Lactobacillus plantarum-derived inorganic polyphosphate and application thereof in promoting cell proliferation

Abstract

The invention relates to lactobacillus plantarum-derived inorganic polyphosphate and application thereof in promoting cell proliferation. The preparation method of the lactobacillus plantarum-derived inorganic polyphosphate comprises the following step: extracting from lactobacillus plantarum by using glass milk. According to the invention, long-chain PolyP (PolyPLp) with the chain length of about 250 phosphate residues is extracted and purified from lactobacillus plantarum by using glass milk. And the effect of promoting cell proliferation is proved. An IL-1beta-induced Caco-2 intestinal epithelial cell barrier injury model is utilized, it is found that PolyP has the function of relieving intestinal inflammation, the effect of PolyP has chain length dependence, and the action effect of PolyPLp is optimal.

Description

Technical Field

[0001] This invention belongs to the technical field of preparation and application of Lactobacillus plantarum-derived inorganic polyphosphates, and particularly relates to Lactobacillus plantarum-derived inorganic polyphosphates and their application in promoting cell proliferation. Background Technology

[0002] Inorganic polyphosphates (PolyP) are linear polymers (3-1000 Pi) composed of several to thousands of phosphate groups (Pi) linked by high-energy phosphoric anhydride bonds. The effects of exogenously purified PolyP depend primarily on chain length and dosage. Microbially extracted PolyP exhibits varying biological functions and applications due to differences in chain length, synthesis mechanism, and physicochemical properties. Currently, there is a lack of synthetic techniques for precisely controlling chain length, making long-chain PolyP (>100 Pi) difficult to obtain. Existing PolyP extraction methods generally utilize radionuclide labeling for separation or strong acid treatment followed by collection, which suffer from drawbacks such as resource consumption, low sensitivity, low extraction rates, and susceptibility to structural damage or the introduction of impurities, thus limiting the practicality of PolyP.

[0003] The intestinal barrier, as a protective barrier between the animal body and the external environment, plays a crucial role in maintaining the body's health. Studies have shown that the integrity of the intestinal barrier function is significant in preventing autoimmune diseases and inflammation. Intestinal barrier dysfunction is associated with intestinal ecological imbalance and various diseases, and genetic defects and environmental factors can also disrupt intestinal balance, thus contributing to the development of chronic diseases. Currently, the drugs used clinically to treat ulcerative colitis are mainly anti-inflammatory or immunomodulatory drugs, lacking products that can directly improve intestinal barrier function. There are currently no reports on the application of *Lactobacillus plantarum*-derived inorganic polyphosphates in promoting cell proliferation. Summary of the Invention

[0004] In view of the problems existing in the prior art, the present invention provides an inorganic polyphosphate derived from Lactobacillus plantarum and its application in promoting cell proliferation.

[0005] The technical solution of the present invention to solve the above-mentioned technical problems is as follows: A method for preparing plant-derived inorganic polyphosphates from Lactobacillus plantarum includes the following steps: extracting inorganic polyphosphates from Lactobacillus plantarum using glass milk.

[0006] This invention utilizes glass milk to extract and purify a long-chain PolyP (PolyP) with a chain length of approximately 250 phosphate residues from Lactobacillus plantarum. LpUsing an IL-1β-induced Caco-2 intestinal epithelial cell barrier injury model, it was found that PolyP has the function of alleviating intestinal inflammation, and its effect is chain-length dependent. Lp PolyP exhibits the best therapeutic effect. Metabolomics analysis revealed that PolyP participates in pathways such as glycerophospholipid metabolism, autophagy, amino acid metabolism, TCA cycle, and mTOR signaling by regulating differentially metabolites including lipids, organic acids, amino acids, carbohydrates, hormones, and nucleotides, thereby mediating signal transduction and inflammatory responses. Furthermore, HMDB database analysis showed that PolyP reduces differentially metabolites associated with diseases such as irritable bowel syndrome, colorectal cancer, Crohn's disease, and ulcerative colitis. PolyP inhibits the expression of the IL-1β receptor IL-1R1 and suppresses the activation of the TNF / TNFRSF10D cell death pathway, thereby hindering signal transduction and interfering with the initiation of apoptosis. It also reduces the expression of FADD and CASP10, decreasing the apoptosis cascade and reducing the activation of the downstream NF-κB inflammatory pathway, ultimately alleviating intestinal epithelial cell inflammation and reducing intestinal epithelial barrier damage.

[0007] This invention elucidates the mechanism by which PolyP derived from Lactobacillus plantarum alleviates intestinal epithelial cell inflammation. Specifically, PolyP antagonizes intestinal epithelial cell apoptosis by regulating the TNF apoptosis pathway and alleviates the inflammatory response through the NF-κB inflammatory pathway, thereby repairing damage to the intestinal epithelial barrier.

[0008] Furthermore, the following steps are included: (1) Treat the Lactobacillus plantarum cells with GITC lysis buffer, water bath, and sonicate to disrupt them; (2) Add 10% SDS, 95% ethanol and glass milk, mix well, centrifuge, and discard the supernatant; (3) Add NW buffer to the precipitate on ice, vortex to mix well, sonicate, centrifuge, and discard the supernatant; (4) Add nucleic acid buffer, DNase I and RNase A to the precipitate, incubate, centrifuge, and discard the supernatant; (5) Wash the precipitate, centrifuge, and discard the supernatant; (6) Add Tris-HCl to the precipitate, incubate, centrifuge, and collect the supernatant; (7) Ultrafilter the supernatant, centrifuge, and collect the concentrate.

[0009] Further, in step (1), the mixture was incubated in a 95°C water bath for 5 min and then sonicated for 15 min. Further, in step (2), the volume ratio of 10% SDS, 95% ethanol, and glass milk was 1 mL: 15 mL: 200 μL. Further, in step (4), the mixture was incubated at 37°C for 1 h. Further, in step (5), the precipitate was washed with GITC lysis buffer and 95% ethanol, respectively, and then washed with NW buffer. Further, in step (6), 50 mM Tris-HCl preheated to 90°C was added, and the mixture was incubated at 95°C for 2 min. Further, in step (7), the supernatant was ultrafiltered using a 3 kDa ultrafiltration tube.

[0010] This invention provides the application of *Lactobacillus plantarum*-derived inorganic polyphosphate in promoting cell proliferation, wherein the *Lactobacillus plantarum*-derived inorganic polyphosphate is prepared using the method described above. This invention also provides the application of the above-mentioned *Lactobacillus plantarum*-derived inorganic polyphosphate in the preparation of products that alleviate intestinal inflammation. Furthermore, this invention provides the application of the above-mentioned *Lactobacillus plantarum*-derived inorganic polyphosphate in the preparation of products that repair damage to the intestinal epithelial barrier. These products are not limited to food, pharmaceuticals, or health supplements.

[0011] This invention establishes an intestinal epithelial cell inflammation model, using PolyP (250 Pi) extracted from Lactobacillus as the research object. Metabolomics, proteomics, and various molecular biology methods are employed to elucidate the mechanism by which PolyP alleviates intestinal inflammation. Furthermore, using an established macrophage immune stress model, the effects of PolyP on macrophage morphology and inflammatory responses are investigated, thereby elucidating the immunomodulatory role of PolyP. This invention systematically elucidates the anti-inflammatory and immunomodulatory effects of PolyP and its mechanism, providing a theoretical basis for the application and development of PolyP as an immunomodulator and intestinal anti-inflammatory drug. Attached Figure Description

[0012] Figure 1 The effects of PolyP with different chain lengths on IL-1β-induced Caco-2 cell proliferation. Figure 2 For PolyP Lp Effects on IL-1β-induced intracellular cytokine secretion in Caco-2 cells. Figure 3 To investigate the effects of different chain lengths of PolyP on the expression levels of inflammatory factor genes in intestinal epithelial cells. Figure 4 To investigate the effects of PolyP chains of different lengths on the expression levels of tight junction protein genes in intestinal epithelial cells. Figure 5 For PolyP Lp Effects on the expression levels of cytokine genes induced by IL-1β in Caco-2 cells. Figure 6 For PolyP Lp Effects on the expression levels of tight junction protein genes induced by IL-1β in Caco-2 cells. Figure 7 The effect of PolyP on the expression of apoptosis-related genes induced by IL-1β in Caco-2 cells. Figure 8 The effect of PolyP on the expression of apoptosis-related proteins induced by IL-1β in Caco-2 cells. Detailed Implementation

[0013] The principles and features of the present invention are described below with reference to the accompanying drawings. The examples given are only for explaining the present invention and are not intended to limit the scope of the present invention.

[0014] This invention establishes a method for extracting and purifying *Lactobacillus plantarum* PolyP, and uses an intestinal epithelial cell inflammation model to explore the anti-inflammatory and immunomodulatory effects of *Lactobacillus plantarum* PolyP, elucidating the anti-inflammatory and immunomodulatory regulatory mechanism of *Lactobacillus plantarum* PolyP, providing a new application for PolyP in maintaining and improving intestinal health.

[0015] (1) PolyP was isolated, extracted and purified from Lactobacillus plantarum using glassmilk. Lp The extracted PolyP molecules were identified by Urea-PAGE as having a size of approximately 250 Pi residues.

[0016] (2) An intestinal epithelial barrier injury model was established using IL-1β-induced Caco-2 cells, and the effects of PolyP with different chain lengths on the intestinal epithelial barrier were investigated. The results showed that PolyP with different chain lengths (20 μg / mL) had no toxic effect on Caco-2 cells and did not induce an inflammatory response; in the barrier injury model, PolyP with different chain lengths significantly promoted cell proliferation, and PolyP… Lp The promoting effect was significantly higher than that of PolyP. 45 and PolyP 120 Meanwhile, PolyP of different chain lengths can all reduce NF-κB and IL-1β The expression and promotion Claudin-1 and Occuldin PolyP expresses [the substance], thereby repairing IL-1β-induced intestinal epithelial cell barrier damage; further research found that PolyP […]. Lp It alleviates the inflammatory response in intestinal epithelial cells by regulating the production and expression of pro-inflammatory factors (TNF-α, IL-1β) and anti-inflammatory factors (IL-4, IL-10).

[0017] (3) Non-targeted metabolomics analysis was used to analyze the effect of PolyP on intestinal epithelial cell inflammation at the metabolic level. The results showed that PolyP... LpPolyP participates in signal transduction and inflammatory responses mediated by differential metabolites such as lipids, organic acids, amino acids, carbohydrates, hormones, and nucleotides, as well as glycerophospholipid metabolism, autophagy, amino acid metabolism, TCA cycle, and mTOR signaling pathways. HMDB database analysis revealed that PolyP... Lp Significantly regulates differentially expressed metabolites associated with irritable bowel syndrome, Crohn's disease, ulcerative colitis, and other diseases. These results indicate that PolyP has a role in alleviating intestinal inflammation, and that long-chain PolyP... Lp The effect is most significant.

[0018] (4) Exploring PolyP using proteomics methods Lp The molecular mechanism by which PolyP alleviates intestinal epithelial cell inflammation was analyzed, revealing its role. Lp PolyP primarily alleviates intestinal inflammation through the apoptosis pathway; the Hoechst / PI double staining assay revealed that PolyP... Lp PolyP significantly reduced IL-1β-induced apoptosis and necrosis; further analysis of the expression levels of key genes and proteins in the apoptosis pathway revealed that PolyP... Lp Reduce the receptors acting on IL-1β IL-1R1 It inhibits the expression of [certain substances] and suppresses the activation of the TNF / TNFRSF10D cell death pathway, thereby hindering the transduction of apoptotic signals and by reducing [certain substances]. FADD and CASP10 PolyP's expression reduces the apoptosis cascade, thereby slowing down the activation of the downstream NF-κB inflammatory pathway, ultimately alleviating intestinal epithelial cell inflammation and reducing inflammatory damage to the intestinal barrier. These results indicate that PolyP... Lp This invention alleviates intestinal inflammation by inhibiting the activation of the TNF / NF-κB pathway. It will provide new targets and pathways for the treatment of intestinal-related diseases.

[0019] This invention does not have any special limitations on Lactobacillus plantarum. PolyP can be obtained from Lactobacillus plantarum using the method of this invention. In the examples, the Lactobacillus plantarum used can be obtained commercially.

[0020] The solution preparation method involved in this invention is as follows.

[0021] GITC lysis buffer (4M GITC lysis buffer), for a 200 mL system: 94.4 g GITC, 1.21 g Tris-base, dissolved in 100 mL ultrapure water, pH adjusted to 7.0 with dilute hydrochloric acid, and then brought to a final volume of 200 mL with ultrapure water. This buffer can be stored at room temperature for several months. New Wash buffer (NW buffer), for a 500 mL system: 0.3 g Tris-base, 1.4625 g NaCl, 0.9306 g EDTA-2Na2H2O, dissolved in 150 mL ultrapure water, pH adjusted to 7.5 with dilute hydrochloric acid, 250 mL anhydrous ethanol added, cooled, and then brought to a final volume of 500 mL with ultrapure water. Nucleic acid buffer, for a 200 mL system: 1.21 g Tris-base, 0.19 g MgCl2, dissolved in 100 mL ultrapure water, pH adjusted to 7.4 with dilute hydrochloric acid, and then brought to a final volume of 200 mL with ultrapure water. PolyP eluent, taking a 200 mL system as an example: Tris-base 1.21 g, dissolved in 100 mL of ultrapure water, pH adjusted to 8.0 with dilute hydrochloric acid, and ultrapure water brought to a final volume of 200 mL.

[0022] Glass milk: Weigh 0.1 g of silica into 1 mL of 0.5% KH2PO4 (pH=7.0), vortex to mix, incubate at 4℃ in the dark for 2 h, centrifuge at 3000 rpm for 5 min, discard the supernatant, wash twice with 0.5% KH2PO4, resuspend the precipitate in 1 mL of 0.5% KH2PO4, and store at -20℃ in the dark. HEPES buffer is 20 mM HEPES-NaOH buffer (pH 7.5). DAPI solution: Weigh 0.0035 g of DAPI, dissolve in ultrapure water, and bring the volume to 100 mL. 80% methanol-water internal standard extraction reagent: Dissolve the internal standard in 80% methanol-water solution. The internal standard is L-2-chlorophenylalanine, and the final concentration of the internal standard is 1 μg / mL. Complete culture medium: Mix DMEM F / 12 high glucose medium with fetal bovine serum at a volume ratio of 9:1.

[0023] Unless otherwise specified, all methods and conditions used in the embodiments are conventional or performed according to the techniques or conditions described in the literature in this field, or according to the product instructions. Reagents and instruments used without specified manufacturers are all conventional products that can be purchased through legitimate channels. Percentages in this invention refer to the mass-volume percentage of a solid solute dissolved in a liquid; for example, a mass-volume percentage of 1% means that 100 mL of solution contains 1 g of solute. Percentages refer to the volume percentage of a liquid solute dissolved in a liquid. Unless otherwise specified, all solutions in this invention are prepared using water as the solvent. The primers involved in this invention were synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0024] Example 1: Isolation and purification of PolyP and chain length analysis

[0025] In this embodiment, PolyP was extracted from Lactobacillus plantarum and its molecular size was identified by Urea-PAGE electrophoresis, and then the structure and function of exogenous PolyP were analyzed.

[0026] Activation and culture of Lactobacillus plantarum: Glyceryl-preserved Lactobacillus plantarum was inoculated into sterile MRS broth and incubated at 37°C for 21 h. After two generations of activation, it was used for subsequent experiments.

[0027] (1) Extraction of Lactobacillus plantarum PolyP PolyP was extracted and separated using glass milk. The specific procedure was as follows: ① Take 20 mL of overnight cultured Lactobacillus plantarum culture (OD200). 600 = 1.5) Centrifuge in a 50 mL sterile centrifuge tube at 12000 rpm / min for 10 min, and discard the supernatant; ② Add 15 mL of GITC lysis buffer preheated to 95℃, vortex to mix, incubate at 95℃ for 5 min, and sonicate for 15 min; ③ Add 1 mL of 10% SDS, 15 mL of 95% ethanol, and 200 μL of glass milk (for PolyP binding) to the tube from step ②, vortex to mix, centrifuge at 12000 rpm / min for 5 min, and discard the supernatant; ④ Add 10 mL of NW buffer (ice bath), vortex to mix, sonicate for 5 min, centrifuge at 12000 rpm / min for 5 min, and discard the supernatant, repeat twice; ⑤ Add 1 mL of nucleic acid-free buffer, 60 μL of DNase I, and 20 μL of RNase A to the precipitate, incubate at 37℃ for 1 h, centrifuge at 12000 rpm / min for 10 min, and discard the supernatant; ⑥ Dilute the precipitate with 5 mL of GITC lysis buffer and 5 mL of... Wash once with 95% ethanol, then twice with NW buffer. Centrifuge at 5000 rpm / min for 5 min and discard the supernatant. Add 1 mL of 50 mM Tris-HCl preheated to 90℃ to the precipitate and incubate at 95℃ for 2 min to elute PolyP. Centrifuge at 13000 rpm / min for 5 min, collect the supernatant, and store at -80℃ or concentrate. Transfer the above eluent to a 3 kDa ultrafiltration tube, centrifuge to collect the concentrate, and remove low molecular weight components including ATP and short-chain PolyP. Place the above concentrate in a vacuum protein concentrator for concentration and drying, and store the dried sample at -80℃ for later use.

[0028] Quantitative detection of intracellular PolyP in Lactobacillus plantarum: Intracellular PolyP in Lactobacillus plantarum was directly quantified by DAPI staining, referring to the method of Wang Xin et al. (Wang Xin, Zhang Wen, Chen Xu, Zhu Wen, Yang Liuyan, 2015. Direct quantitative determination of intracellular polyphosphate in bacteria by DAPI staining [J]. Environmental Monitoring Management and Technology, 27(06): 40-43.). The specific operation is as follows. ① Pretreatment of bacterial culture: Take Lactobacillus plantarum cultured for 21 h and measure OD. 600 Regulate OD 600 = 1.0. Take 1.5 mL of bacterial suspension, centrifuge at 12000 rpm / min for 10 min, wash the precipitate once with HEPES buffer, and resuspend the precipitate in 1 mL of HEPES buffer for determining PolyP content; ② Prepare PolyP standard curves: Prepare the PolyP standard series according to Table 1, where ru refers to the volume ratio of standards in 900 μL (HEPES buffer + PolyP standard) mixture, and 1 ru represents 3 μmol of orthophosphate. After vortexing and mixing, incubate at room temperature in the dark for 10 min. After incubation, use the fluorescence module of the microplate reader, set the excitation wavelength to 415 nm and the emission wavelength to 550 nm, and measure the fluorescence value.

[0029] Table 1 Preparation of PolyP45 Standard

[0030] Based on the above detection method, the content of monomer Pi in the solution prepared from PolyP extracted by Lactobacillus plantarum (1 mg / mL, dissolved in 20 mM HEPES buffer) was calculated to be 1.30 μmol / L Pi.

[0031] (2) Determination of PolyP chain length from Lactobacillus plantarum The distribution of chain lengths of extracted *Lactobacillus plantarum* PolyP was analyzed using Urea-PAGE. This example utilizes reagent-grade PolyP... 45 and PolyP 120 As a control, the polymer chain length of PolyP derived from *Lactobacillus plantarum* was estimated using 8% Urea-PAGE. The results showed that three different chain lengths of PolyP migrated and separated in the PAGE gel, producing different migration rates. Compared to the control, the migration distance of PolyP extracted from the strain was shorter than that of PolyP. 120 Therefore, estimate PolyP Lp The average chain length is approximately 250 Pi residues. Based on the classification of PolyP chain length (short chain 3-10 Pi, medium chain 10-100 Pi, long chain >100 Pi), the PolyP extracted in this invention belongs to the long chain, suggesting that it has immunomodulatory and anti-inflammatory functions.

[0032] Example 2: Study on the Immunomodulatory Effect of PolyP on the Intestinal Epithelial Cell Barrier

[0033] This embodiment uses IL-1β to stimulate Caco-2 cells to establish an intestinal epithelial cell barrier injury model, and studies the effects of PolyP on intestinal epithelial cells and the immunomodulatory role of intestinal epithelial cell barrier function.

[0034] In this embodiment, all experimental results were statistically analyzed using one-way ANOVA in SPSS 24.0 software for one-way ANOVA and Duncan's multiple comparison test for analysis of significant differences between groups (*). P < 0.05, ** P < 0.01, *** P < 0.001), all experiments were performed in at least three biological replicates. Data are expressed as mean ± SD. Statistical analysis graphs were created using GraphPad Prism 8 software. Caco-2 cells were cultured in DMEM F / 12 high-glucose medium containing 10% fetal bovine serum at 37°C in a 5% CO2 incubator, with medium changes every other day. Once the cells reached a monolayer and were in the logarithmic growth phase, they were passaged and subsequent experiments were performed. PolyP LP The cells were prepared using the method described in Example 1. Detection of cytokine secretion levels: Cytokine secretion levels were detected using an ELISA kit. After cell treatment, the culture supernatant was collected and centrifuged for analysis. The protein content of the cell pellet was determined using the BCA method to calculate the cytokine content. This test was performed in triplicate.

[0035] 2.1 Effects of PolyP on Caco-2 cell proliferation The effects of different concentrations and chain lengths of PolyP on the proliferation of intestinal epithelial cells were analyzed using the CCK-8 assay. Cultured Caco-2 cells were inoculated at 1.0 × 10⁶ cells / cells. 5 Cells were seeded at a density of 100 μL / well, with a final cell density of 1.0 × 10⁶ cells / mL into 96-well plates. 4 The plate was set up with 6 replicates, and a blank control group was set up with only serum-free cell culture medium. The culture plate was placed in a cell culture incubator (37°C, 5% CO2) and cultured overnight. When the cells reached 50% confluence, 100 μL of different chain lengths (PolyP) was added to the 96-well plate. 45 PolyP 120 PolyP LpPolyP serum-free cell culture medium with different concentrations (final concentrations of 5, 10, 20, 40, 80, and 160 μg / mL) was used. No PolyP was added to the control and blank groups. After incubation for 24 h, cck8 solution was added to each well, and the cells were incubated in an incubator for 1 h. The absorbance at 450 nm was measured.

[0036] To determine the effects of PolyP and its chain length on Caco-2 cell proliferation, Caco-2 cells were treated with PolyP of three different chain lengths and at different concentration gradients for 24 h, and cell viability was assessed using the CCK8 assay. The results showed that chain length and concentration did not significantly inhibit cell proliferation overall, indicating that PolyP of different chain lengths had no cytotoxic effects on cells. Regarding PolyP... Lp When the concentration was 20 μg / mL, the cell viability was significantly higher than that of other concentration treatment groups. Therefore, the final treatment concentration of 20 μg / mL was selected for subsequent experiments.

[0037] 2.2 Establishing an IL-1β-induced inflammation model in Caco-2 cells (1) Determine the IL-1β induction time and concentration: Incubate the cultured Caco-2 cells at 2.0 × 10⁻⁶ mg / L. 5 Cells were seeded at a density of 0.5 mL / well in 24-well plates, with a final cell density of 1.0 × 10⁶ cells / mL. 5 Cells / well were cultured in a cell culture incubator (37℃, 5% CO2) overnight. Once the cells reached 50% confluence, different concentration gradients (0, 10, 25, 50 μg / mL) and incubation times (0.5, 3, 6, 12, 24 h) of IL-1β induction were established, with four replicates. After incubation for the corresponding times, cell supernatants were collected, and the levels of TNF-α and IL-6 were measured. The results showed that a concentration of 10 ng / mL of IL-1β induced a stable inflammatory state in Caco-2 cells for 6 h, characterized by significantly elevated levels of the inflammatory factors TNF-α and IL-6. P <0.05), while IL-1β stimulation at concentrations of 25 ng / mL and 50 ng / mL did not produce a more severe inflammatory response, and the inflammatory response gradually disappeared with prolonged stimulation. Therefore, in subsequent experiments, this invention used 10 ng / mL IL-1β treatment on Caco-2 cells for 6 h as a condition for establishing an inflammatory injury model in the experiment on the effect of PolyP on intestinal inflammation.

[0038] (2) Establishment of an IL-1β-induced intestinal epithelial cell inflammation model: The cultured cells were seeded into the corresponding cell culture plates. After sufficient adhesion, the intestinal epithelial cell inflammation model was established according to the following groups for subsequent experiments. Control group: Cells were cultured normally without any treatment; IL-1β group: Cells were treated with 10 ng / mL IL-1β for 6 h; PolyP group: Cells were treated with 20 μg / mL PolyP for 24 h; IL-1β + PolyP group: Cells were first stimulated with 10 ng / mL IL-1β for 6 h, and then incubated with PolyP for 24 h.

[0039] 2.3 Detection of cell viability and cytokine secretion levels in each group like Figure 1 As shown, PolyP of different chain lengths significantly promoted the proliferation of IL-1β-induced Caco-2 cells. Compared with the control group, cell viability was significantly reduced after IL-1β stimulation. P < 0.05), as the PolyP chain length increases (PolyP 45 PolyP 120 PolyP Lp The effect of promoting cell proliferation gradually increased significantly. P < 0.05), PolyP derived from Lactobacillus plantarum Lp The promoting effect of PolyP is most significant, and for normal Caco-2 cells, PolyP... Lp It also has the effect of promoting proliferation. P < 0.05).

[0040] Depend on Figure 2 It can be seen that IL-1β stimulation significantly promotes the secretion of pro-inflammatory factors IL-1β and TNF-α by Caco-2 cells. P <0.05), PolyP Lp After acting on cells, the secretion of IL-1β and TNF-α was significantly reduced. P < 0.05), thereby inhibiting the development of inflammation. In addition, IL-1β stimulation affects the secretion of the cellular anti-inflammatory factor IL-4, while PolyP... Lp After action, it significantly promoted the production of IL-4. P < 0.05), thereby alleviating the cellular inflammatory response.

[0041] 2.4 Detection of target gene expression levels: Real-time quantitative PCR (RT-qPCR) was used to measure the expression level of the target gene in cells. IL-1β, TNF-α, IL-4, IL-10, Occuldin, Claudin-1, GADPHThe expression levels of the above genes are as follows: NM_000576.3, NM_000594.4, NM_000589.4, NM_000572.3, XM_054351382.1, NM_021101.5, and NM_001289746.2.

[0042] (1) Effects of PolyP with different chain lengths on intestinal barrier function Depend on Figure 3 It can be seen that, compared with the control group, different chain lengths of PolyP treated Caco-2 cells have a greater effect on cytotoxic inflammatory factors. NF-κB and IL-1β The expression of IL-1β was not significantly affected, but IL-1β stimulation significantly promoted the expression of inflammatory factors in cells. NF-κB and IL-1β The expression ( P < 0.05), while treatment with PolyP of different chain lengths significantly reduced the expression of inflammatory factors ( P < 0.05). By Figure 4 It can be seen that, compared with the control group, PolyP 45 and PolyP 120 For cell tight junction proteins Claudin-1 , Occuldin The expression of PolyP was not significantly affected, but Lp Significantly promoted Occuldin , ZO-1 The expression ( P < 0.05), IL-1β stimulation reduced the expression of tight junction proteins, while treatment with PolyP of different chain lengths significantly promoted the expression of tight junction proteins. Claudin-1 , Occuldin The expression ( P < 0.05).

[0043] (2) Effects of PolyPLp on the expression levels of inflammatory cytokine genes induced by IL-1β in Caco-2 cells Depend on Figure 5 It can be seen that, compared with the control group, IL-1β stimulation increased the number of pro-inflammatory factors in Caco-2 cells. IL-1β and TNF-α Gene expression levels increased significantly ( P < 0.05), anti-inflammatory factors IL-4 The expression level of PolyP decreased, while PolyP Lp After treatment, the expression levels of cytokines changed significantly, manifested as follows: IL-1β and TNF-α Expression levels were significantly reduced ( P < 0.05), IL-4 The expression level of was significantly increased ( P< 0.05).

[0044] (3) Effect of PolyPLp on the expression level of tight junction protein gene in Caco-2 cells induced by IL-1β By measuring cell tight junction proteins Claudin-1 and Occuldin Gene expression analysis of PolyP on the intestinal epithelial barrier, by Figure 6 It was found that IL-1β stimulation disrupted the expression of tight junction proteins, while PolyP treatment significantly promoted their expression. Claudin-1 and Occuldin Gene expression ( P < 0.05), thus protecting the epithelial cell barrier function.

[0045] This invention found that the chain length and concentration of PolyP had no significant effect on the viability of intestinal epithelial cells, indicating that PolyP has no cytotoxic effect and its function in the intestinal epithelial barrier can be further studied. In an intestinal epithelial cell barrier damage model, PolyP resisted IL-1β-induced slowed cell growth or cell death by increasing cell viability. Preliminary assessment suggests that PolyP can alleviate inflammatory stimulation-induced intestinal epithelial cell barrier damage. This invention found that PolyP of different chain lengths did not induce inflammation in cells and did not damage the intestinal barrier. More importantly, under inflammatory stimulation, PolyP significantly repaired IL-1β-induced barrier damage while inhibiting inflammation. Furthermore, long-chain PolyP derived from *Lactobacillus plantarum* showed significant improvement. Lp The effect is most significant, indicating that PolyP Lp PolyP possesses the potential to protect the intestinal barrier. This invention utilizes PolyP of different chain lengths to analyze its inflammatory regulatory effects on intestinal epithelial cells under normal and inflammatory conditions. The results showed that PolyP can influence the release of cytokines and other inflammatory mediators, such as inhibiting the production of pro-inflammatory cytokines like IL-1β and TNF-α, while simultaneously increasing the concentration of anti-inflammatory cytokines in an inflammatory environment. This dual effect highlights the complexity of PolyP's role in the inflammatory environment and determines its crucial role in inflammation regulation. Moreover, PolyP may significantly influence the severity and duration of inflammation by affecting cytokine levels, making it a key player in the pathophysiology of inflammatory diseases. PolyP can act as a potent regulator of immune signaling pathways, altering the inflammatory environment to protect the host's immune function.

[0046] In summary, in the intestinal epithelial cell barrier injury model, PolyP of different chain lengths significantly promoted cell proliferation, and PolyP... Lp Its effect is significantly higher than that of PolyP 45 and PolyP 120PolyP can reduce the chain length of different chains. NF-κB and IL-1β The expression and promotion Claudin-1 and Occuldin The expression of PolyP thereby repairs IL-1β-induced intestinal epithelial barrier damage. Lp It alleviates IL-1β-induced intestinal inflammation by inhibiting the secretion and expression of pro-inflammatory factors TNF-α and IL-1β and promoting the secretion and expression of anti-inflammatory factors IL-4 and IL-10.

[0047] Example 3: Non-targeted metabolomics analysis of PolyP's anti-inflammatory and immune functions

[0048] This study utilizes non-targeted metabolomics to analyze the effects of PolyP of different chain lengths on the intestinal epithelial cell barrier and intestinal epithelial cell inflammation at the metabolic level, aiming to discover biomarkers and metabolic pathways of PolyP in inflammation regulation.

[0049] In this embodiment, the cell culture was the same as in Example 2. The cultured Caco-2 cells were cultured at 1.0 × 10⁻⁶ cells / mL. 5 Cells were seeded at a concentration of 10 ng / mL into 6 cm culture dishes, with 4 mL of cell suspension added to each dish. The dishes were placed in a cell culture incubator (37°C, 5% CO2) and cultured overnight. Once the cells reached 50% confluence, they were washed and added to fresh complete culture medium. The following groups were then treated, with three replicates per treatment: Control group: cells were cultured normally without any treatment; IL-1β group: cells were stimulated with 10 ng / mL IL-1β for 6 h; PolyP treatment groups with different chain lengths: PolyP was added to each group. 45 PolyP 120 PolyP Lp The final concentration was 20 μg / mL for all groups; IL-1β-stimulated cells were treated with PolyP of different chain lengths: after stimulating cells with 10 ng / mL IL-1β for 6 h, PolyP was added to each group. 45 PolyP 120 PolyP Lp The final concentration was 20 μg / mL.

[0050] After cell treatment, wash twice with pre-cooled PBS buffer, collect cells, centrifuge at 1000 rpm for 10 min at 4°C, discard supernatant, wash with PBS buffer, centrifuge at low speed, and repeat this operation twice. Add 500 μL of 80% methanol-water internal standard extraction reagent to the cell pellet to resuspend the cells. Place the centrifuge tube containing the cells in liquid nitrogen for 5 min, remove and thaw on ice, vortex to mix for 2 min, and repeat this process 3 times. Centrifuge the above sample at 12000 rpm for 10 min at 4°C, then transfer 300 μL of supernatant to a new centrifuge tube and incubate at -20°C for 30 min. Remove the centrifuge tube, centrifuge at 12000 rpm for 3 min at 4°C, and finally transfer 200 μL of supernatant to a sample vial. Detection was performed using a T3 column (Waters ACQUITY Premier HSS T3 Column 1.8 µm, 2.1 mm × 100 mm) and an AB TripleTOF 6600 mass spectrometry system. Raw data were preprocessed before bioinformatics analysis.

[0051] (1) Effects of PolyP of different chain lengths on Caco-2 cell metabolism: Metabolomics data of Caco-2 cells treated with PolyP of different chain lengths were analyzed using the OPLS-DA model. Score plots of each treatment group were obtained to show the differences between the groups. The results showed that the samples in each treatment group could cluster together well, indicating that the samples in the same group had high consistency in metabolic characteristics. At the same time, the samples in different treatment groups could be significantly separated in the model space, showing a trend of complete separation, indicating that there were significant differences in the metabolic levels of these four groups. This experiment combined univariate and multivariate statistical analysis methods to explore differential metabolites, according to P Differential metabolites were identified using a standard screening method with a value < 0.05 and a VIP > 1. Cluster analysis of the differential metabolites from the four treatment groups revealed that different chain lengths of PolyP significantly upregulated carbohydrates and their metabolites, terpenes, benzene and its derivatives, fatty acyls, alkaloids, organic acids and their derivatives, nucleotides and their metabolites, and amino acids and their metabolites in cells. Furthermore, medium- and long chain PolyP (PolyP...) 120 and PolyP Lp PolyP significantly upregulated steroids, sphingolipids, heterocyclic compounds, aldehydes, ketones, esters, lignins and coumarins, coenzymes and vitamins, hormones and hormone-related compounds, flavonoids, and lipids in cells. LpThe treatment group significantly downregulated glycerophospholipids, tryptophan, choline, pigments, and bile acids in cells. Overall, PolyP treatment significantly upregulated the levels of differentially expressed metabolites. KEEG pathway enrichment analysis of these significantly expressed metabolites revealed that they are mainly involved in retrograde endocannabinoid signaling, glycerophospholipid metabolism, Kaposi's sarcoma-associated herpesvirus infection, autophagy, glycosylphosphatidylinositol (GPI) anchor biosynthesis, pathogenic Escherichia coli infection, α-linolenic acid metabolism, choline metabolism in cancer, phenylalanine metabolism, adrenergic signaling in cardiomyocytes, fructose and mannose metabolism, circadian rhythm regulation, drug metabolism-other enzymes, estrogen signaling pathways, phagocytosis, folic acid biosynthesis, pancreatic secretion, gastric acid secretion, and long-term potentiation.

[0052] (2) Effects of PolyP of different chain lengths on the metabolism of IL-1β-stimulated Caco-2 cells: The metabolomics data of IL-1β-stimulated Caco-2 cells treated with PolyP of different chain lengths were analyzed using the OPLS-DA model. The scores of each treatment group were obtained, indicating that the samples in the same group had high consistency in metabolic characteristics, and that the four groups of samples had significant differences in metabolic levels. P Differential metabolites were selected based on the criteria of -value < 0.05, VIP > 1 for clustering and enrichment analysis. The results showed that only PolyP... Lp The metabolites of the treatment group changed significantly, PolyP Lp It significantly upregulated alkaloids, heterocyclic compounds, alcohol amines, aldehydes, ketones, esters, nucleotides and their metabolites, amino acids and their metabolites, flavonoids, bile acids, fatty acyls, hormones and hormone-related substances, benzene and its derivatives, phenolic acids, carbohydrates and their metabolites, glycerophospholipids, steroids, glycerides, sphingolipids, and terpenes in IL-1β-stimulated intestinal epithelial cells, while significantly downregulating only four types of metabolites: tryptamine, choline, pigments, and organic acids and their derivatives. KEEG pathway enrichment analysis of significantly different metabolites revealed that these metabolites are mainly involved in metabolic pathways such as glycerophospholipid metabolism, retrograde endocannabinoid signaling, autophagy, glycosylphosphatidylinositol (GPI) anchor biosynthesis, pathogenic Escherichia coli infection, Kaposi's sarcoma-associated herpesvirus infection, lipids and atherosclerosis, cholesterol metabolism, choline metabolism in cancer, synthesis, secretion and action of parathyroid hormone, glycolysis / glycogenogenesis, D-amino acid metabolism, primary bile acid biosynthesis, alanine, aspartate and glutamate metabolism, ABC transporters, serotonergic synapses, steroid biosynthesis, oxidative phosphorylation, and 2-oxocarboxylic acid metabolism.

[0053] (3) PolyP LpMultivariate statistical analysis of metabolites that alleviate intestinal inflammation: Analysis using the OPLS-DA model revealed that CON, PolyP Lp IL-1β, PolyP Lp The samples from the four IL-1β treatment groups clustered together, and the sample distribution between groups showed significant dispersion, indicating that there were significant differences in metabolic levels among the four groups. The statistical analysis will then proceed accordingly. P By combining the -value and FC value, a differential volcano plot was drawn, indicating that PolyP Lp A total of 2060 metabolites were detected in the +IL-1β vs CON comparison group, of which 128 metabolites were significantly upregulated and 31 metabolites were significantly downregulated; PolyP Lp A total of 2155 metabolites were detected in the +IL-1β vs IL-1β comparison group, of which 44 metabolites were significantly upregulated and 20 metabolites were significantly downregulated.

[0054] (4) Analysis of differentially expressed metabolites among different groups: Metabolites were sorted according to their log2FC values, and then a distribution map of the sorted metabolites was plotted, showing the top 10 significantly differentially expressed metabolites. The results showed that PolyP LpIn the vs CON comparison group, the top 10 differentially upregulated metabolites included PA (20:0 / 24:0) (glycerophospholipids), methyl 4,7-dehydro-2,3-dideoxy-7-phenylheptanoate, glycerides, N-phenylacetylglycine, nucleotides and their metabolites including (R)-NADH-Hydrate, Prfar, NADH, and Carnitine. C14:0 (fatty acyls), phorbol 12-tegyl ester 13-decanoate; while the top 10 differentially regulated metabolites were also mainly PA (16:0 / 22:1(13Z)), PC (14:0 / 15:0), PC (18:0 / 18:0), PE (18:0 / 20:5(5Z,8Z,11Z,14Z,17Z)), 1-(1Z-octadecenyl)-2-(4Z,7Z,10Z,13Z,16Z,19Z-docohexanoyl)-sn-glycerol-3-phosphocholine and other glycerophospholipids, as well as alcohols, amines, 6,8-trianedione, TG (14:0 / 22:2(13Z,16Z) / 14:1(9Z)) (glycerides), fluorescein, 2-methylguanosine (nucleotides and their metabolites). In the IL-1β vs CON comparison group, the top 10 differentially expressed metabolites that were significantly upregulated included glycerophospholipids such as PE (14:0 / 22:2(13Z,16Z)), 1-stearoyl-2-arachidonicyl PC-D8, PA (20:0 / 24:0), PE (22:1(13Z) / 14:0), and PE-NMe2 (18:2(9Z,12Z) / 18:2(9Z,12Z)), as well as methyl 4,7-dehydro-2,3-dideoxy-7-phenylheptanoate (alcohols and amines) and 3-(11,12-Dihydroxy-15,19-dotriacontadienyl)-5-methyl-2(5H)-furanone. 9CI (fatty acyl derivatives), N-oleoyl-D-erythrosphoylphosphocholine (sphingolipids), methionyl-tryptophan-glutamate (amino acids and their metabolites), and levothyroxine (organic acids and their derivatives); while the top 10 differentially regulated metabolites were also mainly PE-NMe2 (18:1(9Z) / 22:6(4Z,7Z,10Z,13Z,16Z,19Z)) and 1-(1Z-octadecenyl) PolyP contains glycerophospholipids such as 4Z, 7Z, 10Z, 13Z, 16Z, 19Z-docosahexaenoyl)-sn-glycero-3-phosphocholine, PC (18:0 / 18:0), PA (22:0 / i-15:0), and glycero-3-phosphocholine, as well as 1-stearoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine, folic acid (organic acids and their derivatives), and glycerol lipid metabolites. LpIn the +IL-1β vsIL-1β comparison group, the top 10 differentially expressed metabolites that were significantly upregulated included glycerophospholipids such as PC (22:0 / 15:0), PC (15:0 / 16:1(9Z)), PA (20:0 / 22:0), 1-stearoyl-2-linoleyl-sn-glycero-3-phosphoethanolamine, PA (22:0 / i-15:0), and PC (18:1(11Z) / 18:4(6Z,9Z,12Z,15Z)), as well as undecadiene phosphate (organic acids and their derivatives), pyromethoxyphylla chlorophyll b, N-ceramide-D-erythrosphocholine (sphingolipids), and trans- and octa-depentenylphospho-β-D-furanose (terpenes); while those that were significantly downregulated... The top 10 differentially metabolites also mainly include ent-16-Kauren-19-ol acetate (aldehydes, ketones, esters), NG-amino-L-arginine (amino acids and their metabolites), bacterial tetrazygosaccharide glucosamine (alcohols, amines), glycerides, 3-Phenyl-1-[(3R)-3-[(2R)-piperidin-2-yl]piperidin-1-yl]prop-2-en-1-one (heterocyclic compounds), 1,2-dispalmitoyl-sn-glycerol-3-phosphocholine (glycerophospholipids), pectin toxin-1 (heterocyclic compounds), 1-stearoylglycerol phosphate glycerol (glycerophospholipids), amino acids and their metabolites. PolyP Lp In the +IL-1β vs CON comparison group, the top 10 differentially upregulated metabolites included glycerophospholipids such as PE (14:0 / 22:2(13Z,16Z)), PA (20:0 / 24:0), PE (22:1(13Z) / 14:0), and PE-NMe2 (18:2(9Z,12Z) / 18:2(9Z,12Z)), as well as methyl 4,7-dehydro-2,3-dideoxy-7-phenylheptanoate (alcohols, amines), levothyroxine (organic acids and their derivatives), N-oleoyl-D-erythrosphoylphosphocholine (sphingolipids), cholesterol acetate (aldehydes, ketones, esters), stearoylcarnitine (fatty acyls), and bacteriophage-acetaminophen. Glucosamines (alcohols, amines); the top 10 differentially regulated metabolites included PE-NMe2 (18:1(9Z) / 22:6(4Z,7Z,10Z,13Z,16Z,19Z)), 1-stearoyl-2-docosahexaenoyl-sn-glycerol-3-phosphocholine, glycerol-3-phosphocholine, 1-(1Z-octadecenyl)-2-(4Z,7Z,10Z,13Z,16Z,19Z-docosahexaenoyl)-sn-glycerol-3-phosphocholine and other glycerophospholipids, as well as glycerides, erythromycin A (benzene and its derivatives), folic acid (organic acids and their derivatives), and 2-methylguanosine (nucleotides and their metabolites).

[0055] (5) Intestinal diseases associated with differentially expressed metabolites in the HMDB database: Based on the KEGG and HMDB databases, intestinal diseases associated with differentially expressed metabolites were identified, mainly including irritable bowel syndrome, colorectal cancer, Crohn's disease, and ulcerative colitis. The analysis results showed that after IL-1β stimulation, 13 differentially expressed metabolites in cells were associated with intestinal diseases. Among them, 9 differentially expressed metabolites were significantly upregulated, namely deoxycholic acid, pantothenic acid, 6-methyl-5-hepten-2-one, 1-palmitoyl-2-oleoyl-sn-glycero-3-(phospho-rac-(1-glycerol)) (glycerophospholipids), betaine, creatine, γ-glutamylcysteine ​​(amino acids and their metabolites), acetyl-L-carnitine (fatty acyls), and citrate. 4 differentially expressed metabolites were significantly downregulated, namely cytidine (nucleotides and their metabolites), LPE (16:0 / 0:0) (glycerophospholipids), carnitine C3:0, and xanthine. Lp After treatment of IL-1β-induced Caco-2 cells, the number of differentially expressed metabolites associated with intestinal disease decreased to six compared to the IL-1β group, and all of them were upregulated. These were L-pyroglutamate, N-acetyl-L-alanine, 2-phenylethanol, N,N-dihydroxy-L-tryptophan, glycocholic acid, and 3-oxotetradecanoic acid, most of which belong to the amino acid and its metabolites. Lp Compared to the CON group, the +IL-1β group showed 29 differentially expressed metabolites associated with intestinal disease, mostly belonging to the amino acid and its metabolites, and nucleotide and its metabolites. These results indicate that IL-1β induces an inflammatory response in intestinal epithelial cells by regulating lipid metabolism, while PolyP... Lp It can exert anti-inflammatory effects by regulating metabolites such as amino acids and nucleotides, thereby alleviating intestinal inflammation.

[0056] (6) PolyP Lp Cluster analysis of differentially expressed metabolites that alleviate intestinal inflammation: Cluster analysis of differentially expressed metabolites revealed that PolyP... Lp Treatment of IL-1β-stimulated Caco-2 cells significantly upregulated the levels of flavonoids, benzene and its derivatives, steroids, glycerides, hormones and hormone-related substances, coenzymes and vitamins, aldehydes, ketones, esters, terpenes, heterocyclic compounds, alkaloids, lignans and coumarins, carbohydrates and their metabolites, sphingolipids, nucleotides and their metabolites.

[0057] (7) PolyP Lp Enrichment analysis of differentially metabolites for alleviating intestinal inflammation: Enrichment pathways and their corresponding primary classifications were plotted using KEGG enrichment analysis of differentially metabolites. Results showed that IL-1β+ PolyP... LpThe differential metabolite enrichment between the vs. CON groups mainly involved six aspects, including cellular processes such as autophagy and necrosis; environmental information processing mainly included ABC transport, phosphatidylinositol signaling system, sphingolipid signaling pathway, AMPK signaling pathway, mTOR signaling pathway, HIF-1 signaling pathway, cAMP signaling pathway, NF-κB signaling pathway, MAPK signaling pathway, and ras signaling pathway; genetic information processing mainly included aminoacyl-tRNA biosynthesis and sulfur transfer systems; in human diseases, it mainly included pathways related to cancer and the metabolism of substances in cancer and infectious processes; in metabolism, it mainly included metabolic pathways, including lipid metabolism, nucleotide and amino acid metabolism, and metabolic pathways in the TCA cycle; and in biological systems, it mainly included retrograde endocannabinoid signaling, digestion, absorption, metabolism, and secretion, as well as multiple immune processes such as B / T cell receptor signaling pathway and immune cell differentiation. IL-1β+PolyP Lp The differential metabolite enrichment between the IL-1β group and the IL-1β group mainly involves six aspects: cellular processes, primarily autophagy and necrosis; environmental information processing, primarily sphingomyelin signaling pathways, phosphatidylinositol signaling systems, and ABC transporters; genetic information processing, primarily aminoacyl-tRNA biosynthesis; human diseases, primarily choline metabolism in cancer, pathogenic E. coli infection, Kaposi's sarcoma-associated herpesvirus infection, insulin resistance, lipids, and atherosclerosis; metabolism, primarily metabolic pathways, glycerophospholipid metabolism, glycosylphosphatidylinositol metabolism, α-linolenic acid metabolism, arachidonic acid metabolism, linoleic acid metabolism, glycerol ester metabolism, sphingolipid metabolism, cofactor biosynthesis, tryptophan metabolism, nicotinic acid and nicotinamide metabolism, phenylalanine metabolism, and inositol phosphate metabolism; and biological systems, primarily retrograde endocannabinoid signaling, cholesterol metabolism, fat digestion and absorption, adipocyte lipolysis regulation, vitamin digestion and absorption, and thermogenic processes, including digestion, absorption, metabolism, and secretion.

[0058] (8) PolyP Lp Pathway enrichment of differentially expressed metabolites alleviating intestinal inflammation: Differentially expressed metabolites from different comparison groups were enriched using the KEGG pathway, and then DA-Score lollipop plots were generated to analyze the regulation of metabolic pathways. Results showed that IL-1β stimulation of Caco-2 cells upregulated pathways such as autophagy, pathogenic E. coli infection, TCA cycle, interconversion of pentose and glucuronic acid, and choline metabolism in cancer, while downregulating metabolic pathways such as lipid and atherosclerosis, one-carbon metabolism, and cholesterol metabolism; PolyP Lp By regulating autophagy and E. coli infection pathways, and upregulating lipid metabolism, PolyP promotes cellular metabolism and alleviates the development of inflammation. LpTreatment of IL-1β-stimulated Caco-2 cells significantly upregulated, compared with the control group, pathways involved in cancer including central carbon metabolism, D-amino acid metabolism, glutathione metabolism, protein digestion and absorption, diabetic cardiomyopathy, 2-oxocarboxylic acid metabolism, arginine and proline metabolism, insulin resistance, TCA cycle, mTOR signaling pathway, Kaposi's sarcoma-associated herpesvirus infection, fever, autophagy, tryptophan metabolism, neurotrophic factor signaling pathway, and adipokines signaling pathway, while significantly downregulating one-carbon metabolism pathways.

[0059] This invention uses non-targeted metabolomics to identify intestinal diseases associated with differentially expressed metabolites related to PolyP's role in alleviating intestinal inflammation. Results showed significant changes in metabolites associated with irritable bowel syndrome, colorectal cancer, Crohn's disease, and ulcerative colitis in IL-1β-stimulated intestinal epithelial cells. These changes were observed after PolyP... Lp Treatment significantly reduced the amount of metabolites associated with intestinal diseases, indicating that PolyP... Lp It is a potential adjunctive medication for relieving or treating inflammatory bowel diseases.

[0060] PolyPs alleviate inflammatory responses by modulating pro-inflammatory cytokines and enhancing anti-inflammatory pathways, thus exhibiting anti-inflammatory and immune-boosting potential. This expands the therapeutic applications of PolyPs to various inflammatory diseases, including autoimmune diseases, metabolic syndromes, and chronic inflammations such as rheumatoid arthritis, inflammatory bowel disease, and type 2 diabetes. Combining PolyPs with existing therapies can improve treatment efficacy, reduce side effects, and enhance overall disease management. As research continues to elucidate the mechanisms of action of PolyP-related metabolites, developing targeted therapies using PolyPs may revolutionize the treatment of inflammation-related diseases.

[0061] The non-targeted metabolomics analysis results of this invention indicate that PolyPs of different chain lengths all affect the metabolism of lipids, amino acids, nucleotides, and other substances in intestinal epithelial cells, and mainly participate in the regulation of cellular function through key metabolic pathways such as glycerophospholipid metabolism, autophagy, glycosylphosphatidylinositol (GPI) anchor biosynthesis, pathogenic E. coli infection, α-linolenic acid metabolism, choline metabolism in cancer, and phenylalanine metabolism. However, under inflammatory stimulation, only long-chain PolyPs, i.e., PolyP... LpPolyPLP influences the metabolism of cellular compounds such as lipids, organic acids, amino acids, carbohydrates, hormones, and nucleotides, and regulates the metabolism of these substances through pathways including glycerophospholipid metabolism, autophagy, glycosylphosphatidylinositol (GPI)-anchored biosynthesis, lipid and atherosclerosis, cholesterol metabolism, parathyroid hormone synthesis, secretion and action, D-amino acid metabolism, alanine, aspartate and glutamate metabolism, and glycolysis. Therefore, PolyPLP has a significant regulatory effect on the metabolism of intestinal epithelial cells and may alleviate the inflammatory process of intestinal epithelial cells through metabolic pathways such as lipid metabolism, amino acid metabolism, and glycolysis.

[0062] In summary, non-targeted metabolomics results indicate that PolyPs of different chain lengths regulate the metabolism of lipids, amino acids, and nucleotides in healthy intestinal epithelial cells through metabolic pathways including glycerophospholipid metabolism, autophagy, glycosylphosphatidylinositol (GPI) anchor biosynthesis, pathogenic E. coli infection, α-linolenic acid metabolism, cancer choline metabolism, and phenylalanine metabolism. Long-chain PolyPs (PolyP...) Lp PolyP regulates the metabolism of lipids, organic acids, amino acids, carbohydrates, hormones, and nucleotides in IL-1β-induced inflammatory cells through pathways such as glycerophospholipid metabolism, autophagy, lipid and atherosclerosis, cholesterol metabolism, and amino acid metabolism. Analysis of differentially metabolites in the HMDB database revealed that PolyP... Lp It alleviates intestinal epithelial cell inflammation by regulating the amount of metabolites in cells associated with diseases such as irritable bowel syndrome, colorectal cancer, Crohn's disease, and ulcerative colitis.

[0063] Example 4 PolyP Lp Mechanisms for alleviating intestinal epithelial cell inflammation This experiment used IL-1β to induce Caco-2 cells to establish an intestinal epithelial cell inflammation model, and employed proteomics and molecular biology detection methods to explore the mechanism by which PolyP derived from Lactobacillus plantarum regulates intestinal inflammation.

[0064] In this embodiment, cell culture was performed as in Example 2. Experimental grouping was the same as in 2.2(2) of Example 2. In this embodiment, statistical analysis of all experimental results was performed using one-way ANOVA in SPSS 24.0 software for one-way ANOVA and Duncan's multiple comparison test for inter-group significance analysis (*). P < 0.05, ** P <0.01, *** P < 0.001), all trials were performed in at least three biological replicates. Data are expressed as mean ± SD. Statistical graphs were generated using GraphPad Prism 8 software.

[0065] 4.1 Proteomics Analysis of PolyP Protein extraction: After cell treatment in each experimental group, cell pellets were collected, and an appropriate amount of protein lysis buffer was added. After thorough lysis, the supernatant was collected for subsequent experiments. DIA mass spectrometry was used for detection, and DIA data analysis and bioinformatics analysis were performed.

[0066] Differential protein analysis revealed that PolyP... Lp A total of 8401 differentially expressed proteins were observed between the treatment group and the control group, of which 117 were upregulated and 428 were downregulated; PolyP Lp A total of 8,401 differentially expressed proteins were found between the treatment groups and the IL-1β treatment group, with 152 upregulated and 382 downregulated. Principal component analysis (PCA) showed that the samples from each treatment group clustered together, indicating good sample reproducibility. Based on the PCA scores, it can be seen that PolyP... Lp The treatment and control groups were not clearly distinguishable; the two groups clustered together, indicating that the differences between the two groups were small. (This is in contrast to PolyP.) Lp The treatment group and the IL-1β treatment group were clearly distinguishable and far apart, indicating that there was a large difference between the two groups of samples.

[0067] Differential protein annotation: GO functional annotation was performed on differentially expressed proteins from three aspects: involved biological processes (BP), molecular functions (MF), and cellular components (CC). The results showed that IL-1β+PolyP Lp Compared with IL-1β, the annotated biological processes mainly included cellular processes, biological regulation, metabolic processes, developmental processes, stress responses, localization, multicellular biological processes, reproductive processes, and immune system processes. Cellular components were mainly annotated as cytoskeleton components and protein complexes. Molecular functions included adhesion, catalytic activity, transport regulation activity, molecular functional regulation activity, transport activity, structural molecular activity, ATP-dependent activity, and molecular converter activity. Annotation of differentially expressed proteins using the KEGG pathway database revealed involvement in biological systems, environmental information, genetic information, human diseases, and cellular processes. Cellular processes primarily included transport and degradation, cell populations, cell growth, and cell death; human diseases mainly included cancer and infectious diseases; environmental information processes involved signal transduction; and biological systems included endotoxin systems, the immune system, and the nervous system.

[0068] GO enrichment analysis of differentially expressed proteins: In the GO functional enrichment analysis profile, IL-1β+PolyP Lp The differentially expressed proteins in the / IL-1β comparison group showed significant changes in biological processes such as the Wnt signaling pathway, cell surface signaling pathway, Snrna transcription, G protein-coupled receptors, adrenergic receptor signaling pathway, and typical Wnt signaling pathway; in terms of cellular components, proteins such as intrinsic proteins and the β-catenin-TCF complex showed significant changes; and in terms of molecular function, proteins such as molecular sensor activity, signal receptor activity, G protein-coupled receptor activity, DNA binding-related proteins, and transcriptional regulatory activity showed significant changes.

[0069] KEGG enrichment analysis of differentially expressed proteins: IL-1β+ PolyP Lp KEGG pathway enrichment analysis of differentially expressed proteins in the / IL-1β comparison group revealed significant enrichment in immune-related pathways such as the MAPK signaling pathway and cancer pathway.

[0070] Cluster analysis of apoptosis-related differentially expressed proteins: Cluster analysis of differentially expressed proteins effectively distinguished between different treatment groups, and the relative expression levels of some differentially expressed proteins showed low similarity. Therefore, the target protein set could be screened for cluster analysis. Cluster analysis of apoptosis-related differentially expressed proteins revealed that, compared with the IL-1β-induced group, PolyP... Lp In treated inflammatory cells, differentially expressed proteins associated with apoptosis, such as CASP10, CASP2, NFKB1, MAPK, BCL2L1, CASP9, and TNFRSF10D, were significantly downregulated. Cluster analysis of differentially expressed TNF-related proteins revealed that, compared with the IL-1β-induced group, PolyP... Lp In the treated inflammatory cells, differentially expressed proteins associated with TNF, such as CASP2, PI3K, NFKB1, MAPK, and TNFRSF, were significantly downregulated.

[0071] 4.2 Mechanism of IL-1β-induced apoptosis in Caco-2 cells (1) Apoptosis and necrosis detection: PI / Hoechst 33342 double staining was used to detect apoptosis and necrosis. Caco-2 cells were injected with 1×10⁻⁶ cells / cells. 5Cells were seeded at a density of [number] cells / well in 12-well plates and cultured overnight in a cell culture incubator until 50% confluence. Cells were then divided into groups for treatment, followed by staining observation. The specific procedure was as follows: cells were washed three times with PBS buffer, 1 mL of staining buffer was added, followed by 5 μL of Hoest33342 staining solution and 5 μL of PI staining solution, respectively, and incubated at 4°C for 30 min in the dark. After staining, cells were washed once with PBS, and then observed under a fluorescence microscope to detect apoptosis and necrosis. Hoest33342 can penetrate the cell membrane, and cells in the early stages of apoptosis emit strong blue fluorescence. PI cannot penetrate the cell membrane, but the cell membrane is already damaged in the late stages of apoptosis, therefore, after staining, it emits strong red fluorescence. Therefore, cells emitting weak red fluorescence + weak blue fluorescence are normal cells, those emitting weak red fluorescence + strong blue fluorescence are apoptotic cells, and those emitting strong red fluorescence + strong blue fluorescence are necrotic cells.

[0072] Experimental results showed that control group cells emitted weak blue fluorescence or no fluorescence after staining, with a slight red fluorescence, indicating a good cell condition. IL-1β-stimulated Caco-2 cells, after Hoechst 33342 / PI double staining, emitted strong blue fluorescence + strong red fluorescence, indicating apoptosis. In contrast, cells stained with PolyP... Lp After treatment, the number of cells emitting blue and red fluorescence was significantly reduced, indicating a reduction in apoptosis.

[0073] (2) Detection of expression levels of apoptosis-related genes Quantitative fluorescence detection IL-1R1 , FasL , TNFRSF10D , CASP10 , Bcl2 , FADD, GADPH The expression levels of the target genes are as follows. The Acc. Nos corresponding to the above target genes are NM_001288706.2, NM_000639.3, NM_003840.5, NM_001306083.2, NM_000657.3, NM_003824.4, and NM_001289746.2, respectively.

[0074] Based on proteomics and cell staining observations, PolyP... Lp PolyP has the effect of alleviating IL-1β-induced apoptosis, therefore RT-qPCR was used to analyze the effect of PolyP on the expression levels of apoptosis-related genes at the gene level. IL-1R1 is a high-affinity receptor for IL-1β, and PolyPLp regulates downstream inflammatory responses by inhibiting the expression of IL-1R1. Figure 7b). FADD is a death domain protein that recruits promoters such as CASP10 to activate the TNFR-1 receptor, thereby activating a cascade of downstream apoptotic proteins and ultimately leading to cell death. Figure 7 It is known that after Caco-2 cells are stimulated by IL-1β, the death receptor TNF receptor superfamily member 10D (TNFRSF10D), factor-related apoptosis (FAS) molecules, and their ligands are involved. TNF-α , FAS-L The expression of was significantly increased ( P < 0.05), promoted the transmission of apoptosis signals, and the apoptosis promoter CASP10 and CASP3 It is also activated, ultimately leading to cell death, manifested as FADD The expression level of was significantly increased ( P <0.05). However, when PolyP Lp PolyP acts on IL-1β-stimulated Caco-2 cells. Lp It blocks the transmission of apoptosis signals, inhibits the activation of the TNF (TNF-α / TNFRSF10D) and FAS (Fas / Fas-L) apoptosis pathways, and hinders downstream cascade reactions and the initiation of apoptosis, i.e., apoptosis-related proteins (… CASP10 , CASP3 , FADD The expression level was significantly reduced. P < 0.05%, thereby alleviating IL-1β-induced excessive cell death ( P < 0.05).

[0075] (3) Detect the expression level of apoptosis-related proteins To further clarify PolyP Lp The mechanism of action of PolyP in alleviating IL-1β-induced apoptosis was analyzed using Western blot. Lp The effect on the expression level of apoptosis-related proteins was investigated as follows: Caco-2 cells were inoculated at a concentration of 1×10⁻⁶ cells / cells. 6 Cells were seeded at a density of [number] cells / dish in 6 cm cell culture dishes and cultured overnight until 50% confluence. After cell treatment, the supernatant was discarded, and the cells were washed twice with pre-cooled PBS buffer. Then, Western blotting and IP cell lysis buffer containing protease inhibitors were added (volume as per product instructions). The mixture was thoroughly mixed by pipetting, and the lysis buffer was collected in 1.5 mL EP tubes and centrifuged at 14000 rpm for 5 min. The supernatant was collected, and protein content was determined using the BCA method. The protein concentration in the supernatant was adjusted and mixed with the loading buffer in the correct proportion. The mixture was then incubated in a boiling water bath at 100°C for 10 min to fully denature the protein, and then cooled for later use.

[0076] Electrophoresis was performed using 10% PAGE. Appropriate transfer conditions were selected based on the size of the target protein molecule. After transfer, the PVDF membrane containing the target protein was blocked with TBST containing 5% BSA, followed by overnight incubation with primary antibody at 4°C. After incubation, the PVDF membrane was washed five times with TBST. The membrane was then incubated with HRP-labeled secondary antibody (goat anti-rabbit IgG) at room temperature for 1 h, followed by five washes with TBST. The bands were placed in chemiluminescent solution, developed using a gel imaging system, and images were acquired. The grayscale values ​​of each protein band were calculated using ImageJ software. The experiment was performed in triplicate.

[0077] Depend on Figure 8 It was found that IL-1β stimulation significantly increased the expression of TNF and CASP10 proteins, while PolyP treatment of IL-1β-stimulated Caco-2 cells significantly inhibited the expression of TNF and CASP10 proteins. P < 0.05). In addition, IL-1β stimulation simultaneously activated the expression of NF-κB protein downstream of the apoptosis pathway ( P < 0.05), while PolyP Lp Significantly reduced the expression levels of total NF-κB-ρ65 protein and phosphorylated protein. P < 0.05), which alleviated the inflammatory response.

[0078] This invention links IL-1β-induced inflammatory responses with death receptor-mediated apoptosis pathways, speculating that the process may involve TNFR receptor activation by its ligand TNF-α, initiating apoptosis signal transduction, recruitment of CASP10 via the connector protein FADD, and CASP10 self-activation, which in turn activates a series of downstream apoptotic cascades, such as NF-κB, thereby inducing apoptosis. Excessive production of inflammatory factors and abnormal apoptosis can lead to abnormal inflammatory and immune responses, ultimately resulting in inflammatory diseases. Therefore, identifying regulators of inflammatory cytokines or death receptor membrane transport is helpful in understanding inflammation and immune disorders. Based on this, it is inferred that PolyP... Lp By regulating the expression of death receptors, excessive apoptosis of intestinal epithelial cells induced by IL-1β stimulation is reduced, ultimately alleviating intestinal inflammation. This provides clues to reveal the mechanisms of cell death occurring in the exogenous death pathway and the resulting inflammatory immune diseases.

[0079] This invention successfully isolated PolyP from Lactobacillus plantarum. Lp And utilize proteomics and various molecular biology methods to study PolyP Lp The mechanism by which PolyP alleviates intestinal inflammation was revealed. LpThe biological functions of extracellular PolyP were elucidated, revealing its therapeutic potential in immune-related diseases. The results of this study indicate that PolyP… Lp After acting on IL-1β-stimulated intestinal epithelial cells, the expression level of IL-1R1, the receptor for IL-1β, was significantly reduced, indicating that PolyP may target IL-1R1 to regulate the occurrence of inflammation in intestinal epithelial cells. Further research revealed that PolyP... Lp By inhibiting the activation of the receptor IL-1R1-dependent signaling pathway, namely the NF-κB pathway, and simultaneously reducing the production and expression of pro-inflammatory cytokines (IL-1β and TNF-α), PolyP ultimately alleviates the IL-1β-induced inflammatory response in intestinal epithelial cells. Lp By inhibiting the expression of IL-1R1, the transduction of inflammatory signals is suppressed, thereby alleviating downstream inflammatory responses.

[0080] In this experiment, proteomics analysis predicted that PolyP Lp PolyP can inhibit the activation of the TNF / TNFRSF10D death pathway and reduce the production of FADD and CASP10 proteins during the cascade reaction, thereby reducing IL-1β-induced intestinal cell apoptosis. Molecular and cellular level assays indicate that PolyP... Lp PolyP inhibited the activation of the TNF / TNFRSF10D and Fas / Fas-L apoptosis pathways, thereby suppressing the activation of downstream CASP10 and reducing FADD production. Ultimately, this alleviated the activation of the apoptosis-mediated NF-κB pathway, resulting in reduced intestinal cell apoptosis and decreased inflammatory response. These results indicate that PolyP... Lp By inhibiting IL-1β signaling, the inflammatory cascade and epithelial cell death are reduced. Specifically, this is achieved by regulating signal transduction through the TNF pathway to inhibit the initiation of apoptosis, while interfering with the expression of IL-1R1 to inhibit the activation of the inflammatory response. Through these two pathways, the activation of the NF-κB inflammatory pathway is jointly regulated, thereby alleviating intestinal epithelial cell inflammation and reducing damage to intestinal barrier function.

[0081] In summary, the Hoechst / PI double staining results of cells indicate that PolyP... Lp PolyP significantly reduced IL-1β-induced apoptosis and necrosis. Proteomics analysis results showed that PolyP... Lp PolyP can inhibit the activation of the TNF / TNFRSF10D and Fas / Fas-L cell death pathways, reduce the expression of FADD and CASP family proteins in the cascade reaction, thereby reducing IL-1β-induced intestinal cell apoptosis. Key signaling pathway analysis results indicate that PolyP... Lp By inhibiting the TNF / NF-κB pathway, it antagonizes IL-1β-induced apoptosis of intestinal epithelial cells, thereby alleviating the inflammatory response and reducing damage to intestinal barrier function.

Claims

1. A method for preparing Lactobacillus plantarum-derived inorganic polyphosphate, characterized in that, Includes the following steps: Inorganic polyphosphates were extracted from Lactobacillus plantarum using glass milk.

2. The method for preparing Lactobacillus plantarum-derived inorganic polyphosphate according to claim 1, characterized in that, Includes the following steps: (1) Treat the Lactobacillus plantarum cells with GITC lysis buffer, then in a water bath and sonicate. (2) Add SDS, 95% ethanol and glass milk, mix well, centrifuge, and discard the supernatant; (3) Add NW buffer solution in an ice bath to the precipitate, shake to mix, sonicate, centrifuge, and discard the supernatant; (4) Add nucleic acid buffer, DNase I and RNase A to the precipitate, incubate, centrifuge, and discard the supernatant; (5) Wash the precipitate, centrifuge, and discard the supernatant; (6) Add Tris-HCl to the precipitate, incubate, centrifuge, and collect the supernatant; (7) After ultrafiltration of the supernatant, centrifuge and collect the concentrate.

3. The method for preparing Lactobacillus plantarum-derived inorganic polyphosphate according to claim 1, characterized in that, In step (1), the mixture is placed in a 95°C water bath for 5 minutes and then subjected to ultrasonic disruption for 15 minutes.

4. The method for preparing Lactobacillus plantarum-derived inorganic polyphosphate according to claim 1 or 2, characterized in that, In step (2), the volume ratio of 10% SDS, 95% ethanol and glass milk is 1 mL: 15 mL: 200 μL.

5. The method for preparing Lactobacillus plantarum-derived inorganic polyphosphate according to claim 1 or 2, characterized in that, In step (4), the precipitate was incubated at 37°C for 1 h. In step (5), the precipitate was washed with GITC lysis buffer and 95% ethanol, and then washed with NW buffer.

6. The method for preparing Lactobacillus plantarum-derived inorganic polyphosphate according to claim 1 or 2, characterized in that, In step (6), add 50 mM Tris-HCl preheated to 90°C and incubate at 95°C for 2 min.

7. The method for preparing Lactobacillus plantarum-derived inorganic polyphosphate according to claim 1 or 2, characterized in that, In step (7), the supernatant is ultrafiltered using an ultrafiltration tube.

8. The application of *Lactobacillus plantarum*-derived inorganic polyphosphate in promoting cell proliferation, characterized in that... The Lactobacillus plantarum-derived inorganic polyphosphate is prepared by the method described in any one of claims 1-7.

9. The application of *Lactobacillus plantarum*-derived inorganic polyphosphates in the preparation of products for relieving intestinal inflammation, characterized in that... The Lactobacillus plantarum-derived inorganic polyphosphate is prepared by the method described in any one of claims 1-7.

10. The application of *Lactobacillus plantarum*-derived inorganic polyphosphates in the preparation of products for repairing intestinal epithelial barrier damage, characterized in that... The Lactobacillus plantarum-derived inorganic polyphosphate is prepared by the method described in any one of claims 1-7.

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