Tanshinones Inhibit NLRP3 Inflammasome Activation To Prevent And Treat Inflammatory Diseases

Background:Salvia miltiorrhiza Bunge,as traditional medicinal herbs have been widely used for treating human diseases,including cardiovascular disease,cerebrovascular disease,cancer,and immune system diseases.The main active ingredients of Salvia miltiorrhiza Bunge include tanshinone IIA(TNA),isocryptotanshinone(ICT),dihydrotanshinone Ⅰ(DHT),cryptotanshinone,methyltanshinone and so on.Salvia miltiorrhiza Bunge and its main components have anti-inflammatory effect in animal model and clinic.NLRP3 inflammasome,which is responsible for the maturation and secretion of IL-1β,has a role of vital importance to play in immune response and disease development.Suppressing the activation of NLRP3 inflammasome may be a strategy for treating those inflammatory diseases.To identify whether tanshinones inhibit NLRP3 inflammasome activation or not,we collected available 15 different tanshinones and found those of compounds have the strongest effect in the inhibition of NLRP3-dependent IL-1βrelease.In this study,we will further study the pharmacological mechanism of tanshinones against NLRP3 inflammasome activation,and investigate the potency in related-inflammatory disease animal models.Purpose:This study designed to investigate the effects and mechanism of tanshinones on NLRP3 inflammasome activation and NLRP3-driven inflammatory diseases.Methods:Part 1:Macrophages were stimulated with LPS/Nigericin or LPS/ATP to promote NLRP3 inflammasome activation to establish cell-screening model.The level of IL-1βsecretion with 15 different tanshinones in LPS/Nigericin stimulated J774A.1cells detected by ELISA Assay Kit;The survival of 15 tanshinones at 24 hours in J774A.1 cells were detected by CCK-8 Assay Kit.Three available and representative tanshinones(TNA,DHT,and ICT)were further studied for the mechanisms.(1)The survival of tanshinones(TNA,DHT,and ICT)at 2 hours or 24 hours in J774A.1 cells were detected by CCK-8 Assay Kit.(2)The level of IL-1βsecretion in macrophages were detected by ELISA Assay Kit,and immunoblot analysis of IL-1βand caspase-1(P20)in the supernatants and NLRP3,Pro-IL-1β,Pro-caspase-1 and ASC in cell lysate;The release of LDH was analyzed by LDH Assay Kit.(3)In J774A.1 cell,NLRC4 inflammasome activation was stimulated with LPS/FLA-ST Ultrapure and AIM2 inflammasome activation was treated with LPS/poly(d A:d T),ELISA Assay Kit detected the secretion of IL-1β.Part 2:(1)J774A.1 cells were treated as usual and then loaded with DHE or DCFH-DA and the production of ROS was detected by Muse Cell Analyzer or fluorescence micro plate reader.(2)J774A.1 cells were treated as usual,and then were loaded with Mito SOX probe and the mitochondrial ROS were observed by FACS and fluorescence micro plate reader.(3)J774A.1 cells were incubated Mito Q or DPI and then the production of ROS and the level of IL-1βsecretion were detected and explored the source of ROS regulate the activation of NLRP3 inflammasome.Part 3:(1)J774A.1 cells were treated as usual,and then loaded with JC-1 probe,mitochondrial membrane potential(MMP)measurement detected by flow cytometry and fluorescence micro plate reader.(2)J774A.1 cells were loaded with were loaded Mito Tracker Green or Mito Tracker Red probe to evaluate mitochondrial function by fluorescence micro plate reader or microscope.(3)The activity changes of complexes I,II and III were detected by commercial kits.(4)Quantitative analysis of the ATP/NAD~+production by commercial kits.Part 4:(1)Immunoblot analysis of LC3B expression in lysates of LPS/Nigericin-treated J774A.1 cells.(2)Macrophages were incubated with 3-MA or chloroquine(CQ),ELISA of IL-1βlevel was evaluated and caspase-1 expression was measured by western blotting.(3)Immunoblot analysis of AMPK and ACC1phosphorylation in the treatment of J774A.1 cells.Part 5:(1)Cytokines from serum and peritoneal fluid in LPS-induced septic mice were evaluated by ELISA Assay Kit.Immunoblot analysis of the expression of caspase-1,LC3B and AMPK in the liver of LPS-induced septic mice.Histopathology analysis of the liver from LPS-induced septic mice survival of C57BL/6 mice intraperitoneally injected with LPS.(2)Serum IL-1βlevels from MSU-challenged gouty rats were detected by ELISA Assay Kit.ELISA Assay Kit was used to assess caspase-1 in the synovial tissue;Histopathology analysis of the knee joint from MSU-induced gouty rats to observe the pathological characteristics.Results:Part 1:15 different tanshinones remarkably suppressed NLRP3-dependent IL-1βsecretion in J774A.1 cells.The viability of J774A.1 cells incubated with 15tanshinones for 24 hours showed largely difference.(1)The viability of J774A.1 cells treated with tanshinones(DHT,ICT and TNA)at indicate dose(0.625-10μM)for 2hours or 24 hours showed no significant changes compared with control group;(2)Tanshinones(DHT,ICT and TNA)can dose-dependently inhibit IL-1βsecretion induced by LPS/Nigericin or LPS/ATP in macrophages.Tanshinones(DHT、ICT and TNA)can dose-dependently block the expression of caspase-1 and IL-1βin the supernatants,and the expression of NLRP3,Pro-IL-1β,Pro-caspase-1 and ASC in cell lysate were not changed;Tanshinones(DHT,ICT and TNA)can dose-dependently inhibit the release of LDH.(3)Tanshinones(DHT,ICT and TNA)had no effect on AIM2 inflammasome activation,but inhibited NLRC4 inflammasome activation.Part 2:(1)DHE or DCFH-DA fluorescence results displayed that tanshinones(DHT,ICT and TNA)significantly suppressed ROS production induced by LPS/Nigericin in J774A.1 cells.(2)Mito SOX fluorescence results displayed that tanshinones(DHT,ICT and TNA)remarkblely attenuated mt ROS production induced by LPS/Nigericin in J774A.1 cells.(3)With the treatment of Mito Q or DPI,the fluorescence and ELISA results showed that the reduction of mitochondrial-sourced ROS may be the reason why tanshinones could inhibit NLRP3 inflammasome activation.Part 3:(1)Treatment with tanshinones(DHT,ICT and TNA)mitochondrial membrane potential was improved by LPS/Nigericin-induced in J774A.1 cells.(2)DHT,ICT,and TNA did not affect the staining with Mito Tracker Green,but enhanced the staining with Mito Tracker Red in LPS/Nigericin-induced J774A.1 cells.(3)DHT,ICT and TNA effectively prevented the decline of the activities of complexes I,II and III were reduced induced by LPS/Nigericin in J774A.1 cells.(4)With the treatment of tanshinones(DHT,ICT and TNA)both of the production of ATP and NAD~+were enhanced induced by LPS/Nigericin in J774A.1 cells.Part 4:(1)Immunoblot results showed that tanshinones(DHT,ICT and TNA)promoted LC3 cleavage and make the conversion of LC3-I to LC3-II in LPS/Nigericin-treated J774A.1 cells.(2)DHT,ICT and TNA-induced inhibition of IL-1βrelease were reversed by autophagy inhibitors 3-MA and CQ;Western blot results showed that DHT,ICT and TNA-mediated inhibition of cleaved caspase-1(P20)in J774A.1 macrophages was significantly blocked by 3-MA.(3)Tanshinones(DHT,ICT,and TNA)enhanced the phosphorylation of AMPK and ACC1 in LPS/Nigericin insulted J774A.1 cells.Part 5:(1)Compared to the vehicle group,treatment with DHT significantly decreased different cytokines release(IL-1β,IL-6 and TNF-α),both in serum and peritoneal fluid induced by LPS-induced peritonitis;Caspase-1(P20)level was obviously decreased with DHT administrated accessed by western blot assay in the mice liver.Western blot results showed that DHT could also enhance AMPK phosphorylation and LC3-II expression in the liver upon the mouse septic shock model.Histological analyses in the liver revealed DHT decreased infiltration of inflammatory cells;DHT significantly improved the survival of LPS-challenged mice.(2)DHT can decrease the serum IL-1βand the activity of caspase-1 in joint tissues from MSU-induced gout model;Histological analysis revealed DHT suppressed inflammatory response in the ankle joint and synovial hyperplasia.Conclusions:In this study,we found that 15 different tanshinones could suppress NLRP3inflammasome activation and different structures of tanshinone have different effects on NLRP3 inflammatory activation.Three tanshinones(tanshinone IIA,isocrypto-tanshinone,and dihydrotanshinone Ⅰ)reduced mitochondrial reactive-oxygen species production in lipopolysaccharide(LPS)/Nigericin-stimulated macrophages and correlated with altered mitochondrial membrane potentials,mitochondria complexes activities,and adenosine triphosphate and protonated-nicotinamide adenine dinucleotide production.The tanshinones may confer mitochondrial protection by promoting autophagy and the AMP-activated protein kinase pathway.Importantly,our finds demonstrate that dihydrotanshinone Ⅰ improved the survival of mice with LPS shock and ameliorated inflammatory responses in septic and gouty animals.Our results suggest a potential pharmacological mechanism whereby tanshinones can effectively treat inflammatory diseases,such as septic and gouty inflammation.