Mechanism Of Caffeine Inhibiting NLRP3 Inflammasome Activation In Lipopolysaccharide Induced THP-1 Macrophages

Objective:Bronchopulmonary dysplasia is a respiratory complication that seriously threatens the life and health of premature infants.Previous studies have confirmed that lung inflammation caused by NLRP3 inflammasome activation is closely related to BPD.Knocking out the NLRP3 gene significantly attenuated lung inflammation in LPS and hyperopia BPD animal models,and improved alveolar simplification.Caffeine is a safe and effective drug for clinical prevention of BPD,and its specific mechanism of action to protect lung development is still unclear.Recent studies have found that caffeine has a certain anti-inflammatory effect,which can inhibit the infiltration of inflammatory cells in the lungs of premature infants and the release of inflammatory mediators.Whether the anti-inflammatory effect of caffeine is related to the inhibition of NLRP3 inflammasome activation,there is no corresponding report.Therefore,this study intends to construct a LPS induced THP-1 macrophage NLRP3 inflammasome activation model in vitro,to explore the effect of caffeine on NLRP3 inflammasome activation,and to explore its mechanism.Therefore,provide a certain experimental basis for clarifying the mechanism of caffeine anti-inflammatory and protecting lung development,it also provides a new idea for the development of BPD preventive drugs based on NLRP3 inflammasome inhibitory effect.Methods:1.Establishment of LPS-induced NLRP3 inflammasome activation model in THP-1macrophages and caffeine safety assessment.PMA induces the differentiation of THP-1 monocytes into macrophages.After treatment with different concentrations of LPS,RT-PCR and western blot were used to detect NLRP3 gene transcription and protein expression.After treatment of cells with different concentrations of caffeine,the apoptosis rate was detected by flow cytometry,and the activation level of caspase3 was detected by western blot.2.To determine the effect of caffeine on the secretion of IL-1? and IL-18 induced by LPS/ATP in THP-1 macrophagesAfter pretreatment with different concentrations of caffeine,the secretion of IL-1? and IL-18 in cell culture supernatant was detected by ELISA,IL-1? and IL-18 gene transcription level was detected by RT-PCR in LPS/ATP-induced THP-1macrophages.3.Verify the inhibition effect of caffeine on LPS/ATP or LPS/Nigericin-induced NLRP3 inflammasome activation in THP-1 macrophages.LPS/ATP or LPS/Nigericin induces NLRP3 inflammasome activation in THP-1macrophages after pretreatment with different concentrations of caffeine.The gene transcription and protein expression levels of NLRP3,ASC and caspase1 were detected by RT-PCR and western blot,and intracellular ASC speck formation was observed.4.To investigate the effect of caffeine on the activation of MAPK/NF-?B signaling pathway induced by LPSAfter pretreatment with different concentrations of caffeine,LPS induced MAPK/NF-?B signaling pathway activation in THP-1 macrophages.The gene transcription and protein expression of TNF-? and IL-6 were detected by RT-PCR and ELISA.The phosphorylation level of MAPK/NF-?B signaling pathway was detected by western blot.The nuclear translocation of p65 was observed by immunofluorescence.5.To verify the inhibition of caffeine on LPS/ATP-induced A2 aR associated ROS production in THP-1 macrophages.si RNA was used to interfere A2 a R in LPS/ATP-induced THP-1 macrophages,interference effects were detected by RT-PCR and western blot.Western blot was used to detect LPS/ATP-induced NLRP3 inflammasome activation after si RNA interference,and intracellular ROS production was detected by flow cytometry.After pretreatment with different concentration gradient caffeine,LPS/ATP co-treated THP-1 macrophages,RT-PCR and western blot to detect A2 a R gene transcription and protein expression levels,and flow cytometry to detect intracellular ROS production.Results:1.THP-1 monocytes were mostly differentiated into macrophages after induction with 50 n M PMA overnight.After induction of THP-1 macrophages for 3 h at 1 ?g/ml LPS,the gene transcription and translation level of NLRP3 increased significantly.When the concentration of caffeine under 800?M,it's safe to treat THP-1macrophages.2.The release of IL-1? and IL-18 in THP-1 macrophages were significantly increased after LPS and ATP co-treatment.Caffeine inhibited IL-1? transcription and IL-1? and IL-18 protein expression in a dose-dependent manner.3.Caffeine significantly inhibited LPS/ATP induced NLRP3 gene transcription and protein expression but had no significant effect on ASC and caspase1 gene transcription and protein expression.In addition,caffeine significantly inhibited caspase(p20)protein expression and intracellular ASC specks formation.4.In LPS induced THP-1 macrophages,caffeine pretreatment reduced the phosphorylation level of JNK,ERK and p38 in MAPK pathway,the phosphorylation levels of I?B-? and p65 in NF-?B pathway,and inhibited p65 nucleus translocation.5.A2 a R si RNA effectively inhibits A2 a R gene transcription and protein translation in LPS/ATP-induced THP-1 macrophages,in addition ROS production was also been significantly reduced.The expression of caspase1(p20)was decreased after knockdown of A2 a R expression,and the expression of NLRP3,ASC and caspase1 was not significantly changed.Caffeine dose-dependently reduced A2 a R gene transcription and protein expression as well as intracellular ROS production.Conclusion:In conclusion,our results showed that caffeine can effectively inhibits NLRP3 inflammasome activation in LPS induced THP-1 macrophages via suppressing MAPK/NF-?B signaling and A2 a R associated ROS production.