LPS) in Escherichia coli.

7. Use of sodium butyrate in the preparation of an immunomodulator.

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