Ganoderma lucidum polysaccharide inhibits HSC activation and liver fibrosis via targeting inflammation, apoptosis, cell cycle, and ECM-receptor interaction mediated by TGF-β/Smad signaling.

• GLP attenuates CCl 4 -induced liver injury, inflammation, and fibrosis in mice. • GLP inhibits HSCs activation and collagen formation in vivo and in vitro. • GLP elicits dual effects on apoptosis in vivo (inhibit) and in vitro (promote). • GLP inhibits ECM-receptor interaction pathway including integrin expressions. • GLP alleviates liver fibrosis via suppressing the TGF-β/Smad signaling pathway. Ganoderma lucidum polysaccharide (GLP) has many biological properties, however, the anti-fibrosis effect of GLP is unknown at present. This study aimed to examine the anti-fibrogenic effect of GLP and its underlying molecular mechanisms in vivo and in vitro. Both CCl 4 -induced mouse and TGF-β1-induced HSC-T6 cellular models of fibrosis were established to examine the anti-fibrogenic effect of a water-soluble GLP (25 kDa) extracted from the sporoderm-removed spores of G. lucidum.. Serum markers of liver injury, histology and fibrosis of liver tissues, and collagen formation were examined using an automatic biochemical analyzer, H&E staining, Sirius red staining, immunohistochemistry, immunofluorescence, ELISA, Western blotting, and qRT-PCR. RNA-sequencing, enrichment pathway analysis, Western blotting, qRT-PCR, and flow cytometry were employed to identify the potential molecular targets and signaling pathways that are responsible for the anti-fibrotic effect of GLP. We showed that GLP (150 and 300 mg/kg) significantly inhibited hepatic fibrogenesis and inflammation in CCl 4 -treated mice as mediated by the TLR4/NF-κB/MyD88 signaling pathway. We further demonstrated that GLP significantly inhibited hepatic stellate cell (HSCs) activation in mice and in TGF-β1-induced HSC-T6 cells as manifested by reduced collagen I and a-SMA expressions. RNA-sequencing uncovered inflammation, apoptosis, cell cycle, ECM-receptor interaction, TLR4/NF-κB, and TGF-β/Smad signalings as major pathways suppressed by GLP administration. Further studies demonstrated that GLP elicits anti-fibrotic actions that are associated with a novel dual effect on apoptosis in vivo (inhibit) or in vitro (promote), suppression of cell cycle in vivo , induction of S phase arrest in vitro , and attenuation of ECM-receptor interaction-associated molecule expressions including integrins ITGA6 and ITGA8. Furthermore, GLP significantly inhibited the TGF-β/Smad signaling in mice, and reduced TGF-β1 or its agonist SRI-011381-induced Smad2 and Smad3 phosphorylations, but increased Samd7 expression in HSC-T6 cells. This study provides the first evidence that GLP could be a promising dietary strategy for treating liver fibrosis, which protects against liver fibrosis and HSC activation through targeting inflammation, apoptosis, cell cycle, and ECM-receptor interactions that are mediated by TGF-β/Smad signaling. [Display omitted] [ABSTRACT FROM AUTHOR]