Lactobacillus fermentum for inhibiting cytotoxicity of methylglyoxal and application of lactobacillus fermentum

Abstract

The invention relates to lactobacillus fermentum for inhibiting cytotoxicity of methylglyoxal, and belongs to the technical field of biology. The invention provides a strain of lactobacillus fermentum, which can be used for remarkably inhibiting cytotoxicity induced by methylglyoxal (MGO) and improving the cell survival rate. The lactobacillus fermentum can remarkably inhibit Caco-2 cell apoptosis and reduce oxidative stress level and inflammatory response, so that MGO-induced cytotoxicity is relieved. Further research finds that the lactobacillus fermentum plays a role in relieving MGO-induced cytotoxicity by activating autophagy. Besides, the conjoint analysis of transcriptomics and metabonomics shows that the lactobacillus fermentum promotes the generation of a key metabolite pyruvic acid and activates an AMPK signal channel related to autophagy. Therefore, the lactobacillus fermentum activates the AMPK pathway by promoting the generation of a key metabolite pyruvic acid, further activates autophagy, and relieves MGO-induced cytotoxicity.

Description

TECHNICAL FIELD

[0001] The present application relates to a Lactobacillus fermentum for inhibiting methylglyoxal cytotoxicity, and belongs to the technical field of biotechnology. BACKGROUND

[0002] Methylglyoxal (MGO) is an intermediate product of the Maillard reaction, which is widely present in various processed foods. Its generation pathway is closely related to food storage and heat treatment, caramelization and oxidative cleavage of monosaccharides (such as monosaccharides contained in honey), lipid peroxidation of unsaturated fatty acids, and activation of glycolysis bypass in microbial metabolic activity (especially in fermented foods). Abnormal glucose metabolism, glucose-modified proteins, nucleotide degradation, and lipid peroxidation in the human body also promote the production of endogenous MGO. The serum MGO content of chronic disease patients (such as diabetes and chronic kidney disease) is significantly higher than that of healthy people.

[0003] Previous studies have shown that MGO can cause mitochondrial dysfunction, endoplasmic reticulum stress, inflammation cascade activation, and oxidative stress, ultimately triggering cytotoxicity. In addition, as a highly active dicarbonyl compound, MGO can covalently modify nucleophilic sites in nucleic acids and amino acids, promoting the formation of advanced glycation end products (AGEs). The accumulation of AGEs in the body can increase free radicals, promote the release of cellular molecules, and cause oxidative stress and inflammatory reactions. Therefore, it is crucial to develop effective methods to alleviate MGO-induced cytotoxicity.

[0004] Although existing dicarbonyl compound scavengers, such as metformin and aminoguanidine, have the ability to scavenge MGO in theory, these substances can cause various side effects, including gastrointestinal reactions, lactic acidosis, and other side effects, limiting their clinical application. In addition, although certain natural products such as flavonoids, phenolic acids, polysaccharides, and terpenoids are considered to have the potential to scavenge MGO, their extraction and purification processes are complex, and their yield is low, resulting in high cost, making it difficult to be applied on a large scale. Based on this, the development and application of natural MGO inhibitors without toxic side effects are imminent. SUMMARY

[0005] To solve the above problems, the present application provides a lactic acid bacteria for inhibiting methylglyoxal cytotoxicity.

[0006] The Lactobacillus fermentum HBUTFMNH4 (denoted as Lactobacillus fermentum 2-14) has been preserved in the China Center for Type Culture Collection on June 10, 2025, and the preservation number is CCTCC NO: M20251327.

[0007] In an embodiment, the Lactobacillus fermentum has one or more of the following functions: (1) can significantly inhibit methylglyoxal-induced apoptosis of Caco-2 cells, reduce ROS and MDA production, increase CAT, SOD and GSH-Px activity, and reduce inflammatory response; (2) promotes the production of key metabolite pyruvate, activates the AMPK signaling pathway, and then activates Caco-2 cell autophagy and promotes autophagosome formation.

[0008] A second object of the present application is to provide a microbial agent comprising at least one of the bacterial cells, culture solution, and metabolites of the Lactobacillus fermentum described above.

[0009] In an embodiment, the viable bacterial concentration of the microbial agent is 1x10 6 ~ 1x10 9 CFU / mL.

[0010] In an embodiment, the microbial agent is a resuspension of Lactobacillus fermentum, and the resuspension is selected from PBS, LB containing 20% glycerol, or MRS glycerol mixture.

[0011] A third object of the present application is to provide the use of the Lactobacillus fermentum described above, or the microbial agent described above, in the preparation of a product for alleviating methylglyoxal toxicity.

[0012] In an embodiment, the product includes a food or a drug.

[0013] In an embodiment, the dosage form of the drug includes a solid, a liquid, or a powder.

[0014] In an embodiment, the drug further includes a pharmaceutically acceptable carrier or excipient.

[0015] In an embodiment, the food includes a food containing the Lactobacillus fermentum, or a dairy product, a soy product, a meat product, or a fruit and vegetable product produced by a starter culture containing the Lactobacillus fermentum or a fermentation product of the Lactobacillus fermentum.

[0016] Advantages The Lactobacillus fermentum treatment can significantly inhibit MGO-induced cytotoxicity and improve cell survival rate. The Lactobacillus fermentum treatment can significantly inhibit Caco-2 cell apoptosis, reduce ROS and MDA production, improve CAT, SOD and GSH-Px activity, and reduce inflammatory response. Further research found that the Lactobacillus fermentum activated Caco-2 cell autophagy and promoted autophagic lysosome formation. In addition, joint analysis of metabolomics and transcriptomics found that Lactobacillus fermentum treatment promoted the production of key metabolite pyruvate and activated the AMPK signaling pathway related to autophagy. Therefore, Lactobacillus fermentum activates autophagy by promoting the production of key metabolite pyruvate to activate the AMPK pathway, thereby alleviating MGO-induced cytotoxicity.

[0017] Biological material preservation A strain of Lactobacillus fermentum, designated as Lactobacillus fermentum HBUTFMNH4, is classified as Lactobacillus fermentum, and has been preserved in the China Center for Type Culture Collection on June 10, 2025, with a preservation number of CCTCC NO: M20251327 and a preservation address of Wuhan University, Wuhan, China. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 Cell viability (A) and cytotoxicity (B) in MGO and lactic acid bacteria + MGO treatment groups compared with the control group. According to the unpaired t test, * * indicates significant difference at the level of p<0.01, and * * * indicates significant difference at the level of p<0.0001.

[0019] Figure 2 TUNEL (apoptosis marker) staining images (A) and red fluorescence intensity statistics (B) in different treatment groups, scale, 50 μm. (C-E) Compared with the control group, the amount of ROS (C), the content of pro-inflammatory factor IL-1β (D) and the content of anti-inflammatory factor IL-10 (E) in MGO and Lactobacillus fermentum 2-14 + MGO treatment groups. According to the unpaired t test, n.s., no significant difference; * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001 level of significant difference.

[0020] Figure 3(A) and green fluorescence intensity chart (B) of LC3-GFP staining of different treatment groups after transfection of LC3-GFP fusion protein into Caco-2 cells. (C) Effect of L. fermentum 2-14 treatment on the expression of autophagy marker genes. Effect of L. fermentum 2-14 treatment on the content of autophagy marker proteins LC-3 (D) and P62 (E) in cells. (F) GFP and RFP staining chart of different treatment groups after transfection of GFP and RFP double-labeled LC3 into cells. Green fluorescence and red fluorescence coexist, representing autophagosomes, and only red fluorescence represents autolysosomes. Scale bar, 10 μm. (G) Number of autophagosomes and autolysosomes in control group, positive control group and L. fermentum 2-14 treatment group. According to the unpaired t test, n.s., no significant difference; * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001 level of significant difference.

[0021] Figure 4 (A) and green fluorescence intensity chart (B) of LC3-GFP staining of different treatment groups after transfection of LC3-GFP fusion protein into Caco-2 cells. (C) Effect of L. fermentum 2-14 treatment on the expression of autophagy marker genes. Effect of L. fermentum 2-14 treatment on the content of autophagy marker proteins LC-3 (D) and P62 (E) in cells. (F) GFP and RFP staining chart of different treatment groups after transfection of GFP and RFP double-labeled LC3 into cells. Green fluorescence and red fluorescence coexist, representing autophagosomes, and only red fluorescence represents autolysosomes. Scale bar, 10 μm. (G) Number of autophagosomes and autolysosomes in control group, positive control group and L. fermentum 2-14 treatment group. According to the unpaired t test, n.s., no significant difference; * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001 level of significant difference.

[0022] Figure 5 (A) and green fluorescence intensity chart (B) of LC3-GFP staining of different treatment groups after transfection of LC3-GFP fusion protein into Caco-2 cells. (C) Effect of L. fermentum 2-14 treatment on the expression of autophagy marker genes. Effect of L. fermentum 2-14 treatment on the content of autophagy marker proteins LC-3 (D) and P62 (E) in cells. (F) GFP and RFP staining chart of different treatment groups after transfection of GFP and RFP double-labeled LC3 into cells. Green fluorescence and red fluorescence coexist, representing autophagosomes, and only red fluorescence represents autolysosomes. Scale bar, 10 μm. (G) Number of autophagosomes and autolysosomes in control group, positive control group and L. fermentum 2-14 treatment group. According to the unpaired t test, n.s., no significant difference; * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001 level of significant difference.

[0023] Figure 6Transcriptional levels (A-C) and protein content (D-F) of pro-inflammatory cytokines IL-1 b (A, D), IL-6 (B, E) and anti-inflammatory cytokine IL-10 (C, F) in cells after control, MGO treatment, L. fermentum 2-14 + MGO treatment and L. fermentum 2-14 + MGO + autophagy inhibitor Cq treatment. According to unpaired t-test, n.s., no significant difference; * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001 level of significant difference.

[0024] Figure 7 (A) Principal component analysis (PCA) of sample transcriptome information. (B) Heatmap of differential gene expression abundance between each sample. (C) Venn diagram showing unique and shared transcripts of different treatments. (D) Volcano plot showing differential expression of genes between control and MGO treatment groups. (E) Volcano plot showing differential expression of genes between MGO treatment group and L. fermentum 2-14 + MGO treatment group. (F) Number statistics of differential genes between different treatment groups. (G) Bubble plot showing KEGG significantly enriched pathways of differential genes in MGO treatment group and L. fermentum 2-14 + MGO treatment group. (H) TPM values of key genes in AMPK signaling pathway under different treatment conditions.

[0025] Figure 8 (A) Principal component analysis (PCA) of sample metabolome information. (B) Heatmap of differential metabolite abundance between each sample. (C) Venn diagram showing unique and shared metabolites of different treatments. (D) Volcano plot showing differential metabolites between MGO treatment group and L. fermentum 2-14 + MGO treatment group. (E) Bubble plot showing KEGG significantly enriched pathways of differential metabolites in MGO treatment group and L. fermentum 2-14 + MGO treatment group. Relative abundance of intracellular NAD + (F) and pyruvic acid (G). (H) Correlation analysis of differential expressed genes, differential metabolites and L. fermentum 2-14 alleviating MGO cytotoxicity. According to unpaired t-test, n.s., no significant difference; * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001 level of significant difference.

[0026] Figure 9 (A) Cell viability after adding MGO, L. fermentum 2-14, autophagy inhibitor Cq and AMPK inhibitor Dd, respectively, compared with control group. (B) Relative abundance of intracellular NAD +(C) Cell viability after addition of MGO, pyruvate, and autophagy inhibitor Cq, respectively, compared with the control group. (D) Cell viability after addition of MGO, pyruvate, and AMPK inhibitor Dd, respectively, compared with the control group. According to the unpaired t test, n.s., no significant difference; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 significant difference at the level. DETAILED DESCRIPTION

[0027] The scheme of the present application is further illustrated below by combining with the implementation examples, which are not a limitation of the present application.

[0028] Bacterial suspension preparation: activated lactic acid bacteria were inoculated into 10 mL MRS medium at an inoculation amount of 2% (V / V) and cultured at 37°C for 18 h. 1 mL of bacterial solution was centrifuged at 12,000 rpm and 4°C for 10 min, and the supernatant was discarded and the bacterial body was resuspended in PBS buffer (0.01 mol / L, pH 7.4), and centrifuged again under the above conditions. Repeat three times, finally resuspend the bacterial body in complete medium without double antibody, and dilute the bacterial suspension to 10 9 CFU / mL for subsequent experiments.

[0029] Example 1 Determination of Caco-2 cell survival rate and cytotoxicity Caco-2 cells were digested and inoculated into a 96-well plate using complete medium without double antibody (penicillin-streptomycin solution) and cultured for 24 h. The culture medium was aspirated, and 1 mL of bacterial suspension-free double-antibiotic-free medium was added to the Lactobacillus fermentum 2-14 treatment group, and an equal amount of bacterial suspension-free double-antibiotic-free medium was added to the control group and the MGO group, and co-cultured at 37°C and 5% CO2 for 6 h. Then remove the culture medium, add 2.4 mM MGO to the Lactobacillus fermentum 2-14 treatment group and the MGO group, and add an equal amount of double-antibiotic-free medium to the control group, and treat for 24 h. Add 100 μL of LDH detection working solution to each well of the 96-well plate, mix well, and incubate at room temperature (about 25°C) for 10-30 min in the dark. Incubate with aluminum foil wrapped and place on a horizontal shaker for slow shaking. Add 20 μL of stop solution to each well, mix well, and then measure the absorbance at 450 nm and record the data. The cytotoxicity (%) is calculated as follows: , where A1 is the absorbance of the experimental group, A2 is the background value of the experimental group, A3 is the absorbance of the control group, and A4 is the blank background value.

[0030] After the cell inoculation, lactic acid bacteria intervention and MGO exposure were completed by the above method, 100 μL of CCK-8 reagent diluted with MEM was added, and the light was incubated for 2-4 h. The absorbance was measured at 450 nm wavelength by multifunctional enzyme label instrument, and the data was recorded. At least three independent biological replicates were set for each treatment group to ensure the statistical efficiency of the experiment. The cell survival rate (%) was calculated as follows: . In the formula, A1 is the absorbance of the experimental group, A2 is the background value of the experimental group, A3 is the absorbance of the control group, and A4 is the blank background value.

[0031] Compared with the control group, the cell survival rate decreased significantly after MGO treatment, and the cell toxicity increased significantly. Compared with the MGO treatment group, L. fermentum 2-14 treatment significantly improved the cell survival rate and reduced the cell toxicity, and the effect was the most significant Figure 1 ).

[0032] Example 2: Effect of L. fermentum 2-14 treatment on MGO-induced apoptosis, oxidative stress and inflammatory response After the cell inoculation, lactic acid bacteria intervention and MGO exposure were completed by the method shown in Example 1 in the 12-well plate, all the culture medium was removed. According to the standard process of TUNEL detection: 4% paraformaldehyde fixation for 30 min, 0.1% Triton X-100 permeation treatment for 10 min, TdT enzyme reaction system (37°C, avoid light incubation for 1 h), DAPI re-stain cell nucleus for 5 min. Randomly select more than 3 fields of view using a fluorescence microscope for image acquisition, set three independent biological replicates for each experimental group and set an apoptosis induction positive control. Image J was used to analyze the image to measure the average fluorescence intensity.

[0033] According to the instructions of the reactive oxygen species detection kit (Beyotime Biotechnology Co., LTD, Shanghai, China), the content of ROS in Caco-2 cells was detected. According to the method shown in Example 1, the cell inoculation, lactic acid bacteria intervention and MGO exposure were completed, the probe was loaded according to the kit instructions, and the fluorescence intensity was detected at Ex=488 nm, Em=525 nm by multifunctional enzyme label instrument, and the data was recorded. The ROS fluorescence intensity (%) was calculated as follows: . In the formula, A1 is the absorbance of the experimental group, A2 is the absorbance of the control group.

[0034] The content of IL-1β and IL-10 in Caco-2 cells was determined according to the instructions of the ELISA detection kit (SenBeiJia Biological Technology Co., Ltd, Nanjing, China). After completing cell inoculation, lactobacillus intervention and MGO exposure according to the method shown in Example 1, pre-cooled sterile PBS was used for two washes. The lysis system containing protease inhibitors was prepared at a ratio of 100:1 (v / v), and 200 μL of ice-precooled lysis solution was added to each well. The culture plate was placed on ice for phased lysis: initial 5 min static lysis, horizontal shaker 50 rpm shaking for 3 min, secondary static lysis for 2 min, and during which sterile cell scraper was used to collect adherent cells along the fixed direction (clockwise one-way scraping). The lysis mixture was collected and transferred to a pre-cooled centrifuge tube, centrifuged at 4°C 12,000 rpm for 15 min, and the supernatant was carefully aspirated into a labeled frozen tube. Protein quantification analysis was performed using the BCA protein quantification kit (Beyotime Biotechnology Co., LTD, Shanghai, China), and the quantified protein samples were detected according to the standard process of the ELISA kit, and finally the data was read at 450 nm wavelength, and the target protein was accurately quantified by four-parameter logistic curve fitting.

[0035] Compared with the control group, after MGO treatment, the level of TUNEL staining, the amount of ROS production, and the content of pro-inflammatory factor IL-1β significantly increased, and the content of anti-inflammatory factor IL-10 significantly decreased. Lactobacillus fermentum 2-14 treatment significantly reduced the level of TUNEL staining, the amount of ROS production, and the content of pro-inflammatory factor IL-1β, and increased the content of anti-inflammatory factor IL-10 Figure 2 . This indicates that lactobacillus fermentum 2-14 may improve MGO-induced Caco-2 cell toxicity by regulating apoptosis, oxidative stress and inflammation.

[0036] Example 3 Effect of lactobacillus fermentum 2-14 on autophagy of Caco-2 cells Autophagy marker protein LC3 was labeled with GFP (green) and RFP (red) fluorescence by cell transfection technology. After 24 h of plating in 12-well plates, the cell density should be observed under a microscope at 50% to 80%. On the day of transfection, replace the old culture medium with fresh complete culture medium. Prepare the DNA: Lipo2000 complex. Pre-warm Lipo2000, plasmid DNA, and Opti-MEM medium to room temperature. Move the Opti-MEM medium into a sterile microcentrifuge tube, add 1.0 μg of plasmid DNA, and thoroughly mix with a pipette. Add 2.0 μL of Lipo2000 reagent to the diluted DNA mixture, and mix the complex thoroughly with a pipette gun. Incubate the complex at room temperature for 15 min. Add the complex to the cell culture medium. Distribute the complex to the cells by dropping the complex into different areas of the well, and gently shake the culture container back and forth to evenly distribute the Lipo2000: DNA complex, and incubate for 24 h. Then replace the growth culture medium, and wait for 18-24 h before proceeding to the subsequent experiment.

[0037] Remove the culture medium, and add 1 mL of double-antibody-free culture medium containing the bacterial suspension to the Lactobacillus fermentum 2-14 treatment group, and add an equal amount of double-antibody-free culture medium to the control group, and treat for 24 h. Remove the culture medium, and wash with pre-cooled PBS on a shaker for 5 min each time for three times. Fix with 4% paraformaldehyde solution at room temperature for 20 min, and wash with pre-cooled PBS on a shaker for 5 min each time for three times. Permeate with 0.2% Triton X-100 at room temperature for 20 min, and dilute the Triton X-100 with PBS. Wash with pre-cooled PBS on a shaker for 5 min each time for three times. Dilute DAPI with PBS, add to the well plate, and incubate for 5 min. Wash with pre-cooled PBS for 5 min each time for three times. Mount with an anti-fluorescence quencher, and observe under a fluorescence microscope or a laser confocal microscope.

[0038] The transcription level of autophagy-related genes in Caco-2 cells was detected by real-time quantitative fluorescence PCR (RT-qPCR). The Caco-2 cells were inoculated and intervened with lactic acid bacteria according to the steps in Example 1, the RNA of the cells in the six-well plate was extracted by the Trizol method, and after detecting the RNA concentration and purity and verifying the integrity, the RNA was reverse transcribed into cDNA for RT-qPCR experiment.

[0039] Cell seeding and lactic acid bacteria intervention were performed on Caco-2 cells according to the steps in Example 1. The contents of LC3 and P62 in Caco-2 cells were determined according to the ELISA detection kit (SenBeiJia Biological Technology Co., Ltd, Nanjing, China) instructions, and the total protein content and LC3 and P62 contents in Caco-2 cells were determined according to the steps in Example 2.

[0040] Compared with the control group, the autophagy fluorescence after L. fermentum 2-14 treatment was significantly increased, indicating that L. fermentum 2-14 enhanced autophagy. Figure 3 A-B). In addition, L. fermentum 2-14 treatment significantly increased the expression of autophagy marker factors LC3, Beclin-1 and ATG16L1, increased the intracellular LC3 protein content, and reduced the transcription level and protein content of autophagy negative regulator P62. Figure 3 C-E). In the autophagosome, the green and red fluorescence of the RFP-GFP-LC3 double-labeled fluorescent protein exists at the same time. When the autophagosome fuses with the lysosome to form the autolysosome, the pH decreases, causing the green fluorescence to be quenched, and only red fluorescence exists. Therefore, the formation of autolysosome can be judged by the ratio of red and yellow fluorescence. The results are shown in Figure 3 F-G). Compared with the control group, the red fluorescence and yellow fluorescence after L. fermentum 2-14 treatment were significantly increased. The above experimental results show that L. fermentum 2-14 effectively activates autophagy and promotes the formation of autolysosome.

[0041] Example 4 Effect of simultaneous inhibition of autophagy on cell survival rate, cytotoxicity and apoptosis during L. fermentum 2-14 treatment Cell seeding, lactic acid bacteria intervention and MGO exposure were completed according to the method in Example 1, and the cells in the 2-14+MGO+Cq group were treated with L. fermentum 2-14 and autophagy inhibitor Cq at the same time. Cell survival rate and cytotoxicity were determined according to the steps in Example 1. TUNEL fluorescence level was determined according to the steps in Example 2. The transcription level and protein content of apoptosis marker proteins Bad, Caspase-3 and Beclin-2 were determined according to the steps in Example 3.

[0042] The results show that the simultaneous inhibition of autophagy during L. fermentum 2-14 treatment reduces the cell survival rate Figure 4 A), enhances cytotoxicity Figure 4 B), and enhances TUNEL staining level Figure 4C-D). Compared with the control group, MGO treatment increased the transcriptional level and protein content of pro-apoptotic factor Bad and Caspase-3, and decreased the transcriptional level and protein content of anti-apoptotic factor Beclin-2. L. fermentum 2-14 treatment could inhibit the expression of Bad and Caspase-3 induced by MGO, while promoting the expression of Beclin-2. Compared with the L. fermentum 2-14 treatment group, the inhibition of autophagy at the same time as the L. fermentum 2-14 treatment promoted the expression of Bad and Caspase-3 and inhibited the expression of Beclin-2 (Figs. 2E-J). Figure 4 E-J). The above results show that L. fermentum 2-14 alleviates MGO-induced apoptosis through autophagy to reduce cell toxicity.

[0043] Example 5 Effect of Inhibition of Autophagy at the Same Time as L. fermentum 2-14 Treatment on Oxidative Stress Cell seeding, lactic acid bacteria intervention, autophagy inhibition and MGO exposure were completed according to the method in Example 4. The total protein content in the cells was determined according to the method in Example 2. After quantification, the protein samples were determined for CAT, SOD and GSH-Px activities in the samples according to the instructions of the CAT, SOD and GSH-Px kits (Beyotime Biotechnology Co., LTD, Shanghai, China). The amount of ROS produced in the cells was determined according to the method in Example 2. After cell seeding, lactic acid bacteria intervention, autophagy inhibition and MGO exposure according to the method in Example 4, the total protein content in the cells was determined according to the method in Example 2, and the content of MDA in the samples was determined according to the instructions of the MDA content detection kit (Beyotime Biotechnology Co., LTD, Shanghai, China).

[0044] Compared with the control group, the activities of antioxidant enzymes such as CAT, SOD and GSH-Px in the MGO group cells were significantly decreased, and the contents of ROS and MDA were significantly increased. L. fermentum 2-14 treatment significantly increased the activities of antioxidant enzymes and reduced the production of ROS and MDA. Compared with the L. fermentum 2-14 treatment group, the inhibition of autophagy at the same time as the L. fermentum 2-14 treatment decreased the activities of antioxidant enzymes and promoted the production of ROS and MDA (Figs. 3A-D), which indicates that L. fermentum 2-14 alleviates the damage to cells caused by oxidative stress by activating autophagy. Figure 5

[0045] Example 6 Effect of Inhibition of Autophagy at the Same Time as L. fermentum 2-14 Treatment on Inflammation Level ​Cell seeding, lactic acid bacteria intervention, autophagy inhibition and MGO exposure were performed according to the method in Example 4. The transcription level and protein content of inflammatory factors IL-1β, IL-6 and IL-10 were determined according to the steps in Example 3.

[0046] Compared with the control group, MGO treatment increased the transcription level and protein content of pro-inflammatory factors IL-1β and IL-6, and decreased the protein content of anti-inflammatory factor IL-10. Lactobacillus fermentum 2-14 treatment could inhibit the expression of IL-1β and IL-6 induced by MGO, while promoting the expression of IL-10. Compared with the lactobacillus fermentum 2-14 treatment group, inhibiting autophagy at the same time promoted the expression of IL-1β and IL-6 and inhibited the expression of IL-10 in the lactobacillus fermentum 2-14 treatment group. Figure 6 The above results show that lactobacillus fermentum 2-14 alleviates MGO-induced inflammation through autophagy to reduce cytotoxicity.

[0047] Example 7 Transcriptome explores the mechanism of lactobacillus fermentum 2-14 improving MGO-induced cytotoxicity After cell seeding, lactic acid bacteria intervention and MGO exposure according to the steps in Example 1, the culture solution was discarded, and two gradient washes were performed with pre-cooled PBS. Then 1 mL trypsin-EDTA digestion solution (0.25%, containing 0.02% EDTA) was added at 37°C for 2 min, and the cells were confirmed to be completely detached under a microscope, and then an equal volume of complete culture medium was immediately added to terminate the digestion reaction. The cell suspension was centrifuged at 1,000 rpm for 5 min (4°C) to completely remove the supernatant, and the cell pellet was resuspended with sterile PBS (pH 7.4), and the centrifugal washing step was repeated to eliminate residual culture medium components. The final cell pellet was added with 1 mL TRIzol lysis solution (1×10 6 cells / mL), transferred to a pre-cooled RNase-free cryotube, immediately frozen in liquid nitrogen, then transferred to a -80°C refrigerator, and stored in dry ice for shipment to Shanghai Meiji Biopharmaceutical Technology Co., Ltd. for eukaryotic transcriptome sequencing.

[0048] The expression of genes in Caco-2 cells was analyzed by principal component analysis, differential gene heat map analysis and Venn analysis. Principal component analysis showed that compared with the control group, MGO group and lactobacillus fermentum 2-14 treatment group, there were differences in the transcription profile Figure 7 A). The differential gene heat map showed that the expression patterns of the three groups were quite different, the similarity of gene expression patterns was small, and some differentially expressed genes had significant clustering phenomenon Figure 7B). As can be seen from the Veen diagram, the number of genes common to the three groups is 12,629, and the number of genes unique to the control group, MGO treatment group and L. fermentum 2-14 treatment group is 858, 363 and 331, respectively. The number of genes common to the control group and the MGO treatment group is 820, the number of genes common to the control group and the L. fermentum 2-14 treatment group is 178, and the number of genes common to the Caco-2 cells in the MGO group and the L. fermentum 2-14 treatment group is 242. Figure 7 C). Therefore, L. fermentum 2-14 treatment changed the transcriptional profile of Caco-2 cells. In addition, compared with the control group, the expression of 1,105 genes in Caco-2 cells in the MGO treatment group was significantly changed, of which 525 genes were significantly up-regulated and 580 genes were significantly down-regulated; compared with the MGO group, the expression of 1,985 genes in Caco-2 cells in the L. fermentum 2-14 treatment group was significantly changed, of which 1,054 genes were significantly up-regulated and 931 genes were significantly down-regulated. Figure 7 D-F).

[0049] In addition, enrichment analysis was performed on the differentially expressed genes based on the KEGG database. The AMPK signaling pathway of the differentially expressed genes in the MGO treatment group and the L. fermentum 2-14 treatment group showed significant enrichment. Figure 7 G), AMPK is an important energy sensor in cells and also participates in the pathophysiological process of metabolic diseases such as diabetes and obesity. These findings suggest that lactic acid bacteria may regulate autophagy function through the AMPK signaling pathway, thereby affecting the metabolic state of cells. By analyzing the changes in the TPM values of multiple genes in different treatment groups (control group, MGO treatment group, and L. fermentum 2-14 treatment group) shown in the figure, we observed that the expression of genes such as SIRT1 and PGC-1α, which are closely related to mitochondrial function and energy metabolism, increased in the L. fermentum 2-14 treatment group, indicating that L. fermentum 2-14 may alleviate MGO-induced energy stress by enhancing mitochondrial function. In addition, the expression of PPARP and PFKFB4 genes related to lipid metabolism decreased in the MGO treatment group, but increased in the L. fermentum 2-14 treatment group, which may mean that L. fermentum 2-14 helps to restore normal lipid metabolism and reduce MGO-induced metabolic disorders. At the same time, the expression of PCK1 and PCK2 genes involved in gluconeogenesis decreased in the MGO treatment group, but increased in the L. fermentum 2-14 treatment group, indicating that L. fermentum 2-14 may alleviate MGO-induced energy stress by promoting gluconeogenesis. Figure 7H). In summary, the gene expression changes in the figure suggest that L. fermentum 2-14 can alleviate MGO-induced cytotoxicity by regulating the AMPK signaling pathway. This regulation involves multiple aspects such as enhancing mitochondrial function, improving lipid and carbohydrate metabolism, thereby improving the adaptability and resistance of cells to energy stress.

[0050] Example 8 Metabolome explores the mechanism of L. fermentum 2-14 improving MGO-induced cytotoxicity Refer to the steps in Example 7 for cell seeding, lactic acid bacteria intervention, and MGO exposure of Caco-2 cells. After the end of culture, the culture medium was aspirated, and the cells were collected into enzyme-free EP tubes after trypsinization and centrifugation. After aspirating the supernatant, it was quickly frozen in liquid nitrogen and sent to Shanghai Meiji Biomedical Technology Co., Ltd. for non-targeted metabolite detection.

[0051] The difference in metabolites in the three groups of Caco-2 cells was analyzed by principal component analysis, differential metabolite heat map analysis, and Venn analysis. Principal component analysis showed that compared with the control group, MGO group and L. fermentum 2-14 treatment group, there were differences in metabolites (P<0.001) Figure 8 A). The differential metabolite heat map showed good intra-group parallelism and inter-group differences among the three metabolome samples (P<0.001) Figure 8 B). Venn analysis of differential metabolites between samples was performed, and common and unique metabolites in different groups were counted. The number of metabolites common to the three groups was 1,281, and the number of metabolites unique to the control group, MGO treatment group and L. fermentum 2-14 treatment group was 1, 11 and 3, respectively. The number of metabolites common to the control group and MGO treatment group was 1, the number of metabolites common to the control group and L. fermentum 2-14 treatment group was 14, and the number of metabolites common to MGO group and L. fermentum 2-14 treatment group was 11 Figure 8 C). In addition, compared with the MGO group, the content of 354 metabolites in the L. fermentum 2-14 treatment group Caco-2 cells changed significantly, of which 154 metabolites were significantly up-regulated and 200 metabolites were significantly down-regulated (P<0.05) Figure 8 D).

[0052] Based on the KEGG database, the differential metabolites in Caco-2 cells were analyzed to explore the metabolic pathways regulated by the differential metabolites. In the analysis of MGO treatment group vs. L. fermentum 2-14 treatment group, the AMPK signaling pathway had a darker color, indicating that the enrichment of this pathway in differential metabolites had a higher significance (P<0.05) Figure 8 E). In addition, L. fermentum 2-14 treatment significantly increased the two key metabolites NAD + ( Figure 8 F) and pyruvate in the AMPK signaling pathway (P<0.05) +Figure 8 To investigate the effects and correlations between differentially expressed genes and metabolites on the cytotoxicity of *Lactobacillus fermentum* 2-14 in alleviating MGO cytotoxicity, we performed correlation analysis on cell viability, cytotoxicity, apoptosis, autophagy genes LC3B and P62, apoptosis genes Beclin-2, Bad, Bax, Caspase-3, and Caspase-9, inflammatory genes IL-1β, IL-6, IL-10, TNF-α, and NF-κB, antioxidant markers ROS, MDA, CAT, SOD, and GSH-Px, as well as 12 differentially expressed genes and 2 differentially expressed metabolites in the AMPK pathway. The results showed that cell viability was correlated with LC3B, Beclin-2, IL-10, CAT, SOD, GSH-Px, SIRT1, PCK2, PPARG, CREB5, PFKFB3, IRS2, and NAD+ levels. + Pyruvate showed a significant positive correlation with apoptosis. Apoptosis and cytotoxicity were significantly negatively correlated with Beclin-2, CAT, SOD, GSH-Px, SIRT1, PCK1, PPARG, IRS2, and pyruvate, and significantly positively correlated with P62, Bad, Bax, Caspase-3, Caspase-9, IL-1β, IL-6, TNF-α, NF-κB, ROS, MDA, and FASN, suggesting that apoptosis may be caused by inflammation and oxidative stress. Furthermore, the differential metabolite NAD... + Pyruvate showed a significant positive correlation with cell viability, LC3B, Beclin-2, IL-10, SIRT1, CREB5, and IRS2. Figure 8 H).

[0053] Example 9: Lactobacillus fermentum 2-14 promotes pyruvate production to activate the AMPK signaling pathway and autophagy, thereby alleviating MGO-induced cytotoxicity. Following the steps in Example 4, Caco-2 cells were seeded, treated with lactic acid bacteria, subjected to autophagy inhibition, and exposed to MGO. Cells in the 2-14+MGO+Dd group were treated with Lactobacillus fermentum 2-14 for 5 h, followed by incubation with the AMPK inhibitor dosomorphin dihydrochloride (Dd) for 1 h. Cell viability was determined following the steps in Example 1.

[0054] The results showed that inhibiting the AMPK signaling pathway significantly reduced cell viability. Figure 9 A). Compared to the MGO treatment group, adding NAD+ Cell viability was not improved (Fig. 2A, B), while the addition of pyruvate significantly alleviated the decrease in cell viability induced by MGO (Fig. 2C, D), and this alleviating effect was inhibited when autophagy or AMPK signaling pathway was inhibited (Fig. 2E, F). Figure 9 B), while the addition of pyruvate significantly alleviated the decrease in cell viability induced by MGO (Fig. 2C, D), and this alleviating effect was inhibited when autophagy or AMPK signaling pathway was inhibited (Fig. 2E, F). Figure 9 C-D). The above results showed that L. fermentum 2-14 promoted pyruvate production to activate AMPK signaling pathway and autophagy, thereby alleviating the decrease in cell viability induced by MGO.

[0055] Although the present application has been disclosed with reference to the preferred embodiments, it is not intended to limit the present application, and any person skilled in the art can make various modifications and modifications without departing from the spirit and scope of the present application, and the scope of protection of the present application should be defined by the claims.

Claims

1. A Lactobacillus fermentum strain that inhibits the cytotoxicity of methylglyoxal, characterized in that, Lactobacillus fermentum HBUTFMNH4, which was preserved in China Center for Type Culture Collection on June 10, 2025, has a preservation number of CCTCC No. M20251327.

2. The Lactobacillus fermentum strain for inhibiting methylglyoxal cytotoxicity according to claim 1, characterized by, The Lactobacillus fermentum has one or more of the following functions: (1) can significantly inhibit methylglyoxal-induced Caco-2 cell apoptosis, reduce ROS and MDA production, increase CAT, SOD and GSH-Px activity, and reduce inflammatory response; (2) promotes the production of key metabolites pyruvate, activates the AMPK signaling pathway, and then activates Caco-2 cell autophagy and promotes autophagolysosome formation.

3. A microbial inoculant, characterized in that, The microbial agent comprises at least one of the cell bodies, culture solution, and metabolic products of the Lactobacillus fermentum according to claim 1 or 2.

4. The microbial inoculant of claim 3, wherein, The viable cell concentration of the microbial inoculant is 1 x 10 6 ~ 1 x 10 9 CFU / mL.

5. The microbial inoculant of claim 3, wherein the Bacillus amyloliquefaciens is B. amyloliquefaciens strain F727. The microbial agent is a resuspension of Lactobacillus fermentum 2-14, and the resuspension is selected from PBS, LB containing 20% glycerol, or MRS glycerol mixed solution.

6. Use of the Lactobacillus fermentum according to claim 1 or 2, or the microbial agent according to any one of claims 3-5 in the preparation of a product for alleviating methylglyoxal toxicity.

7. Use according to claim 6, characterized in that, The product includes food or medicine.

8. Use according to claim 7, characterized in that, The dosage form of the medicine includes solid, liquid, or powder.

9. Use according to claim 7, characterized in that, The medicine further includes a pharmaceutically acceptable carrier or excipient.

10. Use according to claim 7, characterized in that, The food includes food containing the Lactobacillus fermentum, or dairy products, bean products, meat products, or fruit and vegetable products produced by a fermenting agent containing the Lactobacillus fermentum or the fermentation product of the Lactobacillus fermentum.

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