Neutral Polysaccharide from Bletilla striata (Thunb. ex A. Murray) Rchb. f. Alleviates Liver Fibrosis through Modulation of TGF- β /SMAD Signaling Axis and Remodeling of Gut Microbiota

Abstract Hepatic fibrosis remains a global health concern that urgently requires development of new anti-fibrotic compounds with high efficacy and low toxicity. Bletilla striata, known for its high polysaccharide content and well-established safety profile, shows therapeutic potential. In this study, we isolated a novel neutral glucomannan (BsPsIII1-1, approximately 74.8 kDa) from B. striata. Structural analysis showed that the proposed main chain repeating unit was →{4)-β-D-Glcp-(1}x→{4)-β-D-Manp-(1}n→{4)-β-D-Glcp-(1}y→4)-β-D-2Ac-Manp-(1→4)-β-D-3Ac-Manp-(1→4)-β-D-2Ac-Manp-(1→{4)-β-D-Manp-(1}m→4)-β-D-3Ac-Manp-(1→{4)-β-D-Manp-(1}i→ (where x+y=8 and n+m+i=4). BsPsIII1-1 attenuated hepatic fibrosis both in vitro, by reducing TGF-β1-induced α-smooth muscle actin (α-SMA) and collagen type I (COL-1) in LX2 cells, and in vivo, by mitigating CCl₄-induced weight loss, AST and ALT elevation, and hepatic MDA and GSH imbalance in mice, with histopathology confirming decreased collagen deposition. Mechanistically, BsPsIII1-1 inhibited TGF-β/SMAD signaling by inhibiting TGF-β1 secretion, suppressing phosphorylation of SMAD2 and SMAD4, and increasing SMAD7. It also modulated gut microbiota by enhancing beneficial bacteria, suppressing pathogens, and promoting production of short-chain fatty acids. These actions synergistically downregulated α-SMA and COL-1 through the gut-liver axis and TGF-β/SMAD signaling. In our study, we identified BsPsIII1-1, a novel biomacromolecule isolated from B. striata, as a potential anti-fibrotic agent with hepatoprotective properties, demonstrating promise as a functional food or therapeutic candidate for the treatment of fibrotic liver diseases.