Rhein alleviates renal interstitial fibrosis by inhibiting Smad3 phosphorylation in TGF-β/Smad signalling pathway

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- β 1-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 interferometry 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 therapeutic development in renal fibrosis. These findings provide a new mechanistic insight for further clinical research and drug development.