Influence Of The NLRP3and NOD2Receptor Activated On Expression Of Inflammatory Cytokines In THP-1

Objective：NOD-Like Receptors （NLRs） with nucleotide binding oligomerizationdomain （NOD） is an important pattern recognition receptors （PRRS） in mammaliancells. The NLRs, as a group of signal transduction cytosolic, participate in inflammatoryresponses broadly. The NLRs family, including five members as follows, NODs、NALPs、ICE、CIITA、IPAF/NAIP. NLRP3and NOD2, two important members of theNLRs, play a key role in the inflammatory response. ATP is the specific ligand ofNLRP3. The expression of NLRP3is enhanced when neutrophils and macrophageswere stimulated by ATP. Activated NLRP3can lead to caspase-1activation, and finallycut inflammation, at the same time cause the secretion of IL-1β and IL-18precursor toincrease and send to the extracellular[1]. MDP is specific ligand of NOD2. NOD2isactivated after MDP stimulate monocytes and macrophages, then classical NF-κBsignaling pathway is started, make non-active inflammatory precursor gene transcriptionincrease significantly. But also caspase-1is activated by NLRP1pathway, whichprovides the reactions platform to activation of non-active inflammatory precursor.There is less report about the effect of NLRP3and downstream factors expression afterMDP and ATP jointly stimulate cell. This experiment observed that mRNA expressionof NLRP3，caspase-1and the change of IL-1β, IL-18expression in cell supernatantssolution by intracellular receptor-specific ligand MDP, ATP alone and the jointstimulated human monocytic cell line （THP-1）, to explore The Influence of Activatingthe NLRP3and NOD2Receptor on Expression of Inflammatory Cytokines in THP-1Methods: Human monocytic cell line （THP-1） was purchased from the CASShanghai Institutes for Biological Sciences drug intervention trial. Four bottles ofTHP-1in good condition were added to the medium to10ml after cell-count.Concentration of MDP was0.5μM, ATP was100μM. The required drug MDP25μl, ATP256μl should be added to10ml liquid after the calculation. They were divided intofour groups in the experiment: control group （added to PBS100μl then incubated intothe constant temperature of37℃CO₂incubator for24h）, TMDPgroup （added to0.5μMconcentration of MDP25μl and then incubated into the constant temperature of37℃CO₂incubator for24h）, TATPgroup （added to100μM concentration of ATP256μl andthen incubated into the constant temperature of37℃CO₂incubator for24h）, TMDP+ATPgroup （added to0.5μM concentration of MDP25μl to pre-stimulate and incubated intothe constant temperature of37℃CO₂incubator for24h, then added to100μMconcentration of ATP256μl and incubated into the constant temperature of37℃CO₂incubator for24h）. After thermostat incubation, these cells were transferred to15mlcentrifuge tubes. They were centrifuged for7minutes （1500rpm/min）in thermostat thenthe solution were divided into supernatant and sediment layer.200μl of the supernatantwere to be determined expression of IL-1β and IL-18by enzyme-linked immunosorbentassay （ELISA）, the remaining supernatant were absorbed entirely by tip then abandoned,and the remaining cells pellet were used to RNA extraction to determine the expressionof NLRP3and caspase-1by real time-PCR.Results:1.Compared to THP-1stimulated by MDP and ATP respectively and thetwo jointly, the expression of IL-1β and IL-18in the supernatant of mononuclear cellsas follows: both group of TATPand TMDPwere greater than blank group, the differencewas statistically significant （p <0.05）, TMDP+ATPgroup was obviously higher thanblank group, TMDPgroup, TATPgroup, respectively,the difference was statisticallysignificant （p <0.05）.2.Compared to THP-1stimulated by MDP and ATP respectively and the twojointly, the expression of NLRP3mRNA of mononuclear cells as follows: the TATPgroup slightly higher than blank group, the difference was statistically significant （p <0.05）, TMDP+ATPgroup was obviously higher than blank group, TMDPgroup, TATPgroup, respectively,the difference was statistically significant （p <0.05）.3.Compared to THP-1stimulated by MDP and ATP respectively and the twojointly, the expression of caspase-1mRNA of mononuclear cells as follows: both groupof TATPand TMDPwere greater than blank group, the difference was statisticallysignificant （p <0.05）, TMDP+ATPgroup was obviously higher than blank group, TMDPgroup, TATPgroup, respectively,the difference was statistically significant （p <0.05）.Conclusion:1.The expression of caspase-1and downstream factors was weakwhen THP-1stimulated by NOD2’s specific ligand，the MDP，whereas NLRP3is not have expression.2.the expression of NLRP3and the downstream factors was weak when THP-1was stimulated respectively by NOD2’s specific ligand, the MDP, and NLRP3’s specificligand, the ATP. whereas significant increase when MDP and ATP jointly, promotionand synergy to be between the two.