| Purpose:To observe the effect of levulinin on the activity of Lactacystin-induced Parkinson's disease model cells and the inflammatory factors hs-CRP and MMP-9 in the cell supernatant,and to explore the mechanism of levulinin to protect Parkinson's disease model cells.Clinical application of solitary living treatment of Parkinson's disease provides experimental and theoretical support.Material and method : 1.Preparation of drug-containing serum: rats were randomly divided into four groups: normal group,Tween group,celecoxib group and Duhuo coumarin group.The dose formula of rats in each group was that the adult clinical dose was multiplied by 0.018.According to the previous experimental study,the dose in the coumarin group was 3 times of the conventional dose.Except for the normal group(n = 20),the other 10 rats(n = 10).The concentrations of each group were as follows: normal group: normal saline;Tween group: 0.08% Tween 80;celecoxib group: 20 mg / kg;coumarin group: 155mg/kg(Tween 80 to help dissolve).The drug was given by intragastric administration once a day in the morning,2ml/rat,continuously for 7 days.On the 7th day,the blood was taken and the supernatant was separated.After inactivation and sterilization,freeze it in the refrigerator at-20 ? and set aside.2.Differentiation and culture of neuron-like PC12 cells:PC12 cells in logarithmic growth phase were induced to differentiate into neuron-like PC12 cells by adding nerve growth factor NGF(the final concentration was 50ng/ml).After complete differentiation,the culture medium was replaced by a cell medium containing 15% horse serum and inoculated into a 96-well plate with a density of 1 × 106/ml and a system of 100 ? l.3.Determine the concentration of Lactacystin: the neuron-like PC12 cells in logarithmic growth phase were cultured with 15% horse serum as cell culture medium at 37 ? for 24 hours in 5%CO2 box.After the cells were attached,diluted Lactacystin,was added to each group for 24 hours to detect the cell survival rate and select the concentration of the model drug.4.Determine the concentration and action time of drug-containing serum: the neuron-like PC12 cells in logarithmic growth phase were divided into 16 groups:blank control group(horse serum),normal control group,model group,Tween group,celecoxib group and coumarin blood group.The cells were pre-protected for24 hours and 48 hours according to the above groups.except for the blank control group and each normal control group,the other 12 groups were treated with 5 ? mol/ LLactacystin for 24 hours to detect the cell survival rate and screen the appropriate concentration and time of drug-containing serum.5.Experimental cell treatment:the experiment was divided into the following five groups.Normal control group,model group,Tween group,celecoxib group and Duhuo coumarin group all selected the final concentration of 15% rat serum DMEM,to protect neuron-like PC12 cells for 24 hours.Except for the normal control group,all the other groups were treated with 5 ? mol / L Lactacystin,37 ? and 5%CO2 incubator for 24 hours,and then left for follow-up detection.6.Index detection and statistical processing: MTT method was used to detect the cell viability of each group,and ELISA method was used to detect the expression of hs-CRP and MMP-9 protein in the cell supernatant of each group.All the experimental data were analyzed by SPSS25.0 software,and the difference was statistically significant.Results : 1.The determination of the concentration and action time of the experimental drug 1.1 the concentration of the model drug Lactacystin was determined the absorbance value detected by MTT method showed that the OD value decreased with the increase of Lactacystin concentration,and the inhibition of cell activity was significantly dose-dependent.When the concentration of Lactacystin was 10% ? mol / L or more,the cell proliferation was significantly inhibited and the cell proliferation rate was significantly decreased.Therefore,the concentration of 5% umol / LLactacystin,for 24 h was selected as the stimulating condition for the establishment of Parkinson's disease cell model.1.2 when the concentration and time of action of drug-containing serum were the same as that of action time,there was no significant difference in cell OD value and relative survival rate between model group and Tween group(P > 0.05).Except that the OD value and relative survival rate of 20% of the normal control group were higher than those of the blank control group,the OD value and relative survival rate of the drug-containing serum group were lower than those of the blank control group(p <0.01).When the action time was 24 hours and 48 hours,the relative survival rates of normal control groups with 15% concentration were 96.96% and 97.99%,respectively,which were closest to those of the blank control group(p < 0.01).Considering that the scientific research experiment is more efficient and economical,the concentration of drug-containing serum in each group is 15%,and the protection for 24 hours is the protection condition of neuron-like PC12 cells.2.The effect of drug-containing serum on the activity of neuron-like PC12 cells induced by Lactacystin after the intervention of drug-containing serum and model drugs with definite concentration and time,the cell activity of neuron-like PC12 cells was detected by MTT method.The results showed that the OD value of Duhuo coumarin group was slightly lower than that of normal control group,but higher than that of celecoxib group,model group and Tween group.The OD value of celecoxib group was lower than that of normal control group and Duhuo coumarin group(p < 0.01),but higher than that of model group and Tween group(p < 0.01),but there was no significant difference between model group and Tween group(p >0.05).The OD value of cells in celecoxib group was significantly lower than that in normal control group,Duhuo coumarin group and celecoxib group(p < 0.01).It is suggested that compared with the normal control group,the cell activity was damaged in the model group and Tween group,and the model was replicated successfully,and there was no neuroprotective effect in the Tween group.Compared with the model group and Tween group,both coumarin and celecoxib had obvious protective effects on cell viability,and the protective effect of coumarin was better than that of celecoxib.3.Effect of drug-containing serum on the expression of hs-CRP protein in the supernatant of neuron-like PC12 cells induced by Lactacystin after the intervention of drug-containing serum and model drugs with definite concentration and action time,the content of hs-CRP in the supernatant of neuron-like PC12 cells was detected by ELISA method.The results showed that the content of hs-CRP protein in the supernatant of Duhuo coumarin group was slightly higher than that of normal control group.It was lower than that in celecoxib group,model group and Tween group(p < 0.01).The content of hs-CRP protein in the supernatant of celecoxib group was higher than that in normal control group,coumarin group and celecoxib group,but lower than that in model group and Tween group.There was no significant difference in the content of hs-CRP protein in supernatant between model group and Tween group,but it was higher than that in normal control group,Duhuo coumarin group and celecoxib group.4.Effect of drug-containing serum on the expression of MMP-9 protein in the supernatant of neuron-like PC12 cells induced by Lactacystin after the intervention of drug-containing serum and model drugs with definite concentration and action time,the content of MMP-9 in the supernatant of neuron-like PC12 cells was detected by ELISA method.The results showed that the content of MMP-9 protein in the supernatant of Duhuo coumarin group was slightly higher than that of normal control group.It was lower than that in celecoxib group,model group and Tween group(p < 0.01).The content of MMP-9 protein in the supernatant of celecoxib group was higher than that in normal control group,coumarin group and celecoxib group,but lower than that in model group and Tween group.There was no significant difference in the content of MMP-9 protein in supernatant between model group and Tween group,but it was higher than that in normal control group,Duhuo coumarin group and celecoxib group.Conclusion:1.As a modeling agent,proteasome inhibitor Lactacystin successfully established the PD model of neuron-like PC12 cells.The most suitable modeling condition is that the concentration is 5% umol / LLactacystin,for 24 hours.After successful replication of the model,the activity of neuron-like PC12 cells decreased and the expression of inflammatory cytokines hs-CRP and MMP-9 in the supernatant increased.2.The best protective conditions of coumarin on PD model cells induced by Lactacystin were as follows: the concentration of coumarin in serum was 15% and the action time was 24 hours.Coumarin-containing serum can improve the relative survival rate of PD model cells,which is better than celecoxib.Tween 80,a coumarin cosolvent,has no protective effect on cells.3.Duhuo coumarin has the effect of inhibiting nerve inflammation.The drug-containing serum of coumarin may down-regulate the expression of inflammatory factors hs-CRP and MMP-9 in the supernatant of PD model cells,inhibit the inflammatory response of PD model cells induced by Lactacystin,and then have a protective effect on neuron-like PC12 cells induced by Lactacystin. |
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