Saponins are secondary metabolites that aid in plants’ defense against herbivory and have been used in ancient medical practices. Ginsenosides from Panax ginseng C.A.MEY, Curcumin from Curcuma longa L. and many more were explored for their anti-inflammatory, anti-cancer, and anti-fungal properties. Especially triterpenoid saponins like ginsenosides are well characterized whereas steroidal saponins like DT-13 are not well explored. Therefore, in this study we investigated the mechanism and targets of DT-13 as an anti-inflammatory compound. To achieve this, lipopolysaccharide (LPS) stimulated RAW264.7 mouse macrophages were used as in vitro inflammatory model. Cells pretreated with DT-13 were compared with those pretreated with Rk1 (ginseng saponin) and dexamethasone (synthetic anti-inflammatory drug). DT-13 inhibited pro-inflammatory cytokines more efficiently when compared to Rk1 and dexamethasone. DT-13 downregulated gene expression of tumor necrosis factor-alpha (TNFα), cyclooxygenases-2 (COX-2) by 85% and 99% respectively. It also inhibited interleukin-6 (IL-6) release from cells by 89% as measured by enzyme linked immunosorbent assay (ELISA). Like Rk1 and dexamethasone, DT-13 inhibited nuclear localization of phosphorylated-nuclear factor kappa B (p-NFκB), as observed with immunofluorescence microscopy. These results indicate that DT-13 acts as anti-inflammatory agent via NFκB signaling pathway. The confirmation of interference of NFκB signaling by DT-13 lead to investigation of other genes regulated by NFκB activation. Interestingly, it was observed that DT-13 reduced the expression of nod like receptor family pyrin binding domain 3 (NLRP3) and interleukin 1 beta (IL-1β) gene by 73% and 92% respectively. It also significantly inhibited NLRP3-inflammasome induced Caspase-1 activation and IL-1β release. Membrane attack complex formation was also inhibited as checked by alternate pathway (AP) complement assay. This concludes that it is involved in attenuation of NLRP3 inflammasome formation as well. It is known that ligand activated peroxisome proliferator activated receptor inhibits LPS stimulated NFκB gene activation by a mechanism called as ‘trans-repression’. DT-13 and Rk1 were explored as ligands for PPARγ activation using HEK transfection model system. DT-13 and Rk1 both significantly induced the expression of luciferase conjugated PPARγ response elements and hence the activation of PPARγ. This was also concluded by in silico study that shows the binding affinity of DT-13 in PPARγ ligand binding pocket similar to that of rosiglitazone. To summarise, all the results successfully concluded anti-inflammatory and anti-inflammasome properties of DT-13 via ligand activation of PPARγ in mouse macrophages.
