Studies On The Inhibitory Effects And The Underlying Mechanism Of Scutellarin On PANoptosis In Macrophages

Aim:As an important kind of innate immune cells,macrophages are one of the key immune cells that regulate a variety of inflammatory responses,with a complex signaling network of regulated cell death.Macrophages are not only the body’s first line of defense against pathogen infection,but also involved in the pathological processes of a variety of inflammatory diseases.Recent studies have revealed that macrophages can simultaneously undergo pyroptosis,apoptosis,and necroptosis,a complex pattern of cell death known as PANoptosis,when can be induced by multiple viral,bacterial,or inflammatory factors,or when the transforming growth factor βactivated kinase 1(TAK1)activity is lost.PANoptosis may play an important role in various inflammatory diseases,thus making it a potential therapeutic target for the prevention and treatment of various inflammatory diseases.However,there is currently no inhibitor for inhibiting this form of cell death.Scutellarin is a natural flavonoid with a variety of pharmacological activities,including anti-inflammatory,antioxidant,anti-apoptotic,protecting endothelial cells and myocardium,etc.,and is clinically used to treat stroke,myocardial infarction and so on.Previous studies have shown that scutellarin can effectively inhibit the activation of canonical and non-canonical NLRP3 inflammasomes,but its effect on PANoptosis of macrophages is currently unknown.Using macrophages as a cell model,TAK1 inhibitor(TAK1i)combined with LPS was used to induce PANoptosis,and the effects and mechanism of action of scutellarin on PANoptosis were explored,which would provide experimental and theoretical basis for further clinical research on PANoptosis-related inflammatory diseases.Methods:1.Induction of the differentiation of C57BL/6 mouse bone marrow cells in vitro to obtain bone marrow-derived macrophages(BMDMs).2.The BMDMs were pretreated with TAK1i(5Z-7-oxozeaenol)followed by LPS stimulation to establish the in vitro cell model of PANoptosis.3.Propidium iodine(PI)and Hoechst 33342 staining was used to analyze the effect of scutellarin on TAK1i+LPS-induced PANoptosis in BMDMs.4.Several critical proteins associated with the pyroptosis(e.g.,Caspase-1,GSDMD),proteins associated with the apoptosis(e.g.,Caspase-3),and necroptosis-related proteins(e.g.,p-MLKL)were detected by using Western blotting.5.The formation of ASC speck and its co-localization with p-MLKL and cleaved caspase-3 were analyzed by immunofluorescence microscopy.6.Mitochondrial damage and reactive oxygen series(ROS)were analyzed by means of mitochondrial membrane potential detection kit(JC-1),TMRE,DCFH-DA,mitochondrial superoxide probe(Mito SOX)staining combined with immunofluorescence microscopy and flow cytometry,respectively.7.The effect of scutellarin on related inflammatory diseases in vivo were explored in Con A-induced acute hepatitis in mice.Results:1.TAK1 i in combination with LPS induced PANoptosis in primary murine BMDMs,thus establishing a cellular model of PANoptosis in vitro.2.Scutellarin was able to dose-dependently inhibit the occurrence of TAK1i+LPS-induced PANoptosis in BMDMs.3.Scutellarin simultaneously inhibited expression of hallmarks for three cell death pathways,including pyroptosis,apoptosis and necroptosis,in TAK1i+LPS-activated BMDMs.4.Scutellarin significantly inhibited the assembly of PANoptosome in TAK1i+LPS-induced BMDM.5.TAK1i+LPS-induced PANoptosis was accompanied by intracellular mitochondrial function damage and ROS production,whereas scutellarin was able to significantly protect mitochondrial function and decrease intracellular levels of ROS and superoxide,suggesting that this drug inhibited PANoptosis by scavenging ROS.6.Scutellarin reduced the severity of Con A-induced hepatitis in mice.Conclusion:A macrophage PANoptosis model was induced by TAK1 inhibitor combined with LPS.Scutellarin exhibited a dose-dependent inhibitory effect on PANoptosis of macrophages,simultaneously inhibiting the cell death pathways of pyroptosis,apoptosis and necroptosis.Scutellarin inhibited the assembly of PANoptosome.Scutellarin-mediated inhibition of macrophage PANoptosis was associated with its protection of mitochondrial function and lowering ROS levels.Scutellarin significantly alleviated symptoms of acute hepatitis in Con A-induced hepatitis in mice.These results suggest that scutellarin is an inhibitor of PANoptosis and has clinical applications for the treatment of inflammatory diseases associated with PANoptosis.