Use of mangiferin in preparation of products for preventing and treating staphylococcus aureus mastitis

Abstract

The present application relates to the field of biological medicine, and particularly relates to application of mangiferin in preparation of products for preventing and treating staphylococcus aureus mastitis. The present application discloses application of mangiferin in preparation of products for preventing and treating staphylococcus aureus mastitis. The LDH release, live / dead staining, expression of ferroptosis-related proteins after mangiferin is used for treating staphylococcus aureus infection of mouse mammary epithelial cells mMECs, and the H&E staining relief of mammary gland tissue after mangiferin is used for treating staphylococcus aureus infection of mouse mammary gland model, so as to verify that mangiferin can reduce ferroptosis of staphylococcus aureus targeting host, and verify that mangiferin has a protective effect on staphylococcus aureus infection of mastitis. Due to the abuse of traditional antibiotics and the continuous strengthening of bacterial drug resistance, mangiferin has the characteristics of high cure rate and no drug resistance, so that mangiferin used for treating staphylococcus aureus infection can increase the selectivity of the used drug, and has important significance for developing new drugs.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to the application of mangiferin in the preparation of products for the prevention and treatment of Staphylococcus aureus mastitis. Background Technology

[0002] Mastitis in dairy cows is one of the most common and economically damaging diseases in dairy farming. It not only leads to a sharp decline in milk production and reduced milk quality, but in severe cases, it can cause loss of lactation function in cows, resulting in culling and even death. Among the many pathogenic factors, Staphylococcus aureus (S. aureus) is one of the most prevalent infectious agents. After invading the mammary gland, this bacterium destroys mammary epithelial cells by secreting various virulence factors such as α-hemolysin. It also readily forms biofilms within the mammary ducts, thereby evading immune clearance and antibiotic penetration, leading to persistent and recurrent infections.

[0003] Recent research reveals that Staphylococcus aureus infection can induce ferroptosis in mammary epithelial cells. The mechanism involves the bacterium acquiring large amounts of iron from host cells for its own proliferation. Simultaneously, its virulence factors disrupt the redox homeostasis of host cells, leading to intracellular iron overload, reactive oxygen species accumulation, and suppressed glutathione peroxidase 4 activity, ultimately triggering the cellular ferroptosis program. This pathological process not only causes direct death of mammary tissue parenchymal cells but also exacerbates local and systemic inflammatory responses by releasing large amounts of pro-inflammatory mediators and damage-related molecular patterns, pushing mastitis towards severe stages. This finding suggests that intervention targeting host cell ferroptosis may be a novel and crucial pathway for the prevention and treatment of Staphylococcus aureus mastitis.

[0004] Currently, clinical treatment for Staphylococcus aureus mastitis in dairy cows mainly relies on antibiotics for treatment during the dry period or intraperitoneal infusion during lactation. However, traditional antibiotic therapy faces serious challenges: firstly, the increasing prevalence of drug-resistant Staphylococcus aureus strains, especially methicillin-resistant strains, has significantly reduced the efficacy of conventional antibiotics; secondly, antibiotic residues in milk lead to the disposal of raw milk, resulting in direct economic losses and food safety risks. Therefore, developing novel, highly effective, low-toxicity, non-resistant, and residue-free natural active substances has significant industrial value for the prevention and control of mastitis in dairy cows.

[0005] Mangiferin is a natural xanthonone compound mainly derived from mango trees and plants in the Gentianaceae family. It has been used in traditional medicine and possesses various biological activities, including antioxidant, anti-inflammatory, and immunomodulatory effects. Currently, there are no reports on the use of mangiferin for the prevention and treatment of Staphylococcus aureus mastitis in dairy cows, particularly regarding its therapeutic effects through antiviral activity and biofilm removal mechanisms. Summary of the Invention

[0006] The purpose of this section is to outline some aspects of the embodiments of the present invention and to briefly describe some preferred embodiments. Simplifications or omissions may be made in this section, as well as in the abstract and title of this application, to avoid obscuring the purpose of these documents; however, such simplifications or omissions should not be construed as limiting the scope of the invention.

[0007] To address the aforementioned technical problems, according to one aspect of the present invention, the present invention provides the following technical solution: Application of mangiferin in the prevention and control of Staphylococcus aureus mastitis.

[0008] As a preferred embodiment of the application of mangiferin described in this invention in the prevention and control of Staphylococcus aureus mastitis, the CAS Registry Number of the mangiferin is 4773-96-0.

[0009] As a preferred embodiment of the application of mangiferin described in this invention in the prevention and control of Staphylococcus aureus mastitis, the mangiferin alleviates Staphylococcus aureus-induced ferroptosis in mMECs cells and significantly reduces LDH release.

[0010] As a preferred embodiment of the application of mangiferin described in this invention in the prevention and control of Staphylococcus aureus mastitis, the mangiferin improves the pathological lesions of mouse mammary tissue stained with CFU and H&E by Staphylococcus aureus.

[0011] Compared with existing technologies, the beneficial effects of this invention are: it reveals for the first time a novel application of mangiferin in the prevention and control of Staphylococcus aureus mastitis. Through systematic studies in cell and animal models, this invention confirms that mangiferin can effectively alleviate Staphylococcus aureus-induced damage to mouse mammary epithelial cells, significantly reduce cellular LDH release, and alleviate and reduce pathological damage to mammary tissue in a mouse mammary infection model. Attached Figure Description

[0012] Figure 1 Figure (A) shows the effect of mangiferin on the viability of mMECs, and Figure (B) shows the effect of mangiferin on the growth curve of Staphylococcus aureus. Figure 2 The results of mangiferin assay on LDH release in mMECs cells are shown in (A). Mangiferin staining for live / dead cells in mMECs infected with Staphylococcus aureus is shown in (B). Figure 3 The results of the effect of mangiferin on the expression of ferroptosis protein in Staphylococcus aureus-infected mMECs are shown in Figure (A) and the quantitative results are shown in Figure (B). Figure 4The results of CFU on mammary gland tissue of mice infected with Staphylococcus aureus by mangiferin according to the present invention are shown in Figure (A) and Figure (B) are H&E staining results of mammary gland tissue of mice infected with Staphylococcus aureus by mangiferin. Detailed Implementation

[0013] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings.

[0014] Secondly, the present invention is described in detail with reference to the schematic diagrams. When describing the embodiments of the present invention, for ease of explanation, the cross-sectional views illustrating the device structure may be partially enlarged, not according to the usual scale. Furthermore, the schematic diagrams are merely examples and should not limit the scope of protection of the present invention. In addition, actual fabrication should include the three-dimensional spatial dimensions of length, width, and depth.

[0015] To make the objectives, technical solutions, and advantages of the present invention clearer, the embodiments of the present invention will be described in further detail below with reference to the accompanying drawings.

[0016] This invention discloses the medical use of mangiferin in alleviating Staphylococcus aureus-induced mastitis, and discloses that mangiferin can reduce the pathogenicity of Staphylococcus aureus by significantly reducing cellular LDH release, thereby exerting an effective anti-infective effect, and provides a natural medicine for preventing and treating Staphylococcus aureus-induced mastitis.

[0017] In this embodiment, Staphylococcus aureus ATCC25923 was used as the research object. Growth curve and cytotoxicity assays showed that mangiferin did not significantly inhibit the growth of Staphylococcus aureus and had no toxic effect on mouse mammary epithelial cells within the tested range, significantly reducing LDH release and the expression of ferroptosis-related proteins. Mangiferin treatment effectively alleviated bacterial colonization CFU and pathological lesions in mouse mammary tissue caused by Staphylococcus aureus.

[0018] Example 1 Mangiferin is used as a drug for treating Staphylococcus aureus mastitis in the preparation of drugs for bacterial infectious diseases.

[0019] Example 2 Mangiferin is used as a drug for treating Staphylococcus aureus mastitis and as a pharmaceutically acceptable carrier in the preparation of drugs for treating Staphylococcus aureus infections.

[0020] Example 3 Mangiferin is used as a drug to treat infectious diseases by targeting the ferrodeation process in Staphylococcus aureus infection. Experimental Example 1 Cytotoxicity assay and growth curve determination of mangiferin: mMECs were seeded in 96-well plates (2 × 10⁴ cells / well) and cultured overnight. The next day, the cells were treated with either DMSO or a specific concentration of mangiferin. A cell-free control group served as the blank control, and a 0.1% Triton X-100 treatment group served as the positive control group. Each group was divided into three replicates. After incubation at 37°C for 6 hours, the cells were analyzed according to the CCK8 assay kit instructions. Figure 1 A). Staphylococcus aureus ATCC25923 was cultured in a shaker at 37°C until the OD600 nm value reached 0.3. The culture was then aliquoted and incubated with DMSO solvent or different concentrations of mangiferin (final concentrations of 4 μg / mL, 8 μg / mL, 16 μg / mL, 32 μg / mL, and 64 μg / mL), and cultured at 37°C and 180 rpm. The OD600 value was measured every hour, and a growth curve was plotted based on the absorbance values. Figure 1 B).

[0021] Conclusion: Compared with the solvent control group, mangiferin showed almost no toxicity to mMECs within the concentration range of 4-256 μg / ml. Mangiferin did not affect the growth viability of Staphylococcus aureus within the tested range (see Appendix). Figure 1 .

[0022] Experimental Example 2 Detection of LDH release in *Staphylococcus aureus*-infected *mMECs* by mangiferin, and double staining of *mMECs* live / dead cells by mangiferin. mMECs were seeded at a density of 1 × 10⁶ cells per well in 6-well plates and cultured in DMEM medium containing 10% FBS and 1% penicillin and streptomycin, then incubated at 37°C in a cell culture incubator containing 5% CO₂. ATCC25923 cells were activated by spreading on TSB solid medium. Single colonies were picked and inoculated into TSB liquid medium and cultured until the logarithmic growth phase of OD600 = 0.5. After centrifugation, the bacteria were collected, and Staphylococcus aureus was resuspended in DMEM medium and infected into mMECs cells at a MOI of 200 for 6 h. Simultaneously, different concentrations of mangiferin dissolved in DMSO were added for treatment. LDH release was measured using an LDH detection kit to determine the differences in LDH release between different groups. Figure 2 A). After bacterial infection, mMECs were collected from the culture medium supernatant, centrifuged to obtain a pellet, and adherent cells were digested with trypsin. The digestion was neutralized with complete culture medium, and the cells were collected into centrifuge tubes. The pellet was then centrifuged again, washed once with Assay Buffer, and the pellet was retained. 1 mL of live / dead cell staining solution was added to each centrifuge tube, centrifuged again, and the pellet was retained. The cells were resuspended in PBS, centrifuged again, and the pellet was retained. 3-5 μL of cells were added to a glass slide, which was then mounted and observed under a fluorescence microscope. Figure 2 B) Conclusion: Mangiferin significantly inhibited Staphylococcus aureus-induced LDH release, indicating that it can effectively alleviate bacterial-mediated cell damage. Mangiferin also significantly alleviated Staphylococcus aureus-induced cell death, as evidenced by a significant reduction in the positive rate of dead cells (red fluorescent staining). (See Appendix) Figure 2 .

[0023] Experimental Example 3 Detection of ferroptosis protein expression in mMECs infected with Staphylococcus aureus by mangiferin Cells infected with bacteria were collected by centrifugation. For every 100 μL of cells, 1000 μL of Western lysis buffer was added, along with a mixture of phosphatase inhibitors and 1% benzyl sulfonyl fluoride (PMSF). After centrifugation at 12000 rpm for 10 min at 4°C, the protein concentration in the supernatant was determined using a BCA protein quantification kit. Equal amounts of protein (20 μg) were separated by 10% or 12.5% ​​SDS-PAGE electrophoresis and then transferred to a PVDF membrane. The PVDF membrane was blocked with 5% skim milk powder for 1 h and then incubated overnight at 4°C with primary antibody. After washing with TBST, the membrane was reacted with horseradish peroxidase (HRP)-labeled species-specific secondary antibody for 1 h. Signal detection was performed using ECL chemiluminescence buffer and a Tanon 4200 imaging system.

[0024] Conclusion: Mangiferin significantly inhibited the expression of ferroptosis protein in Staphylococcus aureus-induced mouse mammary epithelial cells (see Appendix). Figure 3 .

[0025] Test Example 4 Analysis of the effects of mangiferin on bacterial colonization CFU and pathological changes in mammary tissue of mice infected with Staphylococcus aureus. Lactating BALB / c mice were anesthetized, and the skin over the fourth pair of nipples was disinfected with 75% ethanol. A small incision was made to expose the mammary ducts, and 50 μL of a solution containing 5 × 10⁻⁶ mg / L of mammary extract was slowly injected using a microsyringe. 6 CFU (Chronic Fumarate) suspension of Staphylococcus aureus was administered. Mice were given mangiferin by gavage every 8 hours post-infection for 48 hours. At the end of the experiment, all mice were euthanized. Bacterial load was quantitatively determined by homogenizing freshly excised mammary tissue and weighing it with 1 mL of TSB solution. The homogenate samples were then diluted proportionally and inoculated onto TSB medium. After 24 hours of incubation, the number of viable bacteria was counted by counting colony-forming units. Figure 4 A) Mouse mammary gland tissue was fixed in 4% paraformaldehyde, embedded in paraffin, and dewaxed by gradient elution with xylene and ethanol. After hematoxylin staining and blue reversion with ammonia, the cytoplasm was stained with eosin. Finally, it was dehydrated with ethanol and xylene, and a gelatin mounting medium was added. The tissue was then observed under a light microscope. Figure 4 B).

[0026] Conclusion: Mangiferin treatment significantly reduced the CFU of bacteria colonizing mammary tissue in mice after Staphylococcus aureus infection, as well as the extensive inflammatory cell infiltration and acinar structure destruction in the mammary tissue. (See appendix) Figure 4 .

[0027] Although the present invention has been described above with reference to embodiments, various modifications can be made and components can be replaced with equivalents without departing from the scope of the invention. In particular, as long as there is no structural conflict, the features in the disclosed embodiments can be combined with each other in any manner. The lack of an exhaustive description of these combinations in this specification is merely for the sake of brevity and resource conservation. Therefore, the present invention is not limited to the specific embodiments disclosed herein, but includes all technical solutions falling within the scope of the claims.

Claims

1. This invention discovers the application of mangiferin in the prevention and control of Staphylococcus aureus mastitis.

2. The application of mangiferin according to claim 1 in the prevention and control of Staphylococcus aureus mastitis, characterized in that, The CAS accession number for mangiferin is 4773-96-0.

3. The application of mangiferin according to claim 1 in the prevention and control of Staphylococcus aureus mastitis, characterized in that, The mangiferin alleviated Staphylococcus aureus-induced ferroptosis in mMECs and significantly reduced LDH release.

4. The application of mangiferin according to claim 1 in the prevention and control of Staphylococcus aureus mastitis, characterized in that, The mangiferin improves the pathological lesions of mouse mammary tissue stained with CFU and H&E by Staphylococcus aureus.

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