The Effect Of BDNF In High Dose On The Neuronal Cells And Its Possible Mechanism

Part One:The influence of brain-derived neurotrophic factor on PC-12 neuronal cells activity and its possible mechanismObjectiveTo observe the effect of brain-derived neurotrophic factor in high dose(BDNF) on the neuronal cell and its possible mechanism.Methods The high differentiated PC-12 cells was cultured in vitro. After dissolved by pancreatin,the cells was counted and inoculated in 96 holes plate,then divided into group BDNF(125μg.L-1,250μg.L-1,500μg.L-1and 1000μg.L-1BDNF were added respectively) and control group(the same dose of solvent were added).To observe the morphology and quantity of the cells after 24 hours of drug addition.Taking the sulforhodamine B(SRB)method to detect the relative number of neurons.The other cells were fixed after 24 hours of drug addition,using immunofluorescence method to detect the change of P62/SQSTM1 expression.Results 1Detecting the relative number of neurons by the sulforhodamine B(SRB)method,the result shows: Compare with control group,There was no obvious change in cell activity in group BDNF(125μg.L-1,250μg.L-1,500μg.L-1)(P>0.05),but the cell activity in group BDNF(1000μg.L-1)was obvious change(P<0.05).2 After 24 hours of drug addition,the immunofluorescence method result shows:Compare with control group,P62/SQSTM1 expression of each BDNF group appear to dose dependent decreation(P<0.05).Conclusion 1000μg.L-1BDNF may cause neuron damage, and the mechanism may be related to the enhancement of autophagy.Part Two:PIK3C3 siRNA-pool inhibits the expression ofP62/SQSTM1 in PC-12 cellsObjective To investigate the dosage and time of action of the siRNA which could most effectively inhibit the expression of PIK3C3 in PC-12 cells,and to observe the regulation of autophagy in the cells.Methods Four siRNAs were chemically synthesized :three of them were used to inhibit PIK3C3 expression in PC-12 cells,the rest was fluorescence-labeled mismatch siRNA asa negative control.They were all transfected into PC-12 cells with Lipofectamine2000.PC-12 cells were randomly divided into 6 groups: normal,vehicle, mismatch, and 25 nmol.L-1、50nmol.L-1、100nmol.L-1 group.After transfection with siRNA for 6 hours, the rate of transfection was calculated under fluorescence microscope,and the cytotoxicity of complex was detected using MTT. The expression of PIK3C3 was detected by real-time PCR;The change of P62/SQSTM1 expression was observed done by immunofluorescence method.Results The transfection ratewas increased with the increase of siRNA dosage.There was no significant difference in the survival rate of the cells.PIK3C3 siRNA 50nmol.L-1、100nmol.L-1 group could dose-dependently inhibit the expression of PIK3C3 mRNA levels.After transfection with PIK3C3siRNA100 nmol.L-1 for 48 hours,the relative expression of P62/SQSTM1 was increased.Conclusion siRNA-pool can effectively inhibit the expression of PIK3C3 in PC-12 cells.And it can increase the expression of P62/SQSTM1,then inhibit the activation of autophagy in PC-12 cells. Part Three:Protective effect of PIK3C3 siRNA on BDNF induced neuronal injury in rats by inhibiting the excessive activation of autophagyObjective Pretreatment with PIK3C3 siRNA transfection, to observe the protective effect of neuronal injury induced by high dose of BDNF and its possible mechanismMethods PC-12 neuronal cells cultured in vitro were randomly divided into six groups:Normal group( group N), Vehicle group(group V), Mismatch group(group M), BDNFgroup(groupB),PIK3C3siRNAgroup(groupS),BDNF+PIK3C3siRNA100nmol.L-1gr oup(group BS).In group B and group BS, the concentration of BDNF was 1000μg.L-1.In the group BS,transfection reagent PIK3C3 siRNA transfect cells at a concentration of 100 nmol.L-1 24 h before adding 1000μg.L-1BDNF. Detect of the changes in neuronalactivity of PC-12 by the method of MTT(n=5);Autophagy related P62/SQSTM1 gene and LC3 gene(n=4) were detected by real-time PCR(n=3); immunocytochemistry and immunofluorescence(n=4) method was used to detect the expression of P62/SQSTM1 protein and LC3 protein.Results The result of MTT showed that compared with group N, the neuronal cells survival rate in group B was significantly decreased(P<0.01), the other cell survival rate had no significant changes(P>0.05); compared with group B, group BS could significantly increase the activity of nerve cells(P<0.01).The result of MTT showed that: compared with group N, the relative expression of P62/SQSTM1 mRNA decreased(P<0.01), LC3 mRNA relative expression increased(P<0.01)in group B,the relative expression of gene P62/SQSTM1 increased(P<0.05), LC3 mRNA expression decreased(P<0.05)in group S; the relative expression of gene P62/SQSTM1 decreased(P<0.05)in BS group, the relative expression level of LC3 mRNA was higher than that of group N(P<0.01);in group V and group M,the difference was not statistically significant(P>0.05). Compared with group B, P62/SQSTM1 mRNA expression increased(P<0.01) and LC3 mRNA expression decreased(P<0.05)in group BS.Immunocytochemistry and immunofluorescence results showed that: compared with group N, P62/SQSTM1 protein expression decreased(P<0.01), LC3 protein relative expression increased(P<0.01)in group B;the expression of P62/SQSTM1 protein increased(P<0.01), LC3 protein expression decreased(P<0.01)in group S; compared with group N, the difference was not statistically significant(P>0.05)in group V, group M and group BS; Compared with group B, P62/SQSTM1 protein expression level increased(P<0.01), LC3 protein levels were significantly decreased(P<0.01)in group BS.Conclusion PIK3C3 si RNA can protect neuronal cell from the BDNF induced neuronal injury in rats by inhibit the excessive activation of autophagy.