| Background and purpose:TLR4 is an important pattern recognition receptor that recognizes pathogen associated molecular patterns,triggers cell signaling cascades,and regulates immune responses.When the body is stimulated by external signals,HMGB1 can be released into the extracellular domain as a damage associated molecular pattern.HMGB1 is an endogenous ligand of TLR4,and the combination of extracellular HMGB1 and TLR4 can induce intracellular signal transduction and mediate inflammation.The effect of inhibiting TLR4 on HMGB1/TLR4 signaling pathway during H.pylori infection has not been reported,so this study aims to investigate the inhibitory effect of TLR4 signaling on HMGB1/TLR4 of gastric mucosa and cells infected with H.pylori,elucidate the interaction between TLR4 and HMGB1,and explore the pathogenic mechanism of H.pylori from a new perspective.Method:?.The effects of TLR4 antibody on the expression of HMGB1 and TLR4 protein in gastric mucosa of BABL/c mice infected with H.pyloriThe gastric mucosa specimens of BABL/c mice were divided into four groups:(1)Control group: BABL/c mice +IgG1(2)Anti-TLR4 group: BABL/c mice +anti-TLR4(3)Hp group: BABL/c mice +IgG1+H.pylori(4)Hp+Anti-TLR4 group: BABL/c mice +anti-TLR4+H.pylori?.The effects of TAK-242 on HMGB1/TLR4 signaling pathway in GES-1cells infected with H.pyloriThe experimental group were as follows:(1)Control group: GES-1 cells(2)Hp group: GES-1 cells+H.pylori(3)Low concentration of TAK-242 group:GES-1cell+0.5?M TAK-242+H.pylori(4)Medium concentration of TAK-242 group:GES-1cell+1.0?M TAK-242+H.pylori(5)High concentration of TAK-242 group:GES-1cell+0.5?M TAK-242+H.pyloriAccording to the above groups,GES-1 cells were pretreated with different concentrations of TAK-242 1 hour in advance and infected with H.pylori(MOI=100).The cells were collected after 24 hours of coculture.?.The effects of GES-1 cells infected with H.pylori by co-culture on the HMGB1/TLR4 signaling pathway of THP-1 cells,and role of TAK-242interventionGES-1 cells were pretreated with 1.0?M TAK-242 1 hour in advance and infected with H.pylori(MOI=100).Then GES-1 cells were co cultured with THP-1cells through Transwel systeml.Two cells and the co-culturing supernatant were collected after 24 hours.The experimental group were as follows:(1)Control group: GES-1 cells+THP-1 cells(2)TAK-242 group: GES-1 cells+ TAK-242+THP-1 cells(3)Hp group:GES-1 cells+ H.pylori+THP-1 cells(4)Hp+TAK-242 group:GES-1 cells+ TAK-242 + H.pylori+THP-1 cells?.Detection methods:(1)Immunohistochemistry was used to detect the protein expressions of HMGB1 and TLR4 in gastric mucosa of BABL/c mice.(2)Real-time PCR was used to detect the mRNA expressions of HMGB1,TLR4 and MyD88 in cells.(3)Western Blot was used to detect the protein expressions of TLR4?MyD88?pNF-?B p65 and HMGB1 in cells.(4)WesTM system was used to detect the The levels of HMGB1 in the co-culturing supernatant.(5)ELISA was used to detect the The levels of TNF-? and IL-10 in the co-culturing supernatant.Results:?.The effects of TLR4 antibody on the expression of HMGB1 and TLR4 protein in gastric mucosa of BABL/c mice infected with H.pylori?The protein expressions of TLR4 in gastric mucosa of BABL/c miceNo matter gland cells or inflammatory cells,there was no difference between Control group and Anti-TLR4 group,TLR4 expressions of Hp group and Hp+Anti-TLR4 group were significantly higher than Control group/Anti-TLR4 group(P<0.001 and P<0.001),TLR4 expressions of Hp+Anti-TLR4 group were lower than Hp group(P<0.01).?The protein expressions of HMGB1 in gastric mucosa of BABL/c miceNo matter gland cells or inflammatory cells,there was no difference between Control group and Anti-TLR4 group,HMGB1 expressions of Hp group were significantly higher than Control group/Anti-TLR4 group(P<0.001 and P<0.01),HMGB1 expressions of Hp+Anti-TLR4 group were lower than Hp group(P<0.05).?.The effects of TAK-242 on HMGB1/TLR4 signaling pathway in GES-1cells infected with H.pylori?The mRNA and protein expressions of TLR4 in GES-1 cellsTLR4 mRNA and protein expressions of Hp group were significantly higher than Control group(P<0.05).TLR4 mRNA expressions of Low TAK-242 concentration group were higher than the Hp group and the protein levels were lower than the Hp group,but there were no significant difference(P>0.05).TLR4 mRNA and protein expressions of Medium and High TAK-242 concentration group were lower than the Hp group,but only the differences of mRNA and protein expressions between Hp group and Medium TAK-242 concentration group were statistically significant(P<0.05).?The mRNA and protein expressions of MyD88 in GES-1 cellsMyD88 mRNA and protein expressions of Hp group were significantly higher than Control group(P<0.05 and P<0.01).MyD88 mRNA expressions of Low TAK-242 concentration group were higher than the Hp group and the protein levels were lower than the Hp group(P<0.05 and P<0.01).MyD88 mRNA and protein expressions of Medium and High TAK-242 concentration group were lower than the Hp group,but only the differences of mRNA and protein expressions between Hp group and Medium TAK-242 concentration group were statistically significant(P<0.05 and P<0.01).?The protein expressions of pNF-?B p65 in GES-1 cellsThe pNF-?B p65 protein expressions of Hp group were significantly higher thanControl group(P<0.01).The pNF-?B p65 protein expressions of all TAK-242 concentration group were higher than the Hp group(P<0.01),but only the difference of Medium and High TAK-242 concentration groups were statistically significant(P<0.05 and P<0.01).?The mRNA and protein expressions of HMGB1 in GES-1 cellsHMGB1 mRNA and protein expressions of Hp group were significantly higher than Control group(P<0.01 and P<0.05).HMGB1 mRNA expressions of Low TAK-242 concentration group were higher than the Hp group(P<0.01)and the protein levels were lower than the Hp group(P>0.05).HMGB1 mRNA and protein expressions of Medium and High TAK-242 concentration group were lower than the Hp group,but only the differences of mRNA and protein expressions between Hp group and Medium TAK-242 concentration group were statistically significant(P<0.01).?.The effects of GES-1 cells infected with H.pylori by co-culture on the HMGB1/TLR4 signaling pathway of THP-1 cells and role of TAK-242intervention?The protein expressions of TLR4 and HMGB1 in GES-1 cellsIn terms of TLR4 and HMGB1 protein expressions in GES-1 cells,Hp group was higher than Control group(P<0.01)and TAK-242 group was lower than Hp group(P<0.01).Control group and TAK-242 group has no difference(P>0.05).?The mRNA and protein expressions of TLR4 in THP-1 cellsIn terms of TLR4 m RNA and protein expressions in THP-1 cells,Hp group was higher than Control group(P<0.05)and TAK-242 group was lower than Hp group(P<0.01 and P<0.05).Control group and TAK-242 group has no difference in protein level(P>0.05).?The mRNA and protein expressions of MyD88 in THP-1 cellsIn terms of MyD88 mRNA and protein expressions in THP-1 cells,Hp group was higher than Control group(P<0.01)and TAK-242 group was lower than Hp group(P<0.05).Control group and TAK-242 group has no difference(P>0.05).?The protein expressions of pNF-?B p65 in THP-1 cellsIn terms of pNF-?B p65 protein expressions in THP-1 cells,Hp group was higher than Control group(P<0.01)and TAK-242 group was lower than Hp group(P<0.01).TAK-242 group was lower than Control group(P<0.01).?The mRNA and protein expressions of HMGB1 in THP-1 cellsIn terms of HMGB1 mRNA and protein expressions in THP-1 cells,Hp group was higher than Control group(P<0.01)and TAK-242 group was lower than Hp group(P<0.05 and P<0.01).Control group and TAK-242 group has no difference in protein level(P>0.05).?The level of HMGB1 in the co-culturing supernatantIn terms of HMGB1 level in the co-culturing supernatant,Hp group was higher than Control group(P<0.05)and TAK-242 group was lower than Hp group(P<0.01).Control group and TAK-242 group has no difference(P>0.05).?The level of TNF-? in the co-culturing supernatantIn terms of TNF-? level in the co-culturing supernatant,Hp group was higher than Control group(P<0.01)and TAK-242 group was lower than Hp group(P<0.01).TAK-242 group was lower than Control group(P<0.01).?The level of IL-10 in the co-culturing supernatantIn terms of IL-10 level in the co-culturing supernatant,Hp group was lower than Control group(P<0.05)and TAK-242 group was higher than Hp group(P<0.05).Control group and TAK-242 group has no difference(P>0.05).Conclusion:1 ? H.pylori can activate HMGB1/TLR4 signaling pathway and induce inflammation of the gastric mucosa.2 ? Inhibiting TLR4 can inhibit the activation of TLR4 signaling and down-regulate expression and secretion of HMGB1,suggesting that HMGB1 not only as the ligand of TLR4 to activate TLR4 signaling pathway,but also may be a downstream molecule of TLR4 signaling pathway.HMGB1 and TLR4 regulate each other and form a positive feedback loop,resulting in enlarge and strengthen of inflammation. |
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