| Backgrounds:Studies have shown that long-term stress can cause laboratory animals'abnormal behavior andalso increase the risk of getting mental diseases.Chronic stimulation of stressful environment can accelerate the aging of brain,attenuate memory ability and eventually lead to the happening nervous system diseases.In addition,Glucocorticoid is widely used as a therapeutic drug in clinical at present,especially for patients with autoimmune diseases and partial endocrine diseases.Since these patients have the needs to use glucocorticoids for a long time.Studies have also shown that the increased level of Cortisol which is one stress hormone is closely related to the atrophy of temporal lobe and cognitive impairment in human aging process.Clinical data also show that the symptoms of cognitive impairment and memory decline in patients with elevated Cortisol levels happened earlier than those in the same age group.In addition to this,long term use of Glucocorticoids,such as Dexamethasone,can also increase the risk of getting neurodegenerative disease and damage memory ability of humans or rodents.At the same time,the level of cortisol in patients with neurodegenerative diseases?such as Alzheimers disease?is higher than that of the same age group.The mechanism of why the long-term use of Glucocorticoid can increase the risk of getting neurodegenerative diseases includes the reduction of neurosecretory factors,the emergence of oxidative stress reactions,and structural changes of the nerve cells.It is worth noting that there are not effective drugs which can slow down the happening of neurondegenerative diseases nowadays.One main reason is that neuron cells are non-renewable when neuron damaged,another reason which cannot be neglected is the blood-brain barrier,since most neuroprotective substances found at present normally have a large molecular structure,meaning that these substances cannot passed through the barrier.Schisandra Chinensis belongs to magnolia plant.It is a traditional traditional Chinesemedicine.The main function of this medicine is to enhance intelligence and improve psychology.It is commonly used in the treatment of viral hepatitis,chemical hepatitis and myocardial disease.Animal models'vivo and vitro experiments show that Schizandrin can protect neurons from many damages including neurotoxicity caused by scopolamine,neurotoxicity induced by amyloid beta protein,oxidative stress induced by cisplatin or neurotoxicity induced by gene expression.The main component playing neuroprotective and antioxidant effects of Schisandra chinensis fruit is biphenyl octyl lignans.deoxyschizandrin,schizandrin and schisandol are the main effective components extracted from Schisandra Chinensis.These components are all biphenyl ocenadiene derivaties.More importantly,these components have small molecular constructure which can also dissolve well with fat substance.These conditions provide Schisandra chinensis with support for passing through the blood-brain barrier.So Schisandra chinensis is hopeful to work as one neuroprotective drug.It is reported that Schisandrin A is the main active ingredient in fruits of Schisandra chinensis,which has the effects of diazepam,anticonvulsant and brain protection.However,it is not clear whether Schisandol has the neuron protective effect against the damage induced by caused by Glucocorticoid,and its possible mechanism is also unclear.Therefore,it is of great importance to study the effects of Schisandrin in protecting neuron damaged by Glucocorticoid.ObjectiveThe aim of this study is to investigate whether Schisandrin can protect neurons damaged byGlucocorticoid,Trying to find out its possible mechanism by doing experiments in vitro and providing a theoretical support for clinic treatment in curing related nervous system injury.MethodsIn this experiment,neurons were extracted from the cerebral cortex of SD rats born within 24hours;then the neurons were identified by NSE experiment to further confirm the morphology of neurons and the purity of nerve cells.The growth condition of neurite was also observed in vitro for 3 days.The optimal cell planting density for different dish sizes were identified,the culture medium for extracted neurons were changed every three days,after cultivating for 9 days in vitro,neurons were divided into different groups inclding the control group?the Dexamethasone treatment group,and the neurons treated with different concentrations of Dexamethasone(10-4,10-5,10-6,10-7mol/L)groups.These groups treated with different conditions and cultivated for another three days.MTT experiments identified the activity of neurons treated by Dexamethasone declined.Results from RT-PCR experoments and immunofluorescence staining experoments show that the expression of MAP2,one microtubule related protein,of different groups were not the same,and the growth state of neurons was also observed.Then one appropriate concentration of Dexamethasone in the test was identified.In the next step,after cultivating in vitro for 9days,neurons were divided into different groups including control groups,Dexamethasone injured groups and the experimental group treated with the mixture of Dexamethasone and different concentrations of Schizandrin?40,80 and 160?mol/L?,after another three days'cultivation with different treatments,protein and RNA from each group were extracted,experimental techniques such as Western blot?Real-time PCR and immunofluorescence assay were used to explore the possible protective mechanism of schisandrin on damaged neurons.Results1 Extracted neurons were cultured in vitro for 3 days.According to the observed formation ofneurite protuberance,the optimal density of nerve cells inoculating in different dishes of different sizes were 107 cells/well for 6 well plate,1.2x106 cells/well for 12 well plate,8x104 cells/well for 24 well plate and 4x104 cells/well for 96 well plate.MTT results show that the 10-44 and 10-5 mol/L Dexamethasone can be seen as effective concentrations which can cause damage to neuron cells.However,low concentration of Dexamethasone such as 10-66 or 10-77 mol/L cause slight damage to neurons and the optimal time for Dexamethasone treatment was 72 hours.MAP2 immunofluorescence staining further demonstrated that when neuron cells were treated with 10-4 and 10-5 mol/L Dexamethasone it is easily to find the obvious neurofibrillary tangles among neurons,With the efforts of 10-4 and 10-55 mol/L Dexamethasone,part of neural network collapse;But when it comes to low concentrations of Dexamethasone?10-66 or 10-77 mol/L?,the situation was quite different,even treated with the same period of time,neural network structure was much better and neurofibrillary tangles among neuron cells was not obvious.2 After cultivated in vitro for 9 days,neurons were treated with the mixture of Dexamethasoneand Schizandrin.the result of MTT experiment show that the survival rate of neuron cells increased,and the results of MAP2 immunofluorescence staining show that the neurite wound of the neurons treated with the mixture was mitigated and the disintegration condition of neural network was less.3 The Western Blot experiment was used to detect the expression of postsynaptic dense protein ofneurons.The results show that the expression of postsynaptic densified protein PSD95 of Dexamethasone treated group decreased,while the level of phosphorylation of tau protein increased,however,the level of phosphorylation happening at different tau sites increased,comparing with blank group and the solvent control group.The results of Real-time PCR experiment show that the expression of MAP2 mRNA and the expression of glucose transporter 3?GLU3?mRNA in Dexamethasone treated group decreased.4 Western blot results show that the expression of PSD95 in these groups which were treated withthe mixture of Dexamethasone and Schizandrin enhanced,while the level of tau protein phosphorylation decreased,the phosphorylation level of tau protein at several loci increased,comparing with the dexamethasone treatment group.When was compared with the Dexamethasone treatment group.The results of Real-time PCR show that the expression of MAP2 mRNA and the expression of glucose transporter 3 mRNA in terms of these groups treated with the mixture of dexamethasone and schizandrin increasedConclusion:Schizandrin can protect the neurons treated with Glucocorticoid.One possible mechanism is thatSchizandrin can increase the expression of glucose transporter 3 mRNA located at the surface of cell membrane and accelerate the transport of glucose,providing more substrates for glycosylation of tau protein and improving the glycosylation level of tau protein and reducing its phosphorylation... |
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