The Protective Effects Of Fasudil Hydrochloride On Injured Rat Cerebral Neurons By LPS

Objective and backgroundApoptosis and the nervous system disease have a very important Contact. In some neurodegenerative diseases such as Alzheimer's disease (Alzheimer Dementia, AD), Parkinson's disease (Parkinson, s disease, PD), progressive spinal muscular atrophy (SMA), Huntington disease (HD), Amyotrophic lateral sclerosis (ALS); Prion infection; ischemic/hypoxic neuronal damage; other central nervous system diseases such as glioblastoma multiforme tumors, traumatic brain injury, HIV encephalitis, Kainic acid-induced seizures under experimental conditions, Rodent model and human status epilepticus Can be found or induced apoptosis. Inhibition of apoptosis can be inhibited and/or delayed the neurological disease, It offers a new theoretical basis for the treatment of neurodegenerative diseases and cerebrovascular diseases Cell signal transduction pathway is an important part or aspect in apoptosis. JNK signaling pathway is one important of cell signal transduction pathways. Cell apoptosis and JNK pathway activation are related. Explore JNK pathway in the central nervous system diseases, Variation of the process of development and a variety of molecular mechanisms of disease development; So that we can take a more reasonable signal transduction inhibitor or activator to treat nervous system diseases provide another means. Fasudil hydrochloride (fasudil hydrochloride, FSD) is a new, multi-target the Rho kinase inhibitors, intracellular Ca2+ antagonist; and it has a significant role in nerve protection and treatment for the acute ischemic brain damage, the clinical effect gradually pay close attention in Cerebrovascular disease. the treatment of Clinical are cerebral infarction, subarachnoid hemorrhage caused by delayed cerebral vascular disease (DINDS), transient ischemic attack (TIA), vertebrobasilar artery insufficiency, cerebral hemorrhage recovery period, brain surgery After surgery and interventional treatment of cerebral vasospasm. Recent studies have shown that fasudil hydrochloride could also inhibit apoptosis, but the exact mechanism is still a lack of depth research.This topic has been proposed in the study based on cultured rat cerebral cortex nerve cells for the study, the use of LPS (Lipopolysaccharides, LPS) induced neuronal cell injury model established, Fasudil hydrochloride as interference factors to detect neuronal apoptosis and JNK1 expression in cells, in order to explore the JNK1 role in apoptosis and fasudil hydrochloride inhibits apoptosis in the process of possible mechanisms. Hope to the nervous system disease control to provide some theoretical basis, For the clinical treatment of nervous system diseases provide experimental evidence.Materials and Methodology:1. The experimental animal is the new born Wistar rat in 24 hour, collected the cerebral cortex nerve cells and primary cultured it in vitro.2. To use 10 mg/L LPS in cultured 7d rat cortical neurons, preparation of nerve cell injury model of apoptosis. At the same time, adding different concentrations Fasudil hydrochloride to observe the protective effect of Fasudil hydrochloride on nerve cell damage caused by of LPS.3. Experimental sub-groups:normal group, LPS group, FSD group 1 (final concentration of 50μmol/L), FSD group 2 (final concentration of 100μmol/L), FSD group 3 (final concentration of 200μmol/L).4. Using inverted microscope observe morphological change in nerve cells; Hoechst33258 staining and acridine orange (AO)/ethidium bromide (EB) fluorescent staining of cell apoptosis; determinate the culture medium of lactate dehydrogenase (LDH) concentration; Expressions of phosphorylated JNK1 were measured by Immunofluorescence.5. SPSS 13.0 statistical package was used for statistical analysis. One-way ANOVA and LSD were used to analyze all experimental data. Differences with P<0.05 were considered statistically significant.Results1. Exposed to 10 mg/L LPS, nerve cells were observed with inverted phase contrast microscope at the same time after exchange for the original culture medium. It showed that damaged cells in LPS model group were much more than in control group. Damaged cells in FSD group were somewhat more than in control group, but significantly less than in LPS model group.2. Exposed to 10mg/L LPS, nerve cells were stained with AO/EB fluorochrome and Hoechst 33258 fluorochrome at the same time after exchange for the original culture medium. It showed that neuronal apoptotic ratios in LPS model group were significantly higher than in control group (all P<0.05). Neuronal apoptotic ratios in FSD group were somewhat higher than in control group, but significantly lower than in LPS model group (all P<0.05).3. LDH activity was detected at the same time after exchange for the original culture medium following neurons transiently exposed to 10mg/L LPS. It showed that LDH activity in LPS model group was significantly higher than in control group (all P<0.05). LDH activity in FSD group was somewhat higher than in control group, but significantly lower than in LPS model group (all P<0.05).4. Exposed to 10mg/L LPS, nerve cells were stained with phosphorylation JNK1 fluorochrome at the same time after exchange for the original culture medium. It showed that fluorescence intensity of nerve cell in LPS model group significantly enhanced contrast to control group (all P<0.05). Fluorescence intensity of nerve cell in FSD group was somewhat enhanced contrast to control group, but significantly attenuated contrast to LPS model group (all P<0.05).Conclusions 1. LPS could induce injury In vitro rat cerebral cortical neurons successfully.2. The expression of phosphorylation JNK1 was up-regulated during apoptosis of rat cerebral cortex neurons induced by LPS.3. FSD could improve the shape of injured neurons, stabilize cell membrane and reduce LPS-induced apoptosis.It may be related to inhibit of phosphorylation JNK1 expression upregulation during apoptosis.