| Part 1 Expression and interaction of ERS and TLR4 in experimental colitis in miceIntroduction:In this part,Trinitro-benzene-sulfonic Acid(TNBS)was used to form experimental colitis in mice to explore the expression of Endoplasmic Reticulum Stress(ERS)and Toll-like receptor 4(TLR4)pathway related proteins and the relationship between the two pathways in IBD mice.Method:The IBD model of Balb/c mice was induced by TNBS and treated with Normal Saline(NS)or endoplasmic reticulum stress inhibitor TUDCA.The mice were divided into normal control group,TNBS + NS group and TNBS +TUDCA group.The disease activity index(DAI)of mice was scored every day since the establishment of the model.Seven days after the establishment of the model,the obvious inflammatory lesions of colon in each group were taken for histopathological score by HE staining,and the expression of ERS marker GRP78 and TLR4 pathway junction protein TLR4 was detected by immunohistochemistry.Then the results were compared between groups.Results:1.Score of DAI and colon histopathology in TNBS+NS group was remarkably greater than those in normal control group.While the score of DAI and colon histopathology in TNBS+TUDCA group was significantly lower than those in TNBS+NS group.2.Compared with the normal control group,the expression of GRP78 and TLR4 in colon tissue of TNBS+NS group was significantly increased,while ERS inhibitor could down-regulate the expression of GRP78 and TLR4.Conclusions:In IBD mouse model,endoplasmic reticulum stress and TLR4 pathway were involved in the formation of experimental colitis in IBD mice,while inhibiting ERS,TLR4 pathway was also inhibited,which alleviated intestinal inflammation.Part 2 Effects of ERS and TLR4 related pathways on inflammatory cytokines and its mechanism in THP-1 monocytesIntroduction:In this part,the effects of the interaction between ERS and TLR4 pathway on the expression of inflammatory cytokines TNF-α,IL-1 β and IL-8in THP-1 monocytes were verified by cell experiments in vitro,and the possible molecular mechanisms were discussed.Method:1.We cultured THP-1 monocytes.ERS was induced by different concentrations of ERS inducer Tunicamycin or Thapsigargin while TLR4 pathway was activated by lipopolysaccharide(LPS).ERS was inhibited by pretreatment with or without ERS inhibitor TUDCA for 1 h.The mRNA transcription levels of inflammatory factors TNF-α,IL-1β and IL-8 were detected by q RT-PCR,their protein expression levels were detected by ELISA,and Phosphorylated MAPK proteins(p-JNK,p-ERK1/2 and p-P38)and Phosphorylated NF-κB protein(p-NF-κB)were detected by WB.2.We cultured THP-1 monocytes.ERS was induced by different concentrations of ERS inducer Tunicamycin or Thapsigargin,Proteins involved in TLR4/MyD88-dependent pathway(TLR4,MyD88,IRAK-4,IRAK-1,IRAK-2 and TRAF6)were detected by WB.3.THP-1 monocytes were cultured.On the basis of the up-regulated expression of p-P38 protein detected by WB,pretreated with different altered of P38 MAPK inhibitor(SB202190)for 1 h,the P38 MAPK pathway was inhibited.The mRNA transcription levels of TNF-α,IL-1β and IL-8 were detected by q RT-PCR,and their protein expression levels were detected by ELISA.Results:1.Co-treatment with ERS inducer and LPS significantly up-regulated the expression of inflammatory cytokines TNF-α,IL-1β and IL-8,while ERS inhibitor TUDCA could down-regulate the expression of TNF-α,IL-1βand IL-8 during co-treatment.2.When ERS was invited by ERS inducer,there was no effect on the expression of TLR4/MyD88-dependent pathway-related proteins,including TLR4,MyD88,IRAK-4,IRAK-1,IRAK-2 and TRAF6.3.Co-treatment of ERS inducer and LPS up-regulated the expression of p-P38 protein,and P38 MAPK inhibitor SB202190 significantly inhibited the mRNA transcription and protein expression of inflammatory cytokines TNF-α,IL-1 β and IL-8 induced by LPS.Conclusions:ERS inducer and LPS may sympathetically advance the production of inflammatory cytokines in THP-1 cells by activating P38 MAPK pathway,but this pathway may not activate through TLR4/MyD88 pathway. |
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