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 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.

[Analysis of hydrochloride 4-methyl-piperazine-1-carbodithioc acid 3-cyano-3,3-diphenyl-propyl ester and its metabolites in rats by HPLC-ESI/ITMSn].

AIM: To identify the main metabolites of hydrochloride 4-methyl-piperazine-1-carbodithioc acid 3-cyano-3,3-diphenyl-propyl ester (TM208) in rats. METHODS: Rat feces, urine and plasma samples were collected after ig 500 mg x kg(-1) TM208, then the samples were extracted and concentrated using ethyl acetate. The treated samples were analyzed by HPLC-ESI/ITMSn. The structures of metabolites were elucidated according to the rules of drug metabolism and disposition in vivo and the characteristic fragmentation behaviors of TM208 in ESI-ITMSn. RESULTS: Eight phase I metabolites were identified existing in rat feces, five of them were also found in rat urine and plasma, but no phase II metabolite was found. CONCLUSION: The HPLC-ESI/ITMSn method is rapid, highly sensitive and specific and it is suitable for the identification of TM208 and its metabolites in rats.

Yifuning postpones ovarian aging through antioxidant mechanisms and suppression of the Rb/p53 signal transduction pathway.

Yifuning is a traditional Chinese medicine recipe that has been used for many years in China for its effects on treating climacteric syndrome in women. The present study aimed to demonstrate the effects and underlying molecular mechanism of Yifuning on the ovaries of aging rats. Selected aging rats were administered different doses of Yifuning (1.0 or 2.0 g/kg by lavage), and after 6 weeks the rats were sacrificed. The activit of indicators of oxidative stress in the serum were measured. The expression levels of 8-oxo-2'-deoxyguanosine (8-OHDG) and p53 in the ovaries were examined using immunohistochemistry. The expression levels of the corresponding genes and proteins were detected by reverse transcription­quantitative polymerase chain reaction and western blotting analyses, respectively. The results indicated that Yifuning significantly prevented ovarian failure, as indicated by improvements in estrous cycling, reproductive organ weights and sex hormone serum levels. Yifuning significantly increased the levels of superoxide dismutase, glutathione peroxidase, catalase and reduced malondialdehyde and hydrogen peroxide levels. Yifuning reduced DNA damage in the ovaries by reducing the expression of 8­OHDG and p53. Treatment with Yifuning significantly reduced the age­induced p19, p53, p21 and Rb activity in the ovaries. The present study demonstrates that Yifuning prevents ovarian failure and the mechanism involved is partly associated with antioxidants and suppression of the Rb/p53 signal transduction pathway.

Screening of chemokine receptor CCR4 antagonists by capillary zone electrophoresis.

CC chemokine receptor 4 (CCR4) is a kind of G-protein-coupled receptor, which plays a pivotal role in allergic inflammation. The interaction between 2-(2-(4-chloro-phenyl)-5-{[(naphthalen-1-ylmethyl)-carbamoyl]-methyl}-4-oxo-thiazolidin-3-yl)-N-(3-morpholin-4-yl-propyl)-acetamide (S009) and the N-terminal extracellular tail (ML40) of CCR4 has been validated to be high affinity by capillary zone electrophoresis (CZE). The S009 is a known CCR4 antagonist. Now, a series of new thiourea derivatives have been synthesized. Compared with positive control S009, they were screened using ML40 as target by CZE to find some new drugs for allergic inflammation diseases. The synthesized compounds XJH-5, XJH-4, XJH-17 and XJH-1 displayed the interaction with ML40, but XJH-9, XJH-10, XJH-11, XJH-12, XJH-13, XJH-14, XJH-3, XJH-8, XJH-6, XJH-7, XJH-15, XJH-16 and XJH-2 did not bind to ML40. Both qualification and quantification characterizations of the binding were determined. The affinity of the four compounds was valued by the binding constant, which was similar with the results of chemotactic experiments. The established CEZ method is capable of sensitive and fast screening for a series of lactam analogs in the drug discovery for allergic inflammation diseases.