Polycyclic-fused cytochalasins with anti-liver fibrosis activity from the endophytic fungus Trichoderma harzianum [TGF-β1+ DMSO or TGF-β1 + 20 μM 8]

A molecular networking-guided investigation of the endophytic fungus Trichoderma harzianum MLJ-4 resulted in the isolation of eight new polycyclic-fused cytochalasins, triharziachalasins A–H (1–8), along with six known analogues (9–14). Their structures including absolute configurations, were elucidated by comprehensive spectroscopic analysis, NMR calculations with DP4+ analysis, and theoretical ECD simulations. Compounds 1–3 feature a rare 5/6/5/8-fused tetracyclic system, while 4–8 possess a novel 5/6/6/7/5-fused pentacyclic scaffold. Notably, compound 1 is the first cytochalasin analogue containing a cis-fused 5/8 ring within the 5/6/5/8 framework. All CYTs were evaluated for anti-liver fibrosis activity in TGF-β1-stimulated LX-2 cells using high content screening assays. The most promising compound, triharziachalasin H (8), dose-dependently suppressed the expression of key fivrotic markers, including fibronectin (FN), collagen I, and α-smooth muscle actin (α-SMA). Mechanism studies revealed that its anti-liver fibrosis effectis mediated via inhibition of the NF-κB signaling pathway. This work expands the structural diversity of bioactive cytochalasins and reveals the anti-liver fibrosis activity of this novel polycyclic-fused architecture, highlighting its potential as a lead compound for anti-liver fibrosis drug development.