Application of honeysuckle extract in preparation of medicine for relieving injury of deoxynivalenol-induced dairy cow mammary epithelial cells

Abstract

The application belongs to the technical field of biological medicine and animal husbandry, and specifically discloses application of honeysuckle extract in preparation of a medicine for relieving damage of deoxynivalenol (DON) induced on dairy cow mammary epithelial cells, and a DON-induced dairy cow mammary epithelial cell oxidative stress damage model is constructed, honeysuckle extract is screened from eight candidate active substances, and 1 μg / mL is determined as an effective concentration for relieving DON damage. The honeysuckle extract can effectively relieve DON-induced cell damage by up-regulating expression of tight junction genes ZO-1 and Occludin, inhibiting mRNA levels of inflammatory factors IL-6, TNF-α and NF-κB, reducing cell apoptosis rate, and regulating expression of MCM5 and MCM6 proteins in DNA replication and cell cycle pathways and PF4 protein in virus protein-cytokine interaction pathways. The application provides a candidate functional substance for preventing and controlling hazards of DON pollution on dairy cow mammary health and dairy product safety.

Description

Technical Field

[0001] This invention relates to the fields of biomedicine and animal husbandry and veterinary technology, and in particular to the application of honeysuckle extract in the preparation of drugs that alleviate deoxynivalenol-induced damage to mammary epithelial cells in dairy cows. Background Technology

[0002] Deoxynivalenol (DON), also known as vomitoxin, is a secondary metabolite produced by fungi of the genus *Fusarium*. It is one of the most widespread mycotoxins found in grains and their products globally. DON is chemically stable and exhibits strong tolerance to high heat, high pressure, and weak acid conditions, making it difficult to remove effectively using conventional feed processing techniques. When dairy cows consume DON-contaminated feed, it not only reduces rumen fermentation function and milk quality but also leads to DON residues in milk and feces. Since mammary gland tissue is the core of milk protein synthesis and secretion, disruption of its functional integrity directly threatens the safety of dairy products.

[0003] Currently, the main detoxification methods for DON contamination include physical detoxification, chemical detoxification, and biological detoxification. Physical detoxification methods (such as adsorbents and high-temperature treatment) have limited efficiency and easily damage the nutritional value of feed; chemical detoxification methods (such as alkali treatment and ozone oxidation) are faster, but suffer from secondary pollution and decreased food quality; biological detoxification utilizes microorganisms or enzymes to degrade DON, which is highly efficient and low in toxicity, but mainly focuses on the degradation of the toxin itself, lacking direct and effective intervention for oxidative stress, inflammatory responses, and cell damage induced in target organs (such as the mammary gland) after entering the animal's body. While there are research reports on using natural active substances (such as pterostilbene and taraxasterol) to alleviate DON damage, the targets and molecular mechanisms of these substances are still unclear, and they mainly focus on single inflammatory or antioxidant indicators, lacking in-depth explanation of their systemic alleviating effects on cell damage. Although honeysuckle extract is known to have antioxidant and anti-inflammatory properties, its ability to alleviate DON-induced damage to bovine mammary epithelial cells, the appropriate concentration, and the specific molecular mechanisms (especially the regulatory targets at the protein level) have not yet been reported.

[0004] Therefore, developing an active substance that can effectively alleviate DON-induced damage to bovine mammary epithelial cells, is safe and low in toxicity, and has a clear mechanism of action is of great practical significance for controlling the harm of DON contamination to bovine mammary health and dairy product safety. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides the application of honeysuckle extract in the preparation of a drug that alleviates deoxynivalenol-induced damage to bovine mammary epithelial cells.

[0006] To achieve the above objectives, the present invention is implemented according to the following technical solution: In a first aspect, the present invention provides the use of honeysuckle extract in the preparation of a medicament for alleviating deoxynivalenol-induced damage to bovine mammary epithelial cells.

[0007] Furthermore, the effective concentration of the honeysuckle extract is 0.25-8 μg / mL; the concentration of the deoxynivalenol is 0.2 μg / mL, and the action time is 16 hours.

[0008] In a second aspect, the present invention provides a medicament for alleviating deoxynivalenol-induced damage to bovine mammary epithelial cells, comprising honeysuckle extract and a pharmaceutically or veterinarily acceptable carrier.

[0009] Compared with the prior art, the present invention has the following beneficial effects: 1) Screening and determination of the effective alleviating effect of honeysuckle extract on DON damage: This invention uses cell viability as an indicator to screen honeysuckle extract from eight natural active substances, including menobiose, eugenol, vitamin A, bamboo leaf flavonoids, honeysuckle extract, inositol, taraxasterol, and astragalus polysaccharide. Furthermore, it determined that 1 μg / mL is the optimal concentration for alleviating DON damage, filling the technical gap of honeysuckle extract in this application field.

[0010] 2) Revealing the multi-pathway molecular mechanism by which honeysuckle extract alleviates DON damage: This invention demonstrates, through multi-level technical means such as qPCR, apoptosis detection, proteomics, and PRM verification, that honeysuckle extract exerts its alleviating effect through the following three pathways: (1) Upregulating the expression of tight junction genes ZO-1 and Occludin, enhancing cell barrier function; (2) Inhibiting the mRNA levels of inflammatory factors IL-6, TNF-α, and NF-κB, reducing the apoptosis rate; (3) Regulating the expression of MCM5 and MCM6 proteins in the DNA replication and cell cycle pathways, as well as PF4 protein in the viral protein-cytokine interaction pathway, systematically reshaping cell homeostasis at the protein level. This provides a complete theoretical basis for understanding the toxic mechanism of DON and developing novel green anti-toxic additives.

[0011] 3) Wide range of raw material sources and high safety: Honeysuckle extract comes from traditional medicinal and edible plants, and has both strong antioxidant properties and low residue characteristics. It can be directly used as a feed additive in dairy farming and has broad industrial application prospects. Attached Figure Description

[0012] Figure 1The following figures illustrate the colony morphology and molecular identification results of Fusarium graminearum in this invention: A is a front view of the colony; B is a back view of the colony; C is an electrophoresis diagram of ITS sequence PCR amplification; and D is a phylogenetic tree.

[0013] Figure 2 The residual plot, standard curve, and MRM chromatogram for the quantitative detection of DON in this invention are shown below: A is the residual plot; B is the standard curve; and C is the MRM chromatogram.

[0014] Figure 3 Effects of different DON concentrations on MAC-T cell viability at 8 h, 16 h, and 32 h: A represents the results of 8 h treatment; B represents the results of 16 h treatment; and C represents the results of 32 h treatment.

[0015] Figure 4 Effects of different DON concentrations on MAC-T oxidative stress after 16 h: A represents intracellular TAS level; B represents intracellular TOS level; C represents intracellular MDA level.

[0016] Figure 5 The figure shows the effect of DON on the apoptosis rate of MAC-T cells in this invention.

[0017] Figure 6 The following graph shows the effects of the eight active substances of this invention on MAC-T cell viability (treated individually): A is menobiose; B is eugenol; C is vitamin A; D is bamboo leaf flavonoids; E is honeysuckle extract; F is inositol; G is taraxasterol; H is astragalus polysaccharide.

[0018] Figure 7 The following figure shows the effect of the eight active substances of this invention on the viability of MAC-T cells when treated with DON: A is the group treated with menobiose; B is the group treated with eugenol; C is the group treated with vitamin A; D is the group treated with bamboo leaf flavonoids; E is the group treated with honeysuckle extract; F is the group treated with inositol; G is the group treated with taraxasterol; and H is the group treated with astragalus polysaccharides.

[0019] Figure 8 The following figure shows the effect of different concentrations of honeysuckle extract on MAC-T oxidative stress indicators: A represents intracellular TAS level; B represents intracellular TOS level; and C represents intracellular MDA level.

[0020] Figure 9 The figure shows the effect of honeysuckle extract and DON co-treatment on MAC-T cell viability according to the present invention.

[0021] Figure 10The following figure shows the effect of honeysuckle extract and DON co-treatment on MAC-T oxidative stress indicators: A is the intracellular TAS level; B is the intracellular TOS level; C is the intracellular MDA level.

[0022] Figure 11 The following figure shows the effect of honeysuckle extract and DON co-treatment on the expression of MAC-T cell permeability genes mRNA (ZO-1, Occludin and Claudin-1): A is the expression level of ZO-1; B is the expression level of Occludin; C is the expression level of Claudin-1.

[0023] Figure 12 The figure shows the effect of honeysuckle extract and DON co-treatment on NF-κB mRNA expression in MAC-T cells.

[0024] Figure 13 The following figure shows the effect of honeysuckle extract and DON co-treatment on the expression levels of pro-inflammatory genes IL-6, IL-1β and TNF-α mRNA in MAC-T cells: A is the expression level of IL-6; B is the expression level of IL-1β; C is the expression level of TNF-α.

[0025] Figure 14 The figure shows the effect of honeysuckle extract and DON co-treatment on the expression level of IL-10 mRNA, an anti-inflammatory gene in MAC-T cells.

[0026] Figure 15 The figure shows the effect of honeysuckle extract and DON co-treatment on the apoptosis rate of MAC-T cells according to the present invention.

[0027] Figure 16 The following figure shows the effect of honeysuckle extract and DON co-treatment on the mRNA expression level of MAC-T apoptosis genes: A is the expression level of the pro-apoptotic gene Bax; B is the expression level of the anti-apoptotic gene Bcl-2; C is the Bax / Bcl-2 ratio.

[0028] Figure 17 This is the result of SDS-PAGE electrophoresis.

[0029] Figure 18 This represents the peptide length distribution.

[0030] Figure 19 The results of the sample repeatability analysis are as follows: A is the PCC analysis result; B is the PCA analysis result.

[0031] Figure 20The following are volcano plots and statistical graphs of differentially expressed proteomic proteins in this invention: A is the volcano plot of the D0.2 / C group; B is the volcano plot of the H1 / C group; C is the volcano plot of the H1D0.2 / C group; D is the volcano plot of the H1D0.2 / D group; E is a bar chart of five histones; and F is a statistical graph of five histones.

[0032] Figure 21 This is a bar chart showing the GO functional enrichment analysis results of this invention.

[0033] Figure 22 The subcellular classification analysis diagram of this invention is as follows: A is group D0.2 / C; B is group H1 / C; C is group H1D0.2 / C; D is group H1D0.2 / D.

[0034] Figure 23 This is a bubble diagram of KEGG pathway enrichment in this invention.

[0035] Figure 24 The following figures illustrate the relative quantitative expression results of proteins verified by PRM in this invention: A shows the relative quantitative expression results of proteins MCM5 and MCM6; B shows the relative quantitative expression results of protein PF4.

[0036] Figure 25 This is a schematic diagram illustrating the molecular mechanism by which the honeysuckle extract of this invention alleviates DON-induced damage to mammary epithelial cells in dairy cows. Detailed Implementation

[0037] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. The specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention.

[0038] Example 1: Preparation of deoxynivalenol (DON) and establishment of a bovine mammary epithelial cell injury model. 1. Test materials The diseased corn silage leaves were collected from Shanxi Province and donated by the Academy of Agricultural Sciences. The experiment was conducted in the Bioreactor Laboratory of the College of Life Sciences, Jilin Agricultural University; the bovine mammary epithelial cells (MAC-T cells) used were preserved and continued to be cultured by the Cell Laboratory of the Ruminant Nutrition Research and Innovation Team of Jilin Agricultural University.

[0039] 2. Isolation and Identification of Fusarium graminearum Infected silage samples were surface-sterilized with 75% ethanol and soaked overnight in sterile PBS at 4°C. The supernatant was spread onto solid culture plates and incubated in the dark at 25°C for 3-14 days. Mycelia at the colony edges were isolated and purified by repeated streaking. The purified strain produced an orange-red pigment after culturing on solid culture medium (see...). Figure 1 A in Figure 1 (B in the text) Fungal genomic DNA was extracted, and the ITS region was amplified by PCR using primers ITS1 (5′-TCCGTAGGTGAACCTGCGG-3′) / ITS4 (5′-TCCTCCGCTTATTGATATGC-3′). The reaction system and amplification conditions are shown in Tables 1 and 2.

[0040] Table 1. PCR reaction system for amplifying fungal ITS sequences

[0041] Table 2. PCR reaction procedure for amplifying fungal ITS sequences

[0042] The amplified product was sent for sequencing (forward 5′→3′). The forward and reverse sequences were assembled into a complete sequence using DNAMAN software (5′→3′). The obtained ITS sequence was submitted to the NCBI GenBank database (https: / / www.ncbi.nlm.nih.gov), and sequence homology was compared using BLASTn. The type strain or reference sequence with the highest similarity was selected, and the strain was identified based on morphological characteristics. A product of 512 bp in length was obtained (see...). Figure 1 The C in the sample, after sequencing comparison, was found to be similar to Fusarium graminearum (C). Fusarium graminearum The ITS sequence similarity reached 100% (see...) Figure 1 (D) The strain was identified as Fusarium graminearum and named... F. graminearum SX-1 .

[0043] 3. Fermentation Extraction and Quantitative Detection of DON Will F. graminearum SX-1 Inoculate into liquid culture medium and activate by shaking at 25℃ and 150 r / min for 3-5 days. Then transfer to 200 mL / 500 mL liquid culture medium and continue culturing for 10-14 days. After fermentation, add twice the volume of n-butanol for extraction, sonicate for 20 minutes, and let stand overnight. Take the upper n-butanol and rotary evaporate to obtain crude extract. Add methanol-water solution (80:20, V / V) and shake for extraction for 60 minutes. After filtration, filter through a 0.22 μm microporous membrane, and collect the filtrate as the DON test extract.

[0044] Quantitative detection was performed using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS). The mobile phase was methanol-0.1% formic acid aqueous solution (30:70, V / V), the flow rate was 0.3 mL / min, the column temperature was 35℃, and the injection volume was 5 μL. Mass spectrometry was performed using an electrospray ionization (ESI) negative ion source. - Multiple response monitoring (MRM) mode. Standard curve as follows: Figure 2 As shown in A, the fitted equation is y = 5.26081x - 32.9014, R0 2 =0.990271. The DON concentration in the sample was measured to be 16.95 μg / mL (see [reference]). Figure 2 (B in the middle).

[0045] 4. Establishment of a model of mammary epithelial cell injury in dairy cows MAC-T cells were used at a rate of 5 × 10 3 Inoculate at a density of 2 × 10⁶ cells / mL into 96-well plates. 5 Cells were seeded at a density of 1 cell / mL in 6-well plates and cultured at 37°C in a 5% CO2 incubator. When the cells reached 80% confluence, they were treated with 0, 0.2, 0.4, 0.6, 0.8, and 1 μg / mL DON for 8, 16, and 32 hours, respectively.

[0046] Cell viability was determined using the CCK-8 assay. Figure 3 As shown, compared with the control group, cell viability decreased in a time- and dose-dependent manner in the DON-treated group. Specifically, after 16 hours of treatment with DON 0.2 μg / mL, the cell viability was 79.09%; after 8 hours and 32 hours of treatment, the viability was 91.77% and 73.57%, respectively.

[0047] Results of oxidative stress index measurement are as follows Figure 4 As shown. Compared with the control group, treatment with DON 0.2 μg / mL for 16 hours significantly increased the MDA content ( P <0.001), TAS levels decreased significantly ( P <0.001), the TOS level shows a downward trend ( P <0.05).

[0048] Apoptosis rate was detected by Annexin V-FITC / 7AAD-A double staining combined with flow cytometry. The results are as follows: Figure 5 As shown in the figure, the proportion of viable cells in the control group was 97.2%, and the total apoptosis rate was 1.66%; the proportion of viable cells in the DON 0.2 μg / mL group was 87.7%, and the total apoptosis rate was 12.10%.

[0049] In summary, treatment with 0.2 μg / mL DON for 16 hours was determined to be the optimal condition for inducing a MAC-T cell injury model.

[0050] Example 2: Screening of active substances that alleviate DON-induced cell damage and determination of the effective concentration of honeysuckle extract. 1. Initial screening of active substances Eight active substances were selected, including menobiose, eugenol, vitamin A, bamboo leaf flavonoids, honeysuckle extract, inositol, taraxasterol, and astragalus polysaccharide. Their sources are shown in Table 3.

[0051] Table 3 Eight active substances

[0052] Reagent preparation (1) MAC-T cell growth culture medium MAC-T cell culture was prepared by adding 10% FBS and 1% penicillin-streptomycin to DMEM high-glucose medium. The medium was prepared fresh for each use.

[0053] (2) Active substance storage solution and working solution Honeysuckle extract, astragalus polysaccharide, bamboo leaf flavonoids, and inositol were dissolved in DMEM high-glucose medium; pinobiose, eugenol, vitamin A, and taraxasterol were dissolved in DMSO. All eight substances were prepared into stock solutions with a concentration of 10 mg / mL. These stock solutions were then serially diluted using complete culture medium to prepare concentrations of 0, 10, and... -4 10 -2 10 -1 10 0 10 1 10 2 The working solution (μg / ml) should be prepared fresh for cell culture.

[0054] (3) Preparation of honeysuckle extract working solution Honeysuckle extract was dissolved in DMEM high-glucose medium to prepare a stock solution with a concentration of 10 mg / mL. The stock solution was then serially diluted with complete medium to prepare working solutions with concentrations of 0, 0.25, 0.5, 1, and 8 μg / mL. The solutions were prepared fresh for use in cell culture.

[0055] MAC-T cells were treated alone or with 0.2 μg / mL DON for 16 hours, and cell viability was detected by CCK-8 assay.

[0056] Individual processing results such as Figure 6 As shown, meliolosylate at 100 μg / mL significantly reduced cell viability (P < 0.0001); eugenol at 10 μg / mL... -4 Cell viability was significantly reduced at concentrations of μg / mL, and significantly decreased at concentrations of 10 μg / mL and above. P<0.001); Vitamin A and taraxasterol showed a significant concentration-dependent decrease in cell viability at concentrations ≥10 μg / mL; while the cell viability of bamboo leaf flavonoids, honeysuckle extract, inositol and astragalus polysaccharide remained at the control group level within the tested concentration range.

[0057] Results of processing with DON as follows Figure 7 As shown, cell viability was not significantly different from that of the DON group across the entire concentration range of menobiose; however, cell viability was significantly lower in the DON group at the entire concentration range of eugenol. P <0.05); the overall cell viability of the vitamin A co-treatment group was lower than that of the DON group; cell viability was significantly reduced only at 100 μg / mL with bamboo leaf flavonoids ( P <0.001); the cell viability of honeysuckle extract at 1 μg / mL was significantly higher than that of the DON group ( P <0.0001); inositol in 10 -1 Cell viability was significantly higher in the μg / mL group than in the DON group ( P <0.05); cell viability decreased at concentrations of taraxasterol (10-100 μg / mL); and cell viability was significantly lower in all concentrations of astragalus polysaccharide compared to the DON group. Based on these results, honeysuckle extract was selected for further research.

[0058] 2. Determination of the effective concentration of honeysuckle extract MAC-T cells were treated with honeysuckle extract at concentrations of 0, 0.25, 0.5, 1, and 8 μg / mL for 16 hours, and oxidative stress indicators were measured. For example... Figure 8 As shown, when honeysuckle extract was used alone, the TAS level increased in a concentration-dependent manner, and was significantly higher than that of the control group at 1 μg / mL. P <0.05), began to decrease at 8 μg / mL; TOS levels showed a gradual decreasing trend; MDA content showed an increasing trend in the range of 0.25-1 μg / mL. P <0.001).

[0059] MAC-T cells were treated with honeysuckle extract and DON 0.2 μg / mL for 16 hours, and cell viability and oxidative stress indicators were detected. Figure 9 As shown, the cell viability was most significantly improved when the honeysuckle extract concentration was 1 μg / mL. P <0.05). For example... Figure 10 As shown, compared with the DON group, honeysuckle extract at concentrations of 0.25–8 μg / mL could reduce MDA levels ( P <0.05), and at 1 μg / mL, the levels of TAS and TOS were significantly higher than those in the DON group ( P <0.05). Therefore, 1 μg / mL of honeysuckle extract was determined to be the appropriate concentration for alleviating DON damage.

[0060] Example 3: The regulatory effect of honeysuckle extract on DON-induced MAC-T cell mRNA expression and apoptosis. 1. Experimental grouping and treatment The control group (CON), the DON group (DON 0.2 μg / mL), the honeysuckle group (H1 1 μg / mL), and the co-treatment group (H1D0.2, honeysuckle extract 1 μg / mL + DON 0.2 μg / mL) were set up. The tests were performed after each group was treated for 16 hours.

[0061] 2. Real-time quantitative PCR detection Total RNA was extracted from cells and reverse transcribed into cDNA. β-actin was used as an internal reference gene, and qPCR was performed using the SYBR Green fluorescent dye method. Primer sequences are shown in Table 4, Real-Time PCR reaction systems are shown in Table 5, and the quantitative PCR procedure is shown in Table 6.

[0062] Table 4. Primer sequences for real-time PCR

[0063] Table 5 Real-Time PCR Reaction System

[0064] Table 6. Quantitative Fluorescence Reaction Procedure

[0065] 3. Expression of genes related to cell permeability The results are as follows Figure 11 As shown, compared with the control group, the expression levels of ZO-1, Occludin, and Claudin-1 mRNA in the DON group were significantly reduced ( P <0.0001). Compared with the DON group, the expression levels of ZO-1 and Occludin mRNA were significantly increased in the honeysuckle extract co-treatment group ( P <0.05), Claudin-1 showed an increasing trend but no significant difference.

[0066] 4. Expression of inflammation-related genes The results are as follows Figure 12 As shown, the expression level of NF-κB mRNA in the DON group was significantly increased ( P <0.0001); the expression level of NF-κB mRNA was significantly reduced in the group co-treated with honeysuckle extract ( P <0.0001). For example... Figure 13As shown, the expression levels of IL-6, IL-1β, and TNF-α mRNA were significantly increased in the DON group. P <0.0001), the expression levels of the above genes were significantly reduced in the honeysuckle extract co-treatment group ( P <0.0001). For example... Figure 14 As shown, the expression level of IL-10 mRNA in the DON group was significantly increased ( P <0.0001), the expression level of IL-10 in the honeysuckle group was significantly reduced ( P <0.0001), the expression level of IL-10 in the co-treatment group was significantly lower than that in the DON group ( P <0.0001).

[0067] 5. Apoptosis rate and expression of apoptosis-related genes Apoptosis rate was detected by flow cytometry, and the results are as follows: Figure 15 As shown in the figure, the proportion of viable cells in the control group was 87.5%, and the total apoptosis rate was 9.47%; the proportion of viable cells in the DON group decreased to 82.2%, and the total apoptosis rate was 9.63%; the proportion of viable cells in the group treated with honeysuckle extract at 1 μg / mL increased to 90.4%, and the total apoptosis rate decreased to 6.35%.

[0068] The results of apoptosis gene mRNA expression are as follows Figure 16 As shown, the pro-apoptotic gene Bax was significantly higher in the DON group than in the control group, the Bax / Bcl-2 ratio was significantly upregulated, and the anti-apoptotic gene Bcl-2 was significantly lower in the DON group than in the control group. P <0.0001); the Bax / Bcl-2 ratio in the honeysuckle extract co-treatment group decreased significantly and was lower than that in the DON group (P<0.0001).

[0069] Example 4: Molecular mechanism of honeysuckle extract alleviating DON-induced MAC-T cell damage based on proteomics analysis 1. Protein extraction Take the sample from the -80°C freezer and quickly place it on ice for gradient thawing to prevent protein degradation. Add 4 volumes of pre-chilled lysis buffer (8 M urea + 1% protease / phosphatase inhibitor mixture), vortex for 1 min to mix, and incubate on ice for 20-30 min, vortexing every 5 min to enhance lysis. Centrifuge at 12,000 × g for 10 min at 4°C, carefully aspirate the supernatant into a sterile enzyme-free centrifuge tube, record the volume, and incubate on ice for later use.

[0070] 2. Protein concentration determination (BCA method) Dissolve 5 mg / mL BSA standard at room temperature to prepare a series of gradient standard solutions. Add 0 μL, 5 μL, 10 μL, 15 μL, and 20 μL of standard to a 96-well plate, respectively, and bring the volume to 20 μL with sample diluent, resulting in final concentrations of 0, 1.25, 2.5, 3.75, and 5.0 mg / mL. Perform three technical replicates for each concentration. Add 5 μL of the supernatant to be tested, and bring the volume to 20 μL with diluent, again performing three technical replicates. Include a 20 μL blank control well. Add 200 μL of fresh BCA working solution (A:B = 50:1, freshly prepared) to each well, mix thoroughly by pipetting three times, and incubate at 37°C for 30 min. After cooling to room temperature, zero the plate using the blank well and measure the absorbance at 562 nm using a microplate reader. Plot a standard curve (R² ≥ 0.99) with BSA concentration on the x-axis and A562 value on the y-axis, and substitute the values ​​into the curve to calculate the protein concentration of the sample.

[0071] 3. SDS-PAGE Sample preparation: Take 10-20 μg of protein sample into a stoppered centrifuge tube, add 4× Loading buffer at a ratio of 4:1, and make up to 20 μL with 2% SDS. Vortex mix and heat in a boiling water bath for 5 min. Remove and cool to room temperature in an ice bath. It can be stored at -80°C or loaded directly. Sample loading procedure: Load the gel into the electrophoresis tank, add sufficient electrophoresis buffer, add 1 μL of pre-stained protein marker to the first lane, add 20 μL of the treated sample to the target lane, and add 20 μL of 1× Loading buffer to the blank lane. Electrophoresis procedure: Concentrate at a constant current of 15 mA / gel for 15 min. After the sample forms a thin line at the top of the stacking gel, adjust to a constant current of 35 mA and stop when the indicator migrates to 1 cm from the bottom of the gel. Staining and destaining: After electrophoresis, rinse the gel twice with ultrapure water (1 min each time), then immerse it in Coomassie Brilliant Blue R-250 staining solution and stain for 2 h at room temperature on a shaker (80-100 rpm). Discard the staining solution and add destaining solution, then destain on a shaker at room temperature 3-4 times (30 min each time), changing the destaining solution during the process, until the background is colorless and the bands are clear.

[0072] 4. Pancreatic enzyme digestion Take the protein sample to be analyzed and adjust the volume to match the lysis buffer. Add DTT to a final concentration of 5 mM and incubate at 56°C for 30 min. After cooling to room temperature, add IAA to a final concentration of 11 mM and incubate at room temperature in the dark for 15 min. Dilute urea with TEAB to a concentration <2 M, add trypsin at a ratio of 1:50 (m / m), and incubate overnight at 37°C. Then add trypsin again at a ratio of 1:100 (m / m) and continue incubation for 4 h to ensure complete digestion.

[0073] 5. Liquid chromatography-mass spectrometry analysis After enzymatic hydrolysis, the peptide fragments were mixed with mobile phase A (0.1% formic acid + 2% acetonitrile aqueous solution) and separated using a Nano Elute ultra-high performance liquid chromatography (UHPLC) system. Mobile phase B consisted of 0.1% formic acid and acetonitrile solution, and the flow rate was maintained at 500 nl / min. The liquid phase gradient was as follows: 0–14 min, 6%–24% of phase B; 14–16 min, 24%–35%; 16–18 min, 35%–80%; 18–20 min, maintaining 80% of phase B. After separation, the peptide fragments were injected into a Capillary ion source for ionization and analyzed using a Tims TOF Pro 2 mass spectrometer with an ion source voltage of 1.75 kV. Pre-peptide ions and secondary fragment ions were detected. Data acquisition was performed in dia-PASEF mode. The main mass spectrometry scan range was 300–500 m / z, acquiring 20 PASEF modes; the secondary mass spectrometry scan range was 400–850 m / z, detected in 7 m / z window segments.

[0074] The results of protein combination are shown in Table 7, and the SDS-PAGE results are as follows: Figure 17 As shown, the results indicate that the protein did not degrade, and the protein bands were distinct and naturally arranged. Although the sample bands were slightly non-parallel, the electrophoretic behavior of each lane was not significantly different under a certain level of high-density protein, making it suitable for subsequent protein identification or functional analysis.

[0075] Table 7 Results of protein concentration determination

[0076] like Figure 18 As shown, the lengths of the enzyme-digested peptides are mainly distributed between 7 and 25 amino acids. This length distribution conforms to international proteomics standards (such as the HUPO guidelines), indicating that the system quality control from sample processing to instrument calibration is effective, the experimental design and operation are in accordance with regulations, and the results are reliable.

[0077] This embodiment uses statistical analysis methods such as Pearson's Correlation Coefficient (PCC) and Principal Component Analysis (PCA) to evaluate experimental repeatability. The evaluation results are shown in […]. Figure 19 As shown in Figure A, the redder the color, the closer the Pearson correlation coefficient is to 1, indicating a stronger correlation between the two samples. Figure B shows the explained variance of PC1 and PC2 on the x and y axes, respectively, revealing a high degree of clustering among the replicated samples, indicating a relatively concentrated data distribution among the replicated samples. In summary, the results indicate that this experiment has high repeatability.

[0078] The proteomic data of four groups of MAC-T are as follows: Figure 20 As shown. This embodiment utilizes liquid chromatography-mass spectrometry (LC-MS) for proteomics analysis. To accurately determine the significance of differences in protein expression, a T-test was performed on the relative protein quantifications in each treatment group. P A difference of < 0.05 is considered significant. P When < 0.05, differential expression changes > 1.5 are considered upregulation thresholds, and differential expression changes < 1 / 1.5 are considered downregulation thresholds. The volcano plot (red, upregulation; blue, downregulation) shows partial overlap in protein counts across groups, and each group contains unique proteins. Specifically, in the D0.2 / C group (see...),... Figure 20 In group A), a total of 878 differentially expressed proteins were screened, including 696 upregulated proteins and 182 downregulated proteins. (See H1 / C group). Figure 20 In group B), 357 differentially expressed proteins were screened, including 264 upregulated proteins and 93 downregulated proteins. (See H1D0.2 / C group). Figure 20 In the C group, a total of 1015 differentially expressed proteins were screened, including 729 upregulated proteins and 286 downregulated proteins. Finally, the H1D0.2 / D group (see [link to H1D group]) was selected. Figure 20 In step D), a total of 109 differentially expressed proteins were identified, including 38 upregulated proteins and 71 downregulated proteins. To more intuitively compare the differences among the five histone groups, the results are shown below. Figure 20 E and Figure 20 As shown in F in the diagram.

[0079] Blast2Go software (https: / / www.blast2go.com / ) was used to perform GO functional annotation on all significantly differentially expressed proteins. GO functional annotation is mainly divided into three categories: biological process (BP), molecular function (MF), and cellular component (CC). GO functional enrichment analysis was performed on the differentially expressed proteins, and the functional categories of the enriched proteins were identified using Fisher's exact test (p < 0.05). See details in [link to documentation]. Figure 21In DON0.2 / C, differentially expressed proteins were significantly enriched in nucleic acid binding, the nucleolus, and various nucleic acid or nitrogen-containing compound metabolic processes. This indicates that DON primarily induces toxic damage by interfering with cellular nucleic acid metabolism and nuclear function, disrupting the genetic homeostasis of MAC-T cells. In H1 / C, differentially expressed proteins were significantly enriched in DNA damage repair-related biological processes such as DNA double-strand break repair and homologous recombination repair, accompanied by enhanced enzyme inhibitor activity. This suggests that honeysuckle extract can enhance the resistance of MAC-T cells to potential damage by activating endogenous DNA repair pathways. The enrichment results of D0.2H1 / D0.2 showed that the differentially expressed proteins were significantly enriched in secretion- and protein synthesis-related biological processes such as exocytosis, regulation of protein metabolic processes, and translation, accompanied by changes in aldolase activity and extracellular region-related functions. This may be the core reason why honeysuckle extract alleviates DON toxicity, not only by repairing DNA damage, but also by potentially reshaping cellular homeostasis at multiple levels, thus achieving a systemic relief of DON-induced damage.

[0080] By comparing the subcellular localization distribution of the four groups of differentially expressed proteins, it was found that these proteins were mainly located in the cell nucleus, cytoplasm, extracellular matrix, mitochondria, and cell membrane. For details, see [link to relevant documentation]. Figure 22 In the D0.2 / C group (Figure A), after DON treatment alone, differentially expressed proteins were mainly enriched in the nucleus, followed by the cytoplasm, extracellular matrix, and plasma membrane. These results indicate that DON primarily induces cellular stress and inflammatory responses by interfering with the expression of nuclear proteins; it also affects the homeostasis of cytoplasmic signaling proteins and extracellular secretory proteins. The H1D0.2 / D group (Figure D) showed significant upregulation, mainly in the cytoplasm, mitochondria, extracellular matrix, and plasma membrane. Honeysuckle extract may alleviate DON-induced cell damage by enhancing cytoplasmic signaling, mitochondrial energy metabolism, and the expression of extracellular defense proteins.

[0081] KEGG pathway enrichment analysis was performed to investigate the regulatory pathway by which honeysuckle extract (1 μg / mL) alleviates DON 0.2 μg / mL-induced MAC-T damage. (See [link to KEGG pathway analysis]). Figure 23 In the D0.2 / C group, differentially expressed proteins were significantly enriched in pathways such as DNA replication, cell cycle, and complement and coagulation cascades. The expression of key regulatory proteins MCM5 and MCM6 in the DNA replication and cell cycle pathways was significantly upregulated. In the D0.2H1 / C group, although differentially expressed proteins were still enriched in DNA replication and cell cycle pathways, the enrichment level was significantly reduced. Furthermore, while the expression levels of MCM5 and MCM6 proteins were still higher than the control group, they were significantly lower than the DON group, indicating that honeysuckle extract can effectively alleviate DON-induced cell damage by reversing its abnormal activation of DNA replication and immune pathways. Further comparison with the D0.2H1 / D0.2 group revealed that this group did not significantly enrich DNA replication and cell cycle pathways, but instead exhibited novel pathways such as viral protein interaction with cytokines and cytokine receptors, with a significant downregulation of PF4 protein expression. This indicates that the mechanism by which honeysuckle extract alleviates DON toxicity is not limited to reversing abnormal DNA replication, but can also systematically reshape cellular homeostasis from the level of immune regulation by regulating the interaction between cytokines and receptors and inhibiting excessive activation of immune pathways, thereby achieving effective intervention against DON-induced damage.

[0082] pass Figure 23 Proteins and their pathways were significantly enriched and screened in the KEGG pathway, resulting in three target proteins shown in Table 8 for PRM validation. Among them, MCM5 and MCM6, proteins involved in DNA replication and the cell cycle pathway, were significantly enriched. Figure 24 The A in the figure represents the relative quantitative expression of the quantitative result.

[0083] Table 8. Effects of enriched differentially expressed proteins on related pathways.

[0084] The results showed that MCM5 and MCM6 proteins exhibited similar trends in proteomics PRM validation and KEGG pathway analysis. After significant enrichment of PF4 protein in the viral protein-cytokine and cytokine receptor interaction pathways, the relative quantitative expression results are as follows: Figure 24As shown in B in the figure. The results indicate that PF4 protein also showed a similar trend in proteomics PRM validation and KEGG pathway analysis, further demonstrating the rationality and reliability of proteomics analysis. Based on the above experimental data, the mechanism by which honeysuckle extract alleviates DON-induced damage to bovine mammary epithelial cells is relatively clear; for details, see [link to relevant documentation]. Figure 25 This diagram visually illustrates the pathway by which honeysuckle extract exerts its protective effect at the cellular level, contributing to a deeper understanding of the relevant biological processes and providing a theoretical basis for further exploration of strategies for maintaining mammary gland health in dairy cows.

[0085] The results above show that honeysuckle extract at 1 μg / mL can alleviate DON0.2 μg / mL-induced MAC-T cell damage by upregulating the expression of MCM5 and MCM6 proteins in the DNA replication and cell cycle pathways, and downregulating the expression of PF4 protein in the viral protein-cytokine interaction pathway.

[0086] The technical solutions of the present invention are not limited to the specific embodiments described above. Any technical modifications made in accordance with the technical solutions of the present invention fall within the protection scope of the present invention.

Claims

1. Application of honeysuckle extract in the preparation of drugs to alleviate deoxynivalenol-induced damage to bovine mammary epithelial cells.

2. The application according to claim 1, characterized in that, The effective concentration of the honeysuckle extract is 0.25-8 μg / mL; the concentration of the deoxynivalenol is 0.2 μg / mL, and the action time is 16 hours.

3. A drug for alleviating deoxynivalenol-induced damage to bovine mammary epithelial cells, characterized in that, It contains honeysuckle extract and pharmaceutically or veterinarily acceptable carriers.

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