Effects Of Tea Polyphemol On Okadaic Acid-induced Neurotoxicity Of Primary Cultured Cortical Neurons

Alzheimer's disease(AD) is the most common neurodegenerative disorder characterized by the progressive deterioration of cognition and memory in association with the presence of senile plaques(SP),neurofibrillary tangles(NFTs),and massive loss of neurons,primarily in the cerebral cortex and hippocampus.SP are extracellular deposits composed ofβ-amyloid(Aβ) fibrils,surrounded by dystrophic neuritis,reactive microglias and astrocytes.The NFT contain paired helical filaments(PHF),which are mostly composed ofa hyperphosphorylated tau.Tau is a kind of microtubule-associated proteins enriched in the axonal compartment,and is encoded by a single gene located on chromosome17(FTDP-17).In normal brain,the tau protein is involved in the formation and the stabilization of microtubules.Recent studies have suggested that the abnormal hyperphosphorylation of tau could be the results of up- regulation of protein kinases or down-regulation of protein phosphatases.Several experiments have shown that four major-protein phosphatases(PPs),PP1,PP2A,PP2B and PP2C.Okadaic acid(OA) is a extractive of marine life,which showed significant inhibition of PP2A.Several studies have suggested that OA could induce microtubule-associated protein tau hyperphosphorylation,and then results in neurotoxieity,including pathology process of analogic AD.Tea polyphenol is a general term in tea polyphenols,is a strong natrural antioxidants, including at least eight of the essential material catechins(EGCG,ECG,EG,CEC,GCG,GC,CG, C),which have been confirmed that it has a close relationship with the promotion of the human body to achieve health.The phenolic group,which is located on the EGCG and ECGB ring,D ring,EC and B ring,are critical to be the antioxidant activity of tea polyphenols.The more phenolics hydroxyl group are,the stronger antioxidant capacity would be.The phenolic hydroxyl structure of tea polyphenols makes it capbility of strong capturing,getting rid of free radical and Antioxidant functions.Tea polyphenols reduce damages of DNA coursed by free radical.Studies found that Aβcould cause damage of cultured hippocampal neurons.However,if the EGCG(the main component of tea polyphenol) and Aβis co- cultiure,the damage is reduced through mediating the starch kind of precursor protein deal with process,enhancing a-starch hydrolysis activity and reduced tumor necrosis factor-a converting enzyme activity,increasing the producing of soluble amyloid precursor protein(sAPP) and inhibiting the generation of amyloidosis.In vivo,tea polyphenols could reduce the concentration and time-dependent rotational behavior produced 6-OHDA in PD model,low ROS and NO,nitrotyrosine concentration in midbrain and striatum,reduce the expression levels of nNOS and iNOS.Tea polyphenol pretreatment could increase survival neurons in substantia nigra and reduce the apoptotic cells.These results suggest that tea polyphenols play an important role in neurodegenerative disease.The purpose of this study was to observe whether if the tea polyphenol could antagonism neurotoxicity mediated by tau protein hyperphosphorylation by OA including cell apoptosis? The main results as following:1.OA induced insults of primary nenrons including apoptosis.2.Tea polyphenol protects primary cortical neurons against toxicity induced by OA3.Tea polyphenol protects primary cortical neurons against apoptosis induced by OAPartⅠTea polyphenol protects primary cultured cortical neurons against toxicity induced by Okadaic acidIn the first part of experiment,we detected the toxicity of primary cultured cortical neurons induced by OA,and observed TP' s effects on OA-induced toxicity.There are two aspects of experiments,in which cell viability was detected by cck-8 viability assay and Calcein-AM staining.Caicein- AM is a colourant which only dye living cell and cck-8 is a widely accepted cell viability detection assay.The results showed:1.cck-8 activity assay:OA(10 nM) produced significantly decrease of cell viability detected by cck-8 assay. Pretreatment of TP(5μg/ml and 10μg/ml) could not suppress OA-induced toxicity,TP(15μg/ml) could protect neurons against OA-induced toxicity,it could elevate cell viability from 45.41±3.72%to 70.53±3.27%(n=5,P＜0.05);TP(20μg/ml) produced even stronger protection,which can improve cell viability from 45.41±3.72%to 79.34±4.36% (n=5,P＜0.05 ).2.Calcein-AM staining:OA(10 nM)produced significantly decrease of living cells detected by Calcein-AM. Pretreatment of TP(5μg/ml and 10μg/ml) could not protect OA-induced cell death.Pretreatment of TP(15μg/ml) significantly increased the positive staining neurons,while TP(20μg/ml) exhibited even more strong protective effects on OA-induced insulrs. The results demonstrate that:1.OA induced toxicity effect in primary cultured cortical neurons,including cell toxicity increased by cck-8 assay and living cells decreased by Calcein- AM staining;2.TP protected primary cultured cortical neurons against toxicity induced by OA.PartⅡTea polyphenol protects primary cultured cortical neurons against apoptosis induced by Okadaie acidSeveral studies have showed that OA can induce tan hyperphosphorylation by inhibiting PP2A,and then cause cortical neurons apoptosis.In parrt one of the experiment,we showed that the tea polyphenol rescued cortical neurons from OA-induced toxicity,whether Tea polyphenol could inhibit OA-induced apoptosis? Therefore,we designed this experiment in which TUNEL staining and the activity of caspase-3 were used to detecte apoptosis,furthermire the neuroprotection of tea ployphenol on OA-induced apoptosis was observed.The results as followings:1.Detection of activity of Caspase3:The activity of Caspase3 increased after added OA(10nM).It has showed neurons cell body refraction degrade,neurapophsis obviously retract,even break and dissolve into small fragments under inverted microscope.Pretreatment of Tea polyphenol(5μg/ml,10μg/ml) could not antagnoise OA induced apoptosis.Tea polyphenol(15μg/ml) partially reduced the activity of Caspase-3,while the activity of Caspase-3 significantly decreased by pretreatment of tea polyphenol(20μg/ml)2.Examination of TUNEL taining:TUNEL staining show that apoptotic neurons was flavor-green and all cell nueleus were red. After added OA(10 nM) to the cultures,the apoptosis rates of neurons were signigicantly increased compared with the control group(from 6.27±0.35%to 51.25±3.16%p＜0.05). Pretreatment of tea polyphenol(5μg/ml和10μg/ml) could not protect OA induced apoptosis by statistics analysis;Tea polyphenol(15μg/ml ) could partially reduce the apoptosis rates from 51.25±3.16%to 26.83±2.35%(p＜0.05),while the apoptosis rates significantly decreased by pretreatment of tea polyphenol(20μg/ml )(from 51.25±3.16%to 17.50±1.47%,p＜0.05).The results demonstrated that:1.OA induced apoptosis in primary cultured cortieal neurons:2.Tea polyphenol protected primary cultured cortical neurons against apoptosis induced by OA. Conclusiond:1.OA induced neurotoxicity in primary cultured cortical neurons,including cell viability decreased and living cells decreased;2.Tea polyphenol protected primary cultured cortical neurons against neurotoxicity induced by OA;3.OA induced apoptosis in primary cultured cortical neurons;4.Tea polyphenol protected primary cultured cortical neurons against apoptosis induced by OA;...