LAUSR

ABSTRACT SsnB previously showed a promising role to lessen liver inflammation observed in a mouse model of NAFLD. Since NAFLD can progress to fibrosis, studies were designed to unravel its role in attenuating NAFLD associated fibrosis. Using both in vivo and in vitro approaches, the study probed the possible mechanisms that underlined the role of SsnB in mitigating fibrosis. Mechanistically, SsnB, a TLR4 antagonist, decreased TLR4‐PI3k akt signaling by upregulating PTEN protein expression. It also decreased MDM2 protein activation and increased p53 and p21 gene and protein expression. SsnB also downregulated pro‐fibrogenic hedgehog signaling pathway, inhibited hepatic stellate cell proliferation and induced apoptosis in hepatic stellate cells, a mechanism that was LPS dependent. Further, SsnB decreased fibrosis by antagonizing TLR4 induced TGF&bgr; signaling pathway. Alternatively, SsnB augmented BAMBI (a TGF&bgr; pseudo‐receptor) expression in mice liver by inhibiting TLR4 signaling pathway and thus reduced TGF&bgr; signaling, resulting in decreased hepatic stellate cell activation and extracellular matrix deposition. In vitro experiments on human hepatic stellate cell line showed that SsnB increased gene and protein expression of BAMBI. It also decreased nuclear co‐localization of phospho SMAD2/3 and SMAD4 protein and thus attenuated TGF&bgr; signaling in vitro. We also observed a significant decrease in phosphorylation of SMAD2/3 protein, decreased STAT3 activation, alteration of focal adhesion protein and stress fiber disassembly upon SsnB administration in hepatic stellate cells which further confirmed the antagonistic effect of SsnB on TLR4‐induced fibrogenesis. HighlightsSparstolonin B (SsnB), a plant derived TLR4 antagonist reverses NASH‐induced fibrosis.SsnB decreases miR21 and increases PTEN‐induced cell cycle arrest.SsnB attenuates TGF‐beta signaling and SMAD2‐3 colocalization responsible for fibrosis in NAFLD.SsnB increases BAMBI that is known to negatively regulate TGF‐beta.SsnB decreases focal adhesion in the liver and stellate cells that is known for epithelial to mesenchymal transition.