The Establishment Of Cell Sensing Method For Inflammasome And The Study Of Intervention Mechanism Of Glabridin

With the improvement of people's living standard,unhealthy diet and living habits make obesity and metabolic disorders more and more serious,which is a great threat to people's health.It is found that inflammation plays an important role in the development of obesity and metabolic disorders.The introduction of anti-inflammatory intervention is of great significance.NLRP3 inflammasome,as a key node of inflammatory response,plays an important role in obesity,insulin resistance and other metabolic disorders.It is a new idea to control the metabolic disorders with the target of NLRP3 inflammasome.However,there is still a lack of a method or tool for efficient screening of interferes with NLRP3 inflammasome.In this context,a fluorescent cell sensor that reflects the activation of NLRP3 in real time had been established and applied to the screening of NLRP3 inhibitors,and the anti-inflammatory mechanism of polyphenols was discussed from the metabolic angle.First,the cell model stimulated by lipopolysaccharide was established in this paper to evaluate and compare the anti-inflammatory effects of 3 polyphenols on the targets of proinflammatory cytokines such as interleukins-6(IL-6),tumor necrosis factor-a(TNF-a)and interleukin-1beta(IL-1?).The results showed that: glabridin can restrain the NO accumulation,TNF-? production,IL-6 production and IL-1? production,but the effect of phloretin and eugenol was not as good as glabridin.100 ?mol/L eugenol although had a good effect on controlling the IL-6,but that on NO accumulation and IL-1? gene production was the worst,and the effect of phloretin was between glabridin and eugenol.So glabridin has the largest potential application in anti-inflammatory effect than the other two.In order to meet the needs of efficiently screening of NLRP3 inhibitors,a real-time fluorescent cell sensor capable of reflecting NLRP3 activation in real time was established.The core of NLRP3 promoter fused with green fluorescent protein(GFP)was attached to plasmid pHBLV-CMVIE-EF1-Puro.Then we used lentivirus to infect THP-1 cells to put the plasmid in the cells.In this cell sensor,green fluorescence emission is under the control of NLRP3 promotor,that is green fluorescence can indicate the activation of NLRP3 receptor.We used high content screening to capture fluorescence images of RAW 264.7 cells stimulated by LPS,and found that fluorescence intensity reached the maximum value at 9 h.This result was consistent with the up regulation of NLRP3 gene and protein,indicating that the fluorescence cell sensor was successfully constructed.We applied this cell sensor into screening of NLRP3 inhibitor with polyphenols and we found phloretin,glabridin and eugenol had inhibitory effect.Finally,we verified the inhibitory effect of glabridin on NLRP3 inflammasome and preliminarily studied its mechanism.It proves that this sensor can be applied to screen NLRP3 inhibitors.We provides a new method for anti-inflammatory research targeting NLRP3 inflammasome.On the basis of previous studies,we used metabolomics to explore the anti-inflammatory mechanism of glabridin.We selected RAW 264.7 cells and the groups are control group,250 ng/m L LPS stimulation group(LPS group)and glabridin intervention group(intervention group).After collecting extracting metabolites and derivatization,GC-MS was used to analysis the metabolites.We used principal component analysis(PCA),orthogonal partial least squares discriminant analysis(OPLS-DA)analysis and heatmap to analysis the data.We found that the clusters of control groups and intervention groups are more closer and that of LPS is far away from the others.Compared with control groups,27 metabolites were changed significantly in LPS groups and 23 of them were reversed in intervention groups.We also analyzed metabolic pathway changes among three groups.We can conclude that metabolic changes caused by LPS can fall into the these three disorder categories:(a)amino acid and protein disorder and lipid metabolism disorder;(b)fuel and energy metabolism disorder;(c)inflammation and oxidative stress metabolism disorder.Glabridin co-treated with LPS significantly reversed the metabolism changes of LPS in RAW 264.7 cells.Glabridin played its corrective role mainly by impacting on amino acid metabolism and energy metabolism as well as by reversing the LPSeffects on inflammatory process-related metabolites.In this chapter,we further expand the mechanism of cells' s proinflammatory response to LPS,and provide a new idea for the research of anti-inflammatory mechanism of glabridin and other anti-inflammatory substances.