Alleviation Effects Of Apricot Bee Pollen Extract On DON Induced Toxicity In Bovine Granulosa Cells

Deoxynivalenol(DON)is a mycotoxin produced by the Fusarium genus,it can cause vomiting,diarrhea,and abortion reactions of human and animals,seriously cause death.It is one of the most common mycotoxins in feed pollution,has great harm to animal husbandry.Studies showed that DON could induce apoptosis,inflammation,and oxidative stress.Apricot Bee Pollen Extract(ABPE)has been reported to have good antioxidant activity.Therefore,this study aimed to study the toxic effects of DON on bovine ovarian granulosa cells by using flow cytometry,q RT-PCR,Western Blot,ELISA and RNA-seq technologies,and explore the relieving effects of bee pollen on the toxicity by co-treatment with DON and bee pollen.The main results of this study are as follows:1.Toxic effects of DON on bovine granulosa cells(1)Effect of DON on proliferation,cycle,and apoptosis in bovine ovarian granulosa cellsAfter treating bovine granulosa cells with 0.1,0.5,and 1 μmol/L DON for 24 h,CCK-8 results showed 0.5 μmol/L and 1 μmol/L DON exposure could significantly decrease the cell ability by 33.57% and 53.65%,respectively,and 0.5 μmol/L DON could significantly reduce the expression of proliferation cell nuclear antigen(PCNA).Flow cytometry results showed that 1 μmol/L DON could significantly reduce the percentage of G1 phase of granulosa cells,while significantly increase the percentage of S phase of granulosa cells.The results of Annexin-V and PI staining showed that all the treatment groups except 0.1 μmol/L DON group significantly induced the apoptosis of granulosa cells,and the 0.5 μmol/L DON group significantly increased the expression of Bcl-2-related protein X(Bax).(2)Effects of DON on secretion of steroid hormones in bovine granulosa cellsAfter treatment with 0.1,0.5 and 1 μmol/L DON for 24 h,the steroid hormones in the supernatants were detected by ELISA.Results showed that 0.5 μmol/L and 1μmol/L DON groups significantly reduced estrogen levels,and 1 μmol/L DON group also significantly reduced progesterone level.Western Blot results showed that DON group significantly reduced the expression of estrogen synthesis aromatase(CYP19A1)and steroid hormone synthesis acute regulatory protein(St AR).(3)Effect of DON on oxidative stress in bovine granulosa cellsAfter treatment with 0.1,0.5 and 1 μmol/L DON for 24 h,ROS and MDA levels were measured.Results showed that DON treatment groups significantly increased ROS and MDA levels in granulosa cells,this effect was in a dose-dependent manner.(4)Effect of DON on the gene transcriptome in bovine ovarian granulosa cellsThe RNA-seq results showed that a total of 4234 differentially expressed genes were selected between the treatment and control groups after DON treatment,including 2414 up-regulated genes and 1820 down-regulated genes.GO analysis showed that the differential genes were mainly enriched in the functional annotation of translation,cytoplasm,redox process and oxidoreductase activity.KEGG analysis showed that the differential expressed genes were mainly enriched in RNA transport,ribosome,propionate metabolism,steroid biosynthesis pathways.2.Alleviation effects of ABPE on DON-induced cytotoxicity of bovine granulosa cells(1)Alleviation effects of ABPE on DON reducing the viability of bovine granulosa cellsAfter treating bovine granulosa cells with 100,200 and 300 μg/m L ABPE for 24 h,the results of CCK-8 showed that ABPE could significantly increase the activity of bovine ovarian granulosa cells.After co-treatment with 100,200 and 300 μg/m L ABPE and 0.5 μmol/L DON for 24 h,both 200 μg/m L and 300 μg/m L ABPE could significantly alleviate the toxic effect of DON on the viability of bovine ovarian granulosa cells.(2)Alleviation effects of ABPE on DON reducing steroid hormones of bovine granulosa cellsAfter co-treatment with 200 μg/m L ABPE and 0.5 μmol/L DON for 24 h,ELISA results showed that ABPE significantly alleviated the toxic effect of DON on estrogen secretion in bovine ovarian granulosa cells,but had no significant effect on progesterone secretion.(3)Alleviation effects of ABPE on DON-induced oxidative stress in bovine ovarian granulosa cellsAfter co-treatment with 200 μg/m L ABPE and 0.5 μmol/L DON for 24 h,we found that ABPE significantly reduced the DON-induced ROS levels.And ABPE could significantly upregulate the expression of antioxidant related genes HO-1 and NQO1 m RNA,and significantly downregulate the m RNA expression of COX-2 gene.However,there were no significant effects on the MDA levels and antioxidant related genes SOD and CAT expression levels.Treatment ABPE alone can significantly improve the total antioxidant capacity(T-AOC)of bovine ovarian granulosa cells.In conclusion,DON can significantly reduce bovine ovarian granulosa cell activity,arrest the cell cycle,induce apoptosis and oxidative stress,and inhibit secretion of steroid hormones.Moreover,ABPE can effectively alleviate the toxic effects of DON on granulosa cells and inhibit oxidative stress.This study further enriches the theoretical research on the effect of DON on bovine ovarian granulosa cell,and can provide a reference for using Apricot Bee Pollen to prevent DON pollution in dairy cows,which has great significance to promote the healthy development of dairy industry.