| Follicular granule cells are the main functional cells of follicles.The proliferation and apoptosis of GCs are closely related to follicle development and maturation.N-acetylcysteine plays an important role in reducing reactive oxygen species in the body,maintaining cell function and cell morphology.NAC has been widely used as an antioxidant.In this study,the normally developed ovaries of healthy female sheep around 2years old were collected from the slaughterhouse in Wushengyi Town,Gansu Province.The ovaries on both sides were removed immediately after slaughter,stored at 37°C,and sent back to the laboratory within 4 h.GCs were separated by follicular fluid aspiration,and GCs were identified by immunofluorescence technology.Explore the optimal in vitro culture conditions for GCs.The effects of different concentrations of NAC（50,100,500,1000μmol/L）on GCs proliferation,apoptosis,ROS level,hormone secretion and related gene and protein expression levels were analyzed.The molecular mechanism of NAC on the proliferation of GCs and steroid hormone secretion in sheep was explored by treatment with PI3K/AKT inhibitor LY294002.H2O2 was used to treat GCs to construct an oxidative stress model,and NAC was pretreated to investigate whether NAC could inhibit the apoptosis of model cells.The antioxidant capacity of cells and NRF2 and PI3K/AKT signaling pathways were studied to find out whether NAC alleviated H2O2-induced oxidative stress damage through NRF2 and PI3K/AKT signaling pathways.The results are as follows:1.The immunofluorescence identification results showed that the purity of GCs in vitro cultured sheep reached 99.2%.When 15%serum was added to the medium,the effect of cell proliferation was significantly higher than that of other concentration groups（P<0.05）,The proliferation effect of cells in the DMEM/F-12 group was better than that of the DMEM group（P<0.05）.2.The proliferative capacity of GCs in the NAC group at different concentrations was higher than that in the control group（P<0.05）.When GCs were cultured in vitro for 48 h,100μmol/L NAC could significantly improve the proliferation capacity of GCs（P<0.01）.q RT-PCR and WB results showed that the expression levels of CCND1,CDK4 and Bcl-2 in the 100μmol/L NAC group were significantly upregulated（P<0.05）,Bcl-2 protein expression levels were significantly upregulated（P<0.05）,while the expression levels of Bax gene and protein were significantly downregulated（P<0.05）.100μmol/L NAC significantly reduced the content of reactive oxygen species in GCs（P<0.05）.The q RT-PCR results showed that the expression levels of SOD1 and CAT in the 100μmol/L NAC group were significantly upregulated（P<0.01）.The results of ELISA and q RT-PCR showed that the secretion of P4 and E2 in the 100μmol/L NAC group was significantly increased（P<0.01）,and the expression levels of P4 and E2-related synthetic genes 3β-HSD and CYP19A1 were significantly increased（P<0.05）.After pretreatment with NAC,PI3K/AKT-specific inhibitor LY294002 was added,which significantly reduced the proliferation capacity of GCs（P<0.01）and inhibited the secretion of E2 and P4 by GCs（P<0.05）.With NAC treatment of GCs,the expression of p-PI3K/PI3K and p-AKT/AKT proteins was significantly upregulated（P<0.05）.After pretreatment with NAC,LY294002 was able to significantly downregulate the expression of p-PI3K/PI3K and p-AKT/AKT proteins（P<0.05）.3.The oxidative damage model was successfully established when the concentration of H2O2 was 200μmol/L and the action time was 12 h.In H2O2 group,the contents of ROS and MDA increased significantly（P<0.05）,the content of GSH and SOD activity decreased significantly（P<0.05）,and the expression levels of CAT and SOD1 decreased significantly（P<0.05）.The proliferation activity of GCs was significantly reduced（P<0.01）,the expression levels of PCNA and Bcl-2 were significantly downregulated（P<0.05）,the protein levels of Bcl-2 were significantly downregulated（P<0.01）,and the expression levels of Bax genes and proteins were significantly upregulated（P<0.05）.The secretion of E2 and P4 was significantly reduced（P<0.05）,and the content of inflammatory factors IL-18 and IL-1bwas significantly increased（P<0.05）.NRF2 and HO-1 gene and protein expression levels were significantly downregulated（P<0.05）,The expression levels of p-PI3K/PI3K and p-AKT/AKT proteins were significantly downregulated（P<0.05）.After pretreatment with NAC and H2O2 treatment,ROS and MDA content were significantly reduced（P<0.05）,GSH content and SOD activity were significantly increased（P<0.05）,and CAT and SOD1 expression levels were significantly increased（P<0.05）.The proliferative activity of GCs was significantly increased（P<0.01）,the expression levels of PCNA and Bcl-2 were significantly increased（P<0.05）,the protein levels of Bcl-2were significantly upregulated（P<0.01）,and the expression levels of Bax genes and proteins were significantly downregulated（P<0.05）.The secretion of E2 and P4increased significantly（P<0.05）,and the content of inflammatory factors IL-18 and IL-1bdecreased significantly（P<0.05）.Gene and protein expression levels of NRF2 and HO-1 were significantly upregulated（P<0.05）,The protein expression levels of p-PI3K/PI3K and p-AKT/AKT were significantly upregulated（P<0.05）.In summary,the optimal conditions for in vitro culture of sheep GCs were DMEM/F-12 medium containing 15%serum and the culture temperature was 37°C.NAC activates the PI3K/AKT signaling pathway to promote the proliferation of GCs,E2 and P4 secretion of sheep GCs in vitro.NAC alleviates H2O2-induced oxidative stress damage through NRF2 and PI3K/AKT signaling pathways. |
