| IntroductionApoptosis plays a fundamental role in the development of numerous pathophysiological states. The balance between cell growth and apoptosis is likely to determine whether pathological phenomena such as hypertension, atherosclerosis and vascular disease occur. The excess generation of reactive oxygen species in the vascular system during the atherogenic process has been reported to enhance atherosclerotic lesion formation, resulting in the development of cardiovascular disease.Hydrogen peroxide (H2O2), one of the most common reactive oxygen species, can easily penetrate the plasma membrane and cause lipid peroxidation and DNA damage in cells. H2O2 induces apoptosis by disrupting the cell's natural antioxidant defense system. It has been shown that H2O2 can stimulate cytochrome c release from the mitochondria, resulting in the loss of endothelial integrity and subsequent vascular disease.Catalpol, a major chemical constituent of Rehmannia glutinosa Libosch, is an iridoid glucoside which exerts well known anti-apoptotic effects in neuronal cells and other cell lines. Findings from in vitro experiments have revealed that catalpol is able to activate the intracellular signal transduction pathway inducing neuronal differentiation and attenuate H2O2-induced apoptosis in PC 12 cells. The effects of catalpol on cardial oxidative stress have not been described. However a protective effect against oxidative stress damage of neurons in global and focal cerebral has been reported. Catalpol has also been shown to protect against oxidative stress in peripheral tissues such as the kidneys. Therefore, it seems reasonable to investigate whether catalpol is able to protect the cardiovascular from oxidative stress injury. Akt is a critical component of the intracellular signaling pathway involved in regulating cell survival and apoptosis. Various growth and survival factors can activate this protein kinase. It has been reported that Akt can phosphorylate and inactivate Bad, thus inhibiting cell death. Among the Bcl-2 family, several members such as Bcl-2 and Bcl-xL induce cell survival, while other members such as Bad and Bax promote cell death. Further to this, it has been shown that members of the Bcl-2 family, which are located on the mitochondrial membrane, can alter mitochondrial membrane permeability and trigger apoptosis.In the present study, we examined the effect of catalpol on H2O2-induced apoptosis in human umbilical vein endothelial cells (HUVECs) and H9c2 cells. We also examined potential mechanisms underlying catalpol-associated protection, including reactive oxygen species scavenging, alterations in the phosphatidylinositol 3-kinase (PI3K)/Akt-Bad signaling pathway and changes in Bcl-2 and Bax expression.Materials and methods(1) We cultured HUVECs and H9c2 cells, and examined the effect of catalpol and H2O2 on cells. We also examined the effect of wortmannin and LY294002 on H2O2 incubated cells.(2) The cell viablity of HUVECs and H9c2 was determined by MTT analysis.(3) The concentration of malondialdehyde (MDA), lactate dehydrogenase (LDH) as well as the activity of superoxide dismutase (SOD) were were all determined by using commercially available kits.(4) The level of intracellular reactive oxygen species was quantified by 2', 7'-dichlorofluorescein diacetate assay.(5) Apoptotic cells were detected by terminal deoxyribonucleotidyl transferase-mediated deoxyuridine triphosphatebiotin nick end labeling, Annexin V-fluorescein isothiocyanate binding assay and hoechst 33258 assay.(6) Expression of Akt, p-Akt, p-Bad, Bcl-2 and Bax activity was quantified by western blotting analysis. (7) Expression of Akt, Bad, Bcl-2 and Bax mRNA was determined by real-time semiquantitative reverse transcription-polymerase chain reaction method.(8) Expression of p-Akt, Bcl-2 and Bax activity was detected by immunohistochemistry.(9) All data are presented as mean±standard deviation (S.D.). Differences between mean values of multiple groups were analyzed by one-way analysis of variance (ANOVA). The results were considered to be statistically significant when P＜0.05.Results(1) Incubation of HUVECs and H9c2 cells with different concentrations of catalpol (0.1,1,10μg/ml) for 48 h increased the viability of cells in a dose-dependent manner. The protective effect of catalpol was almost inhibited and cell viability reduced compared with the control group at a concentration of 1000μg/ml.(2) Increased rates of cell survival were apparent 24 h after treatment. The magnitude of cell survival peaked at 72 h in HUVEC and at 48h in H9c2.(3) Pre-incubation of cells with different concentrations of catalpol (0.1,1 or 10μg/ml) before H2O2 exposure increased viability in a dose-dependent manner.(4) Compared with the control, treatment of cells with 100μM of H2O2 for 24 h caused significantly less activities of SOD. However, catalpol (0.1,1 or 10μg/ml) pretreatment significantly attenuated the changes of SOD activities in a dose dependent fashion, compared to the H2O2 group. In addition, cells treated with 100μM of H2O2 for 24 h caused more MDA and LDH levels, while pre-incubation of cells with catalpol (0.1,1 or 10μg/ml) markedly attenuated the increases compared to the H2O2 group.(5) As expected, DCF fluorescence in cells exposed to H2O2 was strikingly increased compared to fluorescence in control group. Pre-treatment with catalpol (0.1, 1 or 10μg/ml) significantly inhibited DCF fluorescence caused by H2O2 exposure in a dose-dependent fashion.(6) Few TUNEL-positive nuclei were evident in the control group. Exposure of cells to 100μM H2O2 for 24 h, however, resulted in a marked increase in the number of such nuclei. Pre-treatment with catalpol reduced the K2O2-induced increase in the number of cells with TUNEL-positive nuclei.(7) In the control group were shown as round-shaped nuclei with homogenous fluorescence intensity. However, a marked increase of apoptotic cells which contained heterogeneous intensity, chromatin condensation, and fragmentation appeared after 24 h treatment with 100μM H2O2. Pre-treatment of catalpol completely protected cells from morphological changes by H2O2.(8) Flow cytometry in HUVECs revealed that 78.7±1.2% of cells in the control group were viable. In contrast,35.9±0.6% of cells treated with 100μM H2O2 for 24 h were in early apoptosis or late apoptosis/necrosis. Pre-treatment of cells with catalpol (0.1,1 or 10μg/ml) for 24 h reduced the percentage of apoptotic cells associated with H2O2 exposure from 35.9±0.6% to 27.6±0.6%,22.6±0.8% and 19.1±0.4%, respectively.Exposure of H9c2 cells to 100μM H2O2 for 24 h resulted in an increase in cellular apoptosis as revealed by flow cytometry. Pre-treatment of cells with catalpol (0.1,1 or 10μg/ml) for 24 h prior to H2O2 reduced the percentage of apoptotic cells from 14.30±0.41% to 8.97±0.36%,7.81±0.06% and 6.38±0.43%, respectively, in a concentration dependent manner.(9) The protein phosphorylated Akt, Bad, Bcl-2 and Bax were analyzed by western blotting and immunohistochemistry. Catalpol (0.1,1 or 10μg/ml) induced Akt and Bad protein phosphorylation, increased Bcl-2, decreased Bax in a dose-dependent manner Both wortmannin and LY294002 markedly inhibited catalpol-induced Akt and Bad phosphorylation, increased Bax, decreased Bcl-2.(10) We investigated the mRNA expression of Akt, Bad, Bcl-2 and Bax. Akt, Bad, Bcl-2 mRNA levels were significantly increased in catalpol (0.1,1 or 10μg/ml) pre-treated groups, compared to the control group. While H2O2 exposure significantly reduced Akt, Bad, Bcl-2 levels compared to control. Catalpol (0.1,1 or 10μg/ml) pre-treatment down-regulated Bax levels in cells exposed to H2O2. This effect was partially inhibited by concurrent wortmannin or LY294002 treatment.Conclusions(1) Pre-incubation of cells with 0.1,1 or 10μg/ml of catalpol resulted in a time-and dose-dependent manner variation of cell viability.(2) Catalpol may protect cells from H2O2-induced injury by strengthened the changes of SOD activities and attenuated the levels of MDA.(3) Scavenging of reactive oxygen species is involved in the mechanisms underlying the protective effect of catalpol against H2O2-induced injury.(4) Catalpol may protect cells from H2O2-induced apoptosis at least part by activating the PI3K/Akt-Bad signaling pathway.(5) Bcl-2 and Bax are involved in mediating the anti-apoptotic effects associated with catalpol treatment in HUVECs and H9c2 cells exposed to H2O2.
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