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

Publication date: Available online 7 June 2017
Source:Journal of Ethnopharmacology
Author(s): Xiao-Mei Ling, Xu-Hui Zhang, Yan Tan, Jing-Jing Yang, Bo Ji, Xin-Rong Wu, Yankui Yi, Lei Liang
Ethnopharmacological relevanceOviductus Ranae (OR) is a traditional Chinese medicine derived from Rana temporaria chensinensis, and is known to have a wide variety of pharmacological effects.Aim of the studyThe aim of this study was to investigate the function and mechanism of OR in protecting rat ovarian granulosa cells from hydrogen peroxide (H2O2)-induced oxidative damage.Materials and methodsH2O2-treated granulosa cells were pretreated with OR-containing serum, and their viability and proliferation were evaluated using Cell Counting Kit-8 (CCK-8). Apoptotic granulosa cells were identified microscopically by visualizing chromatin condensation and nuclear fragmentation using 4',6-diamidino-2-phenylindole (DAPI), and the percentage of apoptotic cells was determined by flow cytometry after staining with Annexin V/PI. The levels of reactive oxygen species (ROS) and mitochondrial membrane potential (ΔΨm) in the cells were measured using 2,7-dichlorofluoresceinacetate (DCFH-DA) and rhodamine 123, respectively, and analyzed by flow cytometry. Mitogen-activated protein kinases (MAPKs), including ERK1/2, JNK, and p38 MAPK, and other apoptosis-related proteins, such as p53, Bcl-2, Bax, caspase-3 and caspase-9, were detected by Western blot analysis, and related mRNA expression was analyzed using reverse transcriptase-polymerase chain reaction (RT-PCR).ResultsThe 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 mitochondrial membrane potential, which typically lead to apoptosis. Furthermore, the OR-containing serum down-regulated pro-apoptotic proteins such as p53, Bax, caspase-3 and caspase-9, while up-regulating anti-apoptotic protein Bcl-2. Finally, the OR-containing serum attenuated H2O2-induced phosphorylation of JNK and p38 MAPK, and improved ERK1/2 phosphorylation.ConclusionsOR protected rat ovarian granulosa cells from H2O2-induced apoptosis, by reducing ROS production and improving mitochondrial membrane potential, through down-regulation of negative regulators of proliferation, and activation of MAPK signaling pathways.

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