Melatonin Ameliorates the Toxicity Induced by Deoxynivalenol in Murine Ovary Granulosa Cells by Antioxidative and Anti-Inflammatory Effects

Abstract

Melatonin is an important endogenous hormone that shows antioxidant functions and pleiotropic effects, playing a crucial role in animal reproduction. Ovary granulosa cells (GCs) surround the oocyte, which play an important role in regulating oocytes development. Deoxynivalenol (DON) is a common fusarium mycotoxin contaminant of feedstuff and food, posing a serious threat to human and animal reproductive systems. Herein, murine ovary GCs were studied as a reproduction cell model, aimed to assess the protective effect of melatonin on DON-induced toxicity in murine ovary GCs. The results showed that DON adversely affected the viability and growth of murine ovary GCs and increased the apoptosis rate, while melatonin administration ameliorated these toxic effects. We further reveal that DON exposure increased the intracellular reactive oxygen species level, reduced the mitochondrial membrane potential and ATP, and upregulated Tnfα (tumor necrosis factor α), Il6 (interleukin 6), and Il1β (interleukin 1 β) gene expression. Moreover, DON exposure downregulated reproductive hormone gene expression and significantly increased nuclear factor kappa B (p65) activation and mitogen-activated protein kinase phosphorylation. Melatonin treatment attenuated all these effects, suggesting that melatonin protects GCs from the adverse effects of DON by ameliorating oxidative stress, mitochondrial dysfunction, and inflammation. Overall, these results reveal the mechanisms of DON and melatonin in GCs and provide a theoretical basis for melatonin as a drug to improve mycotoxin contamination.

Keywords: anti inflammatory; antioxidation; apoptosis; deoxynivalenol; melatonin; ovary granulosa cells.

Figure 1

The effect of melatonin and DON on murine ovary GCs and oocytes. (A) The mode pattern of mammalian follicle and cumulus–oocyte complex (COC) (Magnification: 400×). (B) The effects of gradient concentration of melatonin on GCs Cell viability for 24 h. (C) The effects of combined treatment melatonin and DON on GCs cell viability for 24 h. Cell viability was determined using the CCK-8 assay. (D) Oocyte morphologies in the control, Melatonin-exposed, DON-exposed, and melatonin + DON groups. Scale bar: 100 µm. (E) The effects of melatonin on the rate of polar body extrusion in DON-exposed oocytes. The data are expressed as the means ± SEM. ** p < 0.05 compared with the control; # p < 0.05 compared with the DON group. The results showed that appropriate melatonin significantly inhibited DON-induced decreases in cell viability. MEL: melatonin.

Figure 2

Melatonin treatment ameliorates DON-induced mouse ovary GC apoptosis. (A) Apoptosis of GCs was evaluated by measurement of Annexin V using flow cytometry. Apoptotic cells were Annexin V-positive and PI (propidium iodide)-negative. (B) The figure shows representative staining, and the numbers in the quadrants indicate the percentage of cells within the respective subpopulations. (C,D) Western blotting analysis of the changes in the protein levels of related apoptosis factors (Bax, Bcl-2, cleaved caspase-3, caspase-3, caspase9). (E,F) Western blotting analysis of the changes in the protein level of functional genes linked to proliferation (PCNA, CDK1, and CCND2). Each bar represents the mean ± SD from three different experiments, n = 3. * p < 0.05 and ** p < 0.01 compared with the control and melatonin (MEL) groups; # p < 0.05, ## p < 0.01 compared with the DON group. MEL: melatonin.

Figure 3

Effects of melatonin on ROS levels and mitochondrial function of murine ovary GCs upon DON exposure. (A) Representative images of ROS levels in the control, melatonin (MEL)-exposed, DON-exposed, and MEL + DON-treated mouse ovary GCs. Scale bar, 300 μm; (B) Fluorescence intensities of ROS in the mouse ovary GCs of the different groups; (C), qRT-PCR analysis of the relative expression of Sod and Gshpx mRNA levels. (D) Mitochondrial membrane potential levels of the different groups. (E) The ratio of the red over green fluorescence intensity by flow cytometry represents the quantitative ΔΨm in each group. (F) ATP content of the different groups. All values are expressed as the means ± SD (n = 3), * p < 0.05 and ** p < 0.01 versus the control group; # p < 0.05 and ## p < 0.01 versus the DON group. MEL: melatonin.

Figure 4

Expression of mRNAs encoding inflammatory cytokines in DON-exposed mouse ovary GCs measured using qRT-PCR. Ovary GCs were stimulated with DON, melatonin (MEL), and DON+MEL treated for 24 h. (A) The relative expression kevels of Tnfa, Il6, and Il1b were calculated by dividing their values by the Gapdh mRNA expression levels. All data are expressed as the means ± SEM. (B,C) TLR4 (1:1000 dilution) protein levels were examined in GCs after different treatments. GAPDH (1:5000 dilution) was used as internal control. * p < 0.05 and ** p < 0.01 compared with the control and MEL groups; # p < 0.05 and ## p < 0.01 compared with the DON group. MEL: melatonin.

Figure 5

Effect of melatonin on DON-induced expression of reproductive hormone-related genes in mouse ovary GCs, measured by qRT-PCR and western blotting. Ovarian GCs were stimulated with DON, melatonin (MEL), and DON+MEL for 24 h. (A) The relative expression levels of Ar, Fshr, Star, P450scc, P450arom, and Hsf2 were calculated by dividing their values by the Gapdh mRNA expression level. All data are expressed as the means ± SEM. (B,C) The protein levels of AR (1:1000 dilution) and FSHR (1:1000 dilution) in murine ovary GCs, assessed using western blot. GAPDH (1:5000 dilution) as the control. The results from each group are shown as the mean ± SD, n = 3. * p < 0.05 and ** p < 0.05 compared with the control and MEL groups; # p < 0.05 and ## p < 0.05 compared with the DON group. MEL: melatonin.

Figure 6

Melatonin (MEL) inhibits DON-induced NF-κB and MAPK activation in murine ovary granulosa cells. Ovarian GCs were stimulated with DON, MEL, and DON+MEL for 24 h. (A,B) IkBα (1:1000 dilution), p-IκBα (1:1000 dilution), P65 (1:1000 dilution), and p-P65 (1:1000 dilution) were examined using western blotting in GCs treated with DON, MEL, and DON+MEL for 24 h. (C,D) P38 (1:1000 dilution), p-P38 (1:1000 dilution), ERK (1:1000 dilution), p-ERK (1:1000 dilution), JNK (1:1000 dilution), and p-JNK (1:1000 dilution) were examined by western blotting in GCs. All data are shown as mean ± SD, n = 3. ** p < 0.05 compared with the control and MEL groups; # p < 0.05 and ## p < 0.05 compared with the DON group. MEL: melatonin.

Figure 7

Diagram of DON exposure and melatonin administration to murine ovary granulosa cells.