Anti-inflammatory Mechanism Of Panaxadiol Targeting ZFP91 To Regulate IL-1B Secretion

Background:In traditional Chinese medicine,the use of Panax ginseng C.A.Mey.dates back about 5,000 years.It is widely used in agricultural products,dietary,health supplements,and medicines,and has several beneficial and healing properties.The signature bioactive ingredients of ginseng are ginsenosides,which are triterpene saponins.Panaxadiol is a triterpenoid sapogenin monomer found in the roots of Panax ginseng C.A.Mey.and has been proven to have various bio-activities such as anti-inflammatory,anti-tumour and neuroprotective effects.However,the molecular mechanism of panaxadiol-mediated anti-inflammatory effects is still unclear,so in-depth study of the anti-inflammatory effects and underlying mechanisms of panaxadiol is of great significance for the further development of anti-inflammatory drugs.Aim of the study:The present study focuses on investigating the anti-inflammation effect and mechanism of panaxadiol to inhibit IL-1β secretion by regulating zinc finger protein 91-regulated activation of non-canonical caspase-8 inflammasome and MAPKs in macrophages.Methods:1.In vitro experiments,we co-stimulated THP-1 cells and BMDM cells with LPS and ATP,and detected and analyzed the anti-inflammatory mechanism of panaxadiol in macrophages by using various experimental methods.①The binding mode of panaxadiol and IL-1β was detected by homology modeling and molecular docking experiments,and the effect of panaxadiol on the production and secretion of IL-1β in macrophages was detected by western blotting,RT-PCR,ELISA and immunofluorescence experiments.②The effects of panaxadiol on inflammasome components and the expression of ZFP91 were detected by western blotting,RT-PCR and immunofluorescence experiments in macrophages.The plasmid ZFP91 siRNA or ZFP91 overexpression was transfected into THP-1 cells,and the effect of panaxadiol on inflammasome activation by regulating ZFP91 was detected.③The effect of panaxadiol on inflammasome complex assembly by regulating ZFP91 was examined by co-immunoprecipitation.Furthermore,the effect of panaxadiol on the co-localization of inflammasome complexes by regulating ZFP91 was examined by immunofluorescence.④The effect of panaxadiol on the activation of MAPKs was examined by western blotting in macrophages.The plasmids ZFP91 siRNA or ZFP91 overexpressed was transfected into THP-1 cells,and the effect of panaxadiol on the activation of MAPKs by regulating ZFP91 was examined.2.In vivo experiments,a ZFP91 knockdown mouse model was established using a recombinant adeno-associated virus vector(AAV-shZFP91).On this basis,an acute colitis model and an acute peritonitis model were established to further verify the anti-inflammatory mechanism of panaxadiol.①DSS-induced colitis model:The mice(n=32,18-22 g)were randomly divided into the following five groups(n=8/group):the normal group,DSS group,DSS+ZFP91WT+PD group,DSS+ZFP91sh group,and DSS+ZFP91sh+PD group.Compared with control group of mice with drinking water,DSS-treated mice were given drinking water containing 4%DSS for 5 days.With the establishment of the DSS model,disease activity index was assessed by measuring weight loss,stool bleeding and stool consistency.Colon and serum were collected for detection of histological changes in the model and the expression of inflammatory mediators and related proteins by immunohistochemistry,ELISA,and western blotting experiments.②Alum-induced peritonitis model:The mice(n=32,18-22 g)were randomly divided into the following five groups(n=8/group):the normal group,Alum group,Alum+ZFP91WT+PD group,Alum+ZFP91sh group,and Alum+ZFP91sh+PD group.WT or AAV-shZFP91 C57BL/6 mice were intraperitoneally injected with 700 μg alum.After 12 hours,inflammatory cell subsets were analyzed,and the number of peritoneal exudate cells(PECs)were collected and analyzed by flow cytometry.The levels of inflammatory factors in the peritoneal fluid were detected by ELISA,and the expression of inflammasome-related proteins in the model was evaluated by western blotting.Results:1.In vitro experiments,study on the anti-inflammatory mechanism of panaxadiol in macrophages:①The experimental results showed that there is an interaction site between panaxadiol and IL-1β.Panaxadiol inhibited the production and secretion of IL-1β in THP-1 cells and BMDM cells co-stimulated by LPS and ATP.②Panaxadiol inhibits inflammasome components and ZFP91 expression in macrophages.Panaxadiol inhibited IL-1β secretion by inhibiting ZFP91-regulated inflammasome activation in THP-1 cells transfected with ZFP91 siRNA or ZFP91 overexpression plasmid.③Further analysis showed that panaxadiol inhibited the inflammasome complex assembly and inflammasome complex co-localization of the non-canonical caspase-8 inflammasome by inhibiting the expression of ZFP91.④Meanwhile,panaxadiol inhibited the activation of MAPKs in macrophages.Panaxadiol inhibited IL-1β secretion by inhibiting the activation of ZFP91-regulated MAPKs in THP-1 cells transfected with ZFP91 siRNA or ZFP91 overexpression plasmid.2.In vivo experiments,study on the anti-inflammatory mechanism of panaxadiol:①Panaxadiol significantly ameliorated DSS-induced acute colitis in mice,which is consistent with the results in AAV-ZFP91 knockdown mice in the model.Panaxadiol inhibits DSS-induced invasion of colonic inflammatory cells in mice,and panaxadiol inhibited the secretion of IL-1β,IL-6 and TNF-α in the serum of DSS-induced acute colitis mice.Meanwhile,panaxadiol inhibited the expression of DSS-induced inflammation-related proteins pro-IL-1β,ZFP91,NLRP3,ASC,cleaved caspase-8,p-ERK,p-JNK and p-p38 in the colon of mice.②Panaxadiol significantly improved alum-induced peritonitis,which is consistent with the results in AAV-ZFP91 knockdown mice in the model.Panaxadiol inhibits the secretion of IL-1β,IL-6 and TNF-α in the serum of mice with peritonitis,and panaxadiol inhibits the recruitment of inflammatory cells in mouse peritonitis.Meanwhile,panaxadiol inhibited the expression of inflammation-related proteins pro-IL-1β,ZFP91,NLRP3,ASC,cleaved caspase-8,p-ERK,p-JNK and p-p38 in mouse peritonitis.Conclusion:We report for the first time that panaxadiol inhibits IL-1β secretion in macrophages,mainly by inhibiting ZFP91-regulated activation of non-canonical caspase-8 inflammasomes and MAPKs.The findings provide theoretical evidence for the anti-inflammatory mechanism of panaxadiol in the treatment of inflammatory diseases.