Fig 1: Proposed mechanistic model depicting the inhibitory effects of baicalein on lipopolysaccharide (LPS)-induced inflammasome activation in H9c2 cardiomyoblasts. Baicalein exerts multilevel suppression of the LPS-triggered inflammatory cascade in H9c2 cardiomyoblasts. Following LPS exposure, canonical TLR4 signaling activates NF-κB, leading to transcriptional priming of NLRP3, Casp1, and Il1b. Mitochondrial stress and elevated ROS further promote NLRP3 inflammasome assembly, resulting in caspase-1 activation and proteolytic maturation of IL-1β and IL-18, culminating in inflammatory cytokine release and cytotoxic injury. Baicalein, administered prior to LPS challenge, inhibits both the priming arm (NF-κB activation and NLRP3 induction) and the activation arm (mitochondrial dysfunction, ROS overproduction, and caspase-1 activity), thereby limiting downstream IL-1β/IL-18 maturation and reducing inflammation-associated cytotoxicity. The schematic summarizes the integrated points of interference experimentally demonstrated across Figure 1 - 4.
Fig 2: Baicalein modulates inflammasome signaling at transcriptional, protein, and enzymatic levels. A, qPCR analysis of Nlrp3, Casp1, and Il1b expression. Cells pretreated with baicalein (5 - 20 µM, 1 h) were stimulated with LPS for 2 h (the transcriptional peak defined in Figure 1). RNA isolation, cDNA synthesis, and qPCR conditions were identical to Figure 1B. B, Western Blot analysis of inflammasome-related proteins. Protein lysates were prepared in RIPA buffer containing protease/phosphatase inhibitors. Equal protein amounts (25 µg/lane) were resolved by SDS-PAGE and immunoblotted for NLRP3, caspase-1 p20, pro-IL-1β/IL-1β p17, and GAPDH. HRP-conjugated secondary antibodies and ECL substrate were used for detection. Representative blots and densitometry summaries are shown. C, Caspase-1 activity assay. Caspase-1 enzymatic activity was quantified using the Abcam ab39412 colorimetric kit. Cell lysates (200 µg protein) were incubated with YVAD-pNA substrate at 37°C for 2 h, and absorbance was measured at 405 nm.
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