Based Reporter Gene And Nuclear Factor-¦Êb Signaling Pathway Of The Anti-inflammatory Compound Screening And Anti-inflammatory Effects Of Three Active Compounds And Mechanism Study

During the past decade,more and more traditional herbal medicines were claimed to have both anti-inflammatory and immuno-modulatiory activities and have gained widespread popularity. Generally,however,neither the active principles of such medicine nor their molecular targets have been well defined.Therefore,to establish a screening platform for evaluating the activity of herbal medicines on anti-inflammation and immuno-modulation,especially using the technology based on molecular biology and cell biology which is more broadly accepted,is a very important strategy to research and develop herbal medicines.Nuclear factor(NF)-κB is an important transcription factor complex that regulates the expression of many genes involved in immune and inflammatory responses,including cytokines,chemokines,receptors and so on.Thus,the establishment of a screening assay specific for NF-κB activity and to discover potential compounds targeting NF-κB signal pathway will be one of the promising ways to the prevention and therapy against inflammation and cancer.In the present study,NF-κB sequence,reporter gene SEAP and selection gene for neomycin resistance in plasmid pNF-κB-SEAP-NPT were transfected into mouse RAW 264.7 macrophages,therefore,NF-κB activity was able to be evaluated by SEAP expression in the transfected cells.Based on pNF-κB-SEAP-NPT/ RAW 264.7 cells,a screening assay was established by which known NF-κB stimulator lipopolysaccharide(LPS)was demonstrated to activate the NF-κB activity in dose and time dependent manners.This assay is characterized of stable,specific and efficient system.In this assay system,61 compounds were investigated on their effects on LPS-induced NF-κB activation.Several compounds were found to have inhibitory effects on LPS-induced NF-κB activation.NO is one of the markers in inflammation.In order to screen for potential anti-inflammatory compounds,the effects of those compounds on LPS-induced NO production were investigated in LPS-stimulated RAW 264.7 macrophages.Further studies on anti-inflammatory activity and mechanisms were performed on those selected compounds which can inhibit LPS-induced NF-κB activation and NO production.4-Methoxyhonokiol isolated from Magnolia obovata and 21(α,β)-methylmelianodiol isolated from Poncirus trifoliate Rafinesque were found to have significant inhibitory effects on LPS-induced NF-κB activation and NO production.Therefore,the in vivo anti-inflammatory effects of 4-methoxyhonokiol and 21α-methylmelianodiol(21α-MMD)were examined in three mouse models of acute inflammation,and then the mechanisms responsible for the effects were investigated in LPS-stimulated RAW 264.7 macrophages by using cell culture,spectrophotofluorimetry,ultraviolet spectrophotometry,Western blot and RT-PCR techniques. Increased vascular permeability is one of the essential features of the acute inflammatory response, so vascular permeability assay is a typical in vivo model of first-stage inflammatory reactions. Carrageenan-induced paw edema,another classical model of acute inflammation,has been widely used on the study of anti-inflammatory agents.Therefore,in our study,both of acetic-acid-induced vascular permeability assay and carrageenan-induced paw edema assay were performed to investigate whether 4-methoxyhonokiol and 21α-MMD have in vivo anti-inflammatory effects.The results showed that 4-methoxyhonokiol and 21α-MMD,at the doses of 20 and 100 mg/kg(i.p.), significantly inhibited dye leakage and paw swelling in vascular permeability assay and carrageenan-induced paw edema assay,respectively.In LPS-induced systematic acute inflammation model in mice,pretreatment of mice with 4-methoxyhonokiol(20 mg/kg and 100 mg/kg,i.p.)or 21α-MMD(5 mg/kg and 20 mg/kg,i.p.)inhibited plasma nitrite/nitrate production.These results indicate that 4-methoxyhonokiol and 21α-MMD have potential in vivo anti-inflammatory activity.The productions of NO and prostaglandin-E₂(PGE₂)are mainly regulated by the expression and/or the activity of iNOS and COX-2,respectively.Overproduction of NO and PGE₂ is involved in chronic inflammation and cancer.Therefore,the suppression of NO and PGE₂ production by inhibition of iNOS and COX-2 expression and/or enzyme activity can be a very important therapeutic target in the development of anti-inflammatory agents.In LPS-stimulated RAW 264.7 macrophages,1-30μM of 4-methoxyhonokiol and 21α-MMD were demonstrated to inhibit NO and PGE₂ production in a concentration-dependent manner.The inhibitory effects were consistent with their inhibitory effects on the protein and gene expressions of iNOS and COX-2.These data suggest that 4-methoxyhonokiol and 21α-MMD can decrease LPS-induced NO and PGE₂ production by down-regulating iNOS and COX-2 expression,and this effect happens at the transcription level. The expression of the iNOS and COX-2 genes in macrophages is under the control of several transcription factors,which include NF-κB.To investigate the molecular mechanisms involved in 4-methoxyhonokiol,21α-MMD and 21β-MMD-mediated inhibition of iNOS and COX-2 expression,NF-κB transcriptional activity were investigated using a SEAP reporter gene assay system.Then the phosphorylation and degradation of IκB-αwere tested by western blot analysis. The results showed that the three compounds significantly inhibited LPS-induced NF-κB transcriptional activity via the prevention of inhibitorκB(IκB)phosphorylation and degradation.The MAPK pathways are well known to be involved in NO and several pro-inflammatory cytokine productions in LPS-stimulated macrophages.In order to further investigate the pathways involved in the inhibition of 4-methoxyhonokiol,21α-MMD and 21β-MMD on NO production, their effects on LPS-induced MAPKs phosphorylation were studied.The results showed that 4-methoxyhonokiol,21α-MMD and 21β-MMD had no effect on the LPS-induced phosphorylation of extracellular signal-regulated kinase(ERK),whereas it attenuated the phosphorylation of p38 mitogen-activated protein kinase(p38 MAPK)and c-Jun NH₂-terminal kinase(JNK)in a concentration-dependent manner.These results suggest that besides NF-κB inactivation, 4-methoxyhonokiol,21α-MMD and 21β-MMD might inhibit iNOS gene expression via an additional mechanism of JNK and p38 MAPK inhibition in LPS-stimulated RAW 264.7 macrophages.Taken together,our results demonstrate for the first time that 4-methoxyhonokiol and 21α-MMD have potential anti-inflammatory activity both in in vivo and in vitro models.It is suggested that 4-methoxyhonokiol and 21(α,β)-methylmelianodiol is active anti-inflammatory constituent of the bark of M.obovata and P.trifoliate,respectively.The data in the present study support the pharmacological basis of the use of M.obovata and P.trifoliate as traditional herbal medicines for the treatment of inflammation.