In this study,J774A.1 macrophages stimulated by lipopolysaccharide(LPS)and adenosine triphosphate(ATP)were used to establish an in vitro model of pyroptosis,and the intervention mechanism of free total rhubarb anthraquinones(FTRAs)on py-roptosis was investigated.J774A.1 macrophages were cultured in vitro,and the experiment was assigned to the control group and groups with different concentrations of LPS(0.25,0.5,and 1 μg·mL-1)and ATP(1.25,2.5,and 5 mmol·L-1).An in vitro model of macrophage pyroptosis was established by detecting cell viability through CCK-8,propidium iodide(PI)apoptotic cell staining,lac-tate dehydrogenase(LDH),interleukin(IL)-18,and tumor necrosis factor(TNF)-α release.Then,J774A.1 macrophages were ran-domly divided into six groups:blank control group,LPS+ATP group,high-dose FTRA group,and low,medium,and high-dose FTRA pre-protection group.The phenotypic characteristics and key indicators of pyroptosis were detected as the basis for evaluating the effect of FTRAs on pyroptosis induced by LPS and ATP.Western blot and RT-PCR were used to detect the expression levels of protein and mRNA related to the pyroptosis pathway in caspase-1/11 and elucidate the molecular mechanism of the anti-pyroptosis effect.The re-sults showed that the stimulation condition of 0.50 μg·mL-1 LPS+5.00 mmol·L-1 ATP was the most effective in the in vitro model of macrophage pyroptosis.FTRAs pre-protected cells for 24 h and then can increase cell viability under pyroptosis conditions,alleviate cell damage,lower the positive rate of PI staining,and reduce the release of LDH,IL-18,and TNF-α.FTRAs were able to signifi-cantly inhibit the activation of GSDMD proteins and significantly down-regulate the protein expression of the pyroptosis pathway signa-ture molecules,TLR4,NLRP3,cleaved-caspase-1,and cleaved-caspase-11,but they had no significant effect on ASC proteins.FTRAs were also able to significantly inhibit the mRNA expression of caspase-1,caspase-11,and GSDMD.These results indicate that FTRAs have an inhibitory effect on the pyroptosis model induced by LPS and ATP and play an anti-pyroptosis effect by regulating clas-sical and non-classical pyroptosis signaling pathways and reducing the production of inflammatory cytokines.
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