Journal: Frontiers in Microbiology

Article Title: The supernatant of Lactiplantibacillus plantarum 25 is more effective than extracellular vesicles in alleviating ulcerative colitis and improving intestinal barrier function

doi: 10.3389/fmicb.2025.1742486

Figure Lengend Snippet: Effects of LP25-derived extracellular vesicles and LP25 supernatant on the intestinal mucosal barrier in acute ulcerative colitis mice. (A,B) Western blot analysis of barrier-associated proteins ZO-1, Occludin, Claudin-1, and α-SMA in colonic tissues from mice treated with 3% DSS alone, low-dose LP25 EVs (1 mg/kg), high-dose LP25 EVs (2 mg/kg), low-dose LP25 supernatant (125 mg/kg), and high-dose LP25 supernatant (250 mg/kg). (C–F) Quantitative densitometric analysis of the respective protein bands. (G) Representative hematoxylin and eosin (H&E) staining of colonic tissues from each group (100×, scale bar = 50 μm; magnified views at 400×). Black arrows indicate inflammatory cell infiltration. Data are presented as mean ± SD ( n = 3). *** p < 0.001, **** p < 0.0001 indicate significant differences compared to the model group; # p < 0.05, significant difference between low-dose LP25 EVs and low-dose LP25 supernatant groups; && p < 0.01, &&& p < 0.001, significant differences between high-dose LP25 EVs and high-dose LP25 supernatant groups.

Article Snippet: Membranes were blocked with 5% non-fat milk in PBST for 2 h at room temperature and incubated overnight at 4 °C with rabbit primary antibodies against anti-ZO-1 (1: 1000), anti-Claudin-1 (1: 1000), anti-Occludin (1: 1000), anti-α-SMA (1: 500) (Affinity Biosciences, Cincinnati, United States), anti-Villin (1: 2000) (Proteintech, Chicago, United States), anti-intestinal alkaline phosphatase (IAP) (1: 5000) (Abcam, UK), anti-CD86 (1: 1000), anti-Arg-1 (1: 1000), anti-TLR4 (1: 500) (Affinity Biosciences, Cincinnati, United States), anti-NF-κB p65 (1: 2000) (Zenbio, Chengdu, China), anti-phospho-NF-κB p65 (1: 1000) (Abmart, Shanghai, China), anti- IκB-α (1: 5000), anti-phospho-IκB-α (1:2000) (Zenbio, Chengdu, China), and GAPDH (1:10000) (Affinity Biosciences, Cincinnati, United States).

Techniques: Derivative Assay, Western Blot, Staining

Journal: Frontiers in Microbiology

Article Title: The supernatant of Lactiplantibacillus plantarum 25 is more effective than extracellular vesicles in alleviating ulcerative colitis and improving intestinal barrier function

doi: 10.3389/fmicb.2025.1742486

Figure Lengend Snippet: Effects of LP25-derived extracellular vesicles and LP25 supernatant on intestinal mucosal barrier integrity in DSS-induced acute ulcerative colitis mice. (A–F) Immunofluorescence staining of colonic tissues showing tight junction proteins ZO-1, Occludin, and Claudin-1 in each treatment group (magnification 10×; scale bar = 100 μm; insets show magnified images at 20×), along with corresponding fluorescence intensity quantification. Data are presented as mean ± SD ( n = 3). * p < 0.05, *** p < 0.001, **** p < 0.0001 indicate significant differences compared to the model group; # p < 0.05, ## p < 0.01 indicate significant differences between the low-dose LP25 EVs group and the low-dose LP25 supernatant group; & p < 0.05, && p < 0.01 indicate significant differences between the high-dose LP25 EVs group and the high-dose LP25 supernatant group.

Article Snippet: Membranes were blocked with 5% non-fat milk in PBST for 2 h at room temperature and incubated overnight at 4 °C with rabbit primary antibodies against anti-ZO-1 (1: 1000), anti-Claudin-1 (1: 1000), anti-Occludin (1: 1000), anti-α-SMA (1: 500) (Affinity Biosciences, Cincinnati, United States), anti-Villin (1: 2000) (Proteintech, Chicago, United States), anti-intestinal alkaline phosphatase (IAP) (1: 5000) (Abcam, UK), anti-CD86 (1: 1000), anti-Arg-1 (1: 1000), anti-TLR4 (1: 500) (Affinity Biosciences, Cincinnati, United States), anti-NF-κB p65 (1: 2000) (Zenbio, Chengdu, China), anti-phospho-NF-κB p65 (1: 1000) (Abmart, Shanghai, China), anti- IκB-α (1: 5000), anti-phospho-IκB-α (1:2000) (Zenbio, Chengdu, China), and GAPDH (1:10000) (Affinity Biosciences, Cincinnati, United States).

Techniques: Derivative Assay, Immunofluorescence, Staining, Fluorescence

Journal: Frontiers in Microbiology

Article Title: The supernatant of Lactiplantibacillus plantarum 25 is more effective than extracellular vesicles in alleviating ulcerative colitis and improving intestinal barrier function

doi: 10.3389/fmicb.2025.1742486

Figure Lengend Snippet: Effects of LP25-derived EVs and LP25 supernatant on TJ proteins expression in LPS-stimulated Caco-2 cells. (A) Caco-2 cells were stimulated with lipopolysaccharide (LPS) at concentrations of 0, 1, 5, 10, and 50 μg/mL. TNF-α mRNA expression was measured by qPCR to determine the optimal concentration for inducing inflammation. A concentration of 10 μg/mL was selected for subsequent experiments. *** p < 0.001, **** p < 0.0001 indicate significant differences compared to the 0 g/mL group; # p < 0.05 between 5 g/mL and 10 g/mL. (B) Cell viability was assessed by CCK-8 assay following treatment with LP25 EVs at concentrations of 120, 240, 480, and 960 g/mL. * p < 0.05 compared to the 120 g/mL group. (C) Cell viability was assessed after treatment with LP25 supernatant at concentrations of 0.25, 2.5, 25, and 250 g/mL. Concentrations of 25 g/mL and 250 g/mL were selected for further experiments. ** p < 0.01, *** p < 0.001, **** p < 0.0001 vs. 250 g/mL group. (D,E) Western blot analysis of TJ proteins (ZO-1, Occludin and Claudin-1) in Caco-2 cells treated with 10 g/mL LPS alone or in combination with low-dose LP25 EVs (120 g/mL), high-dose LP25 EVs (240 g/mL), low-dose LP25 supernatant (25 g/mL), or high-dose LP25 supernatant (250 g/mL). (F–H) Densitometric quantification of TJ proteins levels. (I) Representative immunofluorescence staining of ZO-1 in each treatment group, with DAPI staining for nuclei (scale bar = 50 m). (J) Quantitative analysis of ZO-1 fluorescence intensity. Data are presented as mean ± SD from three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 indicate significant differences compared to the LPS group; # p < 0.05, ## p < 0.01 indicate significant differences between the low-dose LP25 EVs group and the low-dose LP25 supernatant group; & p < 0.05, && p < 0.01, &&& p < 0.001 indicate significant differences between the high-dose LP25 EVs group and the high-dose LP25 supernatant group.

Article Snippet: Membranes were blocked with 5% non-fat milk in PBST for 2 h at room temperature and incubated overnight at 4 °C with rabbit primary antibodies against anti-ZO-1 (1: 1000), anti-Claudin-1 (1: 1000), anti-Occludin (1: 1000), anti-α-SMA (1: 500) (Affinity Biosciences, Cincinnati, United States), anti-Villin (1: 2000) (Proteintech, Chicago, United States), anti-intestinal alkaline phosphatase (IAP) (1: 5000) (Abcam, UK), anti-CD86 (1: 1000), anti-Arg-1 (1: 1000), anti-TLR4 (1: 500) (Affinity Biosciences, Cincinnati, United States), anti-NF-κB p65 (1: 2000) (Zenbio, Chengdu, China), anti-phospho-NF-κB p65 (1: 1000) (Abmart, Shanghai, China), anti- IκB-α (1: 5000), anti-phospho-IκB-α (1:2000) (Zenbio, Chengdu, China), and GAPDH (1:10000) (Affinity Biosciences, Cincinnati, United States).

Techniques: Derivative Assay, Expressing, Concentration Assay, CCK-8 Assay, Western Blot, Immunofluorescence, Staining, Fluorescence

Journal: Frontiers in Microbiology

Article Title: The supernatant of Lactiplantibacillus plantarum 25 is more effective than extracellular vesicles in alleviating ulcerative colitis and improving intestinal barrier function

doi: 10.3389/fmicb.2025.1742486

Figure Lengend Snippet: Effects of LP25-derived extracellular vesicles and LP25 supernatant on the intestinal mucosal barrier in acute ulcerative colitis mice. (A,B) Western blot analysis of barrier-associated proteins ZO-1, Occludin, Claudin-1, and α-SMA in colonic tissues from mice treated with 3% DSS alone, low-dose LP25 EVs (1 mg/kg), high-dose LP25 EVs (2 mg/kg), low-dose LP25 supernatant (125 mg/kg), and high-dose LP25 supernatant (250 mg/kg). (C–F) Quantitative densitometric analysis of the respective protein bands. (G) Representative hematoxylin and eosin (H&E) staining of colonic tissues from each group (100×, scale bar = 50 μm; magnified views at 400×). Black arrows indicate inflammatory cell infiltration. Data are presented as mean ± SD ( n = 3). *** p < 0.001, **** p < 0.0001 indicate significant differences compared to the model group; # p < 0.05, significant difference between low-dose LP25 EVs and low-dose LP25 supernatant groups; && p < 0.01, &&& p < 0.001, significant differences between high-dose LP25 EVs and high-dose LP25 supernatant groups.

Article Snippet: Membranes were blocked with 5% non-fat milk in PBST for 2 h at room temperature and incubated overnight at 4 °C with rabbit primary antibodies against anti-ZO-1 (1: 1000), anti-Claudin-1 (1: 1000), anti-Occludin (1: 1000), anti-α-SMA (1: 500) (Affinity Biosciences, Cincinnati, United States), anti-Villin (1: 2000) (Proteintech, Chicago, United States), anti-intestinal alkaline phosphatase (IAP) (1: 5000) (Abcam, UK), anti-CD86 (1: 1000), anti-Arg-1 (1: 1000), anti-TLR4 (1: 500) (Affinity Biosciences, Cincinnati, United States), anti-NF-κB p65 (1: 2000) (Zenbio, Chengdu, China), anti-phospho-NF-κB p65 (1: 1000) (Abmart, Shanghai, China), anti- IκB-α (1: 5000), anti-phospho-IκB-α (1:2000) (Zenbio, Chengdu, China), and GAPDH (1:10000) (Affinity Biosciences, Cincinnati, United States).

Techniques: Derivative Assay, Western Blot, Staining

Journal: Frontiers in Microbiology

Article Title: The supernatant of Lactiplantibacillus plantarum 25 is more effective than extracellular vesicles in alleviating ulcerative colitis and improving intestinal barrier function

doi: 10.3389/fmicb.2025.1742486

Figure Lengend Snippet: Effects of LP25-derived extracellular vesicles and LP25 supernatant on intestinal mucosal barrier integrity in DSS-induced acute ulcerative colitis mice. (A–F) Immunofluorescence staining of colonic tissues showing tight junction proteins ZO-1, Occludin, and Claudin-1 in each treatment group (magnification 10×; scale bar = 100 μm; insets show magnified images at 20×), along with corresponding fluorescence intensity quantification. Data are presented as mean ± SD ( n = 3). * p < 0.05, *** p < 0.001, **** p < 0.0001 indicate significant differences compared to the model group; # p < 0.05, ## p < 0.01 indicate significant differences between the low-dose LP25 EVs group and the low-dose LP25 supernatant group; & p < 0.05, && p < 0.01 indicate significant differences between the high-dose LP25 EVs group and the high-dose LP25 supernatant group.

Article Snippet: Membranes were blocked with 5% non-fat milk in PBST for 2 h at room temperature and incubated overnight at 4 °C with rabbit primary antibodies against anti-ZO-1 (1: 1000), anti-Claudin-1 (1: 1000), anti-Occludin (1: 1000), anti-α-SMA (1: 500) (Affinity Biosciences, Cincinnati, United States), anti-Villin (1: 2000) (Proteintech, Chicago, United States), anti-intestinal alkaline phosphatase (IAP) (1: 5000) (Abcam, UK), anti-CD86 (1: 1000), anti-Arg-1 (1: 1000), anti-TLR4 (1: 500) (Affinity Biosciences, Cincinnati, United States), anti-NF-κB p65 (1: 2000) (Zenbio, Chengdu, China), anti-phospho-NF-κB p65 (1: 1000) (Abmart, Shanghai, China), anti- IκB-α (1: 5000), anti-phospho-IκB-α (1:2000) (Zenbio, Chengdu, China), and GAPDH (1:10000) (Affinity Biosciences, Cincinnati, United States).

Techniques: Derivative Assay, Immunofluorescence, Staining, Fluorescence

Journal: Frontiers in Microbiology

Article Title: The supernatant of Lactiplantibacillus plantarum 25 is more effective than extracellular vesicles in alleviating ulcerative colitis and improving intestinal barrier function

doi: 10.3389/fmicb.2025.1742486

Figure Lengend Snippet: Effects of LP25-derived EVs and LP25 supernatant on TJ proteins expression in LPS-stimulated Caco-2 cells. (A) Caco-2 cells were stimulated with lipopolysaccharide (LPS) at concentrations of 0, 1, 5, 10, and 50 μg/mL. TNF-α mRNA expression was measured by qPCR to determine the optimal concentration for inducing inflammation. A concentration of 10 μg/mL was selected for subsequent experiments. *** p < 0.001, **** p < 0.0001 indicate significant differences compared to the 0 g/mL group; # p < 0.05 between 5 g/mL and 10 g/mL. (B) Cell viability was assessed by CCK-8 assay following treatment with LP25 EVs at concentrations of 120, 240, 480, and 960 g/mL. * p < 0.05 compared to the 120 g/mL group. (C) Cell viability was assessed after treatment with LP25 supernatant at concentrations of 0.25, 2.5, 25, and 250 g/mL. Concentrations of 25 g/mL and 250 g/mL were selected for further experiments. ** p < 0.01, *** p < 0.001, **** p < 0.0001 vs. 250 g/mL group. (D,E) Western blot analysis of TJ proteins (ZO-1, Occludin and Claudin-1) in Caco-2 cells treated with 10 g/mL LPS alone or in combination with low-dose LP25 EVs (120 g/mL), high-dose LP25 EVs (240 g/mL), low-dose LP25 supernatant (25 g/mL), or high-dose LP25 supernatant (250 g/mL). (F–H) Densitometric quantification of TJ proteins levels. (I) Representative immunofluorescence staining of ZO-1 in each treatment group, with DAPI staining for nuclei (scale bar = 50 m). (J) Quantitative analysis of ZO-1 fluorescence intensity. Data are presented as mean ± SD from three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 indicate significant differences compared to the LPS group; # p < 0.05, ## p < 0.01 indicate significant differences between the low-dose LP25 EVs group and the low-dose LP25 supernatant group; & p < 0.05, && p < 0.01, &&& p < 0.001 indicate significant differences between the high-dose LP25 EVs group and the high-dose LP25 supernatant group.

Article Snippet: Membranes were blocked with 5% non-fat milk in PBST for 2 h at room temperature and incubated overnight at 4 °C with rabbit primary antibodies against anti-ZO-1 (1: 1000), anti-Claudin-1 (1: 1000), anti-Occludin (1: 1000), anti-α-SMA (1: 500) (Affinity Biosciences, Cincinnati, United States), anti-Villin (1: 2000) (Proteintech, Chicago, United States), anti-intestinal alkaline phosphatase (IAP) (1: 5000) (Abcam, UK), anti-CD86 (1: 1000), anti-Arg-1 (1: 1000), anti-TLR4 (1: 500) (Affinity Biosciences, Cincinnati, United States), anti-NF-κB p65 (1: 2000) (Zenbio, Chengdu, China), anti-phospho-NF-κB p65 (1: 1000) (Abmart, Shanghai, China), anti- IκB-α (1: 5000), anti-phospho-IκB-α (1:2000) (Zenbio, Chengdu, China), and GAPDH (1:10000) (Affinity Biosciences, Cincinnati, United States).

Techniques: Derivative Assay, Expressing, Concentration Assay, CCK-8 Assay, Western Blot, Immunofluorescence, Staining, Fluorescence