| Background:A series of 4-substituted-benzoxazolone derivatives were synthesized in our research,and the majority of compounds exhibited potent anti-inflammatory effects through the primary screening.4-o-methyl-benzenesulfonyl-2?3H?-benzoxazolone?MBB?,3-?2'-phenyl?ethyl-4-?2'-phenyl?ethoexy-benzoxazolone?W6I?and 4-?5'-dimethylamino?-naphthalenesulfonyl-2?3H?-benzoxazolone?W3D?showed the best anti-inflammatory activities.However,the anti-inflammatory mechanisms and the target are still unclear.Objective:The aim of the study is to evaluate the anti-inflammatory effects of the novel 4-substituted-benzoxazolone derivatives on LPS-induced RAW264.7 cells and to discuss the possible mechanism and target of the compound W3D.Method:Part?:Anti-inflammatory activity of 4-substituted-benzoxazolone derivatives MBB,W6I and W3D1.The cell viability of MBB,W6I and W3D on RAW264.7 were detected by MTT assay.2.The RAW264.7 cells were treated with LPS and a series of 4-substituted- benzoxazolone derivatives with different concentrations,then the expression of NO in the cell supernatant was analyzed with Griess reagent,the contents of TNF-?,IL-6,IL-1?and COX-2 were detected using ELISA method.3.The anti-inflammatory activities in vivo of W3D,W6I and MBB were evaluated by the xylene induced ear swelling model.Part?:Regulation of the compound W3D on TLR4-MyD88-NF-?B signal pathway in LPS-induced RAW264.7 cells1.The RAW264.7 cells were cultured with different concentrations W3D and LPS. The protein expression of TLR4,MyD88,p-IRAK4 and NF-?B were examined by Western blot.2.The RAW264.7 cells were treated with with different concentrations of W3D and LPS.The protein expressions of p-P38 and p-ERK were examined by Western blot.3.The RAW264.7 cells were treated with different concentrations of W3D and LPS. The protein expression of IL-6 and iNOS were examined by Western blot.4.The RAW264.7 cells were cultured with different concentrations of W3D and LPS. The mRNA expressions of TLR4,MyD88 and NF-?B were investigated using fluorescent quantitation PCR?FQ-PCR?.Part?:Preliminary anti-inflammatory target study of the compound W3D in LPS-induuced RAW264.71.To clarify the anti-inflammatory target of 4-substituted-benzoxazolone derivatives, the structure-activity relationship of TLR4 was studied.The impact of the 4-substituted-benzoxazolone derivatives W3G,MBB,W3E,W3A,W6I and W3D on the protein expression of TLR4,p-NF-?B in LPS-induced RAW264.7 cells were analyzed by Western blot.2.The interference effect of W3D on the connection between FITC-LPS and RAW264.7 cells was tested by Flow cytometry.3.The specific TLR4 inhibitor TAK242 was used to investigate the effect of W3D on TLR4-NF-?B signaling pathway.The content of NO,IL-1?,IL-6,TNF-?in supernatant was analyzed with Griess reagent,and the protein expression of TLR4,p-IRAK4,p-NF-?B were measured by the Western blot.Part?:Research on RNA-seq transcriptome to analyze the effects of rhe compound W3D on LPS-induced RAW264.7 cells1.To study the effect of W3D on LPS-induced RAW264.7 cells at the gene level,DEGs?differential expression genes?werescreened byRNA-seq. Molecular function,biological processes,and cellular components of these DEGs were investigated with GO program.Furthermore,pathway enrichment analysis of the DEGs were analyzed based on KEGG database.2.The expressions of major DEGs were verified by FQ-PCR method.Result:1.The compound W3D,W6I and MBB has no significant cytotoxicity on RAW264.7 cells within the selected concentration range of 0 to 100?mol·L-1.They could decrease the release of NO,IL-1?,IL-6,TNF-?and iNOS in the cell supernatant induced by LPS in a dose-dependent manner,and they also presented inhibitory activity aginst xylene induced mouse ear welling.Among these compounds,the compound W3D showed the best ancitvity.NO inhibitory effect of compound W3D was equivalent with celecoxib at the dosage of 25?mol·L-1,and it showed the stronger inhibitory activity against IL-6 than celecoxib.Meanwhile,the compound W3D showed equivalent activity with celecoxib.2.The compound W3D could obviously inhibited LPS-induced TLR4,MyD88,p-P38, p-ERK,IL-6,iNOS production and the NF-?B transportation to the nucleus in a dose-dependent manner?p<0.01?.FQ-PCR result showed that W3D could significantly reduce the mRNA expression of TLR4 and MyD88,but had no effect on NF-?B.3.4-substituted-benzoxazolone derivatives W3G,MBB,W3E,W3A,W6I and W3D had various degrees of inhibitory effects on LPS-induced TLR4 and p-NF-?B protein expression,and this result was coincidence with anti-inflammatory activity of preliminary screening.4.Flow cytometry result showed that the compound W3D could interrupt the connection between FITC-LPS and cell membrane TLR4 of RAW264.7 cells.5.Compared with LPS group,the TLR4 specific repressor TAK-242 could partially counteract the NO,IL-1?,IL-6,TNF-?inhibitory activity induced by the compound W3D and the TLR4,p-IRAK4 and p-NF-?B protein expression inhibitory activity of W3D.6.The RNA-seq results showed that 9021 genes were down-regulated and 8968 genes were up-regulated in the LPS group compared with control group.Compared with LPS group,there were 8650 up-regulated genes and 9253 significant down-regulated genes in compound W3D(25?mol·L-1)group.GO analysis indicated that these genes were mainly related to functions such as cell apoptosis,cell metabolism and the immune stress response.KEGG pathway analysis showed that these genes were involved in multiple signal pathways,such as MAPK signaling pathway,TNF-?signaling pathway,Toll-like receptor signaling pathway.The FQ-PCR result was concordant with the sequencing result.Conclusion:1.The novel 4-substituted-benzoxazolone derivative W3D could significantly inhibit LPS-induced RAW264.7 inflammatory response.W3D displayed obvious anti-inflammatory activity on the xylene-induced mouse ear swelling.2.The novel 4-substituted-benzoxazolone derivative W3D could exert anti-inflammatory effect in LPS-induced RAW264.7 cells by regulating of TLR4-MyD88-MAPK-NF-?B signal pathway.3.The results suggested that TLR4 was likely to be an ideal candidate of anti-inflammatory target of the compound W3D. |
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