Effect Of Caffeic Acid On ZEA-induced Apoptosis And Autophagy In Mouse Ovarian Granulosa Cells

Objective: The effects of caffeic acid on the apoptosis and autophagy of mouse ovarian granulosa cells induced by zearalenone.Methods: Three-week-old Kunming mouse ovarian granulosa cells were separated mechanically,and the isolated cells were identified by indirect immunofluorescence.The effect of CA on the viability of normal mouse ovarian granulosa cells was detected by MTT.Granulosa cells were co-treated with CA(200,100,50 μg/m L)and ZEA(60 μmol/L),and the cell control group and ZEA group were set up at the same time.After 24 hours,the cytopathic condition was observed under the microscope,and the cell viability was detected by MTT.The apoptosis rate of ovarian granulosa cells was detected by TUNEL.Dansylcadaverine staining detects the formation of autophagosomes.The relative m RNA expression of caspase-3,Bax,Bcl-2,GRP 78,LC3,Beclin 1 was detected by q RT-PCR.Western blot was used to detect the protein expression levels of cleaved-caspase-3,cleaved-PARP,Bax,Bcl-2,GRP 78,LC 3 and Beclin 1.Results: 1.Follicle-Stimulating Hormone Receptor(FSHR)positive staining appeared in the cytoplasm of the test group.This result indicated that the isolated cells are mouse ovarian granulosa cells,which can be used for subsequent experimental studies.2.At the tested concentration,CA has no toxic effect on mouse ovarian granulosa cells and the cell viability exceeds 90%(P>0.05).3.Compared with the cell control group,the cells in the ZEA group shrank,the cell body became smaller,the adhesion was poor,the intercellular space was enlarged,the cell viability was significantly reduced(P<0.05),and the cell apoptosis rate was significantly increased(P<0.05).Compared with the ZEA group,after co-treatment with CA,the cell shrinkage and the gap was reduced,the adhesion was tight,the cell viability was significantly increased(P<0.05),and the cell apoptosis rate was significantly decreased(P<0.05).4.Compared with the control group,the relative expression of caspase-3 and GRP 78 m RNA in the ZEA group increased significantly(P<0.05),and the ratio of Bax/Bcl-2 was significantly increased(P<0.05).The protein expression levels of cleaved-caspase-3,cleaved-PARP and GRP 78 increased significantly(P<0.05),and the ratio of Bax/Bcl-2 increased significantly(P<0.05).Compared with the ZEA group,the relative m RNA expression of caspase-3 and GRP 78 in the CA group was significantly reduced(P<0.05),the ratio of Bax/Bcl-2 was significantly down-regulated(P<0.05),and the protein expression levels of cleaved-caspase-3,cleaved-PARP and GRP 78 were significantly reduced(P<0.05).5.MDC staining showed that both the ZEA group and the caffeic acid group had autophagosome formation.Compared with the cell control group,the relative expression of Beclin 1 m RNA and protein in the ZEA group were significantly increased(P<0.05).The relative m RNA expression of LC3 was significantly increased(P<0.05).The ratio of LC3 II/LC3 I was significantly increased(P<0.05).Compared with the ZEA group,the relative expression of Beclin 1 mRNA and protein in the 200 μg/mL CA group were significantly increased(P<0.05).The relative mRNA expression of LC3 was significantly increased(P<0.05)and the ratio of LC3 II/LC3 I was significantly increased(P<0.05),while the relative m RNA expression of Beclin 1 and LC3 in the 100 μg/m L CA group were significantly reduced(P<0.05).The protein expression of Beclin 1 was significantly down-regulated(P<0.05),LC3 II/LC3 I ratio was no significant difference(P>0.05).The relative m RNA expression of Beclin 1 in the 50 μg/m L CA group had no significant change(P>0.05),the LC 3 m RNA expression was significantly reduced(P<0.05).The protein expression level of Beclin 1 was significantly reduced(P<0.05),and the ratio of LC3 II/LC3 I did not change significantly(P>0.05).Conclusion: CA and ZEA act together on granulosa cells for 24 hours,and resist ZEA-induced granulosa cell apoptosis by affecting the expression of mitochondrial pathway and endoplasmic reticulum stress pathway related proteins.200 μg/m L CA may alleviate ZEA-induced injury by promoting autophagy to restore the viability of granulosa cells.100 μg/m L and 50 μg/m L CA may reduce cell damage through other ways and reduce the level of intracellular autophagy.