Anti-inflammatory glycosides from plants: Identification of cellular targets and examination of structure–function relationships
Biochemistry
Final Report Abstract
Saponins are a very diverse group of glycosylated steroids or triterpenoids. Thousands of structures are known, in particular of saponins isolated from officinal plants. It is an undoubted finding that certain saponins have a strong anti-inflammatory potential; however, most investigations applied saponin mixtures of semi-purified fractions or total saponins and did not provide mechanistic insights. The project therefore aimed at elucidating structure–function relationships of saponins regarding their known anti-inflammatory nature. Four major objectives were defined for the project, (1) selection of anti-inflammatory saponins suitable for the investigations, (2) identification of primary cellular targets to understand their interaction with antiinflammatory saponins, (3) determination of structural characteristics of saponins that are required to exhibit their anti-inflammatory effects, and (4) identification and structural elucidation of new saponins with similar behavior to generalize the molecular mechanisms. For our main studies, we selected three substances, one well known triterpenoid saponin from Panax ginseng C. A. MEY (Rk1), one steroidal saponin isolated from Liriope muscari L. H. BAILEY with very less information available yet (DT-13), and for comparison, an anti-inflammatory drug regularly used in the clinic, the glucocorticoid dexamethasone. LPS stimulated RAW264.7 mouse macrophages were used as in vitro inflammatory model. DT-13 inhibited pro-inflammatory cytokines more efficiently when compared to Rk1 and dexamethasone and downregulated gene expression of TNFα and COX-2 by 85% and 99% respectively. It also inhibited IL-6 release from cells by 89% as measured by ELISA. Like Rk1 and dexamethasone, DT-13 inhibited nuclear localization of p-NFκB, as observed with immunofluorescence microscopy. This indicates that DT-13 acts as anti-inflammatory agent via NFκB signaling pathway. The confirmation of an interference of NFκB signaling by DT-13 led to investigations of other genes regulated by NFκB activation. It was observed that DT-13 reduced the expression of NLRP3 and 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. This concludes that it is involved in attenuation of NLRP3 inflammasome formation as well. DT-13 and Rk1 were explored as ligands for PPARγ activation using a 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 studies that show the binding affinity of DT-13 in PPARγ ligand binding pocket similar to that of rosiglitazone. To summarize, all the results successfully concluded anti-inflammatory and anti-inflammasome properties of DT-13 via ligand activation of PPARγ in mouse macrophages.
Publications
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DT-13 attenuates inflammation by inhibiting NLRP3-inflammasome related genes in RAW264.7 macrophages. Biochemical and Biophysical Research Communications, 708, 149763.
Raina, Shikha; Hübner, Emely; Samuel, Esther; Nagel, Gregor & Fuchs, Hendrik
