Study On Anti-inflammatory Activity Of A Novel Oxicam Nonsteroidal Drug XK01 In Vitro

OBJECTIVE:Inflammation is one of the most common diseases.Traditional non-steroidal anti-inflammatory drugs have gastrointestinal or cardiovascular adverse reactions during clinical use,which limits their use and development.Oxicams have good anti-inflammatory effects,such as meloxicam,but considering the long-term use and the selectivity to cyclooxygenase 2 of the drug may cause cardiovascular problems.Therefore,to develop new non-steroidal compounds has practical significance.The purpose of this study was to evaluate the anti-inflammatory activity and clarify the underling mechanism of the novel nonsteroidal drug XK01 by constructing an cell inflammation model in vitro,and to provide a new experimental basis for the development of new nonsteroidal drugs.METHODS:Establishing inflammatory cell model of mouse mononuclear macrophages RAW264.7 induced by lipopolysaccharide.Experimental cells were divided into control group,LPS induction group,drugs groups(XK01 with different concentrations groups,and meloxicam group).Firstly,the viability of RAW264.7 cells with different concentrations of drugs were detected by the MTT method,to determin the concentration of the drug used in subsequent experiments;the NO content of the supernatant of RAW264.7 cells was measured by the Griess;ROS production was measured by ROS kit;Use ELISA kit and RT-PCR to detect the content of inflammatory mediators such as TNF-α,IL-1β and IL-6 and their transcription expression levels;Western Blot and RT-PCR experiment to detect the related protein expression;Finally,Western Blot and Immunofluorescence experiments were used to detect NF-κB pathway proteins such as p-p65 and p-IκBα,and the MAPK signaling pathway proteins such as ERK,p38 and JNK total protein and their phosphorylated proteinsRESULTS:The viability of RAW264.7 cells with different concentrations of drugs were detected,and XK01 with a concentration of 0±100μg/ml was selected for subsequent experiments.Different concentrations of XK01 can reduce the content of NO,clear ROS,and inhibit the transcription levels of TNF-α,IL-6,and IL-1β in cells induced by LPS in a dose-dependent manner.XK01 has no significant inhibitory effect on COX-1,but it inhibits the expression of COX-2 mRNA and protein to achieve anti-inflammatory effect.In addition,about signaling pathways,XK01 prevents the transfer of NF-κB p65 protein from the cytoplasm to the nucleus,it also inhibits the phosphorylation of p65,IκB,and MAPKs proteins,and high concentration of XK01 inhibits p-JNK and p-ERK has stronger effect than meloxicam.CONCLUSION:The new oxicam nonsteroidal drug XK01 can inhibit the expression of inflammatory mediators and cyclooxygenase 2 in cell inflammation model.Its anti-inflammatory mechanism may be related to two signaling pathways of NF-κB and MAPK.This study provides experimental basis for exploring the anti-inflammatory activity and mechanism of the novel oxicam nonsteroidal drug XK01,which can be used as a theoretical basis for further development.