| NLRP3(NACHT,LRR and PYD domains-containing protein 3)play an important role in the innate immune system and is an important intracellular pattern recognition receptor(PRR).The inflammatory complex formed by NLRP3 is mainly a macromolecular multiprotein complex composed of a nucleotide-binding oligomerization domain-like receptor family member NLRP3,a linker protein ASC,and an effector protein Caspase-1.When stimulated by exogenous microorganisms or endogenous danger signals,the NLRP3 core protein recruits the downstream adaptor protein ASC and the effector protein Caspase-1,assembles into an NLRP3 inflammasome,and mediates Caspase-1 activation.Intracellular activated caspase-1(also known as interleukin-converting enzyme)cleaves the proinflammatory cytokine precursors pro-IL-1β and pro-IL-18 into mature IL-1β and IL-18 and secretes them to the outside Activation of other immune cells induces more inflammatory factors,chemokines,and adhesion molecules to form a cascade of amplified inflammatory responses.Berberine,also known as berberine,is an alkaloid in Chinese medicine C optis chinensis,which has a wide range of anti-inflammatory effects.Previous studies have found that berberine can effectively reduce the mortality rate of infected mice and improve the inflammatory lesions of lung tissue in influenza virus-infected mouse model of viral pneumonia.In particular,it can effectively reduce the levels of inflammatory cytokines TNF-αand MCP-1 in the lungs of mice with influenza virus pneumonia and reduce the expression of adhesion molecules such as ICAM-1 and VCAM-1,which will reduce inflammation.Exudation of cells from blood vessels,berberine may play a role in the treatment of influenza virus pneumonia to suppress the immune pathological damage caused by excessive inflammation.In the process of influenza virus infecting lung tissues,virus-activated alveolar macrophages become central effector cells of the lung antiviral inflammatory response,and release a variety of pro-inflammatory factors including IL-1β and TNF-α.Our previous in vitro experiments also confirmed that berberine effectively reduces the intracellular Caspase-1 activity of influenza virus-stimulated macrophages(RAW264.7).Therefore,whether berberine inhibits excessive pulmonary inflammation is through intervention of macrophage activation.And the activity to achieve whether the specific berberine can directly inhibit macrophage-mediated caspase-1 mature NLRP3 inflammasome activity,intervene IL-1β and IL-18 maturation and release.In order to answer these questions,this article starts from the following two aspects.First of all,in order to directly investigate the intervention of berberine on NLRP3 inflammasome activation,we transiently overexpress mouse NLRP3 inflammasome key group using eukaryotic expression vectors in human HEK293T cells lacking endogenous NLRP3 inflammasome.The three genes(NLRP3,ASC and pro-Casp1)and the interleukin precursor gene(pro-IL-1β)establish the activated NLRP3 inflammasome(NLRP3/ASC/pro-Caspl)to cleave pro-IL-1β production.An in vitro cell model of active IL-1[beta]was used and this cell model was used to directly assess the inhibitory effect of berberine on NLRP3 inflammasome activity.It was found that under the intervention of berberine,the level of extracellularly secreted IL-1β was significantly reduced,and intracellular Caspase-1 activity was also significantly reduced.Berberine seems to directly inhibit intracellular NLRP3 inflammasome-mediated pro-Caspse-1 activation.Second,in order to analyze the effect of berberine on the extracellular secretion of mature IL-1β,the mouse IL-1β gene was transiently over-expressed in human HEK293T cells,and the levels of extracellular IL-1β before and after berberine intervention were analyzed.There was no significant change in the content of extracellular IL-1β,indicating that berberine does not affect the extracellular secretion of mature IL-1β.Again,in order to investigate the effect of berberine on the assembly of P20 and P10 subunits(forming active caspase-1)and the cleavage of pro-IL-1β by the P20/P10 assembly(Caspae-1),in human HEK293T cells We transiently over-expressed the coding genes of P20 and P10 of Caspase-1 in mice.The level of extracellular IL-1β before and after berberine intervention was detected and it was found that the content of extracellular IL-1β was not significantly decreased,indicating that berberine does not affect P20.The assembly of/P10 also does not interfere with the cutting of pro-IL-1β by the P20/P10 assembly.Finally,in order to reverse the evidence that berberine can further inhibit the activation of NLRP3 inflammasome with the NLRP3 inflammasome as a direct target,and interfere with NLRP3 inflammasome-mediated inflammatory signal transduction,we constructed GFP fluorescence and Neo resistance.Double-selection shRNA targeting NLRP3 gene shRNA expression vector,after stable transfection of RAW264.7 cells,using G418 resistance screening and GFP fluorescence-based flow cytometric sorting enrichment,obtained a stable knockdown of NLRP3 gene(This is the NLRP3 inflammasome-deficient mouse macrophage cell line(named RAWNKD).Follow-up verification experiments are still in progress.The above results indicate that berberine can directly inhibit the activation of intracellular NLRP3 inflammasome,reduce intracellular Caspase-1 activity,and decrease the level of IL-1βobserved outside the cell,not because berberine inhibits the secretion of mature IL-1β.Nor is it because berberine inhibits the assembly of P20/P10 subunits(the formation of active caspase-1)and P20/P10 assembly cleavage and processing of pro-IL-1β.Berberine inhibits the activity of NLRP3 inflammasome may be achieved by direct inhibition of NLRP3 inflammasome-mediated activation of pro-Caspase-1.In addition,the established RAWNKD cell line,a NLRP3 inflammasome-deficient mouse macrophage model,can be used to investigate the role of NLRP3 inflammasome in macrophage inflammatory responses,particularly NLRP3 inflammasome-mediated Inflammation signal transduction facilitates. |
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