Ginsenoside Rb1 normalizes mitochondrial dynamic homeostasis and alleviates glufosinate-ammonium-induced testicular damage through targeting Drp1.

Glufosinate-ammonium (GLA), a broad-spectrum non-selective herbicide widely used in contemporary agriculture, raises concerns due to its potential reproductive toxicity. Ginsenoside Rb1, a triterpenoid glycoside, exhibits antioxidant and anti-inflammatory activities. Therefore, this study employs network toxicology and pharmacology to comprehensively analyze GLA-induced testicular toxicity mechanisms and the protective mechanism of ginsenoside Rb1. Results show that GLA exposure causes testicular injury accompanied by vacuolization of Sertoli cells, spermatogenic cell detachment and disorganized spermatids, and the action in a dose-dependent manner. Network toxicology and pharmacology identified mitochondrion, autophagy and apoptosis as key target pathways mediating GLA toxicity, while ginsenoside Rb1's protection also occurred mainly by regulating mitophagy. We observed that GLA exposure causes excessive mitochondrial fragmentation and autophagosome. Mechanistically, GLA disturbs mitochondrial dynamical homeostasis via significantly upregulating mitochondrial fission proteins (Drp1/Fis1) and inhibiting mitochondrial fusion proteins (Opa1/Mfn2), eventually causing excessive mitochondrial fission and mitophagy. Inversely, Mdivi-1 (Drp1 inhibitor) reduced GLA-induced mitochondrial fragmentation and mitophagy, while the addition of ginsenoside Rb1 greatly improved GLA-induced testicular damage by suppressing Drp1-mediated excessive mitochondrial fission and mitophagy. These findings provide a first glimpse into the molecular mechanisms of GLA-induced male reproductive toxicity in mice and demonstrate ginsenoside Rb1's potential to protect Sertoli cells from GLA exposure.