Silibinin inhibits VEGF secretion and age-related macular degeneration in a hypoxia-dependent manner through the PI-3 kinase/Akt/mTOR pathway.

BACKGROUND AND PURPOSE: Hypoxia-mediated neovascularization plays an important role in age-related macular degeneration (AMD). There are few animal models or effective treatments for AMD. Here, we investigated the effects of the flavonoid silibinin on hypoxia-induced angiogenesis in a rat AMD model. EXPERIMENTAL APPROACH: Retinal pigmented epithelial (RPE) cells were subjected to hypoxia in vitro and the effects of silibinin on activation of key hypoxia-induced pathways were examined by elucidating the hypoxia-inducible factor-1 alpha (HIF-1α) protein level by Western blot. A rat model of AMD was developed by intravitreal injection of VEGF in Brown Norway rats, with or without concomitant exposure of animals to hypoxia. Animals were treated with oral silibinin starting at day 7 post-VEGF injection and AMD changes were followed by fluorescein angiography on days 14 and 28 post-injection. KEY RESULTS: Silibinin pretreatment of RPE cells increased proline hydroxylase-2 expression, inhibited HIF-1α subunit accumulation, and inhibited VEGF secretion. Silibinin-induced HIF-1α and VEGF down-regulation required suppression of hypoxia-induced phosphatidylinositol 3-kinase/Akt/mammalian target of rapamycin (mTOR) pathway. In the rat model of AMD, silibinin administration prevented VEGF- and VEGF plus hypoxia-induced retinal oedema and neovascularization. CONCLUSION AND IMPLICATIONS: The effects of silibinin, both in vitro and in vivo, support its potential as a therapeutic for the prevention of neovascular AMD.

Effects of cartap on isolated mouse phrenic nerve diaphragm and its related mechanism.

Cartap, a nereistoxin analogue pesticide, is reported to have no irritation to eyes in rabbits. However, we have demonstrated recently that cartap could actually cause acute death in rabbits via ocular exposure. Our preliminary study with isolated mouse phrenic nerve diaphragms has shown that instead of neuromuscular blockade, cartap caused muscular contracture. The objective of the study was to examine the effect of cartap on the neuromuscular junction in more detail and to investigate its possible underlying mechanism with isolated mouse phrenic nerve diaphragms and sarcoplasmic reticulum (SR) vesicles. Cartap or nereistoxin at various concentrations was added in the organ bath with isolated mouse phrenic nerve diaphragm and both nerve- and muscle-evoked twitches were recorded. Instead of blocking the neuromuscular transmission as nereistoxin did, cartap caused contracture in stimulated or quiescent isolated mouse phrenic nerve diaphragm. Both the cartap-induced muscular contracture force and the time interval to initiate the contracture were dose-dependent. The contracture induced by cartap was not affected by the pretreatment of the diaphragm with the acetylcholine receptor blocker alpha-bungarotoxin; the Na(+) channel blocker tetrodotoxin; or various Ca(2+) channel blockers, NiCl(2), verapamil, and nifedipine. On the contrary, the contracture was significantly inhibited when the diaphragm was pretreated with ryanodine or EGTA containing Ca(2+)-free Krebs solution or in combination. This suggested that both internal and extracellular Ca(2+) might participate in cartap-induced skeletal muscle contracture. Moreover, cartap inhibited the [(3)H]-ryanodine binding to the Ca(2+) release channel of SR in a dose-dependent manner. Additionally, cartap could induce a significant reduction in Ca(2+)-ATPase activity of SR vesicles at a relatively high dose. The results suggested that cartap might cause the influx of extracellular Ca(2+) and the release of internal Ca(2+), with subsequent induction of muscular contracture in the isolated mouse phrenic nerve diaphragm. Based on these findings, we propose that the acute death of rabbits following ocular exposure to cartap might have resulted from respiratory failure secondary to diaphragm contracture.

Effects of boldine on mouse diaphragm and sarcoplasmic reticulum vesicles isolated from skeletal muscle.

The effects of boldine [(S)-2,9-dihydroxy-1,10-dimethoxyaporphine], a major alkaloid in the leaves and bark of boldo (Peumus boldus Mol.), on skeletal muscle were studied using mouse diaphragm and isolated sarcoplasmic reticulum membrane vesicles. Boldine, at 10-200 microM, has little effect on the muscle-evoked twitches; however, the ryanodine-induced contracture was potentiated dose-dependently. At higher concentrations of 300 microM, boldine by itself induced muscle contracture of two phases, which were caused by the influx of extracellular Ca2+ and induction of Ca2+ release from the internal Ca2+ storage site, the sarcoplasmic reticulum, respectively. When tested with isolated sarcoplasmic reticulum membrane vesicles, boldine dose-dependently induced Ca2+ release from actively loaded sarcoplasmic reticulum vesicles isolated from skeletal muscle of rabbit or rat which was inhibited by ruthenium red, suggesting that the release was through the Ca2+ release channel, also known as the ryanodine receptor. Boldine also dose-dependently increased apparent [3H]-ryanodine binding with the EC50 value of 50 microM. In conclusion, we have shown that boldine could sensitize the ryanodine receptor and induce Ca2+ release from the internal Ca2+ storage site of skeletal muscle.

Modulation of microsomal cytochrome P450 by Scutellariae Radix and Gentianae scabrae Radix in rat liver.

The present study has determined the effects of Scutellariae Radix (Huangqin) and Gentianae scabrae Radix (Longdan) on liver microsomal cytochrome P450 (P450)-dependent mono-oxygenases using rats pretreated with crude extracts of medicinal herbs. Scutellariae Radix resulted in a 53% decrease of pentoxyresorufin O-dealkylase activity in liver microsomes. In contrast, Gentianae scabrae Radix caused a 50% increase of benzo(a)pyrene hydroxylase activity. Immunoblotting analysis of liver microsomes revealed that Scutellariae Radix induced and suppressed the levels of P450 1A and 2B proteins, respectively. Scutellariae and Gentianae scabrae Radixes had no effects on microsomal aniline hydroxylase activity and P450 2E1 protein.