Paeoniflorin Is Used To Study The Related Effects Of NLRP3 Inflammasome Against Fungal Infections

ObjectiveTo investigate whether lipopolysaccharide(LPS)combined with fungal glucan(curdlan)activates NLRP3 inflammasome in human bronchial epithelial cells(16HBECs),and whether Peanoflorin influences this activiation of NLRP3 inflammasome,as well as the underlying mechanism.MethodsLPS combined with fungal dextran established infection model group:?blank control group:without any treatment factors;?LPS group:adding 5?g/ml LPS culture 12 hours;?curdlan group:adding final concentration of 10 ?p/ml,100 ?g/Ml,1mg/ml curdlan co-culture for 12 hours respectively;?LPS+curdlan group:the final concentration of 5?g/ml LPS pre-acting for 3 hours,then remove the LPS,adding the final concentration of 10?g/ml,100?g/ml,1mg/L curdlan coculture for 12 hours.?NAC+ LPS+curdlan group:NAC dissolved in PBS,the final concentration of lmmol/L and 5mmol/L respectively pre-acting for 1h,LPS activing for 3 hours,1 mg/ml curdlan co-culture for 12 hours?PF+LPS+curdlan group:paeoniflorin dissolved in RMPI1640,the final co-culture concentrations were 10?M,30?M,100?M respectively pre-acting for 1 hour,then LPS for 3 hours,lmg/ml curdlan co-culture 12 hours.RT-PCR was used to detect the expression of NLRP3,Caspase-1 and IL-1? mRNA in the cells.The levels of IL-1? were detected by ELISA Kit.The activity of caspase-1 was detected by caspase-1 activity assay.The changes of reactive oxygen species(ROS)were detected by using a FACS caliburinstrument.The protein expression of NLRP3,caspase-1 and IL-1? were detected by Western Blot.Results1)LPS combined with fungal dextran promoted the expression of NLRP3,caspase-1 and IL-1? mRNA in 16HBE cells,and the secretion of IL-1? was significantly increased in the supernatant of cells.The expression of NLRP3,caspase-1 and IL-1? protein in the supernatant was significantly higher than that in the control group,The difference was statistically significant(P<0.05).3)LPS combined with fungal dextran activiated the intracellular NLRP3 inflammasome through ROS activation,when given the inhibitor of ROS,NAC,for pre-action,intracellular ROS production was significantly reduced,as well as NLRP3,caspase 1 and IL-1? synthesis and expression,and the difference was statistically significant(P<0.05).4)paeoniflorin can inhibit the activation of NLRP3 inflammasome induced by LPS combined with fungal dextran by inhibiting the production of ROS in human brochial epithelial cells.Along with the increase of paeoniflorin concentration,the amount of intracellular ROS gradually decreased,consistent with the changes of NLRP3,caspase-1 and IL-1?,the difference was statistically significant(P<0.05).ConclusionLPS combined with fungal glucan could effectively activate the NLRP3 inflammasome in human bronchial epithelial cells,while paeoniflorin can effectively inhibit the intracellular ROS generation by inhibiting the NLRP3 inflammasome activation induced by fungal glucan.