| NLRP3 inflammasome are macromolecular protein complexes formed by the assembly of intracytoplasmic NOD-like receptor protein 3(NLRP3),apoptosis-associated speck-like protein containing a CARD(ASC)and cysteinyl aspartate specific proteinase-1(Caspase-1).Abnormal activation of NLRP3 inflammasome,mediated cell pyroptosis,is involved in the inflammatory process in a variety of diseases.Selaginella moellendorffii Hieron,a perennial herb of the Selaginella genus,was first published in "Ben Cao Tu Jing" in Song Dynasty.Flavonoids are the characteristic active ingredients of S.moellendorfii.The preliminary study of our group showed that the total flavonoids of S.moellendorfii have anti-inflammatory effects,but their antiinflammatory substances and mechanisms of action have not been fully elaborated.Therefore,this paper intends to explore the anti-inflammatory mechanism of the flavonoids in S.moellendorfii based on the previous study at the molecular and cellular levels,using NLRP3 inflammasome as the target.Objective:To systematically extract and isolate the flavonoid components of S.moellendorfii and screen the anti-inflammatory potent substances of S.moellendorfii using NLRP3 inflammasome as the target,as well as to study the mechanism of action of the main active ingredients,so as to provide new ideas for the treatment of related inflammatory diseases.Methods:(1)Taking human macrophage THP-1 as the experimental subject,LPS and NLRP3 inflammasome activators,such as nigericin,adenosine triphosphate(ATP),and sodium urate(MSU),were used to construct a cellular inflammation model.The levels of LDH and IL-18 in cell culture supernatant were determined by lactate dehydrogenase(LDH)kit and ELISA method,and the anti-inflammatory activities of ethanol extract,water portion,n-butanol portion and ethyl acetate portion were investigated.ultraviolet spectrophotometry,high performance liquid phase(HPLC)and quadrupole tandem time-of-flight mass spectrometry(UPLC-QTOF-MS/MS)were used to analyze the chemical composition of ethanol extract and extraction portion of S.moellendorfii.Fluo-8 AM probe was used to detect intracellular Ca2+ changes;Sequencing technology was used to find the differentially expressed gene and the anti-inflammatory molecular mechanism of active portion,then immunoblotting was used to verify the protein expression level.(2)Atmospheric pressure silica column chromatography,gel column chromatography,semi-preparative liquid chromatography and other technical means were used to separate and purify the active portion of S.moellendorfii,and the compound structure was identified by LC-MS,1H-NMR and 13C-NMR,and the compounds were screened for anti-inflammatory activity by examining LDH,ROS,and IL-1β levels.(3)The cell pyrophoris rate and the level of inflammatory factors were detected by propidium iodide(PI)staining and ELISA method separately,which was used to further verify the anti-inflammatory activity of amentoflavone;Fluo-8 AM probe was used to detect intracellular Ca2+ changes,and the potential molecular mechanism of amentoflavone was explored and verified by transcriptomics technology,real-time PCR technology and western blotting technology.THP-1 cells were transfected using NLRP3 siRNA and pcDNA3.1(+)-NLRP3 separately to construct knockdown and overexpression models to verify the target of amentoflavone.Results:(1)10 μM Nigericin stimulation for 2.5 h,150 μg/mL MSU stimulation for 5 h or 5 mM ATP stimulation for 40 min could significantly reduce cell viability,promote LDH release,and upregulate IL-1β3 and Caspase1 protein levels(P<0.05,P<0.01),causing cell pyroptosis.The release of LDH,IL-18 and IL-1β(P<0.05,P<0.01)was significantly inhibited in the NLRP3 activation model,and the activity of ethyl acetate portion was relatively stronger,while the water portion had no significant activity(P>0.05).The flavonoid contents of water,n-butanol and ethyl acetate portion were 0.23%,6.23%and 28.65%,respectively.The HPLC and UPLCQTOF-MS/MS showed that the ethyl acetate portion mainly contained flavonoids and a small amount of flavonoid glycosides.ethyl acetate portion can inhibit Ca2+ influx in a concentration-dependent manner.Transcriptomics analysis showed that there were 505 differentially expressed genes in the PMA group,ATP group and ethyl acetate portion group,of which 29 were highly correlated with inflammatory response.In the response of cells to lipopolysaccharides,it mainly involves NLRP3 inflammasome aggregation,regulation of IL-1β production and other functions;The antiinflammatory mechanism of ethyl acetate portion mainly involves signaling molecules and interactions,signal transduction,immune system and cell growth pathways,among which NF-κB signaling pathway and NOD-like receptor signaling pathway are the most related pathways to NLRP3 inflammasomes.Western blotting showed that the ethyl acetate portion of S.moellendorfii significantly inhibited the expression(P<0.05,P<0.01)of NLRP3 inflammasome initimate-stage protein(p-p65/p65,NLRP3)and activation stage protein(GSDMD-N/GSDMD,IL-1β).(2)A total of 11 compounds were separated and identified from ethyl acetate portion of S.moellendorfii by column chromatography and semipreparative high performance liquid chromatography,namely 2,3dihydroamentoflavone(F1),bilobetin(F2),7-O-methylamentoflavone(F3),podocarpusflavone A(F4),ginkgetin(F5),isoginkgetin(F6),amentoflavone(F7),kayaflavone(F8),robustaflavone 4’-methyl ether(F9),7,4 ’,7 ’’-triO-methyrobustaflavone(F10),podocarpusflavone B(F11).Among them,amentoflavone can significantly reduce LDH,ROS and IL-1β levels induced by various inducers(P<0.01)more stable and effective,while compounds F1,F2,F4 and F8 are selective for reducing LDH,ROS and IL-1 β levels.(3)Compared with the model group,amentoflavone could significantly inhibit the positive rate of cell PI and the level of inflammatory factors(IL-18,IL-1β)(P<0.05,P<0.01),but had no significant effect on TNF-α(P>0.05);Similar to ethyl acetate portion,amentoflavone can significantly inhibit Ca2+influx,and its anti-inflammatory mechanism mainly involves signaling molecules and interactions,signal transduction,immune system and cell growth and other pathways,among which,NF-κB signaling pathway and NOD-like receptor signaling pathway are the most related pathways to NLRP3 inflammasomes,and use Cytoscape software to mine Hub gene TOP6:TNF,MYD88,STAT1,CASP1,DDX58,TLR2,IRF7,CXCL11;Real-time PCR and immunoprotein blot verification of major Hub genes and NLRP3 inflammasome-related genes showed that amentoflavone could significantly downregulate IL-1β,IL-18,NLRP3,TNF-α,Caspase-1,p-p65/p65,GSDMDN/GSDMD gene and protein expression(P<0.05,P<0.01);after siRNA downregulated NLRP3 expression,amentoflavone can significantly reduce the expression of Pro-IL-1β protein upstream of NLRP3 inflammasomes(P<0.05),while the inhibitory effect on downstream related proteins GSDMD-N/GSDMD disappears.However,it can still significantly inhibit the increase of supernatant IL-1β in cell culture supernatant(P<0.01),and after overexpression of NLRP3 gene,the inhibitory effect of amentoflavone on NLRP3 protein is weakened,but it can still significantly reverse the increase of Caspase-1(P<0.01).Conclusion:Ethyl acetate portion of S.moellendorfii is an effective portion for inhibiting the activation of NLRP3 inflammasomes.Amentoflavone,the main component,can inhibit the release of LDH,IL-18 and IL-1β in a more stable and effective in a broad-spectrum manner,protect cell membrane integrity,and reduce cell pyroptosis by regulating Ca2+ homeostasis,reducing ROS accumulation,and targeting the activation of NLRP3 inflammasomes. |
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