Neuroprotective Effects Of Icarriin Against Corticosterone-induced Neurotoxicity In Primary Cultured Rat Neuronal Cells

The experience of stress is a common to all living organisms.The main components of the stress system are Hypothalamic-pituitary-adrenal axis and locus ceruleus-norepinephrine (LC/NE)-autonomic systems and their peripheral effectors, the pituitary-adrenal axis, and the limbs of the autonomic system which are over-triggered under stress.A primary endocrine response to stress is the secretion of glucocorticoids.Integration of the hippocampus on the stress reaction is very important on the secretion of GC. The hippocampus is a high regulate centre of HPA axis and the sensitive areas to chronic stress response. Apoptosis is one of the mechanisms of which GC lead to hippocampal neuronal damage. Previous studies have been reported that protective effects of icariin against learning and memory deficits induced by aluminium in ratsAim of the study :To investigate icariin's neuroprotective effect against the toxicity induced with corticosterone in primary cultured rat hippocampal neuronal cells,as well as the underlying mechanisms.Materials and methods:1 .Cultured hippocampal neurons were from newborn Sprague-Dawley rats in 24h2.After various treatments, cell viability was determined by incubating with MTT assay and evaluated by measuring the amount of cytoplasmic lactate dehydrogenase (LDH) released into the medium.3.We performed TUNEL staining, Hochest33258 staining, Rhodaming 123 staining to establish an assay to identify the neuroprotective effects of icarrin against neuronal damages induced by CORT. We also examined caspase-3 activity as a spfaciapoptotic marker in hippocampal neuronal cultures exposed to CORT alone or incombination with icarrin.4.The intracellular [Ca2+]i in hippocampal neurons were tested in the study byfluorospectrophotometry5.To determine whether CORT induce ROS generation, and whether and the extent towhich icarrin pretreatment affects ROS generation,we assayed changes in ROS levelin neuron subjected to various treatment, we also SOD level in neuron subjected toCORT alone or in combination with icarrin6. The key regulators of MAPK and PI3K/Akt signal pathway which maymediated with protective effect of icariin against CORT-induced neuron damage weretested by western blot assay.Results1 Neuronal morphological results showed that cultured neuronal cells were treated without or with icariin in the absence or presence of CORT for 24 h. Cells treated with vehicle (control), icarrin exhibited large vacuole-free cell bodies with elaborate networks of neurites. Exposure of cells to CORT at 1μM and 10uM for 24 h resulted in obviously cell loss and neurites injury. These changes were counteracted by icaritin.2. MTT assay and LDH release results showed cell viability were 58.1% and 44.7% after co-incubation of hippocampal neurons with CORT at 1μM and 10uM for 24h. When 48h, cell viability were 46.3% and 16.8% respectively in time- and concentration dependent manner. After pretreatment of icarrin in 10^-5-10^-6mmol/L for 2h, then co-incubation with CORT for 24h, cell viability were increased.3.Furthermore, We performed TUNEL staining, Hochest33258 staining, Rhodaming 123 staining to establish an assay to identify the neuroprotective effects of icarrin against neuronal damages induced by CORT. We also examined caspase-3 activity as a specific apoptotic marker in hippocampal neuronal cultures exposed to CORT alone or in combination with icarrin. Hochest33258 staining results showed significant nuclear condensation, chromatin condensation, formation of apoptotic bodies after CORT treatment for 24h. Tunel staining results showed CORT-induced the production of TUNEL-positive cell. Pretreatment of icariin can reversed the effects. Mitochondria is not only a major intracellular source of ROS but also a sensitive target for the toxic actions of radicals. Pretreatment of icariin following CORT induced decreases in Rh123 fluorescence intensity compared with the model level.4.The studies results showed the intracellular [Ca2+]i in hippocampal neurons significantly increased after CORT treatment campared to the normal level (P<0.01) . Pretreatment of icariin following CORT induced decreases Fura 2-AM fluorescence intensity compared with the model level (P<0.05) .5. The studies results showed the intracellular ROS leved in hippocampal neurons significantly increased after CORT treatment campared to the normal level. Pretreatment of icariin following CORT not induced decreases DCFH-DA fluorescence intensity compared with the model level (P>0.05 ) .6.The mitogen-activated protein kinases(MAPK)signaling cascades have been implicated in cerebral ischemia.They are mainly composed of the extracellualr signal-regulated kinase1/2,the c-Jun N-terminal kinase(JNK) and p38 cascades.CORT strikingly induced p38 activation within 60min and that of JNK and ERK1/2 within 30min,whereas icariin blocked the activation of p38 MAPK, but not JNK and ERK-1/27. The activation of (PI3-K)/Akt signal pathway by CORT in cultured hippocampal neurons was measured by phospho-Akt levels. Western blot test revealed a decrease of phospho-Akt at 30min following the treatment with CORT, whereas icariin enhanced the activation of Akt. The increase in phospho-Akt levels sustained for at least 2h. while the total non-phosphorylated Akt levels under the experimental conditions remained the same in the control and in the CORT treatment.Conclusions1. The present results indicated that the protective effects of icariin on apoptosis in rat primary hippocampal neuron may be,at least in part,attributable to the its potent anti-Intracellular calcium overload but not antioxidative ability.2. Mitogen-activated protein kinase/extracellular signal-regulated kinase pathway and PI3K/Akt signal pathway may also be involved in and partly contributed to the neuroprotective effects of icaritin.