New C21-steroidal aglycones from the roots of Cynanchum otophyllum and their anticancer activity.

Naturally occurring C21-steroidal aglycones from Cynanchum exhibit significant antitumor effects. To expand the chemical diversity and get large scale C21-steroidal aglycones, the extracts of the roots of Cynanchum otophyllum were treated with 5% HCl in aqueous and the resulting hydrolysate was investigated. Nine new C21-steroidal aglycones (1-9) namely cynotogenins A-I, along with seventeen known analogous (10-26), were isolated from the hydrolysate. The structures of compounds 1-9 were elucidated by spectroscopic analysis (IR, HR-ESI-MS, 1D and 2D NMR) and comparison of observed spectroscopic data with those of reported in the literature. Aglycones 2-5 with rare cis-cinnamoyl group as well as 8 and 9 with 5ß,6ß-epoxy group were found from the genus of Cynanchum for the first time. The cytotoxicities of compounds 1-26 toward human cancer HeLa, H1299, HepG2, and MCF-7 cells were evaluated and preliminary structure-activity relationship (SAR) was discussed. Moreover, compound 20 inhibits HepG2 cell apoptosis and induces of G0/G1 phase arrest in a dose dependent manner.

Activation of α1B-adrenoceptors contributes to intermittent hypobaric hypoxia-improved postischemic myocardial performance via inhibiting MMP-2 activation.

Inhibition of matrix metalloproteinases-2 (MMP-2) activation renders cardioprotection from ischemia/reperfusion (I/R) injury; however, the signaling pathways involved have not been fully understood. Intermittent hypobaric hypoxia (IHH) has been shown to enhance myocardial tolerance to I/R injury via triggering intrinsic adaptive responses. Here we investigated whether IHH protects the heart against I/R injury via the regulation of MMP-2 and how the MMP-2 is regulated. IHH (Po2 = 84 mmHg, 4-h/day, 4 wk) improved postischemic myocardial contractile performance, lactate dehydrogenase (LDH) release, and infarct size in isolated perfused rat hearts. Moreover, IHH reversed I/R-induced MMP-2 activation and release, disorders in the levels of MMP-2 regulators, peroxynitrite (ONOO(-)) and tissue inhibitor of metalloproteinase-4 (TIMP-4), and loss of the MMP-2 targets α-actinin and troponin I. This protection was mimicked, but not augmented, by a MMP inhibitor doxycycline and lost by the α1-adrenoceptor (AR) antagonist prazosin. Furthermore, IHH increased myocardial α1A-AR and α1B-AR density but not α1D-AR after I/R. Concomitantly, IHH further enhanced the translocation of PKC epsilon (PKCε) and decreased the release of mitochondrial cytochrome c due to I/R via the activation of α1B-AR but not α1A-AR or α1D-AR. IHH-conferred cardioprotection in the postischemic contractile function, LDH release, MMP-2 activation, and nitrotyrosine as well as TIMP-4 contents were mimicked but not additive by α1-AR stimulation with phenylephrine and were abolished by an α1B-AR antagonist chloroethylclonidine and a PKCε inhibitor PKCε V1-2. These findings demonstrate that IHH exerts cardioprotection through attenuating excess ONOO(-) biosynthesis and TIMP-4 loss and sequential MMP-2 activation via the activation of α1B-AR/PKCε pathway.

Berbamine protects the heart from ischemia/reperfusion injury by maintaining cytosolic Ca(2+) homeostasis and preventing calpain activation.

BACKGROUND: Berbamine, a natural compound from Barberry, was reported to protect myocardium from ischemia/reperfusion (I/R) injury, but the underlying mechanisms are largely unknown. METHODS AND RESULTS: Berbamine pretreatment from 10 to 100nmol/L concentration-dependently improved post-ischemic myocardial function. Similar protection was confirmed in isolated cardiomyocytes characterized by the attenuation of I/R-induced intracellular free Ca(2+) concentration ([Ca(2+)](i)) overloading and the depression of cell shortening and Ca(2+) transients, which were partially mimicked but not augmented by calpain inhibitor calpeptin and abolished by mitochondrial ATP-sensitive potassium (mitoK(ATP) channel inhibitor 5-hydroxydecanoate (5-HD) and phosphoinositide 3-kinase (PI3K) inhibitor wortmannin. Consistently, I/R-induced increase of calpain activity and decrease of sarcoplasmic reticulum Ca(2+) ATPase (SERCA2) activity; and protein expression of SERCA2a, desmin, calpastatin and Akt was significantly attenuated by berbamine. In addition, I/R-decreased Akt protein was reversed by calpeptin. Moreover, berbamine further increased I/R-enhanced phosphorylation of Akt and glycogen synthase kinase-3ß (GSK3ß). These protections were abolished by wortmannin. Furthermore, berbamine significantly attenuated I/R-induced lactate dehydrogenase release, infarct size and contractile dysfunction, and such cardioprotective actions were abolished by wortmannin and 5-HD or mimicked by glycogen synthase kinase-3ß (GSK3ß) inhibitor SB216763 but without additive effect. CONCLUSIONS: These findings suggest that berbamine confers cardioprotection against I/R injury by attenuating [Ca(2+)inf(i) overloading and preventing calpain activation through the activation of the PI3K-Akt-GSK3ß pathway and, subsequently, opening of the mitoK(ATP) channel.

Intermittent hypobaric hypoxia improves postischemic recovery of myocardial contractile function via redox signaling during early reperfusion.

Intermittent hypobaric hypoxia (IHH) protects hearts against ischemia-reperfusion (I/R) injury, but the underlying mechanisms are far from clear. ROS are paradoxically regarded as a major cause of myocardial I/R injury and a trigger of cardioprotection. In the present study, we investigated whether the ROS generated during early reperfusion contribute to IHH-induced cardioprotection. Using isolated perfused rat hearts, we found that IHH significantly improved the postischemic recovery of left ventricular (LV) contractile function with a concurrent reduction of lactate dehydrogenase release and myocardial infarct size (20.5 ± 5.3% in IHH vs. 42.1 ± 3.8% in the normoxic control, P < 0.01) after I/R. Meanwhile, IHH enhanced the production of protein carbonyls and malondialdehyde, respective products of protein oxidation and lipid peroxidation, in the reperfused myocardium and ROS generation in reperfused cardiomyocytes. Such effects were blocked by the mitochondrial ATP-sensitive K(+) channel inhibitor 5-hydroxydecanoate. Moreover, the IHH-improved postischemic LV performance, enhanced phosphorylation of PKB (Akt), PKC-ε, and glycogen synthase kinase-3ß, as well as translocation of PKC-ε were not affected by applying H(2)O(2) (20 µmol/l) during early reperfusion but were abolished by the ROS scavengers N-(2-mercaptopropionyl)glycine (MPG) and manganese (III) tetrakis (1-methyl-4-pyridyl)porphyrin. Furthermore, IHH-reduced lactate dehydrogenase release and infarct size were reversed by MPG. Consistently, inhibition of Akt with wortmannin and PKC-ε with εV1-2 abrogated the IHH-improved postischemic LV performance. These findings suggest that IHH-induced cardioprotection depends on elevated ROS production during early reperfusion.

Berbamine increases myocardial contractility via a Ca2+-independent mechanism.

Berbamine (BM), a natural compound derived from Berberis vulgaris L, has been reported to inhibit cardiac contractile function at higher concentrations. Here, we report that BM had concentration-dependent biphasic effects on myocardial contraction in Langendorff-perfused rat hearts, that is, at lower concentrations (30-100 nM), it displayed positive inotropic and lusitropic effects, whereas at a higher concentration of 1 µM, it caused a negative inotropic effect after an initially weak increase. These effects were further confirmed in cardiomyocytes isolated from the left ventricles of rats. Moreover, the increased cell shortening by BM at concentrations from 0.1 to 100 nM was not associated with an alteration of intracellular Ca transients. Consistently, at 30 nM, BM shifted the cell shortening--Ca transient relationship curve induced by cumulative elevation of extracellular Ca concentration to the left. Furthermore, BM significantly increased membrane-bound but not filament-bound protein kinase C epsilon (PKCε) in the isolated hearts and cardiomyocytes. Such a translocation was inhibited by PKCε-specific inhibitor PKCε V1-2 concomitant with the abolishment of the BM-induced increase in contraction. These findings reveal the positive inotropic effect of BM in the myocardium and demonstrate that BM increases myocardial contractility by increasing myofilament Ca sensitivity via a PKCε-dependent signaling pathway.