Inhibitory Effect Of Corticosterone On LPS-induced NLRP3 Expression In Murine Macrophages And Its Mechanisms

OBJECTIVE:Body develops an inflammatory response after infection,and glucocorticoids produced by stress response can modulate the inflammatory response to maintain homeostasis.Macrophages are important innate immune cells.In addition to transmembrane TLRs,intracellular NLRP3 is also an important pattern recognition receptor.The NLRP3 inflammasome is a key molecular complex that mediates inflammation.LPS is the main pathogenic component of Gram-negative bacteria.This study investigated the effect of endogenous Corticosteroids(CORT)on the expression of NLRP3 after macrophage stimulation with LPS and its mechanisms,to explain the body's own inflammatory regulatory mechanisms and targets.METHODS:1.The inflammation model of murine macrophage RAW264.7 was constructed with the stimulation of LPS at 1?g/mL.The total protein andmRNA were extracted at 0,0.5,1,1.5 and 2 h,respectively.The protein expression levels of NLRP3 were detected by Western blot,and the mRNA expression of NLRP3 was detected by real-time PCR.2.Total cellular protein of RAW264.7 was extracted after macrophages were stimulated by LPS of 1?g/mL for 1h,pretreated with CORT at different concentrations(0~900ng/mL).The protein expression levels of NLRP3 and cleaved-caspase-1 were detected by Western blot respectively.3.Macrophages were treated with CORT at 700ng/mL and LPS of1?g/mL.Total protein and mRNA were extracted at 0,0.5,1,1.5 and 2h,respectively.The protein expression levels of NLRP3 and XO were analyzed by Western blot,and the real-time PCR was used to detect the mRNA expression levels of NLRP3 and XO,then XO activity was measured.4.Macrophages were treated with Allopurinol at 250?g/mL and LPS of 1?g/mL.The total protein and mRNA were extracted at 0,0.5,1,1.5 and2 h,respectively.The protein expression level of NLRP3 was measured by Western blot,and the real-time PCR detect the mRNA expression level of NLRP3.5.Drug combination experiments were performed with macrophages at the end.Macrophages were treated with LPS of 1?g/mL,CORT of700ng/mL and LPS of 1?g/mL,Allopurinol of 250?g/mL and LPS of1?g/mL,RU486 of 1?mol/L and CORT of 700ng/mL plus 1?g/mL LPS,respectively.Western blot was used to detect the protein expression levels of NLRP3 and XO.The activity of XO was also measured.RESULTS:1.LPS induced the expression of NLRP3 in macrophages.After stimulation with LPS of 1?g/mL,the protein expression level of NLRP3 increased from 0.5h to 2h,which was statistically significant compared with control group(P?0.05).The mRNA expression of NLRP3 from 1h to2 h was higher than that of the control group(P?0.05).2.CORT modulated the expression of NLRP3 and cleaved-caspase-1in a concentration-dependent manner.When the concentration of CORT was lower than 300ng/mL,the protein expression level of NLRP3 was significantly up-regulated compared to the control(P ? 0.05),when the concentration of CORT above 300ng/mL,the protein expression level of NLRP3 was gradually decreased,with the lowest expression level at the700ng/ml of CORT(P?0.05),and the expression of cleaved-caspase-1 was also decreased(P?0.05).3.CORT down-regulated NLRP3 expression while inhibiting the expression and activity of XO.Compared with CORT group,CORT plus LPS gradually decreased the protein expression level of NLRP3 in macrophages at different time points,and the expression level was the lowest at 2h(P?0.05),while protein expression level of XO at 1.5h and 2hwas significantly decreased(P ? 0.05).The mRNA expression level of NLRP3 from 0.5h to 2h was significantly lower than that of CORT group,lowest at 2h(P?0.05).The mRNA expression level of XO at 1.5h to 2h was also significantly lower than that of CORT group(P?0.05).The activity of XO was significantly down-regulated from 1h to 2h compared with CORT group(P?0.05).4.Allopurinol down-regulated the expression of NLRP3 in macrophages.Compared with LPS group,the expression of NLRP3 protein from 0.5h to 2h was significantly decreased(P?0.05),and the expression level of mRNA in 1h to 2h was lower than that in LPS group(P?0.05).5.RU486 can antagonize the inhibitory effect of CORT on NLRP3 and XO activity in LPS-induced macrophages.Compared with LPS group,CORT significantly inhibited the expressions of NLRP3 and XO,and also the activity of XO(P?0.05);the expression levels of NLRP3 and XO,the activity of XO in RU486 group were significantly increased(P ? 0.05)compared with CORT group.CONCLUSIONS:1.LPS induces and up-regulates the expression of NLRP3 in murine macrophages.2.Higher concentrations of CORT at 700ng/mL significantly down-regulate the expression of NLRP3 and cleaved-caspase-1 in murine macrophages.3.Higher concentrations of CORT at 700ng/mL inhibits the expression levels of NLRP3 and XO,also the activity of XO in macrophages.4.Antagonists of XO can affect the expression of NLRP3 in macrophages.5.CORT may inhibit the expression of NLRP3 by down-regulating XO activity.In summary,CORT inhibits LPS-induced NLRP3 expression in macrophages.Decrease in NLRP3 expression is associated with the down-regulation of XO,so CORT may inhibit LPS-induced NLRP3 expression in macrophages by down-regulating XO.