Study On The Anti-Inflammatory Effect Mediated Of A Novel Oxicam Non-Steroidal Compound XK02 In Regulating NF-κB Signaling Pathway

Objective:Non-steroidal anti-inflammatory drugs（NSAIDs）are widely used for treatment of inflammation.Among them,oxicams show better curative effects and smaller side effects.The mechanisms underlying anti-inflammatory effects of NSAIDs is to inhibit the production pro-inflammatory mediators such as IL-1βby suppressing cyclooxygenase activity,thereby directly inhibiting the development of inflammatory response.The purpose of this study is to investigate the regulation of NF-κB signaling pathway in the new oxicam non-steroidal anti-inflammatory drug XK02 mediated anti-inflammatory activity,and to verify its anti-inflammatory activity by establishing an inflammatory model of ear swelling and foot swelling in vivo.Methods:The safe concentration range of XK02 and Meloxicam were detected by measuring cell viability via MTT assay.The experiment was divided into four groups,namely the blank control group,the model group,the XK02 group and the Meloxicam group.The in vitro inflammatory model was established by lipopolysaccharide（LPS）stimulation,the content of Nitrogen oxide（NO）was detected by Griess method,and the expression of cyclooxygenase,NF-κB pathway protein,and inflammatory factors were detected by ELISA,RT-PCR,and Western Blot.Knock down the expression of IκBαgene by si RNA transfection,or treat the cells with different concentrations of XK02,or XK02 combined with si RNA transfection,and then detect the content of related proteins and inflammatory factors on the above cells.Through gavage treatment with different concentrations of XK02,observe the swelling of the ears and feet of the mice,calculate the swelling degree and its inhibition ratio to the swelling,and perform H&E staining on the ears and plantar tissues of the mice at the same time.The content of inflammatory factors in the serum is detected by ELISA.Results:XK02 reduced the content of NO in cells,m RNA and the release of inflammatory factors of IL-1β,IL-6,TNF-αwere also significantly reduced in an XK02 dose-dependent manner by ELISA and RT-PCR.Regarding the influence of the inflammatory factor COX-2,the IC₅₀ values of XK02 and Meloxicam are 37.02μg/m L and 36.33μg/m L,respectively,indicating that XK02 and Meloxicam have equivalent inhibitory effects on COX-2.In terms of NF-κB signaling pathway,the expression of total protein of IκBαand p65 remained unchanged by Western Blot,but the level of phosphorylated protein was significantly down-regulated by XK02,which further blocked the activation of NF-κB signaling pathway.Knocking down IκBαcan enhanced XK02（100μg/m L）mediated down-regulation of the phosphorylation of IκBαand p65 and inhibited the transcription levels of IL-1β,IL-6,and TNF-α.In addition,in vivo experiments showed that XK02 reduced the swelling of ear swelling and foot swelling in disease model mice,inhibited the infiltration of inflammatory cells in mouse ears and foot tissues,and reduced serum inflammatory factors concentration.Conclusion:Both in vivo and in vitro experiments have confirmed that XK02 has anti-inflammatory effects,which can reduce the content of inflammatory factors of IL-6.The mechanism of action may be to down-regulate IκBαand block the NF-κB signaling pathway,resulting in anti-inflammatory effects.