Mechanism Underlying Hydrogen Sulfide-induced Inhibition Of NLRP3 Inflammasome-dependent Pyroptosis

Objective: The present study was conducted to investigate the inhibitory effect of the hydrogen sulfide on NLRP3 inflammasome activation and resultant pyroptosis in human monocyte THP-1 cells and further elucidate the molecular mechanis,allowing for a deeper understanding for the anti-inflammatory potential of hydrogen sulfide and providing a new approach in medical treatment of inflammatory diseases driven by NLRP3-dependent pyroptosis pathway.Methods:(1)In order to evaluate the inhibitory activity of hydrogen sulfide on NLRP3 inflammasome activation and pyroptosis,LPS-primed THP-1 cells were incubated with nigericin in the presence of NaHS,an exogenous hydrogen sulfide donor,or AOAA,a pharmacological inhibitor for endogenous hydrogen sulfide synthesis.The NLRP3 inflammasome activation was assessed by detecting the secretion of IL-1β,caspases-1 activity,NLRP3 oligomerization and NLRP3/ASC co-localization using ELISA,FLICA and ICC,respectively.(2)Pyroptosis in THP-1 cells was evaluated by measuring the expression of intracellular GSDMD-NT and the release of LDH into supernatants.(3)The m RNA and protein expression levels of NLRP3,ASC,IL-1β and caspase-1 in cells were detected by real time-PCR and Western blotting,respectively,to investigate the inhibitory effects of hydrogen sulfide on transcription-dependent priming of NLRP3 inflammasome.To explore the potential of hydrogen sulfide on mtROSmediated NLRP3 inflammasome activation,the amounts of mtROS and 8-OHdG,a marker of oxidative DNA lesion were measured by ELISA and the interaction of NLRP3/8-OHdG were determined using co-IP and ICC.Results:(1)After co-incubation of LPS and nigericin,the NLRP3 inflammasome activation in THP-1 cells was observed as shown by increased IL-1β production,caspase-1 activity,NLRP3 oligomerization and NLRP3/ASC co-localization.In the presence of AOAA,the NLRP3 inflammasome activation was further enhanced,whereas it was significantly suppressed when exogenous hydrogen sulfide was added,demonstrating the inhibitory effects of hydrogen sulfide on NLRP3 inflammasome activation.(2)Pyroptosis was elicited in THP-1 cells following the stimulation of LPS/nigericin,characterized by up-regulation of GSDMD-NT protein expression and growing release of LDH.Similarly,AOAA further augmented pyroptosis,which was,however,markedly weakened in the presence of hydrogen sulfide or MCC950,a specific inhibitor of NLRP3,indicating that hydrogen sulfide possessed the capacity to inhibit NLRP3 inflammasome-dependent pyroptosis.(3)Supplement of exogenous hydrogen sulfide substantially suppressed the m RNA expression of NLRP3、IL-1β and caspase-1 and the protein expression of proIL-1β,pro-caspase-1 and ASC.This activity was dramatically attenuated in the presence of mtROS stimulant rotenone,suggesting the activity of hydrogen sulfide to block transcription-dependent priming of NLRP3 inflammasome components was probably arisen from its ROS-scavenging property.(4)Hydrogen sulfide significantly abrogated LPS/nigericin-induced generation of mtROS and mitochondrial 8-OHdG,the binding of8-OHdG to NLRP3,as well as the secretion of IL-1β.However,such activities of hydrogen sulfide were partly abolished by the co-treatment of rotenone.These data collectively suggested that the mtROS-scavenging activity of hydrogen sulfide probably contributed to the inhibition on NLRP3 inflammasome activation.Conclusions: Hydrogen sulfide exerted substantial ability to inhibit NLRP3 inflammasome activation and consequent pyroptosis in LPS/nigericin-stimulated THP-1cells.As for mechanism,the mtROS-scavenging activity enabled hydrogen sulfide to restrict NLRP3 inflammasome activation by suppressing transcription-dependent priming of NLRP3 inflammasome signaling.Moreover,hydrogen sulfide reduced the production of mtROS,as well as its mt DNA oxidized product 8-OHdG and diminished the binding of 8-OHdG to NLRP3,resulting in the failure of NLRP3 inflammasome assembly.