Intracellular ROS levels assayed with dihydroethidium fluorescent probe in porcine granulosa cells treated with zearalenone.

<p>A (A’), Control (untreated); B (B’), 15 μM ZEA; C (C’), 30 μM ZEA; D (D’), 60 μM ZEA. Intracellular ROS was measured by flow cytometry (A-D) and microscope and fluorescence imaging (A’-D’) by detecting the signals of superoxide anion in cells. For better clarity, a guidance line (red) is drawn through histograms for comparison. E, Average intensity of dihydroethidium. The results are expressed as mean ± SD. Asterisk (*) indicates significant difference (P < 0.05)</p

Intracellular ROS levels assayed with DCFH-DA fluorescent probe in porcine granulosa cells treated with ZEA.

<p>A (A’), Control (untreated); B (B’), 15 μM ZEA; C (C’), 30 μM ZEA; D (D’), 60 μM ZEA. Intracellular ROS was measured by flow cytometry (A-D) and fluorescent imaging (A’-D’) using DCFH-DA probe. For better clarity, a guidance line (red) is drawn through histograms for comparison. E, Average intensity from DCFH-DA. The results are presented as mean ± SD. Asterisk (*) indicates significant difference (P<0.05).</p

Intracellular ROS levels assayed using DCFH-DA and DHE fluorescent probes in porcine granulosa cells treated with curcumin.

<p>A-B, Intracellular ROS was measured by flow cytometry (DCFH-DA and DHE). For better clarity, a guidance line (red) is drawn through histograms for comparison. C-D, Average intensity of DCFH-DA and DHE fluorescence in porcine granulosa cells treated with curcumin. The results are presented as mean ± SD. Asterisk (*) indicates significant difference (P<0.05)</p

The expression levels of endogenous antioxidative enzymes Sod1, Cat and Gpx1 in porcine granulosa cells treated with ZEA for different times (8–24 h).

<p>A-C. Gene expression in ZEA-treated cells at 8 h, 16 h and 24 h. The expression level was normalized to that of β-actin as internal control. Compared to the control group, relative fold changes were presented as mean ± SD. All experiments were repeated at least three times independently. Asterisk (*) indicates significant difference (P < 0.05), while asterisk (**) represents highly significant difference (P < 0.01).</p

Effect of zearalenone on the growth of porcine granulosa cells.

<p>The Porcine granulosa cells were treated with ZEA (15, 30 and 60 μM) for 24 h and imagined by microscope on 6-well plates. A. Untreated cells (Control). B. 15 μM ZEA-treated cells. C. 30 μM ZEA-treated cells. D. 60 μM ZEA-treated cells. E. Quantification of dead and floated cells after treatment of various ZEA concentrations for 24 h. Asterisk (*) indicates significant difference (P < 0.05).</p

The expression of endogenous antioxidative enzymes Sod1, Cat and Gpx1 in porcine granulosa cells treated with DMSO (control group); ZEA (60 μM ZEA treatment for 24 h); Cur+ZEA (Co, co-treatment with curcumin and ZEA; Pre, pre-treatment with curcumin for 12 h).

<p>Gene expression was normalized to β-actin as a loading control. Compared to the control group, relative fold changes are presented as mean ± SD. All experiments were repeated at least three times. Asterisk (*) indicates significant difference (P < 0.05) while asterisk (**) indicates highly significant difference (P < 0.01).</p

The rescue of curcumin of oxidative stress induced by ZEA in porcine granulosa cells.

<p>Multiple enzyme systems are involved in the effect of ZEA, SOD1 in the cytoplasm dismutates O₂^- to H₂O₂ and water. GPX1and CAT converts H₂O₂ to water. During this process, GSH is oxidized to the disulfide form (GSSG) by GPX1, which is converted back to GSH through glutathione reductase action (GSR). ZEA stimulated these enzyme systems, resulting in oxidatvie stress in porcine granulosa cells. Curcumin rescued cellular redox balance through inhibition of SOD1 and CAT signal pathway in porcine granulosa cells after pre-treatment for 12 h.</p

The enzymatic activities of SOD1, CAT, GPX1 and GSH in porcine granulosa cells.

<p>After 24 h of ZEA treatment, porcine granulosa cells were collected for measurement. All values were normalized to protein level in a loading control and presented as relative fold changes in comparison to untreated control. All experiments were repeated at least three times. Asterisk (*) indicates significant difference (P < 0.05), asterisk (**) represents highly significant difference (P < 0.01).</p