Regulation And Mechanism Of Dehydrocostus Lactone On Inflammasome

Objective:We established NOD-like receptor family protein 3（NLRP3）、absence of melanoma 2（AIM2）、NLR family,CARD domain containing4（NLRC4）inflammasome activation model and LPS-mediated mouse model,analyzed the effect of dehydrocostus lactone（DCL）on the activation of NLRP3 inflammasome.By exploring the molecular mechanism of DCL on NLRP3 inflammasome,this study would provide potential drugs for the prevention and treatment of NLRP3 inflammasome related diseases.Methods:1.NLRP3 inflammasome activation models induced by ATP,nigericin,MSU,poly（I:C）and other stimulus were established in different cells.Western blot,Caspase-Glo?1 Inflammasome Assay,and ELISA were used to detect the regulatory effect of DCL on NLRP3 inflammasome related indicators.2.The models of c LPS-induced non-classical NLRP3 inflammasome,poly（d A:d T）-induced AIM2inflammasome,and Salmonella-induced NLRC4 inflammasome were constructed in different cells.Western blot,Caspase-Glo?1 Inflammasome Assay,and ELISA were used to detect the regulatory effect of DCL on inflammasome related indicators.3.Western blot and ELISA were used to detect the expression of Caspase-1 and IL-6 in BMDMs before and after treatment with DCL and ultrapure lipopolysaccharide（LPS）stimulation,and to evaluate the effect of DCL on the NF-κB and activation and assembly stage of inflammasome.The effects of DCL with multiple stimulus have been evaluated on ASC oligomerization,NLRP3 and ASC interaction,mitochondrial damage,Ca²⁺influx,and K⁺efflux.4.ELISA and flow cytometry was used to detect the expression levels of IL-1β,TNF-αand the number of neutrophils from C57BL/6 mice pretreated with DCL.Result:1.DCL significantly inhibited the activation of NLRP3 inflammasome with multiple stimulus and slightly inhibited the activation of AIM2 and NLRC4 inflammasome in primary mouse bone-marrow-derived macrophages（BMDMs）;DCL inhibited canonical and noncanonical NLRP3,AIM2 and NLRC4 The activation of inflammasome has obvious inhibitory effect in human myeloid leukemia mononuclear cells（THP-1 cells）;DCL significantly inhibited the activation of NLRP3 inflammasome in h PBMCs in human peripheral blood mononuclear cells（h PBMCs）.2.DCL can suppress both priming and assembly stage of NLRP3 inflammasome activation.DCL remarkably inhibited ASC oligomerization induced by NLRP3inflammasome agonists.Consistent with the inhibitory effects of DCL on AIM2 or NLRC4inflammasome activation,ASC oligomerization was also accordingly attenuated by DCL.DCL can also slightly affect potassium efflux caused by nigericin,but had no affected in the interaction between NLRP3 and ASC,calcium influx and mitochondrial damage.3.DCL can dose-dependently inhibit the production of IL-1βand recruitment of the neutrophils in LPS-mediated inflammatory mice.In contrast,the production of TNF-αwas not affected by DCL administration.Conclusion:DCL can significantly inhibit NLRP3 inflammasome activation by majorly affecting ASC oligomerization.Importantly,DCL significantly inhibited IL-1βsecretion and neutrophils recruitment in the LPS-mediated inflammation mouse model.Taken together,our data suggest that DCL can be used as a novel and potent anti-inflammatory agent in protection against NLRP3-associated inflammatory diseases.