2,4-D Induces PC12 Cell Apoptosis By The Mitochondrial Pathway And Regulation Of Lycium Barbarum Polysaccharides

Objective Set up 2,4-dichlorobenzene oxygen ethanoic acid(2,4-D)virus in vitro model,to observe the mitochondrial membrane potential change and intracellular ROS oxidative damage indexes such as energy,western Blot method to detect cells,the Bax? Bcl-2? CytC? Caspase3? Caspase9 protein expression,validation of 2,4-D could mitochondria induced by the mechanism of action of PC12 cell apoptosis and LBP(Lycium barbarum polysaccharide)adjustment,To provide evidence for the possible mechanism of 2,4-D induced nervous system injury.Methods PC12 cells were cultured in vitro,cell growth curves were plotted and concentrations of 2,4-D and LBP were determined by CCK-8 method.The experiments were divided into blank control group and 2,4-D exposure group(dose 400,800,1200?mol/L respectively)and the intervention group(1200?mol/L2,4-D + 250 mg/L LBP)were exposed to the cells for 24 hours,and then the cells were collected for testing.Inverted phase contrast microscope was used to observe cell morphological changes,transmission electron microscope was used to observe cell mitochondrial ultrastructural changes and apoptotic bodies.2 ',7'-dichlorofluorescein diacetate(DCFH-DA)method was used to determine ROS levels in PC12 cells,Microenzyme method to determine intracellular MDA content,SOD,GSH-Px activity,Hochest33258 staining to qualitatively detect apoptosis,flow cytometry to quantitatively detect apoptosis,JC-1 method to measure changes in mitochondrial membrane potential,and immunoblottingWestern blot was used to detect the changes of Bax,Bcl-2,CytC,Caspase3,and Caspase9 in the cells.Results1.Using conventional culture PC12 cell after its growth curve is found that within 24 h slower cell proliferation.Within 24-48 h into the logarithmic phase cell proliferation and cell apoptosis quantity is less under normal conditions.Within48-84 h cell number in a large number of growth stage,dead cell number is increasing.Within 84-108 h the total number of cells growth,no longer living cells decline,the number of dead cells increased.2.Compared with the control group,the survival rate of cells in each 2,4-D group was reduced(P<0.05).Compared with the 2,4-D high-dose group,the survival rate of cells in the LBP intervention group was increased(P<0.05).3.Observed under an inverted phase-contrast microscope,compared with the control group,the cells in each concentration group of 2,4-D exposure gradually showed morphological shrinkage,rounding,blurred edges,shortened or disappeared synapses,increased cell gap,and surface Smooth;compared with the2,4-D high-dose group,the number of cells in the LBP intervention group was larger and the degree of damage was lighter.4.Observed under the transmission electron microscope,compared with the control group,the damage of the mitochondrial structure of the cells exposed to the concentration of 2,4-D showed that the mitochondria were swollen,the number was reduced,and apoptotic bodies appeared in the later stage;compared with the2,4-D high-dose group,the organelles in the LBP intervention group were intact and mitochondria were clearly visible.5.Compared with the control group,intracellular ROS levels in the 2,4-D group increased(P<0.05);compared with the 2,4-D high-dose group,the intracellular ROS level in the LBP intervention group was reduced(P<0.05).6.Compared with the control group,the MDA content of cells in the2,4-D-exposed group increased,and the activity of SOD and GSH-Px decreased(P<0.05);compared with the 2,4-D high-dose group,the MDA content of cells in the LBP intervention group decreased,SOD and GSH-Px activity increased(P<0.05).7.Observed under an inverted fluorescence microscope,compared with the control group,the cells in the 2,4-D-exposed group were densely condensed,emitting strong blue fluorescence,showing an apoptotic characteristic morphology;compared with the 2,4-D high-dose group,the nucleus of the LBP intervention group was evenly stained with blue fluorescence and clear cell boundaries.8.Compared with the control group,the apoptotic rate of the cells in the2,4-D-exposed group increased(P<0.05);compared with the 2,4-D high-dose group,the apoptotic rate of the cell in the LBP intervention group decreased(P<0.05).9.Compared with the control group,the mitochondrial membrane potentials of the cells in the 2,4-D exposure group were reduced(P<0.05);compared with the2,4-D high-dose group,the mitochondrial membrane potential of the cells in the LBP intervention group increased(P<0.05).10.Compared with the control group,the expression of Bax,Caspase9,Caspase3,and Cyt-C in 2,4-D-exposed histones increased,the expression of Bcl-2decreased,and the ratio of Bcl-2 / Bax decreased(P<0.05);compared with the2,4-D high-dose group,the expression of Bax,Caspase9,Caspase3,and Cyt-C inLBP histones decreased,the expression of Bcl-2 increased,and the ratio of Bcl-2/Bax increased(P<0.05).Conclusions 2,4-D can inhibit PC12 cell proliferation activity,damage mitochondria ultrastructure,decrease the mitochondrial membrane potential,cause elevated ROS levels in the cell,the nucleus pycnosis concentrated dye,a strong blue fluorescence,apoptosis characteristic form,cause Bax,CytC,Caspase3,Caspase9 protein expression quantity increase,the Bcl-2 expression decreased2,4-D can mitochondrial apoptosis induced by LBP on the apoptosis induced by2,4-D with adjustment.