摘要Objective:The anthraquinone compound rhein(1,8-dihydroxy-3-carboxyanthraquinone),derived from Rhei Radix et Rhizoma(rhubarb,Dahuang in Chinese),exhibits notable anti-fibrotic effects.However,the mechanisms underlying these effects have not been fully elucidated.Suppressor of mothers against decapentaplegic 3(Smad3)phosphorylation plays a crucial role in the canonical transforming growth factor-β(TGF-β)/Smad signalling pathway.In this study,we investigated the effect of rhein on the TGF-β/Smad signalling pathway in renal interstitial fibrosis(RIF).Methods:A unilateral ischaemia-reperfusion injury(UIRI)rat model was employed to simulate renal injury and assess the therapeutic effect of rhein in vivo.In vitro,TGF-β1-stimulated NRK-52E rat renal epithelial cells and HK-2 human proximal tubular epithelial cells were used to mimic fibrotic conditions.Rhein's interaction with Smad3 was further explored using molecular docking and bio-layer interferom-etry assays.Additionally,Smad3 knockdown and overexpression studies were performed in HK-2 cells to elucidate the functional role of Smad3 in rhein-mediated anti-fibrotic activity.Results:Rhein treatment significantly improved renal function and reduced fibrosis in UIRI rats,primarily by inhibiting Smad3 phosphorylation.Rhein treatment mitigated aberrant remodelling and extracellular matrix accumulation in both NRK-52E and HK-2 cells and in the UIRI rat model.The anti-fibrotic effects of rhein were attenuated by Smad3 deficiency but enhanced by Smad3 overexpression in HK-2 cells.Conclusion:Rhein exerts its anti-fibrotic effects in renal interstitial fibrosis by targeting the TGF-β/Smad3 signaling pathway.Acting as a natural antagonist of Smad3,rhein offers promising potential for thera-peutic development in renal fibrosis.These findings provide a new mechanistic insight for further clinical research and drug development.