Study Of Inhibitory Effect Of Benzoxazole Derivative K313 On Inflammation And Mechanism Of Macrophages

Inflammation is a normal defensive reaction to various pathogenic factors in the living tissue of body,and excessive inflammatory reactions can induce occurrence of diseases.Nonsteroidal anti-inflammatory drugs(DSAIDs)are currently widely applied anti-inflammatory drugs for inflammatory diseases,but the types of drugs for patients to choose are limited and long-term high-dose use is likely to cause gastrointestinal side effects.Therefore,it is necessary to explore more effective and less side effects of new anti-inflammatory drugs,so as to provide more medication options for patients and expand the scope of clinical medication.Benzoxazole derivatives are one of anti-inflammatory fields in the development of new drugs,because of its wide clinical application value.Currently,many benzoxazole derivatives drugs have been applied in clinical and scientific research.Through establishing small molecule screening platform,our laboratory found that K313 had anti-inflammatory activity among selecting 4 kinds of bioactive small molecule derivatives.However,the anti-inflammatory effect and mechanism of K313 have still not been studied.Therefore,this topic will focus on exploring the anti-inflammatory properties and mechanism of benzoxazole compound K313,which will lay the foundation for the discovery of new anti-inflammatory drugs.Objective By establishing an inflammatory cell model,the anti-inflammatory activity of benzoxazole compound K313 were explored to discover and synthesize new lead compounds.Moreover,exploring anti-inflammatory mechanism of K313,the signaling pathway and potential targets of K313 were revealed,and to provide new ideas for the selection of anti-inflammatory drug targets.Methods Part Ⅰ Establishing and optimizing inflammation model for LPS-induced macrophages(1)Detecting and plotting the growth curve of RAW264.7 macrophages with CCK-8method;(2)Detecting NO production with Griess reagent in LPS-induced macrophages to determine the suitable time(0-48 h)and concentration(0-1 μg/m L)of LPS.Part Ⅱ K313 inhibited inflammatory cytokines in LPS-induced RAW264.7 macrophages(1)NO production in LPS-induced RAW264.7 macrophages was used to screen small molecule compounds(K296,K310,K313,K317)with Greiess Reagent;(2)CCK-8 method was used to detect the effect of K313 on macrophage viability;(3)Greiess Reagent and 3-Nitrotyrosine(3-NT)Enzyme-Linked Immunosorbent Assay were respectively used to detect the effect of K313 on NO and 3-NT production in LPS-induced RAW264.7 macrophages;(4)RT-PCR was used to detect the effect of K313 on m RNA levels of i NOS,IL-6,COX-1,COX-2 and TNF-a in LPS-induced RAW264.7 macrophages;(5)ELISA was used to detect the effect of K313 on IL-6 and TNF-a inflammatory cytokines in LPS-induced RAW264.7 macrophages.Part Ⅲ Studying the anti-inflammatory mechanism of K313 in LPS-induced RAW264.7 macrophages(1)Western Blot was used to detect the effect of K313 on LPS-induced NF-κB,MAPKs(ERK1/2,p38 MAPK)and AKT inflammatory signaling pathways in RAW264.7 macrophages;(2)Western Blot was used to detect the effect of K313 on GSK-3β signaling protein in LPS-induced RAW264.7 macrophages.Results Part Ⅰ Establishing and optimizing inflammation model for LPS-induced macrophages(1)Detecting and plotting the growth curve of RAW264.7 macrophages,and to confirm that the inoculation density of inflammatory model was 5×105/m L;(2)Detecting NO production at 0-48 h in LPS-induced RAW264.7 macrophages.These results showed that NO production at 24 h rapidly increased and reached the plateau,and confirmed the stimulation time of LPS for 24 h;(3)Detecting LPS(0-1μg/m L)induced NO production.The results showed that NO production reached a plateau when LPS at 100 ng/m L induced RAW264.7macrophages,and confirmed the stimulation concentration of LPS for 100 ng/m L.Part Ⅱ K313 inhibited inflammatory cytokines in LPS-induced RAW264.7 macrophages(1)Detecting the effects of four small molecule compounds(K296,K310,K313,K317)on NO production in LPS-induced RAW264.7 macrophages.These results showed that K313 significantly inhibited NO production in a dose-dependent manner,confirming in following study to explore the anti-inflammatory effect and mechanism of K313;(2)Detecting the cytotoxic effects of LPS and K313.These results showed that no cytotoxic effect of K313(0-20 μM)on RAW264.7 macrophages and LPS-induced RAW264.7 macrophages,confirming in following experiment the highest concentration of K313 was 20 μM;(3)Detecting the effect of K313 on inflammatory genes and inflammatory cytokines in LPS-induced RAW264.7 macrophage.These results showed that K313(0-20μM)significantly inhibited the m RNA levels of i NOS,IL-6 and TNF-α,and inhibited NO,3-NT,IL-6 and TNF-α inflammatory cytokines;(4)Detecting the effect of K313 on LPS-induced COX-1 and COX-2 genes in RAW264.7 macrophages.These results showed that K313 inhibited COX-2 genes in a dose-dependent manner,and no effect of K313 on COX-1 genes.Part Ⅲ Studying the anti-inflammatory mechanism of K313 in LPS-induced RAW264.7 macrophages(1)Detecting the effect of K313 on NF-κB,MAPKs(ERK1/2,p38 MAPK)and AKT signaling pathway in LPS-induced RAW264.7 macrophages.These results showed that LPS-induced p-NF-κB p65,p-p38,p-ERK1/2 and p-AKT protein at 30 min were the highest expression,and K313 did not inhibit these phosphorylated proteins,confirming that anti-inflammatory properties of K313 were not regulated by NF-κB,MAPK,and AKT signaling pathways;(2)Detecting the effect of K313 on p-GSK-3β(Ser9)and GSK-3β in LPS-induced RAW264.7 macrophages.These results showed that K313 significantly enhanced LPS-induced p-GSK-3β(Ser9)protein to decrease of GSK-3β protein.ConclusionThe benzoxazole compound K313 significantly inhibited the production of NO,IL-6,TNF-α and COX-2 in the LPS-induced RAW264.7 macrophages model;its antiinflammatory mechanism is via increasing the phosphorylation levels of GSK-3β(Ser9)to decrease GSK-3β activation in LPS-induced RAW264.7 macrophages.