Fig 2: H. pylori-infected macrophage culture medium and chemokine CCL3 disrupt gastric mucosal barrier through P38 phosphorylation. A The P38 phosphorylation level in GES-1 treated with different conditioned media. B IF detected P38 phosphorylation levels in GES-1 treated with different conditioned media. DAPI was used for nuclear staining (blue), P-P38 stained in red. C, D The P38 phosphorylation level in GES-1 cells with recombinant CCL3 protein (160 ng/mL) and CCL3 overexpressing macrophage medium. E, F The P38 phosphorylation level in GES-1 cells treated with CCL3 neutralizing antibody (100 ng/mL) and CCL3 receptor inhibitor (Maraviroc, 100 nM). G Effects of P38 phosphorylation inhibitor (SB203580, 10 μM) on TEER of MKN28 cells. H IF detected the effect of P38 phosphorylation inhibitor on tight junctions between MKN28 cells. DAPI was used for nuclear staining (blue), Occludin was used for tight junction staining (green), arrows showed the disruption of tight junctions between cells. I, J The protein expression level of tight junction in GES-1 cells treated with P38 phosphorylation inhibitor. K, L Apoptosis and proliferation of GES-1 treated with P38 phosphorylation inhibitor (K: Annexin V- PI apoptosis flow cytometry; L: EdU). Abbreviations: NC, normal culture medium; MC, macrophage culture medium; HMC, H. pylori-infected macrophage culture medium; PV, pcDNA vector; IF, Immunofluorescence. The data are presented as the mean ± S.D. after triplicate. Two groups were compared by t-test, multiple groups were compared by one-way analysis of variance (ANOVA). Note: *P < 0.05, **P < 0.01

Fig 3: H. pylori stimulates macrophages to secrete CCL3 through the JAK1-STAT1 pathway. A CCL3 transcription factor prediction (from JASPAR https://jaspar.genereg.net and TF human http://bioinfo.life.hust.edu.cn/HumanTFDB#!/) and the sequence STAT1 binds to the CCL3 promoter (from JASPAR). B The protein expression level of CCL3, P-STAT1, P-JAK1 proteins in THP-1 infected with H. pylori. C The protein and RNA expression level of CCL3 in THP-1 cells treated with STAT1 phosphorylation inhibitor (Fludarabine, 5 μM). D The protein and RNA expression level of CCL3 in THP-1 cells treated with STAT1 enhancer (2NP, 45 μM). E IF detected the CCL3 expression treated with Fludarabine or 2NP. DAPI was used for nuclear staining (blue), CCL3 was stained in red, arrows showed CCL3 secreted by cells. F The protein and RNA expression level of CCL3 in THP-1 cells treated with JAK1 phosphorylation inhibitors (Upadacitinib, 1 μM). G THP-1 cells treated with Fludarabine and 2NP were transfected with CCL3 promoter plasmid for dual-luciferase reporter assays. H, I ChIP verified the binding ability of STAT1 to the CCL3 promoter, and the STAT1 binding sequences were enriched in 250-450 bp and 1345-1545 bp. Abbreviations: IHC, immunohistochemical; IF, Immunofluorescence; ChIP, Chromatin Immunoprecipitation. The data are presented as the mean ± S.D. after triplicate Two groups were compared by t-test, multiple groups were compared by one-way analysis of variance (ANOVA). Note: *P < 0.05, **P < 0.01, ***P < 0.001

Fig 4: CCL3 neutralizing antibody and CCL3 receptor inhibitor improve mucosal damage and inflammatory response. A Effect of CCL3 neutralizing antibody (100 ng/ml) on TEER of MKN28 cells. B The protein expression level of the tight junction in GES-1 cells treated with CCL3 neutralizing antibody in conditioned media. C IF detected tight junction between MKN28 cells treated with CCL3 neutralizing antibody in conditioned media. DAPI was used for nuclear staining (blue), Occludin was used for tight junction staining (green), arrows showed the disruption of tight junctions between cells. D, E Apoptosis and proliferation of GES-1 treated with CCL3 neutralizing antibody in conditioned media (D: Annexin V- PI apoptosis flow cytometry; E: EdU). F Effect of CCL3 receptor inhibitor (Maraviroc, 100 nM) on TEER of MKN28 cells. G The protein expression level of the tight junction in GES-1 cells treated with CCL3 receptor inhibitor. H IF detected the tight junction between MKN28 cells treated with CCL3 receptor inhibitor. I, J Apoptosis and proliferation of GES-1 treated with CCL3 receptor inhibitor (I: Annexin V- PI apoptosis flow cytometry; J: EdU). Abbreviations: MC, macrophage culture medium; HMC, H. pylori-infected macrophage culture medium; IF, Immunofluorescence. The data are presented as the mean ± S.D. after triplicate Two groups were compared by t-test, multiple groups were compared by one-way analysis of variance (ANOVA). Note: *P < 0.05, **P < 0.01

Fig 5: CCL3 disrupts gastric mucosal barrier and promotes inflammation. A The effect of recombinant CCL3 protein (160 ng/mL) on the TEER of MKN28 cells. B, C The protein level of tight junction in MKN28 and GES-1 with recombinant CCL3 or CCL3 overexpressing macrophage medium. D IF detected the effect of recombinant CCL3 protein on the tight junction between MKN28 cells. DAPI was used for nuclear staining (blue), Occludin was used for tight junction staining (green), arrows showed the disruption of tight junctions between cells. E, F The RNA expression level of inflammatory factors in GES-1 with recombinant CCL3 protein or CCL3 overexpressing macrophage medium. G, H Apoptosis of GES-1 and MKN28 with recombinant CCL3 protein or CCL3 overexpressing macrophage medium. I, J Proliferation of GES-1 and MKN28 with recombinant CCL3 protein or CCL3 overexpressing macrophage medium. Abbreviations: PV, pcDNA vector; IF, Immunofluorescence. The data are presented as the mean ± S.D. after triplicate. Two groups were compared by t-test, multiple groups were compared by one-way analysis of variance (ANOVA). Note: *P < 0.05, **P < 0.01