The Effects And Regulation Of Zinc Oxide Nanoparticles In Vitro Cultured Porcine Granulosa Cells

Zinc oxide nanoparticles(ZnO NPs)are one of the most used metal oxide nanoparticles and are widely used as feed additives in animal husbandry because of their antimicrobial properties and their ability to improve animal production performance.With the widespread use of ZnO NPs,their biosafety has raised concerns.ZnO NPs induce reproductive and developmental toxicity in animals through accumulation in the ovaries,uterus and placenta,which in turn induces reproductive and developmental toxicity.However,there are few reports on the effects of ZnO NPs on porcine ovarian granulosa cells(PGCs).In this study,we used CCK8 assay,ELISA,EdU,ROS staining,qRT-PCR,small molecule inhibitors and Western blot to analyze the effects of ZnO NPs on PGCs in terms of steroid hormone secretion,proliferation,ROS,autophagy and apoptosis.The following results were obtained:1.ZnO NPs(4 μg/mL)significantly reduced GCs viability(P<0.01);secretion of estrogen and progesterone was significantly reduced(P<0.05).Mechanistically,ZnO NPs significantly inhibited the expression of CYP11A1 and StAR genes and proteins,important rate-limiting enzymes for steroid hormone synthesis(P<0.05).Further studies revealed that ZnO NPs inhibited EdU-positive proliferating cells;flow cytometric analysis showed that ZnO NPs stimulated an increase in the proportion of S-phase cells(P<0.05)and a decrease in the proportion of G2/M-phase cells(P<0.05).These results suggest that ZnO NPs inhibit GCs steroid hormone synthesis and proliferation by regulating the expression of important ratelimiting enzyme genes and proteins related to steroid hormone synthesis and by modulating changes in the ratio of cells in different cycles.2.ZnO NPs promote autophagy and apoptosis in GCs.ZnO NPs(4 μg/mL)significantly decreased the mitochondrial membrane potential of GCs(P<0.05).ZnO NPs stimulated autophagy(LC3,P62 and ATG7)(P<0.01)and pro-apoptotic(Bax)mRNA expression(P<0.01).Consistent with this,ZnO NPs upregulated the protein expression of autophagic LC3,P62 and ATG7(P<0.05)and the ratio of Bax/Bcl-2 was significantly higher(P<0.05).CQ(autophagy inhibitor)inhibited autophagy and significantly downregulated the protein expression of LC3(P<0.05)and significantly upregulated the protein expression of Cytochrome C(P<0.01)and cleaved caspase3(P<0.05)in the co-treated group of ZnO NPs and CQ compared with the ZnO NPs-treated group,P62,Beclin1 protein expression and the Bax/Bcl-2 ratio all showed a significant upward trend.These results suggest that ZnO NPs trigger autophagy and apoptosis of GCs mediated by the mitochondrial pathway by disrupting the mitochondrial membrane potential;meanwhile,inhibition of autophagy exacerbates ZnO NPs-induced apoptosis.3.ZnO NPs induced autophagy and apoptosis in GCs via oxidative stress.ZnO NPs(4μg/mL)significantly upregulated ROS levels in GCs(P<0.01).ZnO NPs significantly downregulated the antioxidant enzyme GSH-Px enzyme activity and mRNA expression(P<0.05);stimulated SOD2 protein expression(P<0.05).After the removal of reactive oxygen species by NAC(reactive oxygen scavenger),the expression of LC3,Cytochrome C and cleaved caspase3 proteins was significantly inhibited(P<0.05),the Bax/Bcl-2 ratio was significantly decreased(P<0.05)and there was a significant trend of decrease in P62 protein in the cotreated group of ZnO NPs and NAC compared with the ZnO NPs-treated group.These results suggest that ZnO NPs trigger oxidative stress by inhibiting antioxidant enzyme activity and stimulating ROS levels,thereby promoting GCs autophagy and apoptosis.In summary,ZnO NPs induce oxidative stress,autophagy and apoptosis through inhibition of steroid hormone synthesis and proliferation,and induce GCs toxicity in porcine ovaries.