Anti-inflammatory Constituents Of Murraya Paniculata Ameliorated Sepsis Through Regulation Of Macrophage Functions

Monocyte-macrophages were essential components of innate immunity and played a central role in clearance of pathogen, anti-tumor, host defense and tissue repair. Monocyte-macrophages were drived from myeloid stem cells and were characterized by considerable diversity and plasticity. Macrophages were the first line of defense against invading pathogens; however, macrophages might be various phenotype in response to micro-environmental signals. Macrophage polarization was divided into two main groups according to the function phenotype. IFNs/Toll like receptor pathway or IL-4/TL-13pathway induced classical activated macrophages (M1macrophages) and alternative activated macrophages (M2macrophage), respectively. In view of the powerful functions of macrophages and their important roles in the immune process, designing small molecular drugs which regulating macrophage polarization might be the potential strategies for infectious diseases, inflammatory diseases and cancer therapy.In Chapter one, we reviewed the connection among macrophage polarization, inflammasome, autophagy and their roles in inflammatory diseases. Based onthis, we began to study the small molecule compounds which might regulate macrophage function, and further studied their mechanisms.In Chapter two, we screened the anti-inflammatory constituents of murraya paniculata and found that MurO4, a kind of flavonone, showed the inhibitory role in macrophages. MurO4had no to xico logical effects on resting Raw264.7and peritoneal macrophages. Otherwise, this compound could inhibit IL-1beta and IL-6mRNA in LPS-activated Raw264.7cells. We had established macrophage polarization model on Raw264.7, BMDM and THP-1cells, and then we determined the effect of MurO4 on macrophage polarization. Our results showed that MurO4inhibited the Ml-associated pro inflammatory cytokines IL-1beta, IL-6, TNF-alpha, NO, down-regulated M1-macrophages marker CDllc. On the other hand, MurO4increased the M2-associated cytokines Fizzl, Yml, CD206mRNA level. Further study showed that, MurO4down-regulated STAT1/IRF5pathway, and up-regulated STAT6/SOCS1/IRF4pathway. We also established M1/M2intermediate macrophage polarization model on Raw264.7cells. MurO4inhibitd the IL-1beta, IL-6mRNA, at the same time, enhanced CD206mRNA in this model. This indicated that MurO4might induce the M1/M2switch. To assess the anti-inflammatory activity of MurO4in vivo, we examined the effect of MurO4on LPS and cecal ligation and puncture induced septic shock in mice. MurO4significantly improved the survival rate of mice, decreasd serum IL-1beta, IL-6, inhibits the lung and liver damage.Caspase-1activated in Ml macrophages by self-cleavage that occured in the inflammasome complex. We had proved that MurO4inhibited M1polarization, so we detected the impact of MurO4on the activation of the inflammasome. Our results indicated that MurO4significantly inhibited IL-1beta secretion and caspase-1activation. Evidence now indicated that autophagy negative regulates inflammasome. Considering these, we detected the effect of MurO4on autophagy. We found that MurO4enhanced the expression of Beclinl, LC-3BII and MDC staining.Autophagy, an evolutionarily conserved cellular process, facilitated the turnover of damaged proteins and organelles such as mitochondrial. Autophagy and innate or adaptive immunity pathway intersected at many different levels, but the exactly mechanism between autophagy and TLR pathway was still controversy. Autophagy negative regulated inflammasome through the machinery clearance of damage mitochondria.In Chapter three, we further determined the relationship among macrophage polarization, autophagy and inflammasome. We have found that M1-macrophage had more autophagy:more expression of LC-3BⅡ and more autophagic vacuole. We constructed a Beclinl interference THP-1cell line (ATG6-siRNA) to explore the relations between autophagy and macrophage polarizatioa Our results showed that autophagy negative regulated M1-polarization. Otherwise, the inhibition of MurO4on inflammasome was attenuated on ATG6-siRNA cells, which meant that the effects of MurO4on inflammasome was mediated by autophagy. Then we focused on the relationship between autophagy and macrophage polarization. We had found that MurO4down-regulated the expression of IFR5leading to the inhibition on Ml polarization, which indicated that the regulation of MurO4on macrophage polarization was depended on autophagy.The mainly protein degradation pathway was mediated by ubiquitination. Our results also showed that M1-macrophage had more ubiquitination as well as autophagy. And, MurO4enhances the ubiquitination of IRF5in M1macrophage. What’s more, the effect of MurO4on ubiquitination of IRF5was blocked in ATG6-siENA Ml macrophage compared withNC-siRNA. So we could conclude that MurO4increased IRF5ubiquitination through autophagy.In summary, our findings suggest that MurO4inhibited inflammasome and M1-polarization through enhancing autophagy. In addition, MurO4significantly ameliorated LPS and CLP-induced septic shock in vivo. The research on the mechanism of MurO4revealed that autophagy inhibited macrophage M1polarization via IRF5ubiquitination. This provided a new mechanism on the regulation of autophagy and macrophage polarization. However, there were some shortcomings that the specific molecular mechanisms of ubiquitination and degradation of IRF5were insufficient, and need further experiments to confirmed.