Application of PANoptosis as target spot in screening medicine for treating mastitis

Abstract

The invention relates to the technical field of veterinary medicine, and discloses application of PANoptosis as a target spot in screening of a medicine for treating mastitis. The activation of a PANoptosis pathway is inhibited, and the expression quantity of inflammatory factors in mastitis and the severity of inflammatory reaction are reduced. According to the application of PANoptosis as a target spot in screening of the medicine for treating mastitis, the PANoptosis is a key mechanism of mastitis deterioration, and the severity of mastitis and blood-milk barrier injury can be relieved by inhibiting a PANoptosis pathway and GSDMD-NT pore-forming activity; meanwhile, the invention provides a plant extract, namely cyanidin-3-O-galactoside, which can obviously inhibit PANoptosis activation and GSDMD-NT pore-forming activity, and can be used for preparing a medicine for treating mastitis.

Description

Technical Field

[0001] The present invention relates to the technical field of veterinary medicine, and in particular to application of PANoptosis as a target in screening drugs for treating mastitis. Background Art

[0002] Mastitis is an inflammatory response to mammary tissue, common in lactating women and dairy livestock. It is triggered by pathogen infection, with Escherichia coli being one of the main pathogens. During mastitis, the blood-milk barrier (composed of tight junctions between mammary epithelial cells) is damaged, leading to leakage of blood components into milk and loss of milk components, exacerbating tissue damage, the spread of infection, and even death. PANoptosis, a novel type of programmed cell death that combines features of pyroptosis, apoptosis, and necroptosis, is central to the development of inflammatory responses. GSDMD is a key executor of the PANoptosis pathway. Upon activation of its NT terminus, it forms pores in the cell membrane, releasing proinflammatory cytokines (IL-1β and IL-18), causing rapid progression of the inflammatory response and damage to the blood-milk barrier. Studies have shown that GSDMD is highly expressed in mastitis, but its direct role in the blood-milk barrier remains unknown. Recent studies have shown that anthocyanins, a flavonoid compound, possess multiple pharmacological activities, including antioxidant, anti-tumor, cardiovascular disease prevention, and anti-inflammatory properties, and can effectively alleviate a variety of inflammatory-related diseases, primarily manifesting in colitis, pneumonia, liver damage, and reproductive damage. However, their application in mammalian mastitis has rarely been reported.

[0003] Existing treatments for mastitis have the following defects: 1) Antibiotic treatments are prone to drug resistance and cannot directly protect the blood-milk barrier function. 2) Existing cures for mastitis only focus on reducing inflammatory responses, but the integrity of the blood-milk barrier also needs to be protected. 3) There are no patents for relieving mastitis by damaging the blood-milk barrier, and there is a lack of clear molecular targets and mechanisms of action. 4) From the perspective of waste utilization, we have demonstrated that a crabapple anthocyanin extract can treat mastitis, in which cyanidin-3-O-galactoside plays a key role. 5) Existing technologies mainly focus on a single pathway, and the integrative effect of PANoptosis has not been taken seriously, resulting in limited therapeutic effects. In order to solve the above technical problems, the present invention provides the use of PANoptosis as a target in screening drugs for the treatment of mastitis. Summary of the Invention

[0004] The purpose of the present invention is to provide the application of PANoptosis as a target in screening drugs for treating mastitis, revealing that PANoptosis is a key mechanism for the worsening of mastitis. By inhibiting the PANoptosis pathway and GSDMD-NT activation, the inflammatory response and blood-milk barrier damage in mastitis can be alleviated. At the same time, a plant extract, cyanidin-3-O-galactoside, is provided, which can significantly inhibit PANoptosis activation and GSDMD-NT pore-forming activity and can be used to prepare drugs for treating mastitis.

[0005] To achieve the above objectives, the present invention provides the use of PANoptosis as a target in screening drugs for treating mastitis.

[0006] Preferably, the activation of the PANoptosis pathway is inhibited to alleviate the expression of pro-inflammatory factors in mastitis.

[0007] Preferably, the PANoptosis pathway and GSDMD-NT are used as targets in screening drugs for treating mastitis.

[0008] Preferably, the pore-forming activity of GSDMD-NT is inhibited to alleviate the damage of the blood-milk barrier during mastitis.

[0009] Preferably, the activation of the PANoptosis pathway is inhibited to alleviate the severity of mastitis.

[0010] Furthermore, the present invention also provides targets for screening drugs for treating mastitis, including PANoptosis and GSDMD.

[0011] Furthermore, the present invention also provides a medicine for treating mastitis, the active ingredient of which is cyanidin-3-O-galactoside.

[0012] Furthermore, cyanidin-3-O-galactoside inhibited PANoptosis activation and GSDMD-NT pore-forming activity, reducing the severity of mastitis and blood-milk barrier damage.

[0013] The advantages and positive effects of using PANoptosis as a target in screening drugs for treating mastitis are:

[0014] 1. This invention reveals that PANoptosis is a key mechanism for the worsening of mastitis. By inhibiting the PANoptosis pathway and Gasdermin D activation, it can reduce the inflammatory response in the mammary gland and damage to the blood-milk barrier, and is used to screen drugs for the treatment of mastitis.

[0015] 2. The present invention provides a plant extract - cyanidin-3-O-galactoside, which can significantly inhibit PANoptosis activation and GSDMD-NT pore-forming activity, and can be used to prepare drugs for treating mastitis.

[0016] The technical solution of the present invention is further described in detail below through the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The HPLC results of the crude extract of anthocyanins from crabapple fruit in the embodiment of the present invention are shown in Figure 1, where A is before purification and B is after purification.

[0018] Figure 2 The results of the CCK-8 assay for evaluating the effect of cyanidin-3-O-galactoside (C3Gal) on the viability of primary GMECs in the examples of the present invention, wherein A is the result of C3Gal treatment for 12 hours, B is the result of treatment with the purified anthocyanin extract (P-C3Gal) for 12 hours, C is the cell viability result after primary GEMCs were treated with LPS for 12 hours and then with C3Gal for 12 hours, and D is the cell viability result after primary GEMCs were treated with LPS for 12 hours and then with P-C3Gal for 12 hours;

[0019] Figure 3 The expression of inflammatory-related factor mRNA was analyzed after LPS, P-C3Gal, and C3Gal treatment of mammospheres in the examples of the present invention, where A is TLR4, B is MyD88, C is IKBα, D is NF-κB, E is IL-6, F is IL-1β, G is TNFα, H is ZO-1, and I is Occludin;

[0020] Figure 4 A is the expression analysis of p-p65 in mammospheres, Bar = 100 μm, B is the immunofluorescence analysis of p-p65 nuclear translocation in primary GMECs, Bar = 100 μm;

[0021] Figure 5 A is the expression analysis of ZO-1 in mammospheres, Bar = 100 μm, B is the immunofluorescence analysis of ZO-1 expression on primary GMEC membranes, Bar = 100 μm, C is the expression analysis of occludin in mammospheres, Bar = 100 μm, D is the immunofluorescence analysis of occludin on primary GMEC membranes;

[0022] Figure 6A is a bubble chart obtained by KEGG analysis of RNA-seq data of the control group and LPS-treated group, and B is a bar chart obtained by GO analysis of RNA-seq data of the control group and LPS-treated group;

[0023] Figure 7 Middle A is a bubble chart obtained by KEGG analysis of RNA-seq data of LPS-treated group and C3Gal-relieved group, and middle B is a bar chart obtained by GO analysis of RNA-seq data of LPS-treated group and C3Gal-relieved group;

[0024] Figure 8 for Figure 7 KEGG classification results in;

[0025] Figure 9 Heat map analysis was performed for the RNA-seq data of the control and LPS-treated groups;

[0026] Figure 10 The expression levels of ZBP1 in the control group, LPS treatment group and C3Gal relief group in the examples of the present invention;

[0027] Figure 11 A is a bubble chart obtained by KEGG analysis of the proteomic sequencing data of the control group and the LPS-treated group, and B is a bar chart obtained by GO analysis of the proteomic sequencing data of the control group and the LPS-treated group;

[0028] Figure 12 A in the middle is a bubble chart obtained by KEGG analysis of proteomic sequencing data of LPS-treated group and C3Gal-relieved group, and B is a bar chart obtained by GO analysis of proteomic sequencing data of LPS-treated group and C3Gal-relieved group;

[0029] Figure 13 The expression levels of the main PANoptosis proteins in the examples of the present invention, where A is GSDMD-NT, B is NLRP3, C is ASC, D is Cle-Caspase1, E is Cle-Caspase3, F is p-MLKL, G is Cle-Caspase8, H is p-RIPK3, I is the anti-apoptotic protein Bcl2, and J is the Western blotting (WB) result;

[0030] Figure 14 A shows the expression analysis of NLRP3 in mammospheres, and B shows the expression analysis of NLRP3 in primary GMECs;

[0031] Figure 15 The immunofluorescence results of apoptosis and necrosis in primary GMECs in the examples of the present invention are shown;

[0032] Figure 16The cell death rate of primary GMECs in each group in the present invention, where A is the control group, B is the LPS-treated group, C is the P-C3Gal group, D is the P-C3Gal+LPS group, and E is the C3Gal+LPS group;

[0033] Figure 17 A is the GMEC viability result after 12 hours of treatment with inhibitors or C3Gal, B is the GMEC viability result after 24 hours of treatment with inhibitors or C3Gal, C is the expression level of ZO-1 mRNA, D is the expression level of Occludin mRNA, and E is the WB result;

[0034] Figure 18 The following table represents the expression levels of the main proteins of PANoptosis and the levels of inflammatory factors after treatment with inhibitors or C3Gal in the examples of the present invention, wherein A represents GSDMD-NT, B represents NLRP3, C represents ASC, D represents Bcl2, E represents Cle-Caspase1, F represents Cle-Caspase3, G represents Cle-Caspase8, H represents p-RIPK3, I represents p-MLKL, J represents WB results, K represents the level of IL-6, L represents the level of TNFα, and M represents the level of IL-1β;

[0035] Figure 19 Figure 2 shows the mortality rate of primary GMECs in each group after treatment with inhibitors or C3Gal in the examples of the present invention, where A is the control group, B is the LPS group, C is the inhibitor + LPS group, and D is the C3Gal + LPS group;

[0036] Figure 20 A is the RMSD analysis of the complex, GSDMD and C3Gal molecular ligand, B is the RMSD of GSDMD in the complex and the RMSD of GSDMD protein alone, C is the principal component analysis of the C3Gal trajectory in the complex, D is the surface electrostatic potential of C3Gal binding to GSDMD protein, E is the free energy landscape analysis, and F is the Rg of the complex and the Rg of GSDMD alone;

[0037] Figure 21 A is the B-factor graph constructed using RMSF values as B factors to analyze the flexibility of amino acids around C3Gal, B is the RMSF of GSDMD in the complex and the RMSF of GSDMD protein alone, C is the distance between the binding sites of GSDMD protein and C3Gal, D is the encapsulation region between C3Gal and GSDMD protein, and E is the superposition of simulated conformations;

[0038] Figure 22A is the analysis result of IL-1β content after adding disulfiram, B is the expression level of ZO-1 mRNA, C is the expression level of Occludin mRNA, and D is the WB result;

[0039] Figure 23 Figure 2 shows the necrosis rate of GMECs in each group after transfection of GMECs with the GSDMD-NT overexpression vector constructed in the examples of the present invention. A is the control group, B is the GSDMD-NT overexpression group, C is the GSDMD-NT overexpression + P-C3Gal group, and D is the GSDMD-NT overexpression + C3Gal group. DETAILED DESCRIPTION

[0040] The technical solutions of the present invention are further described below with reference to the accompanying drawings and embodiments. Unless otherwise defined, technical terms or scientific terms used in the present invention should have the same general meanings as those commonly understood by persons skilled in the art in the field to which the present invention belongs.

[0041] Example 1 Method Steps

[0042] 1.1 Extraction and purification of ACN:

[0043] Crabapple fruits were obtained from the Crabapple Germplasm Resource Garden of Northwest Agriculture and Forestry University and served as experimental material. ACN extraction was performed. The crabapple fruits were frozen at -80°C for 48 hours and then freeze-dried. After freeze-drying, the dried fruits were crushed and sieved. 2.0 g of the freeze-dried powder was mixed with 70% ethanol and extracted. The extract was treated with a rotary evaporator to obtain a crude anthocyanin extract from the crabapple fruit. The crude extract was then purified using AB-8 macroporous resin and characterized by HPLC-DAD to determine its composition.

[0044] 1.2 Cell culture and treatment:

[0045] Primary GMECs were isolated and cultured from lactating Saanen goats using Gibco fetal bovine serum (9%), penicillin-streptomycin (1%), and Gibco DF12 medium (90%). For the cellular mastitis model, GMECs were treated with 5 μg / mL LPS for 12 hours. Inhibitors used to inhibit PANoptosis activation included Z-VAD-FMK (50 μM, 2 hours), MCC950 (50 μM, 2 hours), and Neurensin-1 (50 μM, 2 hours). Disulfiram (10 μM, 1 hour) was used to inhibit GSDMD expression, all purchased from Med Chem Express (USA). C3Gal (purity ≥98%) was purchased from Macklin (Shanghai, China).

[0046] 1.3 CCK8 analysis: The density was 2×10 4Cell suspensions were seeded into 96-well plates containing fresh culture medium. The plates were incubated at 37°C in a 5% CO2 atmosphere. Cell viability was assessed using a CCK-8 assay. Optical density was measured at 450 nm using a microplate reader.

[0047] 1.4 RT-qPCR: RNA was extracted from cells using TRIzol reagent and reverse transcribed into cDNA using a reverse transcription kit. Gene expression was quantified using QuantStudio design and analysis software (Applied Biosystems, USA). The primer sequences are as follows:

[0048]

[0049]

[0050] 1.5 Western Blotting: Cells were lysed using RIPA buffer supplemented with a protease inhibitor cocktail. Protein concentration was quantified using an enhanced BCA protein assay kit. Protein expression levels were detected using an ECL western blotting system, and band density was quantified using ImageJ software.

[0051] 1.6 ELISA: Cytokine analysis reagent sources: Goat IL-6 (MM-35226O2), TNFα (MM-0096O2), and IL-1β (MM-1751O2) were purchased from ELISA (Jiangsu, China).

[0052] 1.7 HE staining: Animal tissues for histological analysis were promptly sent to Shaanxi Yike Biotechnology Service Co., Ltd. (Shaanxi, China). The animal tissues were embedded in paraffin and sectioned at 5 μm thickness using a rotary microtome. The sections were stained with HE and analyzed under a light microscope.

[0053] 1.8 Cell Immunofluorescence: After cell treatment, wash samples three times with PBS and incubate cells with primary antibodies overnight at 4°C. Following primary antibody incubation, incubate with secondary antibodies for 1 hour at room temperature in the dark. Nuclear staining with DAPI is performed for 1-2 minutes. Finally, cells are observed and imaged using a confocal microscope.

[0054] 1.9 GSDMD-NT overexpression: A dairy goat GSDMD-NT expression vector was constructed by ligating the sequence of the goat GSDMD-NT fragment into the pcDNA3.10 vector. GMECs were transfected using Lipofectamine 3000, followed by the addition of C3Gal for intervention, and samples were collected 3 h later for flow cytometry analysis.

[0055] 1.10 Mammospheres: Mammary tissue from dairy goats was placed in a 15 mL centrifuge tube and digested with collagenase at 37°C until no visible tissue fragments remained. The resulting cell suspension was filtered to remove debris and the cells were counted. Subsequently, the cells were plated at 1 × 10 5 Cells were seeded into 24-well plates at a density of 100 cells / mL and cultured under standard conditions. Subsequent experiments were performed after 7 days of culture.

[0056] 1.11 Immunofluorescence Analysis of Mammospheres: Mammospheres were fixed in 4% paraformaldehyde and prepared for immunostaining using cell sheets. After fixation, samples were washed thoroughly with PBS and permeabilized with Triton X-100 for 20 minutes. Samples were then washed again with PBS and incubated with primary antibody dilution at 4°C. Secondary antibodies were then used for further incubation. DAPI staining was performed for 3 minutes. Finally, the stained samples were imaged and analyzed using a fluorescence microscope.

[0057] 1.12 Flow cytometry analysis: Cells were plated at 1×10 6 Cells were seeded in 6-well plates at a density of 10 cells / mL for treatment. After treatment, cells were harvested, washed with PBS, and incubated in 500 μL of staining solution at 37°C in the dark for 20 minutes. Stained cells were analyzed within 1 hour using a FACSAria III flow cytometer.

[0058] 1.13 YO-PRO-1 / PI apoptosis assay: cells were plated at 5×10 5 Cells were seeded at a density of 100 cells / mL in 24-well plates and treated according to the experimental design. Staining was performed using the YO-PRO-1 / PI staining kit. Fluorescence images of cells were photographed and analyzed using a fluorescence microscope.

[0059] 1.15 Molecular dynamics simulation analysis: The simulation was performed using Gromacs 2022, the GAFF force field for small molecules, the AMBER14SB force field for proteins, and the TIP3P force field for water models. The complex system of proteins and small molecule ligands was constructed under constant temperature and pressure conditions with periodic boundary conditions. The LINCS algorithm was used to constrain hydrogen bonds, and electrostatic interactions were calculated using the particle mesh Ewald method with a cutoff of 1.2 nm and a cutoff of 0.001 for non-bonded interactions. Updates were made every 10 steps. The simulation temperature was controlled at 298 K using V-level temperature coupling, and the pressure was maintained at 1 bar using the Berendsen method. The system was equilibrated at 298 K using 100 ps of NVT and NPT simulations, followed by 100 ns of production MD simulations, with conformations saved every 10 ps. Post-simulation analysis was performed using VMD and PyMOL software, and the binding free energy between the MMPBSA protein and the small molecule ligand was analyzed using the g_mmpbsa program. Control simulations of a single protein were performed under the same conditions.

[0060] 1.16 RNA-seq Sequencing: After cell treatment, samples were collected according to the protocol and submitted to Novogene for RNA-seq sequencing. The data have been deposited with NCBI under the Bioproject ID PRJNA1213991. The goat reference genome was obtained from Ensembl. All analyses were performed using the Novogene cloud platform.

[0061] 1.17 Proteomic Sequencing: After cell treatment, samples were collected according to the protocol and submitted to Novogene for sequencing. Data have been deposited in ProteomeXchange with the dataset identifier PXD063072. The goat reference genome was obtained from the Ensembl website. All analyses were performed using the Novogene cloud platform. Differentially expressed proteins were identified using a significance threshold of p < 0.05. Functional analyses included volcano plots, cluster heat maps, and GO and KEGG pathway enrichment analyses to assess the roles of differentially expressed proteins.

[0062] 1.18 Statistical Analysis: SPSS 20 and GraphPad Prism software were used for analysis and plotting. Data from at least three independent replicates are presented as mean ± SEM. Comparisons between two groups were performed using an unpaired t-test. Comparisons between more than two groups were performed using one-way analysis of variance. *: indicates a significant difference (P < 0.05); **: indicates an extremely significant difference (P < 0.01); ns: indicates a not significant difference (P > 0.05).

[0063] Example 2 Analysis of results:

[0064] 2.1 ACN extraction and purification

[0065] The crude extract of Malus and its components were characterized by high performance liquid chromatography-diode array detection (HPLC-DAD). Figure 1As shown, C3Gal was identified as the main component, and cyanidin-3-O-arabinoside, cyanidin-3-5-diglucoside, epicatechin, procyanidin B2, eriodictyol, chlorogenic acid, coumalic acid, gallic acid, and phlorizin were also included. After purification, P-C3Gal was obtained.

[0066] 2.2 C3Gal alleviates the inflammatory response and tight junction damage of goat mammary epithelial cells (GMEC).

[0067] In vivo, C3Gal played an important role in reducing the severity of mastitis and preventing BMB damage. Based on these findings, an in vitro mastitis model was developed using mammospheres to validate the role of C3Gal in alleviating mastitis and maintaining BMB integrity.

[0068] First, mammospheres were successfully established and cultured, and then the effect of C3Gal on the viability of primary GMECs was evaluated using a cell counting kit CCK-8 assay to determine the optimal treatment concentration. Treatment with C3Gal (5-160 μM) or P-C3gal (25-800 μg / mL) for 12 hours did not affect the viability of GMECs ( Figure 2 However, both C3Gal (20-160 μM) and P-C3gal (200-800 μg / mL) significantly alleviated the LPS-induced decrease in GMEC survival (P<0.01) ( Figure 2 Based on these findings, 40 μM C3Gal and 200 μg / mL P-C3gal were selected for subsequent experiments.

[0069] To further evaluate the protective effect of C3Gal, a mastitis model was established in primary GMEC and mammospheres by LPS treatment, followed by intervention with selected concentrations of C3Gal and P-C3Gal. Compared with the LPS-treated group, C3Gal treatment significantly downregulated the expression of TLR4, MyD88, and NF-κB, inhibited the activation of the TLR4 / MyD88 / NF-κB signaling pathway, and reduced the expression of proinflammatory cytokines IL-6, TNFα, and IL-1β (P<0.01). Figure 3 In addition, C3Gal treatment significantly increased the expression of ZO-1 and Occludin (P<0.01) ( Figure 3 ), prevented the nuclear translocation of phosphorylated NF-κB p65 during inflammation ( Figure 4 ), and maintained the distribution of ZO-1 and Occludin on the cell membrane ( Figure 5 Notably, the protective effects observed in the mammosphere model were consistent with those observed in primary GMECs. These results suggest that C3Gal effectively preserves tight junction integrity and permeability during inflammatory responses.

[0070] 2.3 Multi-omics sequencing revealed that the PANoptosis pathway is involved in the process of C3Gal alleviating mastitis remission.

[0071] C3Gal, a key component of ACN extract, has been shown to alleviate mastitis and prevent BMB damage both in vitro and in vivo. However, the precise molecular mechanisms underlying these effects remain unknown. Therefore, we used a cellular mastitis model to conduct RNA-seq and proteomic analyses to uncover potential pathways and molecular targets.

[0072] RNA-seq analysis revealed significant involvement of pathways related to inflammation and immune response in LPS-induced GMEC inflammatory response, including IL-17 signaling pathway, cell adhesion molecules, toll-like receptor signaling pathway, chemokine signaling pathway, necroptosis, apoptosis, TNF signaling pathway, NF-κB signaling pathway, and cytokine-cytokine receptor interaction ( Figure 6 GO analysis showed enrichment of terms in biological processes, such as immune system process, immune response, cell adhesion, extracellular region, signal receptor binding, cytokine activity, cytokine receptor binding, and chemokine activity ( Figure 6 After treatment with C3Gal, KEGG enrichment analysis showed that significantly enriched pathways involved cytokine-cytokine receptor interaction, cell adhesion molecules, toll-like receptor signaling pathway, PPAR signaling pathway, programmed necrosis, tight junction, NF-κB signaling pathway and TNF signaling pathway ( Figure 7 GO analysis showed that there was significant enrichment in immune response, cell adhesion, extracellular region, signal receptor binding and active transmembrane transporter activity ( Figure 7 In addition, KEGG classification showed that the immune system, endocrine system, signal transduction and cell growth and death were the main pathways ( Figure 8 The heat map results showed the significant expression of several pro-inflammatory genes, including IL-6, IL1A, TLR4, CCL19, STAT3, IL18R1, PARP12, IRF1, CD14, IL-1β, CCL5, PARP10, IRF7, IL34, NFKBIA, NFKB2, NOS2 ( Figure 9Notably, genes associated with PANoptosis also showed significant expression, including Caspase3, Caspase8, ZBP1, MLKL, and RIPK3. In addition, C3Gal significantly reduced the expression of ZBP1 at the transcriptional level compared with LPS treatment (P < 0.01) ( Figure 10 shown).

[0073] Proteomic sequencing demonstrated that C3Gal alleviated the LPS-induced inflammatory response in GEMCs. Principal component analysis (PCA) revealed distinct clustering patterns; the first principal component mainly distinguished the control and C3Gal-treated groups, while the second principal component distinguished the LPS-treated group. KEGG enrichment analysis revealed significantly enriched terms involving cell adhesion molecules, calcium signaling pathways, cytokine-cytokine receptor interactions, NF-κB signaling pathways, and necroptosis ( Figure 11 ), while GO analysis revealed significant enrichment mainly in immune response, chemokine activity, receptor binding and calcium ion binding ( Figure 11 After C3Gal intervention, KEGG enrichment analysis showed that significantly enriched pathways included toll-like receptor signaling pathway, apoptosis, necroptosis, cytokine-cytokine receptor interaction, ferroptosis, and NF-κB signaling pathway ( Figure 12 GO analysis revealed significant enrichment terms such as immune response, cell adhesion, calcium ion binding, receptor binding, and chemokine activity ( Figure 12 (shown in B).

[0074] 2.4 C3Gal inhibits PANoptosis activation during inflammatory response.

[0075] Multi-omics sequencing analysis showed that PANoptosis is a key pathway for C3Gal to exert resistance to mastitis. Therefore, primary GMEC and mastosphere mastitis models were constructed in vitro to further verify whether C3Gal inhibits PANoptosis activation, thereby playing a protective role during mastitis. The results showed that compared with the LPS-treated group, C3Gal significantly reduced the expression levels of major PANoptosis proteins, including GSDMD-NT, NLRP3, Cle-Caspase1, ASC, Cle-Caspase8, Cle-Caspase3, p-RIPK3 and p-MLKL (P<0.01) ( Figure 13 Meanwhile, the expression of Bcl2 increased significantly (P<0.01) ( Figure 13 In addition, the expression of NLRP3 in GMECs was significantly reduced after C3Gal treatment, and similar results were obtained in mammospheres and primary GMECs ( Figure 14This indicates that during mastitis, C3Gal regulates the expression of PANoptosis-related genes and inhibits the activation of PANoptosis. YO-PRO-1 / PI staining analysis showed that C3Gal intervention significantly reduced the proportion of green-positive apoptotic cells and red-positive necrotic cells in primary GMECs ( Figure 15 In addition, C3Gal treatment significantly reduced the total cell death rate of primary GMECs from 43.59% to 11.68% and 7.02% ( Figure 16 In summary, PANoptosis activation plays a key role in the progression of mastitis, and C3Gal can effectively inhibit PANoptosis activation, thereby reducing the severity of mastitis.

[0076] 2.5 PANoptosis activation is a key pathway leading to GMEC inflammatory response and tight junction damage.

[0077] To determine the relationship between the protective effects of C3Gal in reducing mastitis severity, preventing BMB damage, and inhibiting PANoptosis activation, a combination of pyroptosis, programmed necrosis, and apoptosis inhibitors was used to inhibit PANoptosis activation. This approach was designed to identify PANoptosis as a key pathway for GMEC inflammation and tight junction disruption. Treatment with inhibitors or C3Gal significantly attenuated the LPS-induced decrease in GMEC viability at 12 and 24 h compared to the LPS-only group (P < 0.01) ( Figure 17 (A and B) and significantly upregulated the expression of ZO-1 and Occludin (P<0.01) ( Figure 17 C and D in the middle), indicating that C3Gal can effectively protect the integrity of tight junctions during inflammation. To further verify whether PANoptosis activation plays a key role, the expression of major PANoptosis proteins was detected. Compared with the LPS group, the expression of major proteins in the inhibitor group and C3Gal relief group was significantly reduced (P<0.05) ( Figure 18 Notably, C3Gal failed to prevent the expression of GSDMD-NT, but significantly reduced the secretion of IL-1β. Furthermore, flow cytometry further confirmed these findings, showing that the mortality rate of primary GMECs was significantly reduced after C3Gal intervention, from 30.36% to 12.56% and 5.21% ( Figure 19 These results indicate that PANoptosis is a key pathway driving inflammatory responses and tight junction damage during mastitis and that by inhibiting PANoptosis activation, C3Gal not only reduces cell death and inflammation but also protects the structural integrity of tight junctions.

[0078] 2.6 GSDMD is a key target of C3Gal to alleviate BMB damage during mastitis.

[0079] In both in vivo and in vitro models, C3Gal inhibited PANoptosis activation during mastitis, thereby alleviating the associated inflammatory response and maintaining tight junction integrity. Furthermore, GSDMD serves as a key executioner in the PANoptosis pathway. Importantly, under inflammatory conditions, C3Gal did not prevent the production of GSDMD-NT but inhibited its activity and significantly reduced the extracellular release of IL-1β (P < 0.01). Based on these findings, we hypothesized that C3Gal directly interacts with GSDMD and inhibits NT activity, thereby protecting tight junction integrity during inflammation. To test this, molecular dynamics simulations were performed to evaluate the potential binding interaction between C3Gal and GSDMD.

[0080] Root mean square deviation (RMSD) analysis was performed to evaluate the binding stability of C3Gal and GSDMD. The results showed that the GSDMD protein structure remained stable throughout the simulation. In addition, as the simulation progressed, the RMSD of the C3Gal-GSDMD complex and GSDMD alone gradually stabilized ( Figure 20 Subsequently, PCA and surface electrostatic potential (SEP) analysis showed that C3Gal mainly adopts a single stable conformation ( Figure 20 C3Gal can bind to the positively charged surface of GSDMD, providing the necessary conditions for the formation of hydrogen bonds ( Figure 20 Then, the radius of gyration (Rg) analysis showed that the Rg of GSDMD and the Rg of the complex were basically stable as the simulation progressed, indicating that C3Gal had little effect on the Rg of the complex ( Figure 20 F in the middle), the complex exists only in the low energy state ( Figure 20 Root mean square fluctuation (RMSF) analysis revealed a decrease in the flexibility of amino acids surrounding the C3Gal binding site ( Figure 21 In addition, the centroid evolution analysis showed that the distance between C3Gal and the GSDMD center, as well as the distance between C3Gal and its initial binding site, fluctuated by less than 0.5 nm throughout the simulation. This confirms that C3Gal always binds to the initial binding site of GSDMD ( Figure 21 Buried solvent accessible surface area analysis confirmed these findings, showing that C3Gal always occupies the primary binding site ( Figure 21 (shown in D), combined with conformational superposition analysis, C3Gal is highly superimposed ( Figure 21In summary, the binding interaction between C3Gal and GSDMD is stable, and a highly stable complex is formed.

[0081] Further analysis revealed that C3Gal has multiple binding sites on GSDMD-NT, suggesting that C3Gal may inhibit GSDMD-NT activity to mediate pore-forming events, thereby protecting the integrity of tight junctions between cells during mastitis. To verify this hypothesis, LPS was used to induce a GMEC mastitis model, and disulfiram was used to inhibit the pore-forming events of GSDMD-NT. The results showed that compared with the normal group, the expression of IL-1β was significantly reduced by the addition of disulfiram (P<0.01) ( Figure 22 (shown in A), indicating that the pore-forming activity of GSDMD-NT in the cell membrane was blocked and the expression of ZO-1 and Occludin was significantly increased ( Figure 22 In addition, a GSDMD-NT overexpression vector was constructed and used to transfect GMECs. P-C3Gal and C3Gal were then used for intervention. Flow cytometry was used to measure cell necrosis rates to demonstrate that C3Gal could inhibit GSDMD-NT activity. The results showed that the necrosis rate in the GSDMD-NT overexpression group was 29.08%. When P-C3Gal and C3Gal were added for intervention, the necrosis rates of GMECs were reduced to 18.72% and 11.77%, respectively. Figure 23 In conclusion, GSDMD is the binding target, and C3Gal effectively regulates intercellular tight junction damage during mastitis by directly and stably binding to GSDMD and inhibiting GSDMD-NT activity.

[0082] Therefore, the present invention uses the above-mentioned PANoptosis as a target in the screening of drugs for the treatment of mastitis, revealing that PANoptosis is a key mechanism for the worsening of mastitis. By inhibiting the PANoptosis pathway and GSDMD-NT activity, the inflammatory response and blood-milk barrier damage in mastitis can be alleviated. At the same time, it provides a plant extract - cyanidin-3-O-galactoside, which can significantly inhibit PANoptosis activation and GSDMD-NT pore-forming activity, and can be used to prepare drugs for the treatment of mastitis.

[0083] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the same. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that they can still modify or replace the technical solutions of the present invention with equivalents, and these modifications or equivalent replacements cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. Application of PANoptosis as a target in screening drugs for the treatment of mastitis.

2. The use according to claim 1, characterized in that: Inhibit the activation of the PANoptosis pathway and reduce the content of pro-inflammatory factors in mastitis.

3. Application of GSDMD, a key executive protein of the PANoptosis pathway, as a target in screening drugs for the treatment of mastitis.

4. The use according to claim 3, characterized in that: Inhibit the pore-forming activity of GSDMD-NT and alleviate blood-milk barrier damage during mastitis.

5. The use according to claim 3, characterized in that: Inhibit the activation of the PANoptosis pathway and alleviate the content of pro-inflammatory factors in mastitis.

6. Screening targets for drugs for treating mastitis, characterized by: Including PANoptosis and GSDMD-NT.

7. A medicine for treating mastitis, characterized in that: The active ingredient is cyanidin-3-O-galactoside.

8. The drug according to claim 7, characterized in that: Cyanidin-3-O-galactoside inhibits PANoptosis activation and GSDMD-NT pore-forming activity, reduces the expression of proinflammatory cytokines and blood-milk barrier damage in mastitis.

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