| Objective:To explore the effect of Afamin protein on testosterone propionate（TP）induced oxidative stress related functions in KGN cells and to explore the regulatory effects of Afamin protein on granulosa cells and its mechanisms in terms of mitochondrial function,apoptosis,and steroid hormone secretion functions.Methods and Results:1.Effect of Afamin protein on the level of oxidative stress in TP-treated KGN cellsThe experiments were divided into the Control group（blank control group）,the TP group,and the Afamin protein+TP group.50μM of TP and 50ng/ml of Afamin protein were selected as the experimental concentrations based on the experience of our group in previous experimental studies and the relevant literature.ROS and superoxide anion levels in KGN cells were measured by flow cytometry and levels of malondialdehyde（MDA）,a biomarker of oxidative stress,were also measured using the appropriate kits.The experimental results showed that the levels of ROS,superoxide anion,and MDA in KGN cells were significantly increased in the TP group（P<0.05）,while the levels of ROS,superoxide anion,and MDA were significantly decreased in the Afamin protein addition group（P<0.05）.The activities of a series of antioxidant enzymes such as glutathione reductase（GR）,glutathione peroxidase（GSH-Px）,catalase（CAT）,and superoxide dismutase（SOD）were further measured by the corresponding antioxidant enzyme kits.The results showed that the antioxidant enzyme activities of SOD,CAT,GSH-Px,and GR decreased in the TP group（P<0.05）;the addition of Afamin protein increased the activities of the above antioxidant enzymes（P<0.05）.2.Effect of Afamin protein on mitochondrial function and apoptosis in TP-treated KGN cellsTo investigate whether the presence of oxidative stress in TP-treated KGN cells also caused impairment of mitochondrial function and the onset of apoptosis in KGN cells.After KGN cells were treated in the same manner as described above,changes in mitochondrial membrane potential（MMP）and mitochondrial O2-levels in KGN cells were analyzed using flow cytometry and found that compared to the control group,the TP group showed a significant decrease in MMP levels（P<0.05）and an increase in intracellular mitochondrial O2-levels（P<0.05）;the addition of Afamin protein restored normal levels of MMP and mitochondrial O2-（P<0.05）.In addition,mitochondria are an important intracellular organelle for ATP synthesis,so ATP levels were measured using the appropriate kit.The results showed that ATP levels,an indicator of mitochondrial function,decreased in the TP group（P<0.05）,while ATP levels increased in the Afamin protein addition group（P<0.05）.It is known that the presence of oxidative stress and damage to mitochondria can cause apoptosis,so apoptosis was detected using flow cytometry,and the results showed that the apoptosis rate of KGN cells increased in the TP group and decreased significantly in the KGN cells with the addition of Afamin protein.Further analysis of genes related to apoptosis in KGN cells was performed by fluorescence quantitative PCR.The results showed that the expression of BAX,a gene that promotes apoptosis,was significantly increased（P<0.05）and the expression of BCL-2,a gene that inhibits apoptosis,was significantly decreased（P<0.05）after TP treatment;however,the expression of BAX was significantly down-regulated after the addition of Afamin protein（P<0.05）,and the expression of BCL-2 was significantly up-regulated（P<0.05）.Caspase-3 plays an irreplaceable role in the pathway of apoptosis promotion,so Caspase-3 activity was measured using the corresponding assay kit,and the results showed that Caspase-3 activity increased in KGN cells in the TP group（P<0.05）,while it decreased in the Afamin protein addition group（P<0.05）.3.Effect of Afamin protein on steroid hormone synthesis in TP-treated KGN cellsIt was shown that excessive oxidative stress induces pathological changes in granulosa cells,which are the main source of steroidal estrogen（E2）and progesterone（P）,so the E2 and P levels in the supernatant of KGN cell culture were measured by ELISA.The results showed that the secretory function of KGN cells in the TP group was abnormal compared to the control group,as evidenced by an increase in E2secretion and a decrease in P secretion（P<0.05）,while Afamin protein was able to restore the abnormal secretion levels of E2 and P in KGN cells（P<0.05）.The changes in the expression of hormone synthesis-related genes STAR,CYP11A1,HSD3B1,and CYP19A1 in KGN cells were further examined by fluorescence quantitative PCR.The results showed that the m RNA expression of STAR,HSD3B1,and CYP11A1 decreased and CYP19A1 m RNA expression increased in the TP group（P<0.05）,while the m RNA expression of STAR,HSD3B1,and CYP11A1 increased and CYP19A1 m RNA expression decreased in the KGN cells with Afamin protein（P<0.05）.m RNA expression was increased in KGN cells with Afamin protein（P<0.05）.Conclusion:1.The afamin protein reduces TP-induced oxidative stress in KGN cells by reducing levels of markers of oxidative damage and enhancing the activity of antioxidant enzymes.2.The afamin protein can repair mitochondrial dysfunction by improving mitochondrial membrane potential and ATP levels and counteract TP-induced apoptosis in KGN cells by reducing caspase-3 activity and up-regulating the expression of anti-apoptotic（BCL-2）and down-regulating the expression of pro-apoptotic gene（BAX）.3.The afamin protein ameliorates TP-induced endocrine dysfunction in KGN cells by affecting the expression of genes related to ovarian steroid hormone synthesis. |
