Study On The Regulatory Effect Of Prostaglandin D₂ On The Activation Process Of Mouse Macrophages Induced By Escherichia Coli

Prostaglandins（PGs）is widely present in various tissues and organs of humans and animals,and have a variety of biological activities.PGs can be synthesized and secreted in large quantities,and then they can play a certain regulatory role in the process of inflammation.The activation of pattern recognition receptors in the host’s innate immune system plays a key role in the secretion of cytokines.The synthesis and secretion of PGs is closely connection to the host’s innate immune response mediated by pattern recognition receptors,such as Toll-Like receptors（TLRs）and NLR pyrin domain-containing3（NLRP3）.However,in the process of inflammation in mice induced by lipopolysaccharide（LPS）,Braun lipoprotein（BLP）and（Escherichia coli）E.coli,the synthesis and secretion of prostaglandin D₂（PGD₂）and pattern recognition receptor.Whether there is a close connection and whether it has an effect on the activation of signaling pathways and the secretion of cytokines is still unclear.In this study,LPS,BLP and E.coli was used to respectively stimulate peritoneal macrophages of C57BL/6J WT,TLR2^-/-,TLR4^-/-,and NLRP3^-/-in mice.The expression of COX-2 and H-PGDS were detected by q PCR and Western blot.The results showed that the expression of COX-2 gene and protein were down-regulated（P<0.01）,and the secretion of PGD₂ was also down-regulated by ELISA（P<0.001）.In addition,analyzed the effects of pretreatment with endogenous PGD₂ synthase inhibitors（H-PGDS and HQL-79）on the secretion of pro-inflammatory cytokines（IL-1βand TNF-α）,chemokines（RANTES）and anti-inflammatory cytokine（IL-10）in peritoneal macrophages of C57BL/6J mice induced by LPS,BLP and E.coli.The results showed that PGD₂ was inhibited,IL-1β,TNF-α,RANTES and IL-10 secreted by peritoneal macrophages were significantly down-regulated by LPS or E.coli（P<0.05）;BLP induced peritoneal macrophages IL-1βand TNF-αsecreted by cells were significantly down-regulated（P<0.001）;while after PGD₂ pretreatment the secretion of RANTES and IL-10 were significantly up-regulated（P<0.001）.In addition,analyzed the phosphorylation of NF-κB p65 and MAPKs（ERK,p38）,caspaes-1activation and secretion of inflammatory mediators in peritoneal macrophages of C57BL/6J mice induced by LPS,BLP and E.coli.The results shown that 15 min and30 min stimulation with LPS or E.coli,compared with the PGD₂ unpretreated experimental group,exogenous PGD₂ pretreatment can significantly up-regulate the phosphorylation of ERK,p38 and p65 in mouse macrophages（P<0.05）,which corresponds to the upward regulation of the secretion levels of TNF-αand IL-1β.After 30 min and 60 min stimulation,exogenous PGD₂ could significantly reduce the phosphorylation levels of ERK,p38 and p65（P<0.05）induced by LPS or E.coli,which could be correlated with the trend of down-regulation of IL-10 and RANTES.Exogenous PGD₂ can significantly down-regulate the phosphorylation of ERK and p38 of macrophages after 15 min,30 min and 60 min of BLP stimulation（P<0.05）,and significantly up-regulate of phosphorylation of p65 of macrophages after 15 min and 30 min stimulation（P<0.001）,which is consistent with the secretion of RANTES.After pretreatment with exogenous PGD₂,LPS,BLP and E.coli could not induce the activation of caspase-1 in mouse macrophages.In the presence of ATP,the activation of caspase-1 in mouse macrophages induced by E.coli infection did not change significantly.In conclusion,TLR2,TLR4 and NLRP3 are involved in the synthesis and secretion of PGD₂ induced by LPS,BLP and E.coli,and exogenous PGD₂ can regulate the activation of mouse macrophages induced by LPS,BLP and E.coli.