Bilirubin stabilizes the mitochondrial membranes during NLRP3 inflammasome activation.

Mitochondria sense both intracellular and extracellular stress, with the subsequence released mt-ROS resulted from the interruption of its membrane integrity being a direct activator for NLRP3 inflammasome activation. Regulating the morphology and function of mitochondria could be a strategy against uncontrolled inflammation. We have previously reported that physiological concentrations of bilirubin exhibit anti-inflammatory effect by inhibiting both NF-κB and inflammasome activation. In the current study, we investigated its anti-NLRP3 inflammasome effect per se by means of detecting releasing of IL-1ß and TNF-α, the formation of ASC oligomers and ASC-specks, as well as pro-caspase-1 recruitment. Mechanistically, with respect to the antioxidant nature of bilirubin, we evaluated the effect of bilirubin on the releasing of mt-ROS from mitochondria. In addition, mitochondrial morphofunction mainly including morphology and membrane potential in contact living macrophages was analyzed by applying a newly developed multiplexed high-content mitochondrial imaging analysis system using live-cell microscopy. We revealed that bilirubin targets and stabilizes mitochondrial membrane during NLRP3 inflammasome activation; defined doses of bilirubin could be considered as a mitochondria targeted medication against inflammasome-related diseases.

Identification of bioactive ingredients from Babaodan using UPLC-QTOF-MS analysis combined with network pharmacology guided bioassays.

Babaodan (BBD) is a traditional Chinese medicine (TCM) prescribed for various inflammatory diseases, including viral hepatitis and acute genitourinary tract infection. Like other TCMs, BBD is a multi-component formula whose chemical composition and mode of action are largely unknown. The current study identified the bioactive ingredients of BBD using ultrahigh-performance liquid chromatography combined with quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS) followed by mass spectrometry molecular networking analysis. Subsequently, network pharmacology analysis was performed to predict the potential targets and pathways regulated by BBD. Eventually, a panel of compounds was selected and examined for their anti-inflammatory effects using lipopolysaccharide-stimulated RAW264.7 cells. Eighty-six compounds, including saponins, bile acids, and fatty acids, were identified. Tumor necrosis factor-alpha was identified as a key molecule. Pathways in cancer, inflammatory bowel disease, and hepatitis were predicted to be the major regulatory pathways. The results from bioassays validated ginsenoside Rb1, ginsenoside Rd, deoxycholic acid, chenodeoxycholic acid, and taurochenodeoxycholic acid as novel bioactive ingredients in BBD with anti-inflammatory effects. In conclusion, our study explains the anti-inflammatory efficacy of BBD from both chemical and biological aspects, which provides a scientific basis for the clinical application of BBD in inflammation-related diseases.