Theaflavin mitigates acute gouty peritonitis and septic organ injury in mice by suppressing NLRP3 inflammasome assembly.

Activation of NLR family pyrin domain-containing 3 (NLRP3) inflammasome plays important role in defending against infections, but its aberrant activation is causally linked to many inflammatory diseases, thus being a therapeutic target for these diseases. Theaflavin, one major ingredient of black tea, exhibits potent anti-inflammatory and anti-oxidative activities. In this study, we investigated the therapeutic effects of theaflavin against NLRP3 inflammasome activation in macrophages in vitro and in animal models of related diseases. We showed that theaflavin (50, 100, 200 μM) dose-dependently inhibited NLRP3 inflammasome activation in LPS-primed macrophages stimulated with ATP, nigericin or monosodium urate crystals (MSU), evidenced by reduced release of caspase-1p10 and mature interleukin-1β (IL-1β). Theaflavin treatment also inhibited pyroptosis as shown by decreased generation of N-terminal fragment of gasdermin D (GSDMD-NT) and propidium iodide incorporation. Consistent with these, theaflavin treatment suppressed ASC speck formation and oligomerization in macrophages stimulated with ATP or nigericin, suggesting reduced inflammasome assembly. We revealed that theaflavin-induced inhibition on NLRP3 inflammasome assembly and pyroptosis resulted from ameliorated mitochondrial dysfunction and reduced mitochondrial ROS production, thereby suppressing interaction between NLRP3 and NEK7 downstream of ROS. Moreover, we showed that oral administration of theaflavin significantly attenuated MSU-induced mouse peritonitis and improved the survival of mice with bacterial sepsis. Consistently, theaflavin administration significantly reduced serum levels of inflammatory cytokines including IL-1β and attenuated liver inflammation and renal injury of mice with sepsis, concomitant with reduced generation of caspase-1p10 and GSDMD-NT in the liver and kidney. Together, we demonstrate that theaflavin suppresses NLRP3 inflammasome activation and pyroptosis by protecting mitochondrial function, thus mitigating acute gouty peritonitis and bacterial sepsis in mice, highlighting a potential application in treating NLRP3 inflammasome-related diseases.

Chen SY ，Li YP ，You YP ，Zhang HR ，Shi ZJ ，Liang QQ ，Yuan T ，Xu R ，Xu LH ，Zha QB ，Ou-Yang DY ，He XH ... -  《-》

Geraniin restricts inflammasome activation and macrophage pyroptosis by preventing the interaction between ASC and NLRP3 to exert anti-inflammatory effects.

Geraniin, a chemical component of the traditional Chinese medicine geranii herba, possesses anti-inflammatory and anti-oxidative activities. However, its anti-inflammatory role in managing NLRP3 inflammasome and pyroptosis remains to be elucidated. To investigate the anti-inflammation mechanism of geraniin, LPS-primed macrophages were incubated with classical activators of NLRP3 inflammasome (such as ATP, Nigericin, or MSU crystals), and MSU crystals were injected into the ankle joints of mice to establish an acute gouty arthritis model. The propidium iodide (PI) staining results showed that geraniin could restrain cell death in the ATP- or nigericin-stimulated bone marrow-derived macrophages (BMDMs). Geraniin decreased the release of lactate dehydrogenase (LDH) and interleukin (IL)-1β from cytoplasm to cell supernatant. Geraniin also inhibited the expression of caspase-1 p20, IL-1β in cell supernatant and N-terminal of gasdermin D (GSDMD-NT) while blocking the oligomerization of ASC to form speck. The inhibitory effects of geraniin on caspase-1 p20, IL-1β, GSDMD-NT, and ASC speck were not observed in NLRP3 knockout (NLRP3-/-) BMDMs. Hence, the resistance of geraniin to inflammasome and pyroptosis was contingent upon NLRP3 presence. Geraniin reduced reactive oxygen species (ROS) production and maintained mitochondrial membrane potential while preventing interaction between ASC and NLRP3 protein. Additionally, geraniin diminished MSU crystal-induced mouse ankle joint swelling and IL-1β expression. Geraniin blocked the recruitment of neutrophils and macrophages to the synovium of joints. Our results demonstrate that geraniin prevents the assembly of ASC and NLRP3 through its antioxidant effect, thereby inhibiting inflammasome activation, pyroptosis, and IL-1β release to provide potential insights for gouty arthritis targeted therapy.

Zhou X ，Qin M ，He L ，Zhang Y ，Liu A ，Chen D ，Pan H ... -  《-》

Corilagin Restrains NLRP3 Inflammasome Activation and Pyroptosis through the ROS/TXNIP/NLRP3 Pathway to Prevent Inflammation.

Corilagin, a gallotannin, shows excellent antioxidant and anti-inflammatory effects. The NLRP3 inflammasome dysfunction has been implicated in a variety of inflammation diseases. However, it remains unclear how corilagin regulates the NLRP3 inflammasome to relieve gouty arthritis. In this study, bone marrow-derived macrophages (BMDMs) were pretreated with lipopolysaccharide (LPS) and then incubated with NLRP3 inflammasome agonists, such as adenine nucleoside triphosphate (ATP), nigericin, and monosodium urate (MSU) crystals. The MSU crystals were intra-articular injected to induce acute gouty arthritis. Here we showed that corilagin reduced lactate dehydrogenase (LDH) secretion and the proportion of propidium iodide- (PI-)stained cells. Corilagin suppressed the expression of N-terminal of the pyroptosis executive protein gasdermin D (GSDMD-NT). Corilagin restricted caspase-1 p20 and interleukin (IL)-1β release. Meanwhile, corilagin attenuated ASC oligomerization and speck formation. Our findings confirmed that corilagin diminished NLRP3 inflammasome activation and macrophage pyroptosis. We further discovered that corilagin limited the mitochondrial reactive oxygen species (ROS) production and prevented the interaction between TXNIP and NLRP3, but ROS activator imiquimod could antagonize the inhibitory function of corilagin on NLRP3 inflammasome and macrophage pyroptosis. Additionally, corilagin ameliorated MSU crystals induced joint swelling, inhibited IL-1β production, and abated macrophage and neutrophil migration into the joint capsule. Collectively, these results demonstrated that corilagin suppressed the ROS/TXNIP/NLRP3 pathway to repress inflammasome activation and pyroptosis and suggest its potential antioxidative role in alleviating NLRP3-dependent gouty arthritis.

Luo T ，Zhou X ，Qin M ，Lin Y ，Lin J ，Chen G ，Liu A ，Ouyang D ，Chen D ，Pan H ... -  《-》

Dimethyl fumarate ameliorates autoimmune hepatitis in mice by blocking NLRP3 inflammasome activation.

Dimethyl fumarate (DMF) is a fumaric acid derivative clinically approved for the treatment of some inflammatory diseases, but the underlying mechanism for its therapeutic effects remains incompletely understood. NLR family pyrin domain containing 3 (NLRP3) inflammasome activation has critical roles in innate immune responses to various infections and sterile inflammations. In this study, we aimed to explore whether DMF affects auto-immune hepatitis (AIH) in mice induced by concanavalin A (Con A) by modulating NLRP3 inflammasome activation. The results showed that DMF suppressed the activation of NLRP3 inflammasome activation in lipopolysaccharide-primed murine bone marrow-derived macrophages upon ATP or nigericin treatment, as evidenced by reduced cleavage of pro-caspase-1, release of mature interleukin-1β (IL-1β) and generation of gasdermin D N-terminal fragment (GSDMD-NT). DMF also greatly reduced ASC speck formation upon the stimulation of nigericin or ATP, indicating its inhibitory effect on NLRP3 inflammasome assembly. Consistent with reduced generation of GSDMD-NT, ATP or nigericin-induced pyroptosis was markedly suppressed by DMF. Moreover, DMF treatment alleviated mitochondrial damage induced by ATP or nigericin. Interestingly, all these effects were reversed by the protein kinase A (PKA) pathway inhibitors (H89 and MDL-12330A). Mechanistically, DMF enhanced PKA signaling and thus increased NLRP3 phosphorylation at PKA-specific sites to attenuate its activation. Importantly, DMF decreased serum levels of inflammatory cytokines and ameliorated liver injury in Con A-induced AIH of mice, concomitant with reduced the generation of caspase-1p10 and GSDMD-NT and alleviating mitochondrial aggregation in the liver. Collectively, DMF displayed anti-inflammatory effects by inhibiting NLRP3 inflammasome activation likely through regulating PKA signaling, highlighting its potential application in treating AIH.

Shi FL ，Ni ST ，Luo SQ ，Hu B ，Xu R ，Liu SY ，Huang XD ，Zeng B ，Liang QQ ，Chen SY ，Qiu JH ，He XH ，Zha QB ，Ouyang DY ... -  《-》

Baeckein E suppressed NLRP3 inflammasome activation through inhibiting both the priming and assembly procedure: Implications for gout therapy.

Baeckein E (BF-2) was isolated from the aerial parts of Baeckea frutescens L., which has a long history of use in traditional medicine in Southeast Asia to treat inflammatory disease. BF-2 was identified to have inhibitory activity on nucleotide oligomerization domain (NOD)-like receptor protein-3 inflammasome (NLRP3) activation. This study aimed to investigate the related signaling cascade of BF-2 in both lipopolysaccharides (LPS)/ATP induced pyroptosis in J774A.1 macrophages and its application in a mouse model of gout induced by monosodium urate crystal (MSU). The effect of BF-2 on NLRP3 inflammasome activation and gouty arthritis was studied in J774A.1 macrophages and male C57BL/6 mice. The J774A.1 macrophages were primed with LPS and stained by propidium iodide (PI) for cell pyroptosis detection. A gout mouse model was established by subcutaneous injection of MSU crystals into the hind paw of C57BL/6 mice. Mice were then randomly divided into different groups. The concentrations of IL-1β and IL-18 in both J774A.1 macrophage and gout mouse model were analyzed by ELISA. The NLRP3 inflammasome related protein expression was detected by western blot analysis. The inhibitory effects of BF-2 on NLRP3 inflammasome assembly were analyzed by immunoprecipitation assay. The roles of BF-2 in mitochondrial damage were imaged by Mito Tracker Green and Mito Tracker Red probes. The inhibitory effects of BF-2 on ROS production were imaged by DCF (2',7'-dichlorofluorescein diacetate) probe. The results demonstrated BF-2 could significantly suppress the cell pyroptosis and IL-1β secretion in macrophages. Furthermore, BF-2 significantly inhibited NLRP3 inflammasome activation and reduced ankle swelling in the gout mouse model. In detail, it alleviated mitochondrial damage mediated oxidative stress and inhibited the assembly of NLRP3 inflammasome by affecting the binding of pro-Caspase 1 and ASC. Moreover, BF-2 blocked NLRP3 activation by inhibiting the MAPK/NF-κB signaling pathways. Results demonstrated BF-2 inhibited NLRP3 inflammasome activation in both LPS primed macrophages and mouse model of gout through blocking MAPK/NF-κB signaling pathway and mitochondrial damage mediated oxidative stress. This study strongly suggests BF-2 could be a promising drug candidate against inflammatory diseases associated with NLRP3 inflammasome activation.

Lin X ，Wang H ，An X ，Zhang J ，Kuang J ，Hou J ，Yan M ... -  《-》