Effect of α-cyperone-containing serum on H₂O₂-induced oxidative stress of ovarian granulosa cell apoptosis in rats

Abstract

Objective

The paper investigated the effect and mechanism of α-Cyperone-containing serum on H₂O₂-induced oxidative stress in ovarian granulosa cell apoptosis in rats. Methods: Female SD rats of 21-25 days old were obtained. Mechanical separation and trypsin digestion method were used to collect rat ovarian granulosa cells. H₂O₂ of 50 μM, 100 μM, 200 μM, 500 μM, and 1000 μM were applied to intervene in ovarian granulosa cells. CCK-8 method was employed to screen appropriate drug concentration and establish rat ovarian granulosa cell oxidative stress model. 5%, 10%, and 20% drug-containing serum intervention were performed, CCK-8 method was used to screen serum concentration and intervention time. 200 nm, 400 nm, and 800 nm JNK signaling pathway inhibitor SP600125 were used to intervene with oxidatively stressed ovarian granulosa cells, and CCK-8 method was applied to screen the appropriate concentration. Ovarian granulosa cells in the logarithmic growth phase were randomly divided into the blank group, the H₂O₂ intervention oxidative stress model group (the model group), the drug-containing serum group, and the JNK pathway inhibitor group. After intervention, the laser confocal microscope was used to observe the expressions of intracellular ROS, and the average optical density of each group was compared. The laser confocal microscope was used to observe TUNEL staining, and the apoptosis rate of each group was compared. Western blot was used to detect the expressions of p-JNK, Bax, and caspase-3 proteins. Results: 200 μM H₂O₂ was used to induce ovarian granulosa cell model of oxidative stress. The optimal concentration of drug-containing serum was 10%, and the intervention time was 24 h. The intervention concentration of JNK signal pathway inhibitor SP600125 was 800 nm. Compared with the blank group, the average optical density of intracellular ROS and apoptosis rate increased in the model group, the drug treatment group, and the JNK pathway inhibitor group. Compared with the model group and the JNK pathway inhibitor group, the intracellular ROS expression and apoptosis rate of the drug group decreased. The Western blot expressions of p-JNK, Bax, and caspase-3 proteins in the model group were higher than that of the blank group, the drug administration group, and the JNK pathway inhibitor group. Conclusion: The serum containing α-Cyperone may inhibit the ROS-JNK signaling pathway and reduce the H₂O₂-induced oxidative stress ovarian granulosa cell apoptosis.

Keywords:
α-Cyperone; ROS-JNK signaling pathway; H₂O₂; rat ovarian granulosa cells; apoptosis