Effect and mechanism of free total rhubarb anthraquinones on pyroptosis of J774A.1 macrophages
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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