Effect Of FHC On Oxidative Stress,Cell Proliferation And Apoptosis In Follicular Granulosa Cells Of Geese

The Ferritin heavy chain（FHC）is a major subunit of ferritin that is highly conserved among most species.FHC has ferrous oxidase activity,which converting Fe²⁺into Fe³⁺and store it inside ferritin,and mainly involved in the rapid absorption and release of iron ions in cells.As a mineral element essential for life,iron is important for oxygen transport,ATP production and DNA biosynthesis in living organisms.However,excessive iron ions generate reactive oxygen species（ROS）through the Fenton reaction and eventually damage the cells.Therefore,maintaining iron homeostasis in cells is particularly important,and FHC plays an important role in this process.Studies have shown that interference with FHC expression levels leads to iron ion levels increase significantly,while the increase in ROS levels in cells leads to cellular oxidative stress.Overexpression of FHC inhibits ROS production and exerts antioxidant function.In addition,our previous studies showed that the expression levels of FHC in follicles and ovulation follicles of Sichuan white goose was significantly higher than that of F1-F5 follicles,suggesting that FHC has an important regulatory role in goose granulosa cell apoptosis and follicular atresia.However,the specific regulatory mechanisms remain unclear.Therefore,in this study,in vitro isolated goose follicular granulosa cells were used as research objects to investigate the effects of FHC on oxidative stress,cell proliferation and apoptosis of granulosa cells by over-expressing and interfering with FHC expression levels.The results showed that:（1）After transfecting si RNA-FHC into granulosa cells for 24h,48 h and 72 h,the expression level of FHC was significantly lower than that in the control group（P<0.05）,which decreased by 79.02%,82.54%and 88.32%,respectively.At the same time,the results of Western blot showed that the expression level of FHC protein was decreased significantly at 48 h and 72 h（P<0.05）,which decreased by50.59%and 60.29%,respectively.This indicates that the expression of the FHC was specifically degraded by si RNA-FHC.（2）The cell viability was significantly decreased after transfected si RNA-FHC for 24h,48 h and 72 h（P<0.05）.There was no significant change in ROS level in granulosa cells after transfected si RNA-FHC for 24 h and 48 h（P>0.05）,but increased significantly at 72 h（P<0.05）;the expression levels of CAT and GSH-Px increased significantly at three time points after interferencing with FHC expression（P<0.05）;SOD expression level increased significantly at 72 h.（P<0.05）.（3）The expression levels of AURORA-A,PCNA,SMAD1 and apoptosis-related genes,BCL-2,TMBIM 4,P53,CASPASE 8 and CASPASE 9 were significantly increased after interfering with FHC expression for 24 h（P<0.05）;the key genes of cell proliferation such as CCND1,PCNA,SMAD1 and apoptosis-related genes BCL-2,TMBIM 4,P53,CASPASE 8 and CASPASE 9 expression levels were significantly increased after interfering with FHC expression for 48 h.After interfering with FHC expression for 72 h,the expression level of AURORA-A was significantly decreased（P<0.05）.The expression levels of PCNA and SMAD1 were significantly increased（P<0.05）,the expression levels of key apoptosis genes BCL-2 and CASPASE 8 were significantly increased（P<0.05）.Flow cytometry showed that there was no significant change in apoptosis rate after 24 h and 48 h of interference with FHC expression（P>0.05）.After 72 h,apoptosis rates increased by 19.05%（P<0.05）.（4）After transfecting with pEGFP-FHC overexpression vector for 24 h,48 h and 72 h,the expression level of FHC in granulosa cells increased significantly（P<0.05）,which were 9.76,7.98 and 3.48 fold of the control,respectively.Cell viability significantly increased after overexpression of FHC for 24 h（P<0.05）.The expression levels of CAT and SOD were significantly decreased after overexpression of FHC for 72 h（P<0.05）.Flow cytometry showed that the apoptosis rate decreased by 59.41%,63.39%and 52.31%,respectively（P<0.05）,after overexpression FHC at 24 h,48 h and 72 h.In summary,Low expression of FHC reduced the activity of goose follicle granulosa cells and induces oxidative stress in granulosa cells by inducing an increase in ROS levels,and increased the expression levels of CAT,SOD and GSH-Px in granulosa cells;Low expression of FHC inhibits the expression level of AURORA-A,promotes the expression levels of apoptotic genes P53,CASPASE 8 and CASPASE 9,and induces apoptosis of granulosa cells.