Administration of Sparstolonin B- a TLR4 antagonist, attenuates hepatic fibrosis by inhibiting TLR4 mediated hepatic fibroblast proliferation.

Abstract

Abstract Previous studies have shown that TLR4 activation accentuates hepatic inflammation and fibrosis in nonalcoholic steatohepatitis (NASH). Here we report an anti-fibrotic activity of Sparstolonin B (SsnB)-a TLR4 antagonist, derived from Chinese herb Sparganium stoloniferum. We hypothesize that, SsnB decreases TLR4 induced inflammation and fibrosis in liver by inhibiting TLR4 signaling, TGFβ signaling and by inducing senescence in fibrogenic hepatic stellate cells. Here we used Bromodichloromethane (BDCM) induced liver fibrosis model where diet induced obese (DIO) mice were administered with BDCM (a CYP2E1 activator) for 4 weeks. A similar group were administered SsnB for 4 weeks along with BDCM. Picrosirius stain and IHC of fibronectin images showed a robust decrease of extracellular matrix deposition upon SsnB administration. Mechanistically, we found that SsnB decreased fibroblast proliferation by inhibiting TLR4-PI3K akt mediated fibroblast proliferation-as was shown by increased PTEN, p53, p21 and decreased cyclin D1, MDM2 protein expression in SsnB treated mice. Interestingly, we found a parallel mechanism in which SsnB increased BAMBI (a TGFβ pseudo-receptor) protein expression. Inhibition was possibly mediated by targeting the TLR4 dependent MYD88-NfkB pathway and TGFβ signaling was decreased following attenuated SMAD2/3-SMAD4 co-localization in the nucleus. SsnB increased gene expression of cell senescence markers (p53, p21), decreased alpha SMA gene expression and increased anti-fibrotic matrix metallo protease 2 (MMP2) gene expression in rat stellate cells in vitro. BAMBI gene and protein expression- a negative regulator of pro-fibrogenic TGFβ signaling, were also increased by SsnB treatment in vitro.