| Ginsenosides are the main components of ginseng and notoginseng which are widely used in Far East for thousands of years. Ginsenoside Rb1 (GRb1), one of the most abundant ginsenosides, has been proved to have anti-oxidant, anti-apoptosis, inflammation modulating and transmitters release enhancing effects. GRbl thus could benefit to many central nervous system (CNS) disorders. Due to a relatively large molecular weight of 1109.46, hydrophilic molecular structure and biotransformation in intestinal tracts, GRbl has a poor bioavailability when administrated orally. It's predicted difficult for GRbl to cross the blood-brain barrier (BBB).Autophagy is a highly conservative process for the degradation of cellular contents, particularly organelles and long-lasting proteins. Low level autophagy is crucial for maintaining intracellular homeostasis under normal conditions. However, autophagy could be activated by deficit of nutrition and growth factors, endoplasmic reticulum (ER) stress, accumulation of abnormal products and organelle injury. The modulation and role of autophagy under pathological conditions is a hot topic in recent years.Intranasal administration is now a widely accepted non-invasive brain targeting delivery method. In the present study, we first investigated the contribution of GRb1 in different brain regions after intranasal administration to evaluate the brain targeting effects of this delivery method. Then, the beneficial effects of intranasal GRb1 on middle cerebral artery occlusion (MCAO) in rats were evaluated. Finally, the influence of GRbl on the activity of autophagy in the border of infarction was monitored.MethodsDifferent doses of glutamic acid were added to the culture of cortical neuron. The excitotoxity was tested by MTT. Then, GRbl was added to the culture right after glutamic acid. The protective effect of GRb1 was evaluated.GRbl was delivered intranasally or intravenously at a dose of 12.5 mg/kg to male Sprague Dawley (SD) rats, respectively. Rats were scarified at 5,15,30,60,120 and 180 min after drug administration. Plasma and brain tissues, i.e. olfactory bulb+olfactory tract, cerebrum, hippocampus, medulla oblongata and cerebellum, were collected. A HPLC-MS/MS method was employed to detect the concentration of GRb1 in plasma and brain regions mentioned above. Drug targeting index (DTI) was used to evaluate the brain targeting effects.GRb1 was administrated immediately after the onset of MCAO. Rats were decapitated 24 h later. TTC, Nissl, TUNEL staining and Evans blue test were performed to evaluate the effects of intranasal Rbl on the infarct volume, neurodegeneration and BBB breakdown after MCAO.In order to investigate the effects of GRbl on the recovery after acute ischemic stroke, GRb1 was administrated right after the onset of MCAO at a dose of 12.5 mg/kg and once daily for 14 consecutive days. Modified neurological severity score (mNSS) was performed on day 1,2,3,5,7 and 14. Rats were scarified on day 14. HE staining was used to evaluate the infarct volume. A confocal laser microscope was used to observe the number of FITC-dextran perfused microvessels in the peri-infarction area.The influence of GRb1 on the state of autophagy after MCAO was investigated. Rats received intranasal GRb1 right after the onset of MCAO and killed at 0,1.5,6, 12,24 h later. Western blot, real-time RT-PCR and transmission electron microscopy was used to monitor the LC3, Beclin 1 levels and ultrastructural change of neuron in the border of infarction.ResultsThe viability of cortical neuron lowered as the concentration of glutamic acid raised in vitro. GRb1 could ameliorate the excitoxicity caused by 5 mM glutamic acid, with a most effective dose at 12μM.The bioavailability (F) in plasma for intranasal GRb1 is approximately 1.40%. However, intranasal GRb1 gained rather fast plasma absorption at as early as 5 min. After i.n administration, the brain GRb1 concentration reached peak level at 5 min, followed by a decline with time. Interestingly, the concentration of GRb1 in some brain regions, such as cerebrum and hippocampus, achieved a second raise at 60 min after intranasal administration. The value of DTI ranged from 7.35 to 23.22 in different brain regions, indicating a significant brain targeting effect.Intranasal GRbl could reduce the infarction volume significantly. GRbl at a dose of 12.5 mg/kg was more efficient than 1.25 mg/kg. Intranasal GRb1 12.5 mg/kg could ameliorate the neuronal injury and reduce the number of TUNEL positive cells in the border of infarction. The Evans blue leakage was also suppressed by intranasal GRb1.After consecutive administration, the mNSS at day 1-3 were significant lower in the GRb1 group. HE staining indicated the infarct volume was smaller than that of the control group. But the number of microvessels in the border of the infarction in the GRb1 group was not different from that of the control group.GRb1 could modulate the activity of autophagy after MCAO in rats. The up-regulation of LC3 and Beclin 1 were amplified at 6 h but suppressed by intranasal GRb1 later.ConclusionsGRb1 could ameliorate the excitotoxity induced by glutamate in cortical neuron culture. Intranasal administrated GRbl could enter CNS with a relative brain targeting effect, reduce the infarct volume, ameliorate neuronal and microvascular injury and improve the neurological function. These results suggest powerful neuronal and vascular protective effects of intranasal GRb1. Moreover, GRb1 could modulate the activity of autophagy in the border of infarction. Intranasal ginsenoside would be a potential brain targeting therapy for CNS disorders.
|
PDF Full Text Request