Abstract: The study aimed to study the effect of DNA oxidative damage induced by cobalt chloride (CoCl₂) on the proliferation of porcine follicular granulosa cells. Porcine follicular granulosa cells were used as experimental materials, and hypoxia model was established by treating porcine follicular granulosa cells with chemical hypoxia model inducer CoCl₂. In the previous study, the optimal treatment concentration of CoCl₂was determined. Porcine follicular granulosa cells were treated with 200 μmol·L^-1 CoCl₂ for 12 h, and the cultured passage cells were divided into 4 groups for treatment, as follows:control group, CoCl₂-induced hypoxia treatment group, GSH treatment group, GSH+CoCl₂ combined treatment group. Cells in control group was cultured in complete medium for 12 h; Cells in CoCl₂-induced hypoxia treatment group was cultured in medium with final concentration of 200 μmol·L^-1 CoCl₂ for 12 h; Cells in GSH treatment group was treated with GSH at a concentration of 2 mmol·L^-1 for 12 h; Cells in the GSH+CoCl₂ combined treatment group was treated with 200 μmol·L^-1 CoCl₂ and 2 mmol·L^-1 GSH for 12 h. After 12 h, the proliferation activity, ROS level, γH2AX protein activity and Oxo-8-G level of each group were detected. CCK-8 assay was used to detect its proliferation activity. ROS levels were determined by 2', 7'-dichlorofluorescin diacetate (DCFH-DA). Western blot was used to detect the protein activity of γH2AX, and FITC fluorescent mouse monoclonal antibody was used to detect Oxo-8-G. In order to further clarify the relationship between CoCl₂-induced granulosa cell proliferation arrest and DNA oxidative damage, this experiment added antioxidant GSH on the basis of CoCl₂ treatment for 12 h to detect cell proliferation, ROS level, DNA oxidative damage and other related indicators. Compared with the control group, the proliferation activity of porcine follicular granulosa cells treated with 200 μmol·L^-1 CoCl₂ was significantly decreased (P<0.000 1), and the ROS level, γH2AX protein expression and Oxo-8-G level in hypoxia group were significantly increased (P<0.000 1). Compared with the hypoxia group, the levels of ROS, Oxo-8-G and γH2AX were significantly decreased by adding GSH on the basis of CoCl₂ treatment (P<0.000 1), and the proliferation activity of cells was significantly recovered by adding GSH (P<0.000 1). CoCl₂ can inhibit the proliferation activity of porcine follicular granulosa cells, and the mechanism is related to the damage of DNA induced by oxidative stress.

Key words: cobalt chloride, chemical hypoxia, porcine follicular granulosa cells, cell proliferation, DNA oxidative damage