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-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 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.