Abstract

Renal fibrosis constitutes a central pathological hallmark of chronic kidney disease (CKD), with its progression largely mediated through the transforming growth factor‑β1 (TGF‑β1)/Smad3 signaling pathway. Ligustilide (LIG) has been reported to possess anti‑fibrotic activity; however, its protective effects and underlying molecular mechanisms in renal fibrosis remain incompletely understood. This study examined the effects of LIG and explored its underlying mechanisms in a unilateral ureteral obstruction (UUO) mouse model and TGF‑β1-stimulated mouse renal tubular epithelial cell (TCMK1) cells. In vivo, LIG (20 and 40 mg·kg⁻ 1 ) significantly alleviated renal pathological injury in UUO mice, reduced tubular dilatation, and downregulated the expression of the renal injury marker KIM‑1 as well as fibrosis‑related proteins, including fibronectin, collagen I, and α‑SMA. In vitro, LIG dose‑dependently inhibited TGF‑β1‑induced fibrotic responses in TCMK1 cells. Target screening using drug affinity responsive target stability (DARTS)‑coupled liquid chromatography–mass spectrometry (LC/MS) identified Fblim1 as a putative target of LIG. Cellular thermal shift assay (CETSA) and DARTS assays further confirmed that LIG directly binds to Fblim1, enhancing its thermal stability and protease resistance. Both in vivo and in vitro, LIG treatment reduced the mRNA and protein levels of Fblim1 and phosphorylated Smad3 (p‑Smad3). Notably, LIG suppressed the nuclear accumulation of Fblim1 and p‑Smad3, thereby attenuating TGF‑β1/Smad3‑mediated profibrotic signaling. The results reveal that ligustilide exerts its anti‑renal fibrotic effects by targeting Fblim1 and regulating the TGF‑β1/Smad3 signaling pathway, particularly its nuclear translocation process.