The Protective Effects Of DL-3-n-Butylphthalide On The Oxidative Stress In MPP~+-Induced SH-SY5Y Cells Via ROS/MLK3 Pathway

Objectives To investigate the effects of DL-3-n-Butylphthalide(NBP)on oxidative stress,proliferation and apoptosis of MPP~+-induced SH-SY5 Y cells;To explore whether the neuroprotective effects of DL-3-n-Butylphthalide is related to the MLK3 pathway regulated by reactive oxygen species(ROS).Methods Human neuroblastoma cells SH-SY5 Y cells were in vitro cell culture flasks,with DMEM/F12=1:1 medium for culture.Experiments were divided into five groups:control group,MPP~+model group,NBP+MPP~+group,NAC+MPP~+group and URMC-099+MPP~+group.the Control group:SH-SY5 Y cells were treated with complete medium normal culture,the culture of the whole process without any drug intervention;MPP~+model group:added 1 mmol/L MPP~+in cultured cells and continued culturing for 24 hours;NBP+MPP~+group:the cells were pretreated with 10?mol/L NBP for 3 hours,then 1mmol/L MPP~+was added and continued culturing for 24 hours;NAC+MPP~+group:the cells were pretreated with 200?mol/L antioxidant NAC for 3 hours,then 1 mmol/L MPP~+was added and continued culturing for 24 hours;URMC-099+MPP~+group:the cells were pretreated with 200 nmol/L MLK3 inhibitor URMC-099 for 3 hours,then 1 mmol/L MPP~+was added and continued culturing for 24 hours.The cell relative survival rate was measured with MTT assays.The apoptosis was quantified under flow cytometry with Annexin V/PI fluorescence staining,and the nuclear morphology was observed with Hoechst 33342 staining.The intracellular ROS was measured using the non-fluorescent probe 2',7'-dichlorofluorescein diacetate(DCFH-DA).The protein expression of p-MLK3,p-JNK and p-ERK1/2 were detected with Western blotting.Results 1 MTT assays showed that the MPP~+model group compared with the control group,cell relative survival rate markedly decreased(P<0.05).NBP+MPP~+group,NAC+MPP~+group and URMC-099+MPP~+group compared with the MPP~+model group,cell relative survival rate markedly increased(P<0.05).2 Inverted phase contrast microscope observed that cells in the control group were fusiform or triange,cell junction was obvious.The number of adherent cells in MPP~+model group had decreased,cell shrank,floated,and aggregated into clusters,the processes retracted and vanished.In the NBP+MPP~+group,NAC+MPP~+group and URMC-099+MPP~+group,the morphology of SH-SY5 Y cells was close to the control group.Cells were plump,and the processes were obvious.3 Annexin V/PI fluorescence staining detected that the MPP~+model group compared with the control group,cell apoptosis rate markedly increased(P<0.05).NBP+MPP~+group,NAC+MPP~+group and URMC-099+MPP~+group compared with the MPP~+model group,cell relative survival rate markedly decreased(P<0.05).4 Hoechst33342 staining found that the control group cells exhibited uniform Hoechst 33342 staining,and fluorescence intensity is weak.By contrast,cells in the MPP~+group were observed nuclear morphology changes,including chromatin condensation,and nuclear fragmentation,the staining was uneven,and the blue fluorescence was enhanced.Pretreatment with NBP,NAC or URMC-099 attenuated this effect mediated by MPP~+.5DCFH-DA staining showed that the MPP~+model group compared with the control group,DCF fluorescence intensity markedly increased(P<0.05).NBP+MPP~+group,NAC+MPP~+group and URMC-099+MPP~+group compared with the MPP~+model group,DCF fluorescence intensity markedly decreased(P<0.05).6 Western blotting detected that the MPP~+model group compared with the control group,p-MLK3 and p-JNK protein expression markedly increased,p-ERK1/2 protein expression markedly decreased(P<0.05).NBP+MPP~+group,NAC+MPP~+group and URMC-099+MPP~+group compared with the MPP~+model group,p-MLK3 and p-JNK protein expression markedly decreased,p-ERK1/2 protein expression markedly increased(P<0.05).Conclusions 1 NBP can resist MPP~+induced cell toxicity,promote the proliferation of MPP~+induced SH-SY5 Y cells,inhibit oxidative stress and cell apoptosis,protect neurons.2 The neuroprotective effect of NBP is related to the inhibition of MLK3 pathway regulated by ROS and the modulation of p-JNK and p-ERK1/2 protein,and the result suggests that ROS/MLK3 signaling pathway is a new target of NBP.