Triptolide-containing Tripterygium wilfordii tablet induces male reproductive toxicity via targeting the TGF-β1/Smad2/SF-1 signaling pathway

Tripterygium wilfordii tablet (TWT) is a widely used therapeutic agent for rheumatoid arthritis, but its clinical utility is restricted by severe reproductive toxicity. This study aimed to elucidate the male reproductive toxic effects of TWT and its underlying mechanisms, focusing on the TGF-β1/Smad2/SF-1 signaling pathway, in which TGF-β1/Smad2 acts as the upstream regulatory module of SF-1. Fifty male Sprague-Dawley rats were randomized into five groups: normal control, collagen-induced arthritis (CIA) model, and low/middle/high-dose TWT-treated CIA groups. CIA was induced via bovine type II collagen immunization, and TWT was administered intragastrically for 21 or 42 days. In vitro, triptolide (TP), the core toxic component of TWT, was used to treat GC-2spd(ts) and TM3 cells. Key assays included Western blot and immunofluorescence for analyzing protein expression (TGF-β1, Smad2, SF-1) and localization; flow cytometry for cell cycle and apoptosis; transmission electron microscopy for ultrastructural changes; and histopathology for reproductive tissue damage. In vivo, TWT induced dose- and time-dependent reproductive dysfunction in CIA rats, characterized by reduced sperm count, increased sperm malformation, testicular/epididymal histopathological damage, decreased serum testosterone, and dysregulated expression of TGF-β1, Smad2, and SF-1. In vitro, TP inhibited cell viability, induced G1 phase arrest, apoptosis, and ultrastructural injury, with consistent perturbations in the TGF-β1/Smad2/SF-1 signaling pathway as observed in vivo. These findings demonstrate that TWT induces male reproductive toxicity in CIA rats and target cells through activating TGF-β1/Smad2 signaling and inhibiting SF-1, providing mechanistic insights into TWT-associated reproductive toxicity.