Protective effects of Oviductus Ranae-containing serum on oxidative stress-induced apoptosis in rat ovarian granulosa cells

Ethnopharmacological relevance: Oviductus Ranae (OR) is a traditional Chinese medicine derived from Rana temporaries chensinensis David, and is known to have a wide variety of pharmacological effects. Aim of the study: To investigate the function and mechanism of OR-containing serum in protecting rat ovarian granulosa cells from hydrogen peroxide (H2O2)-induced oxidative damage. Materials and methods: H2O2-treated granulosa cells were pretreated with OR-containing serum, and viability and proliferation assays were carried out using Cell Counting Kit-8 (CCK-8). Apoptotic granulosa cells were observed microscopically using 4',6-diamidino-2-phenylindole (DAPS), and the apoptotic ratio was quantified via Annexin V/ propidium iodide (PI) staining combined with flow cytometry. The levels of reactive oxygen species (ROS) and mitochondrial membrane potential (Delta Psi m) in the cells were measured using 2,7-dichlorofluorescein diacetate (DCFH-DA) and rhodamine 123, respectively, and analyzed by flow cytometry. Mitogen-activated protein kinases (MAPKs), including ERK1/2, JNK, and p38, and other apoptosis-related proteins (p53, Bcl-2, Bax, caspase-9, caspase-3), were detected by western blot analysis, and the related mRNA levels were detected using reverse transcriptase-polymerase chain reaction (RT-PCR). Results: The results revealed that treatment with OR-containing serum reduced apoptosis and mitochondrial membrane damage in H2O2-treated granulosa cells. The OR-containing serum interfered with H2O2-induced intracellular generation of ROS and loss of Delta Psi m, which typically lead to apoptosis. Furthermore, the OR containing serum down-regulated pro-apoptotic proteins such as p53, Bax, caspase-9, and caspase-3, while up regulating the anti-apoptotic protein Bcl-2. Finally, the OR-containing serum increased phosphorylation of ERK1/2, and reduced JNK and p38 phosphorylation. Conclusions: OR-containing serum protected rat ovarian granulosa cells against H2O2-induced apoptosis, by reducing ROS production and improving mitochondrial membrane potential, through down-regulation of negative regulators of proliferation, activation of ERK1/2, and inhibition of the activity of JNK and p38.