Functional Study Of Bergamot Lactone Inhibiting NLRP3 Inflammasome Activation By Inducing Mitochondrial Autophag

Background and ObjectiveChinese medicine has clear therapeutic effects in the application of inflammatory diseases.Bergapten(BeG)is a furocoumarin phytohormone present in herbal medicines such as Bergamot,Angelica dahurica,Notopterygium incisum,Angelicae Pubescentis Radix,Glehnia littoralis,Ficus carica,Cnidium monnieri and Peucedanum decursivum.BeG has many biological activities,including organ protective,neuro-protective,anti-diabetic and anti-cancer properties.BeG also has shown substantial anti-inflammatory and immunomodulatory actions,but the therapeutic efficacy of BeG against bacterial infection-related inflammation along with the underlying molecular mechanism remain poorly understood.NLRP3 inflammasome pathway is an important part of the first line of defense of the innate immune system and it is essential for the immunity against pathogenic infections.However,the dysregulated activation of NLRP3 inflammasome can lead to various inflammatory diseases.Hence,suppressing NLRP3 inflammasome activation could be a promising strategy for controlling inflammatory diseases.NLRP3 inflammasome is a multi-protein heteromeric complex existing in the cytoplasm,which is composed of intracellular receptor NLRP3,apoptosis-associated speck-like protein containing a CARD(ASC),and the downstream protease caspase-1.After NLRP3 inflammasome activation,pro-IL-1β and gasdermin D(GSDMD)are cleaved by active caspase-1,generating mature-IL-1β and N-terminal cleavage product(GSDMD-N),thereby triggering a specific form of inflammatory cell death called pyroptosis.Additionally,a number of pro-inflammatory factors are released,resulting in an intensified inflammatory response.During inflammatory processes,pathophysiological factors such as mitochondrial dysfunction and oxidative stress contribute to NLRP3 inflammasome activation,while mitophagy is beneficial for the maintenance of mitochondrial homeostasis and can negatively regulate the activation of NLRP3 inflammasome.In this study,we aimed to study the potential capacity of BeG on NLRP3 inflammasome and pyroptosis in vitro and in vivo,and elucidate the underlying anti-inflammatory mechanisms,providing a theoretical basis for its application in bacterial inflammatory disease.MethodsIn vitro and in vivo experiments were conducted to explore the anti-inflammatory effects of BeG.For canonical NLRP3 inflammasome stimulation,BMDMs,immortalised BMDM(iBMDM)cells,J774A.1 macrophages were primed with LPS and then stimulated with ATP or nigericin.To activate non-canonical NLRP3 inflammasome,BMDMs were primed with Pam3CSK4.The medium was replaced and LPS was transfected for 16 h.In addition,the anti-inflammatory effects of BeG on E.coli-induced sepsis and C.rodentium-induced intestinal inflammation were also evaluated.1.Cell experiment part:Experiment 1:The effects of BeG on NLRP3 inflammasome activation:Expression levels of the myeloid cell markers of BMDMs differentiated 7 days were measured by staining with two monoclonal antibodies anti-CD11b and anti-F4/80;the viability of J774A.1 cells was measured using the CCK-8 assay;western blot technology was used to detect cleaved-caspase-1,NLRP3,pro-caspasel and apoptosis-associated speck-like protein containing a CARD(ASC)protein from the cell lysates and culture supernatants;interleukin-1β(IL-1β)production in the culture supernatants of BMDMs was determined by ELISA;immunofluorescence technology was used to detect the ASC specks;qPCR technology was used to detect levels of Il1b,116 mRNA in BMDMs and levels of IL1B,IL6 mRNA in THP-1.Western blot technology was used to detect protein levels of core NLRP3 inflammasome components(NLRP3,Pro-caspasel,ASC)in LPS-primed BMDMs.Experiment 2:The effects of BeG on pyroptosis:PI incorporation and Lactate dehydrogenase(LDH)release assay were used to analyse cell death;using light microscope to observe the cell morphology;western blot technology was used to detect protein levels of GSDMD-N.Experiment 3:The effects of BeG on mitochondrial homeostasis in response to NLRP3 inflammasome activation:Transcriptome sequencing analysis;transmission electron microscope were used to detect Mitochondrial morphology;The mitochondrial mass was detected by staining with JC-1 or MitoTracker Deep Red and MitoTracker Green;mitochondrial DNA(mtDNA)/nuclear DNA(nDNA)ratio were detected by qPCR;mitochondria-associated ROS levels were measured by staining with MitoSOX.Experiment 4:The effects of BeG on mitophagy in response to NLRP3 inflammasome activation:western blot technology was used to detect protein levels of LC3;Confocal microscopy was used to detect the co-localization of mitochondria and LC3.After using 3-MA to suppress autophagy:qPCR technology was used to detect levels of Il1b,Il6,and Tnf mRNA in BMDMs.Interleukin-1β(IL-1β)production in the culture supernatants of BMDMs was determined by ELISA;western blot technology was used to detect cleaved-caspase-1,NLRP3,pro-caspase1 and ASC protein from the cell lysates and culture supernatants;LDH release assay was used to analyse cell death;mitochondria-associated ROS levels were measured by staining with MitoSOX.2.Animal experiment part:Experiment 5:The protective effects of BeG on sepsis induced by E.coli:mice were injected intraperitoneally(i.p.)with BeG(50 mg·kg-1)or PBS 24 h and 1 h before injecting E.coli(1×1011 CFU·kg-1 or 5×1010 CFU·kg-1).The mortality rate of mice was monitored regularly.Serum samples were collected 6 h post-infection and cytokine levels(IL-1β,TNF-α)were measured using ELISA.The lungs were removed for hematoxylin and eosin(HE)staining or western blot(NLRP3,pro-caspase-1,GSDMD,cleaved-caspase-1 and LC3),and the colon and liver were removed for HE staining.Experiment 6:The effects of BeG on C.rodentium-induced intestinal inflammation:mice were intragastrically(i.g.)infected with 25×1010 CFU·kg-1·d-1 bacteria 1 h after BeG administration(50 mg·kg-1).Body weight was measured every 2 days.On the ninth day,the mice were sacrificed,and the colon and jejunum were removed for HE staining.The viscera indices(spleen,kidney,and liver)were also measured.The liver,kidneys and mesenteric lymph nodes were removed and homogenized to measure the bacterial burden.Results1.Resluts of Experiment 1:BeG treatment inhibits cleaved caspase-1 release,IL-1βsecretion(P<0.01),and ASC specks formation(P<0.01)under nigericin and ATP stimulation,but this does not affect non-canonical NLRP3 activation by intracellular LPS stimulation.Upon LPS stimulation,transcription levels of Illb and 116 genes in BMDMs treated with BeG do not show changes.Consistently,similar results were obtained from LPS-stimulated THP-1 cells.Moreover,BeG does not suppress the expression of NLRP3,caspase-1,and ASC,upon LPS stimulation,as determined by western blotting.2.Resluts of Experiment 2:Upon ATP stimulation,BeG dose-dependently decreases the ratio of PI-positive J774A.1 cells(P<0.01).BeG also attenuated cell death in LPS-primed BMDMs in response to ATP or poly(I:C)(P<0.01).BeG decreases LDH release(P<0.01)in response to either nigericin or ATP.Some characteristic morphological changes,including cell swelling,cessation of movement and development of ballooning cell membranes that disintegrate abruptly to yield an atrophied corpse,are observed in LPS-primed BMDMs stimulated with either nigericin or ATP and these morphological changes are reversed by BeG treatment(P<0.01).Moreover,BeG blocks the formation of GSDMD-N.3.Resluts of Experiment 3:The bioinformatics analysis results illustrated that BeG treatment may improve mitochondrial homeostasis in response to NLRP3 inflammasome activation.Swollen mitochondria with severe disruption are noted in BMDMs treated with LPS and ATP,whereas BeG treatment significantly improves mitochondrial morphology.MMP is significantly decreased in LPS-primed BMDMs stimulated with ATP,whereas BeG treatment significantly elevates MMP(P<0.01).Consistent with this observation,the ratio of dysfunctional mitochondria increases in LPS-primed BMDMs in response to either ATP or nigericin,while BeG treatment significantly reverses this change.LPS and ATP stimulation results in lower mtDNA levels;but BeG treatment significantly reverses the reduction in mtDNA levels(P<0.05).Mitochondrial superoxide anion radical increases in J774A.1 cells with LPS and ATP stimulation,while BeG treatment significantly decreases the mitochondrial ROS levels(P<0.01).4.Resluts of Experiment 4:Upon NLRP3 activation,the treatment with BeG enhances the protein level of LC3-II in BMDMs.The degree of co-localization of mitochondria and LC3 increases significantly after BeG treatment.The inhibition of IL-1β release(P<0.05)and its mRNA(P<0.05)expression by BeG treatment is reversed by 3-MA treatment.Consistent with this,BeG-mediated inhibition of cleaved caspase-1 release,LDH release(P<0.05),GSDMD-N formation,and ROS production(P<0.01)is reversed by 3-MA treatment.5.Resluts of Experiment 5:Administration of BeG improve mouse survival after E.coli infection compared to that in E.coli group(P<0.01).Furthermore,IL-1β levels sharply increase in the serum after bacterial infection while BeG administration results in a significant decrease in IL-1β secretion(P<0.01).HE staining also indicates that BeG treatment significantly alleviates E.coli-induced overt infiltration of inflammatory cells in the lungs,colon,and liver of mice.E.coli increases the expression of NLRP3,cleaved caspase-1,and GSDMD-N.Pre-treatment with BeG significantly inhibits E.coli-induced expression of cleaved caspase-1 and GSDMD-N in the lung.Simultaneously,the pre-treatment with BeG also leads to an increase of LC3 II.6.Resluts of Experiment 6:C.rodentium infection resulted in significant weight loss and colon length shortening,which are reversed by BeG treatment(P<0.01,P<0.05).BeG treatment leads to lower bacterial loads(P<0.01)and viscera indices(P<0.01)than that in the model group.HE staining indicates that mice infected with C.rodentium show inflammatory cell infiltration and severe barrier breakdown of the colon and jejunum,whereas BeG treatment substantially attenuates these pathological phenotypes.ConclusionsThese findings collectively suggeste that BeG treatment mitigates NLRP3 inflammasome activation and pyroptosis by promoting mitophagy and maintaining mitochondrial homeostasis.Simultaneously,BeG protects against sepsis induced by E.coli and ameliorates C.rodentium-induced intestinal inflammation,suggesting that BeG can be used as a potential anti-inflammatory drug for bacterial-induced inflammatory diseases.