Activation Of NLRP3 Inflammasome Contributes To Cbnps-induced Inflammation

NLRP3 inflammasome is an intracellular supramolecular complex comprising of NLR family,pyrin domain containing 3,apoptosis-associated speck-like protein and caspase-1.Once activated,NLRP3 recruits the adaptor protein,apoptosis-associated speck-like protein(ASC),leading to cleavage of caspase-1 and subsequent cytokine secretion,including interleukins-1?(IL-1?)and interleukins-18(IL-18).Several studies have documented that activation of NLRP3 inflammasome is associated with acute and chronic inflammatory responses in the lung and can lead to the development of many respiratory diseases.This study aims to discuss the relationship between CBNPs and NLRP3 inflammasome in vivo and in vitro.The results showed that CBNPs contributed to pulmonary inflammation and other extrapulmonary organ inflammation due to NLRP3 inflammasome activation.The occurrence and development of inflammation could be suppressed by inhibiting the activation of NLRP3 inflammasom.This study provides new clues for the molecular mechanism of CBNPs-induced respiratory diseases.The toxicity of CBNPs was confirmed in vivo and in vitro1.RAW264.7 cells were exposured to different concentrations of CBNPs(50,100,200 ?g/m L)for 6,12 or 24 hours.CCK-8 results revealed that the viability of RAW264.7 cells was suppressed in a dose-depedent manner of CBNPs.2.To investigate the toxicity of CBNPs to mice,C57BL/6 mice were exposed to CBNPs in the dynamic inhalation exposure system outfitted with an aerosol generator.After 3 days exposure,an increasing trend in the number of mononuclear macrophages.And after 3 and 7 days exposure,an increasing trend in the number of neutrophils and eosinophilic granulocytes in the whole-blood samples were observed.3.A pulmonary inflammatory response consisting of inflammatory cell infiltration and alveolitis was observed in mice following 3 days CBNP treatment.Notable extent of alveolitis and fibrosis of lung were observed in lung sections of C57BL/6 mice with the increased exposure time.4.Hepatocytes enlarged with narrowed hepatic sinusoids and hepatocytes necrosis with inflammatory cell infiltration were observed in mice liver exposed to CBNPs.Intestinal stress reactions with incomplete colonic mucosa were observed in mice exposed to CBNPs.5.Masson staining revealed that alveoli structures were destroyed and the thickness of collagen fibers around bronchi was increased.6.BAL fluid and serum of mice were collected for ELISA assay.The results showed that the levels of IL-1? in serum and BALF were increased after exposure to CBNPs,and the levels of IL-1? and IL-6 were both increased in serum of mice after CBNPs exposure,compared with control.The mechanism of NLRP3 inflammasome activation induced by CBNPs1.According to the increasing level of IL-1? and alveolitis,IHC staining was used to detect the expression of NLRP3 in lung sections.The results revealed that the expression of NLRP3 and IL-1? were increased dramatically in mice exposed to CBNPs,compared with control.2.The CBNP-induced secretion of IL-1? was increased in a CBNPs concentration-dependent manner at 6 and 12 hours compared to control.3.Western Blot and q RT-PCR assays were used to detect the protein level and gene expression of NLRP3 inflammasome in BMDMs treated with CBNPs.The results showed that the expression of NLRP3,caspase-1 and IL-1? increased significantly after exposure.4.The generation of ROS and the release of Cathespin B are considered as the signals of NLRP3 inflammasome activation.Flow cytometry was used to detect the level of ROS in BMDMs cells after CBNPs exposure.BMDMs were pre-treated with a cathepsin B inhibitor(CA-074Me)prior to treatment with CBNPs and evaluated the changes in NLRP3.The results showed that the realese of ROS was significantly increased after exposure to CBNPs in a concentration-dependent manner.When Cathespin B inhibitors were pre-treated with BMDMs,the expression of NLRP3 inflammassome related gene protein was inhibited.Effect of NLRP3 gene knockout on lung injury induced by CBNPs in miceTo further verify the role of NLRP3 inflammasome in CBNPs-induced lung injury,NLRP3 knockout mice and wild-type mice were exposed to CBNPs for 3 days.1.IHC staining results showed that the protein levels of NLRP3 was inhibited in NLRP3-/-mice which exposed to CBNPs compared to control.However,the expression of NLRP3 in lung sections of mice exposed to CBNPs increased significantly.2.ELISA results revealed that the levels of IL-1? in BAL fluid and serum of NLRP3-/-mice showed no significant difference after exposure to CBNPs.However,CBNPs exposure significantly increased the levels of IL-1? in WT mice.3.H&E results revealed that lung injuries induced by CBNPs were alleviated in NLRP3-/-mice.To sum up,we established a dynamic inhalation model of CBNPs in C57BL/6 mice.Lung injuris and other extrapulmonary organ damages in mice were observed after exposure to CBNPs.NLRP3 inflammasome was activated in lung sections after CBNPs exposure.Acute pulmonary inflammation could be induced by a short-term exposure of CBNPs.The persistent inflammation led to histological pathological damages.The activation of NLRP3 inflammsome was induced by CBNPs exposure in vitro.The release of ROS and Cathespin B were considered as two activating signals of NLRP3 inflammasome in this study.Inhibition of of NLRP3 inflammasome can inhibit the process of pulmonary inflammation induced by CBNPs.Inhibition of of NLRP3 inflammasome has potential to suppress the development of pulmonary fibrosis to a certain extent.This study suggested that NLRP3 can be regarded as a treatment target site for CBNPs exposure and provided a new perspective for the repair and intervention of respiratory injury after CBNPs exposure.