| Purpose:To observe the effect of lactacystin on COX-2 and PGE2 of neuron like PC12 cells,and to explore the mechanism of doxorubicin on the cell protection of Parkinson's disease model.Material and method:1.Preparation of drug-containing serum: 50 rats were randomly divided into four groups,including 20 rats in the normal group,80 rats in tween group,10 rats in celecoxib group and 10 rats in the alone live coumarin group.The drug was given by gavage from 8:00 to 10:00 every day,and the drug was given in normal group: normal saline.Tween 80 group: 0.08% tween 80 by gavage,2ml/ time/only.Celecoxib group:20mg/kg.Alone active coumarin group;0.08% tween 80 solution[12] 155mg/kg.Continuous gavage for 7 days.Blood was taken from the abdominal aorta after the last 2 hours of gavage.After standing for 2h,the serum was centrifuged at 2000r/min for 10 min.Activated at56? for 30 min,0.22?m livm filter membrane was used for filtration and sterilization.-20?refrigerator for use.2.Passage and culture of PC12 cells: after purchase,undifferentiated PC12 cells were transformed into neuron-like PC12 cells by NGF induction.The cells were taken into the logarithmic growth stage and cultured in a 96-well plate with cell medium containing 15% horse serum in a 100?l system with a density of 1×105/ml.Determine Lactacystin and serum concentrations.3.Lactacystin concentration was determined:Lactacystin mother liquor was taken at 1 mmol/l and diluted to the concentration of four groups.After dilution,it was added to the cell culture medium for 24 h.The relative survival rate of cultured cells was determined by MTT.4.To determine the concentration and time of drug serum: PC12 cells of neuron-like type were taken with a logarithmic growth cycle and inoculated into a 96-well plate with a density of 1×105/ml in a 100?l system for 24 h,to be grown adherent to the wall.The 96-well plate was divided into 16 parts and divided into different groups.The grouping conditions were as follows: normal serum control group,model group,tween 80 serum group,celexib serum group,and single active coumarin serum group.The medium was replaced in all the experimental groups except the blank group,and the concentration was divided into 10%,15% and 20%,and three compound Wells were set in each group.The incubation time was 24 h and 48 h respectively.The model group,ttwain 80 serum group,cilioxib serum group,and independent coumarin serum group were added with5?mol/L Lactacystin for 24 h and 48 h respectively.MTT was used to detect the relative survival rate of the cells and to determine the optimal concentration and duration of the serum containing the drug.5.Formal experiment: the experimental cells were divided into 5 groups,and the grouping conditions were as follows: normal serum control group,model group,tween 80 serum group,cilioxib serum group,and single active coumarin serum group.According to the concentration and action time of the above determined drug-containing serum and Lactacystin,the drug-containing serum of 15% of each experimental group was added to protect for 24 h,and then 5%Lactacystin was added to build the model for 24 h.6.Indicator detection and statistical treatment: MTT assay was used to detect the cell activity of the experimental group.The protein expression of COX-2 and PGE2 was detected by ELISA.SPSS25.0 statistical software was used to analyze the experimental results,and p < 0.05 was considered as a statistical difference.Results:1.Lactacystin concentration was determined Lactacystin with concentrations of 0,5,10 and 20 respectively acted on neuron-like PC12 cells for 24 h.MTT method was used to determine its absorbance value and compared with the blank control group(0 living mol/l).The results showed that with the increase of Lactacystin concentration,the relative survival rate of cells decreased gradually and in a dose-dependent manner.Compared with the blank control group,the relative survival rate of 5,10 and 20 groups significantly decreased and the difference was significant(P <0.01).Moreover,when Lactacystin concentration was no less than 10 livability mol/l,the relative survival rate of cells decreased significantly.Accordingly,Lactacystin with a concentration of 5 living mol/l was finally determined to be the stimulating condition of neuron-like PC12 cell injury with a time of 24 h.2.Determine the time and concentration of the serum containing the drug By comparing the OD value and relative survival rate of each concentration group,it was found that the OD value and relative survival rate of the normal serum control group with a concentration of 15% were closer to that of the blank control group regardless of 24 hours.In order to optimize the concentration of the experiment and save the time of the experiment,it was finally determined that the concentration of the drug-containing serum was 15% and the action time was 24 hours,which were the protective conditions for the injury of neuron-like PC12 cells.3.To determine the effect of serum on cell activity in each experimental group The well-differentiated neuronal PC12 cells were treated with the drug serum and Lactacystin(not added to the normal serum control group)for 24 h,respectively,and the results were detected by MTT method.There was no statistical difference between the model group and tween 80 serum group(p >,0.05).The OD values of model group and tween 80 serum group were significantly lower than those of normal serum control group(p <0.01).The cell OD value of the single living coucouin serum group was significantly higher than that of the model group,tween 80 serum group and celexib serum group,but lower than that of the normal serum control group(all p < 0.01).The cell OD value in the cilioxib serum group was higher than that in the model group and tween 80 serum group,and lower than that in the normal serum control group and the single active coucouin serum group(both p <0.01).4.Effects of isolated coumarin containing serum on the expression of COX-2 and PGE2 protein in supernatant of Lactacystin induced neuronal PC12 cells The results of MTT assay showed that there was no significant difference between the model group and tween 80 serum group(p >0.05).The contents of COX-2 and PGE2 in the supernatant of the model group and tween 80 serum group were significantly higher than that of the control group(p < 0.01).The contents of COX-2 and PGE2 in the supernatant of the isolated coumarin serum group were significantly lower than those in the model group,tween80 serum group and celexib serum group,but higher than those in the normal serum control group(all p < 0.01).The content of COX-2 and PGE2 protein in the supernatant of cailoxib serum group was lower than that in model group and tween 80 serum group,but higher than that in normal serum control group and single active coumarin serum group(both p < 0.01).Conclusion:1.Lactacystin proteasome inhibitor was applied to neuron-like PC12 cells in this experiment.Successfully simulated the cell damage model of Parkinson's disease.2.The single active coumarin containing serum could promote the lactacystin-induced proliferation of neuron-like PC12 cells,and the effect was better than that of celecoxib group.The single active coumarin containing serum had a protective effect on lactacystin-induced neuron-like PC12 cells.3.The single active coumarin drug-containing serum can reduce the expression of inflammatory factors COX-2 and PGE2 in the superfluid of model cells,thus inhibiting inflammation,which may be the mechanism of its protective effect on neuron-like PC12 cells,and the effect is better than that of celecoxib group.4.The stimulation conditions of neuron-like PC12 cell injury: Lactacystin with a concentration of 5 living mol/l and an action time of 24 h.5.Protective conditions of neuron-like PC12 cells: the concentration of drug-containing serum was 15%,and the time of action was 24 hours. |
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