Shenfu injection protects human ECV304 cells from hydrogen peroxide via its anti-apoptosis way.

ETHNOPHARMACOLOGICAL RELEVANCE: The pathogenesis of thromboangiitis obliterans (TAO) has not been fully elucidated until now. Shenfu injection (SFI), a traditional Chinese formula has been widely used clinically for the treatment of cardiovascular diseases for more than two decade. Our previous results first suggested that SFI can cause a significant therapeutic effect on experimental TAO model rats. This experiment was designed to further investigate the protective effect of SFI on VEC damaged by hydrogen peroxide (H2O2) oxidative stress in vitro. METERIALS AND METHODS: The cell viability was evaluated by the MTT assay, the activities of SOD and GSH-PX and the content of MDA in the supernatants of the cultured ECV304 cells were evaluated by a colorimetry method, cell apoptosis was detected by flow cytometry and an AO/EB double staining method. The protein expressions of Bcl2, Bax and caspase-3 were examined by Western blotting. RESULTS: When compared with control group, lower survival rate of ECV304 cells was observed in H2O2 group (p<0.01) ; 20µl/ml, 30µl/ml and 40µl/ml SFI increased the survival rate of ECV304 cells under H2O2 oxidative stress (p<0.05 and p<0.01). The activities of SOD and GSH-PX were higher and MDA level was lower in H2O2 group than those in control group. These effects of H2O2 on SOD, GSH-PX activities and MDA content were reversed by SFI in concentration-dependent way (p<0.05 and p<0.01). Flow cytometry and AO-EB double staining discovered that SFI pretreatment inhibited the ECV304 cells apoptosis. The protein expression of caspase3 in 30µl/ml and 40µl/ml SFI groups significantly decreased whereas Bcl2 protein expressions in 20µl/ml, 30µl/ml and 40µl/ml SFI groups were higher than H2O2 group, with Bax protein expression much lower than H2O2 group (p<0.05 and p<0.01). CONCLUSIONS: Our findings suggest that SFI could prevent the ECV304 cells against H2O2 oxidative-stress by enhancing antioxidant enzyme activities, reducing the membrane lipid peroxidation, as well as upregulating antiapoptotic and downregulating apoptosis protein expressions.

Proto-oncogene c-erbB2 initiates rat primordial follicle growth via PKC and MAPK pathways.

BACKGROUND: c-erbB2, a proto-oncogene coding epidermal growth factor receptor-like receptor, also as a chemosensitivity/prognosis marker for gynecologic cancer, may be involved in initiation of growth of rat primordial follicles. The aim of the present study is to investigate the role and signal pathway of c-erbB2 in onset of rat primordial follicle development. METHODS: The expression of c-erbB2 mRNA and protein in neonatal ovaries cultured 4 and 8 days with/without epidermal growth factor (EGF) were examined by in situ hybridization, RT-PCR and western blot. The function of c-erbB2 in the primordial folliculogenesis was abolished by small interfering RNA transfection. Furthermore, MAPK inhibitor PD98059 and PKC inhibitor calphostin were used to explore the possible signaling pathway of c-erbB2 in primordial folliculogenesis. RESULTS: The results showed that c-erbB2 mRNA was expressed in ooplasm and the expression of c-erbB2 decreased after transfection with c-erbB2 siRNA. Treatment with EGF at 50 ng/ml significantly increased c-erbB2 expression and primary and secondary follicle formation in ovaries. However, this augmenting effect was remarkably inhibited by c-erbB2 siRNA transfection. Furthermore, folliculogenesis offset was blocked by calphostin (5 x 10(-4) mmol/L) and PD98059 (5 x 10(-2) mmol/L), but both did not down-regulate c-erbB2 expression. In contrast, the expressions of p-ERK and p-PKC were decreased obviously by c-erbB2 siRNA transfection. CONCLUSIONS: c-erbB2 initiates rat primordial follicle growth via PKC and MAPK pathways, suggesting an important role of c-erbB2 in rat primordial follicle initiation and development.

Ginsenosides stimulated the proliferation of mouse spermatogonia involving activation of protein kinase C / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

The effect of ginsenosides on proliferation of type A spermatogonia was investigated in 7-day-old mice. Spermatogonia were characterized by c-kit expression and cell proliferation was assessed by immunocytochemical demonstration of proliferating cell nuclear antigen (PCNA). After 72-h culture, Sertoli cells formed a confluent monolayer to which numerous spermatogonial colonies attached. Spermatogonia were positive for c-kit staining and showed high proliferating activity by PCNA expression. Ginsenosides (1.0 approximately10 microg/ml) significantly stimulated proliferation of spermatogonia. Activation of protein kinase C (PKC) elicited proliferation of spermatogonia at 10(-8) to 10(-7) mol/L and the PKC inhibitor H(7) inhibited this effect. Likewise, ginsenosides-stimulated spermatogonial proliferation was suppressed by combined treatment of H(7). These results indicate that the proliferating effect of ginsenosides on mouse type A spermatogonia might be mediated by a mechanism involving the PKC signal transduction pathway.

Attenuating effect of daidzein on polychlorinated biphenyls-induced oxidative toxicity in mouse testicular cells / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

The attenuating effect of daidzein (DAI) on oxidative toxicity induced by Aroclor 1254 (A1254) was investigated in mouse testicular cells. Cells were exposed to A1254 alone or with DAI. The oxidative damage was estimated by measuring malondialdehyde (MDA) formation, superoxide dismutase (SOD) activity and glutathione (GSH) content. Results show that A1254 induced a decrease of germ cell number, an elevation in thiobarbituric acid reactive substances (TBARS) but a decrease in SOD activity and GSH content. However, simultaneous supplementation with DAI decreased TBARS level and increased SOD activity and GSH content. Consequently, dietary DAI may restore the intracellular antioxidant system to attenuate the oxidative toxicity of A1254 in testicular cells.