Effect Of Deoxynivalenol And 2,5-hexanedione On Apoptosis Of Porcine Ovarian Granulosa Cells And Transcriptome Analysis

Ovarian granulosa cells are an important class of ovarian cells.Studies have shown that follicular atresia is closely related to the apoptosis of granulosa cells during normal development of the ovarian follicular cycle.Therefore,it is of great importance to elucidate the regulation mechanism and influencing factors that cause the apoptosis ofgranulosa cell.Meanwhile,2,5-hexanedione(2,5-HD)is one of the effective decomposition agents of n-hexane that are widely used in the preparation of adhesives,oil extraction,decontamination and oil-based coatings.At the same time,the contamination of mycotoxin in feed ingredients in 14 provinces across China showed a DON detection rate of 99.1%,and in some areas even reach to 100%.Taking the above into consideration,we used the Porcine ovary granulosa cells(pGCs)as vectors to establish 2,5-HD and DON concentration time models,and performed cell morphology and transcriptome analysis to explore how the 2,5-HD and DON regulate the pGCs.The experiment were performed as follows:(1)We performed cell morphology,apoptosis,cell cycle and cell proliferation analysis to detect how the 2,5-HD and DON affect the biological function of pGCs;(2)Transcriptomic analysis of pGCs by adding 2,5-HD(0 mmol/L and 40 mmol/L groups)and DON(0 ?g/L and 1000 ?g/L groups);(3)According to the results of transcriptomics analysis of 2,5-HD,we pick a key gene(CDKN1A)to detect the biological function of pGCs.The mainly results of the above studies:(1)DON(500?g/L,1000?g/L and 2000?g/L,24h)induced apoptosis of pCGs,and the morphological changes and apoptosis rate of pCGs were dose dependent..While,the proliferation of cells was just reversed.After treatment of pGCs with different concentrations of DON,the percentage of cells in S phase and G2/M phase increased.While the percentage of cells is reduced in G0/G1.In the experiment treated with DON(0 ?g/L and 1000 ?g/L),there were 2620 differentially expressed genes,of which668 differentially down-regulated genes and 1952 differentially up-regulated genes.The differential gene was significantly enriched in 13 pathways(2)2,5-HD(20mmol/L,40mmol/L and 60mmol/L,24h)induced apoptosis of PCGs,and morphological changes and apoptosis of PCGs were dose-dependent on 2,5-HD.After treatment of pGCs with different concentrations of DON,the percentage of cells in S phase and G2/M phase increased.While the percentage of cells is reduced in G0/G1.In the experiment treated with DON(0 ?g/L and 1000?g/L),there were 1387 differentially expressed genes,of which 754 differentially down-regulated genes and 633 differentially up-regulated genes.The differential gene was significantly enriched in 13 pathways.(3)After 48 hours of pGCs transfected with CDKN1A-sscRNA,the apoptosis rate of pGCs transfected with CDKN1 A-siRNA decreased.The number of cells in the S phase after transfection was significantly increased,and the number of cells in the G1 phase was significantly reduced.This indicates that the CDKN1A gene has a pro-apoptotic effect.