| Objective: To investigate the effect of rifampicin on the level of α-synuclein(α-syn)oligomers and its relationship with autophagy.Methods:1.Establishing of PD cell model: SH-SY5 Y cells were treated with different concentrations of rotenone(0uM,2.5uM,5uM,10 uM,25uM)for 24 hours.Proliferation of the cells was deteced by MTT method and the survival rate were calculated.50% ~ 70% of the survival rate was chosen to be the standrd of determing the optimal concentration of rotenone(5uM).2.Seting up of rifampin treatment group and rifampin control group: Different concentrations of rifampicin(0uM,50 uM,100uM,150 uM,300uM)were treated in the PD cell model for 24 hours.Cell proliferation was detected by MTT method to calculate survival rate.The rifampicin treatment group and the rifampicin control group was set according to the highest survival rate.3.Establishing of the optimal working concentration and working time of the autophagy tool drug acting on SH-SY5 Y cells: SH-SY5 Y cells were treated with different concentrations of the autophagy inhibitor chloroquine(0uM,0.01 uM,0.1uM,1uM,10 uM,100uM).SH-SY5 Y cells were treated with different concentrations of the autophagy promoter rapamycin(0uM,0.01 uM,0.1uM,1uM,5uM).Proliferation of the cells was deteced by MTT method 24 hours later.The highest concentration without damaging survival rate was determined as the optimal treatment concentration of the tool drugs chloroquine and rapamycin in SH-SY5 Y cells(10uM and10 nM,respectively).10 uM chloroquine was treated to SH-SY5 Y cells for different time of 0 hour,0.5 hour,1 hour and 2 hours and the autophagy marker protein beclin-1 was detected by Western blotting.The optimal treatment time of chloroquine(1 hour)was determined with the lowest expression of beclin-1.10 nM rapamycin was applied to SH-SY5 Y cells at a time gradient of 0 hour,0.5 hour,1hour,2 hours,3 hours,and 4 hours.The highest expression of beclin-1 was used as the standard to determine the optimal time of rapamycin treatment(2 hours).4.Adjusting autophagy to observe the changes of substrate α-syn oligomers under physiological and pathological conditions: Chloroquine(10uM,1 hour)was applied to normal control SH-SY5 Y cells,and the beclin-1expression and α-syn oligomers were observed by Western blotting to clarify the relationship between autophagy inhibition and substrate α-syn oligomers in dopaminergic cells.Pretreatment with rapamycin(10 nM,2 hours)to SH-SY5 Y cells,and rotenone(5uM,24 hours)was added afterwards.Changes of beclin-1 and α-syn oligomers were observed by Western blotting to clarify the relationship of promotion of autophagy and its substrate α-syn in PD.5.Observing the neuroprotective effect of rifampicin and its effects on autophagy:Research subjects were divided into 4 groups: SH-SY5 Y cell control group,PD model group,rifampin treatment group and rifampin control group.The detections were carried out as: flow rate method was used to detect the apoptosis rate of each group;qRT-PCR technology was used to detect the expression level of pathological marker protein α-syn mRNA(SNCA);α-syn oligmers and autophagy-related proteins beclin-1,LC3-Ⅱ/Ⅰ,p62 were detected by Western blotting.6.After pretreatment with the autophagy inhibitor chloroquine,α-syn oligomers were detected by Western blotting to determine whether the inhibition of autophagy couldcounteract therapeutic effect of rifampicin..7.The autophagy morphology of autophagosomes,autophagolysosomes were directly observe by transmission electron microscope.Results:1.Rotenone inhibited the proliferation of SH-SY5 Y cells in a concentration-dependent manner(P<0.001).With a cell survival rate of 50% to 70% as the standard,it was determined that 5uM rotenone acted for 24 hours to establish a PD cell model.2.Compared with the PD cell model group,rifampicin(150uM,24 hours)treatment effectively increased the cell survival rate by about 30%(P<0.001)and decreased the apoptosis rate by 14%(P<0.001)suggesting neuron protection effect of rifampicin.3.The effect of autophagy inhibitor chloroquine and autophagy inducer rapamycin on the autophagy was associated with concentration and time.Compared with SH-SY5 Y cell control group(0.47±0.02): the relative expression of beclin-1 was the lowest(0.12±0.02,P<0.001)at 10 uM,1 hour of chloroquine treatment.And he relative expression of beclin-1 was the highest(0.66±0.03,P<0.01)at 10 nM,2 hours of rapamycin treatment.4.Compared with the SH-SY5 Y cell control group,chloroquine-treated SH-SY5 Y cells reduced the expression of autophagy marker protein beclin-1(0.31±0.04 vs0.95±0.01,P<0.001),and increased the content of α-syn oligomers(0.67±0.04 vs0.32±0.03,P<0.001),indicating that the autophagy substrate α-syn oligomer increased when autophagy was suppressed in dopaminergic cells.Compared with PD cell model group,rapamycin pretreatment significantly increased beclin-1 expression(0.60±0.05 vs 0.28±0.04,P<0.01),reduce α-syn oligomers(0.44±0.03 vs 0.59±0.03,P<0.05),indicating that it is beneficial to reduce the content of substrate α-syn oligomer in PD by enhangcing autophagy.5.Compared with the PD model group,the relative expression of SNCA in rifampicin treatment group decreased(1.31±0.31 vs 2.58±0.74,P<0.05),and α-syn oligomers decreased significantly(0.39±0.30 vs 0.55±0.36,P<0.05),autophagy-related protein beclin-1 increased(1.05±0.03 vs 0.51±0.01,P<0.001),LC3-Ⅱ/Ⅰ level increased(0.68±0.03 vs 0.31±0.05,P<0.001),The content of p62 decreased(0.38±0.03 vs0.81±0.08,P<0.001).It is suggested that rifampicin’s reduction of α-syn oligomer is related to promoting autophagy and inhibiting α-syn mRNA expression.6.Compared with the rifampicin treatment group,chloroquine pretreatment significantly increased the intracellular α-syn oligomer(0.95±0.03 vs 0.41±0.04,P<0.001).It is suggested that inhibition of autophagy can counteract the protective effect of rifampicin in reducing α-syn oligomers.7.Compared with the PD model group,the rifampicin treatment group showed more autophagosomes and autophagolysosomes under the transmission electron microscope,suggesting that rifampicin treatment may promote autophagy maturation.Conclusion: In the PD cell model induced by rotenone,the level of autophagy decreases and α-syn oligomers increase.The protective effect of rifampicin on dopaminergic cells may be reduced by the promotion of cell autophagy to reduceα-syn oligomers.Inhibition of autophagy can counteract the protective effects of rifampicin. |
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