Scope: To evaluate the chemopreventive efficacy of hesperidin (Hsd) in 1,2-dimethylhydrazine
(DMH)-induced colorectal cancer (CRC) and demonstrate its role in mothers against
decapentaplegic homolog 4(Smad4) and activin A signaling pathways.

Methods and results: A CRC rat model was established by DMH exposure, and the animals
were randomly divided into five groups: Control group, Hsd, DMH, DMHþHsd, and DMH
followed by Hsd. The resected colon was subjected to macroscopic, microscopic, molecular,
histopathological, and immunohistochemical examination. Activin A, Smad4, malondialdehyde
(MDA), nitric oxide (NO), reduced glutathione (GSH), and superoxide dismutase (SOD)
levels in tissues were also measured. The DMH group exhibited a significant increase in the
gene and protein expression of activin A as well as MDA and NO levels in tissues. There
was a significant reduction in the gene and protein expression of Smad4 as well as GSH
and SOD levels in tissues. Administration of Hsd significantly upregulated Smad4 and activin
A gene expressions in both the DMHþHsd and DMH followed by Hsd groups. Moreover,
Hsd improved the antioxidant status of the former two groups.

Conclusion: This study demonstrated the chemopreventive effect of Hsd against CRC by
modulating Smad4 and activin A signaling in vivo. Further studies are needed to demonstrate
its clinical value and explore its possible role in advanced malignancy.