Inhibitory effects of the flavonoids extracted from Pollen Typhae on palmitic acid-induced NLRP3 inflammasome activation in macrophages involving AMPK-mediated lipid metabolism.

Background: Pollen Typhae (PT), a traditional Chinese medicine herb utilized in diabetes management, exerts anti-inflammatory effects through its flavonoids, yet the active constituents and mechanisms remain unclear.

Methods: PT total flavone (PTF) was extracted from PT and identified the compounds by UHPLC-MS. Network pharmacology and molecular docking were used to predict the underlying targets and anti-inflammatory mechanisms of PTF. The prediction was validated in RAW264.7 macrophages. IL-1β and IL-18 in culture supernatants were analyzed by ELISA. The protein and gene expression were checked by western blotting and Real-time PCR, respectively. Intracellular ROS production was detected by DCFH-DA method. Intracellular lipids were analyzed by ELISA and Enzyme assay. The Caspase-1 activity was evaluated by bioluminescent method.

Results: PTF was identified 47 flavonoid compounds, including typhaneoside (TYP). Network pharmacology and molecular docking indicated that the flavonoid compounds might regulate inflammatory response, fatty acid metabolism, and the NOD-like receptor, AMPK pathways. PTF and TYP inhibited palmitic acid (PA)-induced NLRP3 inflammasome activation in lipopolysaccharide-primed RAW264.7 macrophages, leading to decreased secretion of IL-1β and IL-18. Furthermore, PTF and TYP improved intracellular lipid metabolism in PA-induced macrophages, indicating decreased free fatty acid and triglyceride contents, reduced protein expression of CD36, PPARγ, FAS, DGAT1, and CPT-1, as well as declined ROS with increased ATP production. Additionally, PTF and TYP increased the p-AMPK/AMPK ratio and upstream p-LKB1/LKB ratio. Activated AMPK, in turn, ameliorated lipid metabolism dysfunction, thus abolishing PA-induced ROS production and NLRP3 inflammasome activation. Antioxidant and improving lipid metabolism by suppressing ACC also inhibited NLRP3 inflammasome activation, respectively. Importantly, AMPK inhibition attenuated or abolished the inhibitory effects of PTF or TYP on ROS production, IL-1β and IL-18 secretion, and Caspase-1 activity.

Conclusion: The findings highlight the ability of PTF and its active component TYP to inhibit PA-induced NLRP3 inflammasome activation in macrophages involving AMPK-mediated lipid metabolism, implying the potential use of PT flavonoid compounds as anti-diabetic inflammation lead compounds.