Protective Effect Of Epigallocatechin-3-Gallate On The Apoptosis Of Rat Cerebellar Granule Neurons Induced By Acrylamide

Objective:To establish the apoptosis model of rat cerebellar granule neurons induced byacrylamide in vitro and investigate the protective effect and mechanism ofepigallocatechin-3-gallate on apoptosis of cerebellar granule neurons.Methods:Cerebellar granule neurons were prepared from the cerebellar cortex cells of5-7day-old Spragule-Dawley rats.The neurons were identified by neuron-specific enolaseimmunofluorescence staining.After culturing for7-8days, the cells were passaged anddivided randomly into different groups:①control group;②acrylamide modelgroup:cerebellar granule neurons were exposed to acrylamide with different doses of0.5mmol/L,1mmol/L,2mmol/L,4mmol/L and8mmo/L for24hours,The IC50wereevaluated by cell inhibition rate in different groups;③epigallocatechin-3-gallatepretreatment group:cerebellar granule neurons were pretreated with differentconcentrations of5,10,25and50μmol/L of epigallocatechin-3-gallate for24hours,thenthe culture medium was got rid of and the fresh solution with acrylamide IC50wasadded to the above mentioned volume and cultured for another24hours.The neuronalviability was measured by MTT.The morphology of neurons and their nuclei wereobserved by phase-contrast and Hoechst33342staining respectively.The neuronalapoptosis index was observed by TdT-mediated dUTP nick end labeling (TUNEL). Theactivity of SOD and the content of MDA were assayed.The mRNA expression of Baxand Bcl-2mRNA were detected with Semi-quantitative RT-PCR.Results:1.After culturing for7-8days,the proportion of rat cerebellar granule neurons was about90%detected by neuron-specific enolase immunofluorescence staining.2.Compared with blank group,different concentrations of ACR(0.5mmol/L,1mmol/L,2mmol/L,4mmol/L,8mmol/L)were added to cultured cells for24hours, IC50of acrylamide was5mmol/L.ACR showed the significant decline in dose-dependence. 3.MTT assay showed that the time and concentration of epigallocatechin-3-gallatepretreatment had different effects on cerebellar granule neurons.EGCG(10μmol/L,25μmol/L,50μmol/L)groups pretreatment for24hours,5mmol/L ACR was added to thecultures for24hours.The neuronal viability could be elevated to62.3%,76.5%and88.4%(P<0.05or P<0.01);EGCG(10μmol/L,25μmol/L,50μmol/L)groups pretreatmentfor48hours,The neuronal viability could be elevated to67.7%,87.5%and80.5%(P<0.05or P<0.01);EGCG(25μmol/L,50μmol/L,100μmol/L) and ACR treatedCGNs for24hours at the same time,The neuronal viability could be elevated to62.4%,65.3%and78.9%(P<0.05or P<0.01).4.After Hoechst33342staining, the nuclei of normal cells appeared regular contours,round and large in size.After adding acrylamide (5mmol)for24hours, many cellsshowed bright blue fluorescence and nuclear chromatin appearedcompaction,condensation and segregation. Ratio of apoptotic cells was up to40.3%detected by TUNEL. After treating with EGCG, the morphology of apoptotic nucleiimproved and apoptosis rate decreased significantly (P<0.01).5.The activity of SOD decreased and the contents of MDA increased significantly afterbeing treated with ACR.EGCG(10μmol/L,25μmol/L,50μmol/L)could elevate theactivity of SOD and reduced the contents of MDA respectively(P<0.01).6.The results of Semi-quantitative RT-PCR showed that compared with normal group,there were a higher level expression of Bax mRNA and a lower level expression ofBcl-2mRNA in ACR group,but the ratio of Bcl-2/Bax decreased in cerebellar granuleneurons. In EGCG(25μmol/L,50μmol/L) pretreatment group,there were a higher levelexpression of Bcl-2mRNA and a lower level expression of Bax mRNA, the ratio ofBcl-2/Bax increased in cerebellar granule neurons(P<0.05).Conclusions:1.The cultured cerebellar granule neurons treated with0.5mmol/L～8mmol/Lconcentrations of ACR for24hours can induce the damage and apoptosis change indose-dependent manner.2.Protective effect of EGCG is related to its concentration, The10μmol/L～50μmol/Lconcentrations of EGCG significantly block the apoptosis of cerebellar granule neuronsinduced by acrylamide, while no protection for apoptotic cells induced by acrylamide inthe concentration of5μmol/L and100μmol/L.3.For the cells treated by ACR(5mmol/L)EGCG can enhance the cell viability significantly, improve the cells condition in morphology, reduce the apoptosis rate,increase the activity of SOD and decrease the contents of MDA.4. EGCG can reduce the CGNs apoptosis rate induced by ACR,These effects of EGCGmay be related with enhancing the antioxidative ability and increasing the expression ofBcl-2mRNA and reducing the expression of Bax mRNA and increasing the ratio ofBcl-2/Bax.