Timosaponin BII reduces colonic inflammation and alleviates DSS-induced ulcerative colitis by inhibiting NLRP3

Ethnopharmacological relevance: The Timosaponin BII (TBII) is one of the main active components of the traditional Chinese medicine Anemarrhena asphodeloides, and it is a steroidal saponin with various pharmacological activities such as anti-oxidation, anti-inflammatory and anti-apoptosis. However, its role in acute ulcerative colitis remains unexplored thus far. Aim of the study: This study aims to investigate the protective effect of TBII against dextran sulfate sodium (DSS)induced ulcerative colitis in mice and elucidate its underlying mechanisms. Methods: Wild -type (WT) and NLRP3 knockout (NLRP3(-/-)) mice were applied to evaluate the protective effect of TBII in DSS-induced mice colitis. Pharmacological inhibition of NLRP3 or adenovirus-mediated NLRP3 overexpression in bone marrow-derived macrophages (BMDM) from WT mice and colonic epithelial HCoEpiC cells was used to assess the role of TBII in LPS + ATP-induced cell model. RNA-seq, ELISA, western blots, immunofluorescence staining, and expression analysis by qPCR were performed to examine the alterations of colonic NLRP3 expression in DSS-induced colon tissues and LPS + ATP-induced cells, respectively. Results: In mice with DSS-induced ulcerative colitis, TBII treatment attenuated clinical symptoms, repaired the intestinal mucosal barrier, reduced inflammatory infiltration, and alleviated colonic inflammation. RNA-seq analysis and protein expression levels demonstrated that TBII could prominently inhibit NLRP3 signaling. TBII-mediated NLRP3 inhibition was associated with alleviating intestinal permeability and inflammatory response via the blockage of communication between epithelial cells and macrophages, probably in an NLRP3 inhibition mechanism. However, pharmacological inhibition of NLRP3 by MCC950 or Ad-NLRP3 mediated NLRP3 overexpression significantly impaired the TBII-mediated anti-inflammatory effect. Mechanistically, TBIImediated NLRP3 inhibition may be partly associated with the suppression of NF-kappa B, a master pro-inflammatory factor for transcriptional regulation of NLRP3 expression in the priming step. Moreover, co-treatment TBII with NF-kappa B inhibitor BAY11-7082 partly impaired TBII-mediated NLRP3 inhibition, and consequently affected the IL1 beta mature and secretion. Importantly, TBII-mediated amelioration was not further enhanced in NLPR3(-/-) mice. Conclusion: TBII exerted a prominent protective effect against DSS-induced colitis via regulation of alleviation of intestinal permeability and inflammatory response via the blockage of crosstalk between epithelial cells and macrophages in an NLRP3-mediated inhibitory mechanism. These beneficial effects could make TBII a promising drug for relieving colitis.