Exploring The Intervention Effect Of Wogonin On LPS-ATP-induced Macrophage Inflammation Based On The Nrf2-NF-κB Signaling Pathwa

ObjectiveMany diseases are occurring and developing accompanied with the inflammatory reaction,and the inflammatory reaction is closely related to oxidative stress.The inflammatory reaction can induce oxidative stress,and oxidative stress can aggravate the inflammatory reaction,and inhibition to oxidative stress may be a more promising strategy to the inhibition of inflammatory reaction.Nrf2 is a key protein involved in the regulation of oxidative stress.When cells are in oxidative stress,Nrf2 is activated,which could initiate the transcription of certain antioxidant genes,to scavenge ROS and inhibit the inflammatory responses.Wogonin is one of the main monomer components of Astragalus membranaceus.The previous study of the research group showed that wogonin could inhibit the expression of macrophage inflammatory factors infected by influenza virus,but its mechanism has not been further explored.In this study,macrophage cell line(RAW264.7)in mouse stimulated by LPS-ATP was used as an inflammatory model,to investigate whether wogonin can inhibit ROS production by up-regulating Nrf2 pathway and thereby inhibit NF-κB signaling pathway to play an anti-inflammatory role.MethodThe maximum non-toxic concentration of wogonin on macrophage RAW264.7 was 56μM by MTT assay.The experimental control group(no stimulation in normal culture),LPS-ATP combined stimulation group(ATP was added to conduct the continued stimulation for 30 min after the stimulation of 1 μg/mL LPS for 4 hours),and wogonin intervention group(ATP was added to conduct the continued stimulation for 30 min after the stimulation of LPS for 4 hours,while the LPS stimulation,wogonin at 56μM or wogonin at 14 μM,28μM or 56 μM was added.(1)The nuclear translocation situation of nuclear factor NF-κB in macrophages of each group was observed by immunofluorescence under laser confocal microscopy.Fluorescence quantitative PCR was used to detect the mRNA in inflammation-related factors IL-1β,1L-18,IL-6 and TNF-α IL in macrophages of each group as well as the transcription levels of inflammatory body-related NLRP3 and Caspase-1 mRNA.The secretion of extracellular inflammatory factors IL-6 and TNF-α was detected by ELISA,to determine the anti-inflammatory effect of wogonin.(2)The nuclear translocation of nuclear transcription factor Nrf2 in macrophages of each group was observed by immunofluorescence under laser confocal microscopy.Nrf2 mRNA in macrophages of each group as well as the transcription level of antioxidant enzymes GPx,NQO-1,HO-1,SOD,CAT mRNA were detected by real-time PCR,The activity of SOD in macrophages of each group was detected by SOD activity kit,and the ROS level in each group of cells was detected by flow cytometry,to explore whether the wogonin can inhibit inflammation through the antioxidative action.Result(1)Immunofluorescence results showed that NF-κB increased in the nucleus of macrophages after LPS-ATP stimulation,and NF-κB in macrophage nucleus decreased significantly after intervention of wogonin(56 μM).The results of real-time PCR showed that the mRNA transcriptional level of IL-1β,IL-18,IL-6,TNF-α,NLRP3 and Caspase-1 in macrophages increased after LPS-ATP stimulation;and mRNA transcriptional level of IL-1β,IL-18,IL-6,TNF-α,NLRP3 and Caspase-1 in macrophages significantly decreased after intervention of wogonin;the ELISA results showed that IL-6 and TNF-α secreted by macrophages increased after LPS-ATP stimulation,and IL-6 and TNF-α secreted by macrophages decreased after intervention of wogonin.(2)Immunofluorescence results showed that compared with the normal group,the expression of Nrf2 in macrophage nucleus of LPS-ATP stimulation group increased;and compared with the LPS-ATP stimulation group,the expression of Nrf2 in macrophage nucleus was significantly increased in wogonin intervention group;fluorescence quantitative PCR results showed that,compared with the un-stimulated control group,Nrf2 mRNA levels in macrophages were higher in LPS-ATP stimulation group,with significant decrease in mRNA levels of antioxidant enzymes GPx,NQO-1,HO-1,SOD and CAT;and wogonin intervention group could up-regulate the mRNA levels of Nrf2 mRNA and antioxidant enzymes GPx,NQO-1,HO-1,SOD and CAT;the results of SOD activity test kit showed that the activity of SOD in macrophages of LPS-ATP stimulation group significantly decreased,and the intervention of wogonin could significantly up-regulate the activity of SOD.The results of flow cytometry showed that,compared with un-stimulated control group,the ROS content in the cells of LPS-ATP stimulation group significantly increased;and compared with LPS-ATP stimulation group,the ROS content in macrophages of the groups with the intervention of wogonin at different concentrations(14μM,28μM,56μM)decreased significantly.ConclusionWogonin may increase the expression of Nrf2 and nuclear translocation,up-regulate the transcription of its regulated antioxidant genes,reduce the production of macrophage ROS,reduce the nuclear translocation of NF-κB,and reduce the transcription of inflammation-related gene regulated by NF-κB,so as to reduce the production of inflammatory factors and intervene the inflammatory response of macrophages.