Journal: Molecular Medicine

Article Title: Helicobacter pylori VacA modulates TRAF1-mediated 4-1BB/NF-kappaB axis to induce host apoptosis and chronic inflammatory damage

doi: 10.1186/s10020-025-01349-5

Figure Lengend Snippet: TRAF1 promotes the expression of 4-1BB, molecules of NF-κB pathway, apoptosis-related proteins, and downstream inflammatory factors in gastric mucosal epithelial cells. a Construction of stable TRAF1-silenced cell lines. The fluorescence intensity of each cell line was observed by fluorescence microscopy after infection of MKN74 cells with interfering lentivirus and control lentivirus (100X). The silencing effect of TRAF1 in MKN74 stable cell lines was detected by Western blot and qRT-PCR. shTRAF1-1, shTRAF1-2, and shTRAF1-3 represent cells infected with lentivirus corresponding to three different TRAF1 interference sequences, respectively; shCtrl represents cells infected with control virus. GAPDH was used as an internal reference. The relative expression level of TRAF1 (TRAF1/GAPDH): shTRAF1-2 vs. shCtrl, p = 0.022; shTRAF1-3 vs. shCtrl, p < 0.001. b Construction of stable TRAF1-overexpressing cell lines. The fluorescence intensity of each cell line was observed by fluorescence microscopy after infection of HGC27 cells with overexpressing lentivirus and control lentivirus (100X). The overexpression effect of TRAF1 in HGC27 stable cell lines was detected by Western blot and qRT-PCR. The relative expression level of TRAF1 (TRAF1/GAPDH): TRAF1 OE vs. vector, p < 0.001. c-g Transcriptome sequencing analysis of TRAF1 stably overexpressing gastric mucosal epithelial cells and their negative control cells. c Top 20 differential factors obtained by KEGG enrichment analysis. d NF-κB pathway-related GSEA enrichment analysis. e Apoptosis-related GSEA enrichment analysis. f Heatmap of differential genes related to the NF-kappa B signaling pathway. g Heatmap of differential genes related to apoptosis. h Western Blot analysis of the effects of stable silencing and overexpression of TRAF1 on 4-1BB, NF-κB pathway, and apoptosis-related proteins in gastric mucosal epithelial cells. GAPDH was used as an internal reference. ELISA detection of inflammatory factors IL-8, IL-6, and TNF-α in the supernatant of TRAF1 stably overexpressing/silenced gastric mucosal epithelial cell lines. The relative protein expression level of TRAF1, 4-1BB, p-p65, Bcl-xl and Bax were compared between shTRAF1-MKN74 cells and shCtrl-MKN74 cells, with statistically significant differences ( p = 0.0039, p = 0.033, p = 0.007, p = 0.048, p = 0.005, respectively). The relative protein expression level of TRAF1, 4-1BB, p-p65, and Caspase9 were compared between TRAF1 OE-HGC27 cells and vector-HGC27 cells, with statistically significant differences ( p < 0.001, p = 0.041, p = 0.046, p = 0.031, p = 0.042, respectively). The level of IL-8, IL-6, and TNF-α were compared between shTRAF1-MKN74 cells and shCtrl-MKN74 cells, with statistically significant differences ( p < 0.001, p = 0.007, p = 0.042, respectively). The level of IL-8, IL-6, and TNF-α were compared between TRAF1 OE-HGC27 cells and vector-HGC27 cells, with statistically significant differences ( p < 0.001, p = 0.007, p < 0.001, respectively). Note: Comparisons were made between two groups using standard two-tailed Student’s t-test. The figure presents the average of three independent experiments ( n = 3). Data are presented as mean ± SEM. Error bars represent standard error of mean.*: p < 0.05.**; p < 0.01.***; p < 0.001. The phosphorylation site of Phospho-NF-κB p65 is serine 536. shCtrl, short hairpin control; shTRAF1, short hairpin TRAF1; OE, overexpression

Article Snippet: NF-κB inhibitor BAY11-7082 (BAY) (Selleck, USA), Anti-human 4-1BB Blocking Antibody (BioLegend, USA) and Anti- 4-1BB Agonist Antibody (BPS Bioscience, USA) were used at the dose as indicated, cells were pretreated for 2 h.

Techniques: Expressing, Fluorescence, Microscopy, Infection, Control, Stable Transfection, Western Blot, Quantitative RT-PCR, Virus, Over Expression, Plasmid Preparation, Sequencing, Negative Control, Enzyme-linked Immunosorbent Assay, Two Tailed Test, Phospho-proteomics

Journal: Molecular Medicine

Article Title: Helicobacter pylori VacA modulates TRAF1-mediated 4-1BB/NF-kappaB axis to induce host apoptosis and chronic inflammatory damage

doi: 10.1186/s10020-025-01349-5

Figure Lengend Snippet: Effects of VacA on TRAF1/4-1BB/NF-κB pathway/chemokine IL-8 axis- and apoptosis-related protein expression in GES-1 cells. a-d GES-1 cells infected with vacA + Hp strain and Δ vacA Hp strain at different MOIs (0, 10, 20, 50, 100, and 200) for 24 h or different time points (0 h, 6 h, 12 h, 24 h, and 36 h), MOI = 100. a, b Western blot analysis of the target proteins (phospho-p65, p65, TRAF1, 4-1BB, apoptosis-related protein Bcl-xl and Bax) levels. GAPDH was used as an internal reference. c, d the secretion levels of IL-8 from GES-1 cells after infection were measured by ELISA. Comparison between vacA + Hp strain-infected group and Δ vacA Hp strain-infected group under identical MOI conditions (c) or identical intervention time (d) showed statistical significance ( p < 0.001 for all comparisons). e Western blot analysis of the target proteins levels in GES-1 cells incubated with recombinant VacA protein at low concentrations (5 µg/ml and 10 µg/ml) for 48 h or at high concentrations (65 µg/ml) for 12 h. In addition, GES-1 cells incubated with isovolumetric protein buffer and cell culture medium served as a buffer control group and an untreated normal group, respectively. The relative protein expression level of TRAF1 and 4-1BB were compared between recombinant VacA protein-incubated group (10 µg/mL) and the correspondind buffer-incubated group, with statistically significant differences ( p = 0.041, p = 0.037, respectively). The relative protein expression level of p-p65, TRAF1, 4-1BB, Bcl-xl and Bax were compared between recombinant VacA protein-incubated group (65 µg/mL) and the correspondind buffer-incubated group, with statistically significant differences ( p < 0.001, p = 0.039, p = 0.044, p = 0.049, p = 0.047, respectively). f Western blot analysis of VacA, phospho-p65, p65, TRAF1, 4-1BB and apoptosis-related protein (Bcl-xl, Bax) levels in GES-1 cells transfected with pDsRED2-N1-HA/VacA for 48 h. g qRT‒PCR analysis of VacA, TRAF1, 4-1BB and IL-8 levels in GES-1 cells transfected with pDsRED2-N1-HA/VacA for 48 h. GAPDH was used as an internal control. Comparison of relative expression levels of VacA, TRAF1, 4-1BB, and IL-8 between the pDsRED2-N1-HA-VacA-transfected groups and the empty vector pDsRED2-N1-HA-transfected groups showed statistically significant differences ( p < 0.001, p = 0.007, p = 0.009, p = 0.005, respectively). h, i The secretion level of IL-8 in GES-1 cells after incubation with recombinant VacA protein ( h ) or transfection with pDsRED2-N1-HA/VacA ( i ) was measured by ELISA. h Levels of IL-8 were compared between recombinant VacA protein-incubated groups (at concentrations of 5, 10, and 65 µg/mL) and the correspondind buffer-incubated groups, with statistically significant differences ( p = 0.007, p < 0.001, p = 0.005, respectively). i Levels of IL-8 were compared between the pDsRED2-N1-HA-VacA-transfected groups (for 48 h and 72 h) and the empty vector pDsRED2-N1-HA-transfected groups, with statistically significant differences ( p = 0.039 and p = 0.0071). The figure presents the average of three independent experiments ( n = 3). Note: Comparisons were made between two groups using standard two-tailed Student’s t-test. The figure presents the average of three independent experiments ( n = 3). Data are presented as mean ± SEM. Error bars represent standard error of mean. *: p < 0.05. **: p < 0.01. ***: p < 0.001

Article Snippet: NF-κB inhibitor BAY11-7082 (BAY) (Selleck, USA), Anti-human 4-1BB Blocking Antibody (BioLegend, USA) and Anti- 4-1BB Agonist Antibody (BPS Bioscience, USA) were used at the dose as indicated, cells were pretreated for 2 h.

Techniques: Expressing, Infection, Western Blot, Enzyme-linked Immunosorbent Assay, Comparison, Incubation, Recombinant, Cell Culture, Control, Transfection, Plasmid Preparation, Two Tailed Test

Journal: Molecular Medicine

Article Title: Helicobacter pylori VacA modulates TRAF1-mediated 4-1BB/NF-kappaB axis to induce host apoptosis and chronic inflammatory damage

doi: 10.1186/s10020-025-01349-5

Figure Lengend Snippet: TRAF1 promotes the expression of 4-1BB, NF - κ B pathway proteins, apoptosis-related molecules and downstream inflammatory factors in gastric epithelial cells induced by VacA. a, b Western Blot was used to detect the protein expression levels of the target proteins (pIKKα/β, p-P65, p65, TRAF1, 4-1BB, caspase9, Bcl-xl and Bax) after co-culturing TRAF1 stably overexpressing/silenced cell lines with different concentrations of the vacA + Hp strain for 24 h. c, d Western Blot was used to detect the target proteins after co-incubation different concentrations of recombinant VacA protein with TRAF1 stably overexpressing/silenced cell lines for 48 h. e VacA protein (10ug/ml) was incubated with TRAF1 stably overexpressing/silenced gastric epithelial cells for 48 h, using an equal volume of acetic acid buffer as a control. Western blot was used to analyze the expression of various target molecules. f The transfected GES-1 cells were incubated with recombinant VacA protein (10 µg/ml) for 48 h. In addition, the transfected GES-1 cells incubated with isovolumetric protein buffer served as a buffer control group. Western blot was used to detect the effect of TRAF1 silencing or TRAF1 gene overexpression on the expression of the target proteins induced by recombinant VacA protein. g ELISA was used to analyze the secretion of IL-8, IL6, and TNF-αin the TRAF1 stably overexpressing/silenced cells supernatant after co-culturing with vacA + Hp strain or ΔvacA Hp strain at different concentrations for 24 h. Comparison of IL-8, IL-6, and TNF-αlevels between vacA + Hp strain-infected groups and ΔvacA Hp strain-infected groups in HGC27 and MKN74 cells under varying MOI conditions (MOI = 10,20,50,100,200) demonstrated statistically significant differences ( p < 0.001 for all cytokine comparisons across both cell lines and all MOI gradients. * p < 0.05, ** p < 0.01, *** p < 0.001.). When infected with vacA + Hp strain at different multiplicities of infection (MOIs: 10, 20, 50, 100, 200), comparisons of IL-8, IL-6, and TNF-αlevels between TRAF1-OE-HGC27 cells and Vector-HGC27 cells or between shTRAF1-MKN74 cells and shCtrl-MKN74 cells revealed statistically significant differences (all p < 0.05. # p < 0.05, ## p < 0.01, ### p < 0.001.). GAPDH was used as a protein internal reference. Vector-HGC27, TRAF1 OE-HGC27: HGC27 cells infected with control lentivirus or overexpressing lentivirus. shCtrl-MKN74, shTRAF1-MKN74: MKN74 cells infected with control lentivirus or interfering lentivirus. Note: Comparisons were made between two groups using standard two-tailed Student’s t-test. The figure presents the average of three independent experiments ( n = 3). Data are presented as mean ± SEM. Error bars represent standard error of mean. shCtrl, short hairpin control; shTRAF1, short hairpin TRAF1; OE, overexpression

Article Snippet: NF-κB inhibitor BAY11-7082 (BAY) (Selleck, USA), Anti-human 4-1BB Blocking Antibody (BioLegend, USA) and Anti- 4-1BB Agonist Antibody (BPS Bioscience, USA) were used at the dose as indicated, cells were pretreated for 2 h.

Techniques: Expressing, Western Blot, Stable Transfection, Incubation, Recombinant, Control, Transfection, Over Expression, Enzyme-linked Immunosorbent Assay, Comparison, Infection, Plasmid Preparation, Two Tailed Test

Journal: Molecular Medicine

Article Title: Helicobacter pylori VacA modulates TRAF1-mediated 4-1BB/NF-kappaB axis to induce host apoptosis and chronic inflammatory damage

doi: 10.1186/s10020-025-01349-5

Figure Lengend Snippet: Suppression of the NF-κB signalling pathway counteracts changes in the expression of TRAF1/4-1BB/IL-8 and apoptosis-related proteins induced by VacA in GES-1 cells, resulting in a lower apoptosis rate and increased proliferation. GES-1 cells pretreated with BAY11-7082 (5 µM) were then infected with the vacA + Hp strain at different MOIs (0, 10, 20, 50, 100, and 200) for 24 h or incubated with VacA recombinant protein at a low concentration (5, 10 µg/ml for 48 h) or at a high concentration (65 µg/ml for 12 h). Compared with the VacA recombinant protein incubation group, GES-1 cells incubated with isovolumetric protein buffer and cell culture medium served as a buffer control group and an untreated normal group, respectively. a-d The protein levels of the target molecules (TRAF1, 4-1BB, p65, phospho-p65, Bcl-xl and Bax) were determined by Western blot analysis. GAPDH was used as a loading control. e The secretion level of IL-8 in GES-1 cells after incubation with recombinant VacA protein was measured by ELISA. Comparison of IL-8 levels between recombinant VacA protein-incubated groups (at concentrations of 5, 10, and 65 µg/mL) and recombinant VacA protein + BAY11-7082-incubated groups revealed statistically significant differences at all three concentrations ( p = 0.0045, p < 0.001, and p = 0.0034, respectively). f CCK-8 assay was used to assess the viability of GES-1 cells pretreated with BAY11-7082 and treated with recombinant VacA protein. OD450 levels were compared between groups incubated with 65 µg/mL recombinant VacA protein alone and those incubated with 65 µg/mL recombinant VacA protein + BAY11-7082 at 12, 24, and 48 h, statistically significant differences were observed at all time points ( p = 0.041, p = 0.009, and p = 0.003, respectively). g-h Annexin V-FITC staining coupled with flow cytometry analysis of the apoptosis of GES-1 cells pretreated with BAY11-7082 and treated with VacA recombinant protein. (h) Percentage of apoptotic cells were compared between groups incubated with 65 µg/mL recombinant VacA protein alone and those incubated with 65 µg/mL recombinant VacA protein + BAY11-7082, with statistically significant differences ( p = 0.037). FITC, fluorescein isothiocyanate; PI, propidium iodide. Note: Comparisons were made between two groups using standard two-tailed Student’s t-test. The figure presents the average of three independent experiments ( n = 3). Data are presented as mean ± SEM. Error bars represent standard error of mean. * p < 0.05, ** p < 0.01, *** p < 0.001. BAY, BAY11-7082

Article Snippet: NF-κB inhibitor BAY11-7082 (BAY) (Selleck, USA), Anti-human 4-1BB Blocking Antibody (BioLegend, USA) and Anti- 4-1BB Agonist Antibody (BPS Bioscience, USA) were used at the dose as indicated, cells were pretreated for 2 h.

Techniques: Expressing, Infection, Incubation, Recombinant, Concentration Assay, Cell Culture, Control, Western Blot, Enzyme-linked Immunosorbent Assay, Comparison, CCK-8 Assay, Staining, Flow Cytometry, Two Tailed Test

Journal: Molecular Medicine

Article Title: Helicobacter pylori VacA modulates TRAF1-mediated 4-1BB/NF-kappaB axis to induce host apoptosis and chronic inflammatory damage

doi: 10.1186/s10020-025-01349-5

Figure Lengend Snippet: Effects of BAY11-7082 or 4-1BB blocking/agonist antibodies on TRAF1-mediated 4-1BB, NF-κB pathway activation, and apoptosis-related protein expression in the context of VacA action. a, b HGC27 cells (infected with TRAF1-overexpressing lentivirus or control lentivirus) pretreated with BAY11-7082 (5 µM) were incubated with VacA recombinant protein at different concentrations (5, 10 µg/ml) for 48 h. a Western blot analysis of target molecules (TRAF1, 4-1BB, pIKKα/β, p-P65, and apoptosis-related molecules Caspase9, Bcl-xl, Bax). b ELISA analysis of the secretion of inflammatory factors IL-8, IL6, and TNF-α in the cell supernatant. In Vector-HGC27 cells and TRAF1 OE-HGC27 cells, the levels of IL-8 were compared between groups treated with recombinant VacA protein (5 µg/mL and 10 µg/mL) and groups co-incubated with recombinant VacA protein and BAY11-7082; statistically significant differences were observed at both concentrations in Vector-HGC27 cells ( p = 0.046 and p = 0.039, respectively) and in TRAF1 OE-HGC27 cells ( p = 0.035 and p = 0.024, respectively).In Vector-HGC27 cells, the levels of IL-6 and TNF-α were compared between groups treated with recombinant VacA protein (5 µg/mL and 10 µg/mLL) and groups co-incubated with recombinant VacA protein and BAY11-7082; statistically significant differences were observed for both cytokines at the 10 µg/mL VacA protein concentration (IL-6: p = 0.040; TNF-α: p = 0.031). In TRAF1 OE-HGC27 cells, the levels of IL-6 and TNF-α were compared between groups treated with recombinant VacA protein (5 µg/mL and 10 µg/mL) and groups co-incubated with recombinant VacA protein and BAY11-7082; statistically significant differences were observed for both cytokines at both concentrations (IL-6: p = 0.008 and p = 0.041; TNF-α: p = 0.047 and p = 0.033). c HGC27 cells (infected with overexpressing lentivirus or control lentivirus) pretreated with 4-1BB blocking antibody at different concentrations (5, 10, 20ug/ml) were incubated with VacA recombinant protein (10 µg/ml) for 48 h, Western blot was used to analyze the expression of target molecules in the cells. d MKN74 cells (infected with TRAF1-silencing lentivirus or control lentivirus) pretreated with 4-1BB agonistic antibody at different concentrations (5, 10ug/ml) were incubated with VacA recombinant protein (10 µg/ml) for 48 h, Western blot was used to analyze the expression of target molecules in the cells. Note: Comparisons were made between two groups using standard two-tailed Student’s t-test. The figure presents the average of three independent experiments ( n = 3). Data are presented as mean ± SEM. Error bars represent standard error of mean. * p < 0.05, ** p < 0.01. shCtrl, short hairpin control; shTRAF1, short hairpin TRAF1; OE, overexpression; BAY, BAY11-7082

Article Snippet: NF-κB inhibitor BAY11-7082 (BAY) (Selleck, USA), Anti-human 4-1BB Blocking Antibody (BioLegend, USA) and Anti- 4-1BB Agonist Antibody (BPS Bioscience, USA) were used at the dose as indicated, cells were pretreated for 2 h.

Techniques: Blocking Assay, Activation Assay, Expressing, Infection, Control, Incubation, Recombinant, Western Blot, Enzyme-linked Immunosorbent Assay, Plasmid Preparation, Protein Concentration, Two Tailed Test, Over Expression

Journal: Molecular Medicine

Article Title: Helicobacter pylori VacA modulates TRAF1-mediated 4-1BB/NF-kappaB axis to induce host apoptosis and chronic inflammatory damage

doi: 10.1186/s10020-025-01349-5

Figure Lengend Snippet: Constructing models of C57BL/6 mice infected with Hp. a Representative images of Hp, TRAF1 and 4-1BB immunohistochemical staining in gastric mucosal tissue from C57BL/6 mice infected with the vacA + Hp or ΔvacA Hp strains for one month. The black arrows indicate colonized Hp (80×). b Representative immunofluorescence images of the localization of Hp, TRAF1, 4-1BB, and P65 in gastric mucosal tissue of C57BL/6 mice infected with vacA + Hp strains for one month. DAPI (nuclei): blue; Hp: green; TRAF1: red; 4-1BB: orange; P65: pink. Magnification: 40×. c Gastric specimens and HE staining of gastric mucosal tissue from C57BL/6 mice infected with the vacA + Hp and ΔvacA Hp strains for twelve months. Magnification: 30×

Article Snippet: NF-κB inhibitor BAY11-7082 (BAY) (Selleck, USA), Anti-human 4-1BB Blocking Antibody (BioLegend, USA) and Anti- 4-1BB Agonist Antibody (BPS Bioscience, USA) were used at the dose as indicated, cells were pretreated for 2 h.

Techniques: Infection, Immunohistochemical staining, Staining, Immunofluorescence