Pakistan Journal of Pharmaceutical Sciences

Abstract: This research explored the protective effects of Trifolium pratense L. flower extract (TPFE) on hepatocytes against hepatocellular carcinoma (HCC) induced by N-diethylnitrosamine (DEN). The study focused on biochemical, molecular and antioxidant pathways, particularly the HOTAIR/miR-124/Notch1 axis. Fifty Wistar rats were divided into five groups: A normal control, an HCC-induced group (DEN 100mg/kg), two HCC groups treated with TPFE (200 mg/kg and 400mg/kg) and a normal group treated with TPFE (400mg/kg). At the study's end, liver function markers, inflammatory cytokines and oxidative stress parameters were measured. The expression of HOTAIR, miR-124, Notch1, and Jagged1 genes and proteins in liver tissue was assessed, along with immunohistochemical analysis for P53-positive cells. DEN administration led to significant alterations in body and liver weight, serum liver enzymes, antioxidant levels, inflammatory markers and gene/protein expression related to the HOTAIR/miR-124/Notch1 axis. Statistical analysis was performed using ANOVA, Newman-Keuls test and SPSS, with significance at p<0.05; data visualized in GraphPad. TPFE treatment mitigated these changes in a dose-dependent manner, particularly at 400mg/kg, improving weight, liver health, antioxidant capacity and inflammatory responses. Histopathological and immunohistochemical analyses showed structural liver improvements with TPFE. The study concludes that TPFE has potential anti-cancer effects against DEN-induced HCC.