Honokiol Attenuates NLRP3 Inflammasome Via Inhibition Of α-hemolysin Secreted By Staphylococcus Aureus

The gram-positive pathogenic bacterium Staphylococcus aureus is among the most serious human pathogens encountered in healthcare settings worldwide which causes a broad of diseases ranging from superficial infections to deep-seated tissue infection and bacteremia.These infections are complicated to treat owing to the bacterium’s notable ability to acquire resistance to a wide range of antibiotics.The occurrence of S.aureus toxicity largely depends on various extracellular virulence factors produced by the strain,such as α-hemolysin.NLRs(Nod-Like Receptors),including NLRP1(Nucleotide-binding domain and Leucine-Rich repeat containing family Pyrin domain containing 1),NLRP3 and NLRC4.NLRs associate with other proteins to form multi-protein complex termed inflammasome.The NLRP3 inflammasome consists of the NLRP3,ASC(Apoptosis-associated Speck-like protein containing a Caspase recruitment domain)and procaspase-1 proteins.The NLRP3 inflammasome is activated in response to large amounts of pathogen-derived toxins in the cause of infection.Thereafter,the infected host will discover membrane lesions and repair it or trigger immune signaling as soon as possible to shield itself.Active NLRP3 inflammasome is signaling complex which activates procaspase-1,processing and secreting proinflammatory cytokines interleukin 1β(IL-1β)and 18(IL-18),and then initiates the programmed cell necrosis.Although purified Hla has been shown to induce inflammation in mice,rats and rabbits,the mechanisms by which Staphylococcal Hla promotes inflammation in animals have not been fully clarified.Correspondingly,the relatively little knowledge is available related to the intracellular relations between Hla and inflammatory mediators.Honokiol,is primarily isolated from the bark and seed cones of the Chinesemedicinal herb Magnolia officinalis.It has been widely used in medicine for its multiple pharmacological properties,such as anti-angiogenesis,anti-tumor,and anti-inflammatory.So far no detailed studies have been reported on its effects on the cytolytic activity of Staphylococcal Hla.We report here that the minimum inhibitory concentrations(MICs)of Honokiol against S.aureus 8325-4 and DU1090 strains were 32 μg/ml.This implies that Honokiol could be an important compound for the development of an anti-S.aureus drug.When added to S.aureus cultures,Honokiol abrogates significantly the production and hemolytic activity of Staphylococcal Hla within the MICs 32 μg/ml in vitro.In parallel,Honokiol attenuates Staphylococcal Hla-induced inflammatory response through inhibiting NLRP3 inflammasome in the macrophages cells.Based on these in vitro findings,we further investigated the suppressive effect of Honokiol in vivo.The bacterial burden was quantified to evaluate the inhibition efficacy of Honokiol on S.aureus strains survival within the mice liver tissues.The CFUs of S.aureus postinfections from mice treated with Honokiol were significantly lower than the control group treated with S.aureus8325-4.Notably,the amount of CFUs in liver tissues in mice challenged with S.aureus strain DU1090 lacking Hla expression was indistinguishable from that seen in mice challenged with S.aureus strain 8325-4.Moreover,the level of the NLRP3 inflammasome in vivo was further examined by determining the expression levels of NLRP3,ASC and caspase-1 proteins in mice liver tissues by using western blot analysis.With the addition of Honokiol,the secretions of NLRP3,ASC and caspase-1proteins in S.aureus 8325-4 system were all attenuated in a dose-dependent manner,which was similar to the tendency observed in the in vitro experiments.Altogether,these results strongly demonstrate that Honokiol effectively attenuates the NLRP3 inflammasome activation in the in vitro and in vivo experiments relying on the secretion of Hla by S.aureus.Histopathologic analysis uncovers that Honokiol suppresses the inflammation by decreasing the expression of inflammasome proteins NLRP3,ASC and procaspase-1 in S.aureus 8325-4 treatment mice.Subsequently,the biologically active forms of the inflammatory cytokines IL-1β and IL-18 are reduced significantly in the mice infected with S.aureus 8325-4 in response to Honokiol.These results reveal that Honokiol inactivates NLRP3 inflammasome-mediatedsignaling activated by Staphylococcal Hla.These results reveal the important contribution of Honokiol to bacterial pathogenesis and represent a new mechanism for developing a therapeutic approach against S.aureus infections.