Xiao-Mei Ling1, Xu-Hui Zhang2, Yan Tan3, Jing-Jing Yang4, Bo Ji4, Xin-Rong Wu4, Yan-Kui Yi5, Lei Liang6. 1. Department of Pharmacy, Guangzhou General Hospital of Guangzhou Military Command, Guangzhou 510010, China; School of Traditional Chinese Medicine, Guangdong Pharmaceutical University, Guangzhou 510006, China. 2. Department of 2nd Oncology, Guangdong No. 2 Provincial People's Hospital, Guangzhou 510317, China. 3. Department of 1st Geriatrics, Guangzhou General Hospital of Guangzhou Military Command, Guangzhou 510010, China. 4. Department of Pharmacy, Guangzhou General Hospital of Guangzhou Military Command, Guangzhou 510010, China. 5. School of Traditional Chinese Medicine, Southern Medical University, Guangzhou 510515, China. Electronic address: dareyyk@sina.com. 6. Department of Pharmacy, Guangzhou General Hospital of Guangzhou Military Command, Guangzhou 510010, China. Electronic address: lhq_pharm@hotmail.com.
Abstract
ETHNOPHARMACOLOGICAL RELEVANCE: Oviductus Ranae (OR) is a traditional Chinese medicine derived from Rana temporaria 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 (DAPI), 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 (ΔΨm) 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 ΔΨm, 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.
ETHNOPHARMACOLOGICAL RELEVANCE: Oviductus Ranae (OR) is a traditional Chinese medicine derived from Rana temporaria 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 (DAPI), 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 (ΔΨm) 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 ΔΨm, 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.