Abstract
Zearalenone (ZEA) is widely derived from moldy cereal grain, which has adverse effects on animal reproduction. In particular, pigs are more sensitive to ZEA-induced toxicity than other animals. Isorhamnetin has extensive pharmacological activity. However, the role of isorhamnetin in ZEA-induced cytotoxicity remains unclear. This study was designed to investigate the therapeutic effect of isorhamnetin on ZEA-induced damage in porcine ovarian granulosa cells and elucidate its molecular mechanism. Two experiments were conducted, where a minimum of 3 biological replicates were used for each treatment. In Exp. 1, ovarian granulosa cells were treated with different concentrations of isorhamnetin (1, 5, 10, 20 and 30 μmol/L) and ZEA (0, 10, 30, 60, 90 and 120 μmol/L) for 24 h. Our results indicated that 60 μmol/L ZEA (half-maximal inhibitory concentration value) and 20 μmol/L isorhamnetin (the most effective concentration against ZEA-induced cytotoxicity) were optimum concentrations. In Exp. 2, ovarian granulosa cells were treated with isorhamnetin (20 μmol/L) for 2 h, before treatment with ZEA (60 μmol/L) for 24 h. Apoptosis, endoplasmic reticulum stress, oxidative stress, proliferation and hormone secretion of ovarian granulosa cells were detected. Our findings showed that isorhamnetin suppressed (P < 0.05) ZEA-induced apoptosis by altering mitochondrial membrane potential and apoptosis-related proteins (B-cell lymphoma-2 [Bcl-2], Bcl2-associated x [Bax] and cleaved caspase-3 [C-Casp3]). Changes in intracellular Ca2+ levels and C/EBP homologous protein (CHOP), recombinant activating transcription factor 6 (ATF6), glucose regulated protein78 kD (GRP78) indicated that isorhamnetin rescued (P < 0.05) ZEA-induced endoplasmic reticulum stress. Furthermore, isorhamnetin prevented (P < 0.05) ZEA-induced oxidative stress via the mitogen-activated protein kinase (P38) signaling pathway. Mechanistically, isorhamnetin stimulated (P < 0.05) the expression of proliferating cell nuclear antigen (PCNA) and cyclin D, thereby increasing the ratio of S phase cells in response to ZEA-induced apoptosis via phosphatidylinositol 3 kinase/protein kinase B (PI3K/Akt) signaling pathway. Isorhamnetin also recovered (P < 0.05) ZEA-induced steroidogenesis disorder by regulating steroidogenic enzyme gene and proteins (follicle-stimulating hormone receptor [FSHR] and cytochrome P450 family 19 subfamily a member 1 [CYP19A1]). Collectively, these findings show that isorhamnetin protects ovarian granulosa cells from ZEA-induced damage, which promotes proliferation, alleviates apoptosis, endoplasmic reticulum stress, oxidative stress, and steroidogenesis disorder.