The Protective Mechanism Of Bushen Jiannao Decoction Against Aβ_25-35-induced PC12 Cell Injury Via MAPK Pathway

In this study,the signal transduction pathway of mitogen-activated protein kinase/extracellular signal-regulated protein kinase(MAPK/ERK)was used as the main clue.The gene expression of ERK1/2 was detected by preparing rat serum containing drugs,culturing PC12 cells,detecting SOD by Elisa method,MDA kit,RT-PCR method,and detecting ERK1/2 protein expression by Western Blot method.The protective effect of Bushen Jiannao Decoction on PC12 cells injured by A beta 25-35 was studied.The protective mechanism of Bushen Jiannao Decoction on neuronal injury was revealed from the perspective of molecular biology,starting with the signal transduction pathway of MAPK/ERK1/2 and oxidative stress response.It provided preliminary experimental basis and theoretical basis for Bushen Jiannao Decoction in the treatment of Alzheimer’s disease.Material and method:In the first part,rat serum containing drugs was prepared.Thirty SD male rats were randomly divided into blank control group,model group and low,medium and high dose groups of Bushen Jiannao Decoction.According to the equivalent dosage of clinical medication,according to the formula and the weight and weight of rats,Bushen Jiannao Decoction was converted to the dosage of rats.According to the dosage of rats 20 ml/kg per day,the dosage of high dose group was 14.4g/Kg/Day,the dosage of medium dose group was 7.2g/Kg/Day,and the dosage of low dose group was3.6g/Kg/Day.All groups were administered orally once a day in the morning.The blank control group was not treated and was given orally for 5 days.On the fifth day,the rats in each group were given intraperitoneal anesthesia according to the order of blood collection,and blood was collected through the abdominal aorta of the rats.After blood collection,the samples were placed for 2hours,centrifuged for 10 minutes at 3000 rpm.Serum was separated,inactivated and filtered.Serum was packed according to different groups,labeled and stored in a refrigerator at-20 C for reserve.The second part,cell experiment:Cell grouping: PC12 cells were divided into the following five groups according to the experimental requirements(1)Model group: Bushen Jiannao Decoction control group rat serum + A beta 25-35(2)Bushen Jiannao Decoction low dose group: Bushen Jiannao Decoction low dosegroup rat serum + A beta 25-35(3)Bushen Jiannao Decoction middle dose group: Bushen Jiannao Decoction middle dose group rat serum + A beta 25-35(4)Bushen Jiannao Decoction high dose group: Bushen Jiannao Decoction high dose group rat serum + A beta 25-35(5)Control group: Serum of control group ratsThe corresponding serum concentration of each group was divided into 10%,15% and20%.Abeta 25-35 was divided into 10 uLmol/L,20 uLmol/L and 40 uLmol/L for intervention.Using MTT to detect OD of cells is worth screening the best concentration,time and serum concentration of Abeta 25-35.The serum of model group,low,medium and high dose group and blank control group were prepared into 10%,15% and 20% drug-containing serum cell culture media,respectively,for storage.PC12 cells in logarithmic growth phase were selected and cultured in 96-well plates with a density of lX104/ml.Cell adherence growth was observed 24 hours after culture.Cell starvation for 12 hours(i.e.continuous culture with fetal bovine serum medium for 12 hours)synchronized the cells.After that,the original culture medium was absorbed and the corresponding cell culture medium containing rat serum was added to each group.In addition to the blank control group,the final concentration of 10umol/L,20 umol/L and 40 umol/L of A beta 25-35 was added to the cell culture medium,and the cell absorbance,or OD value,was detected by MTT method for 12 hours,24 hours and 48 hours,respectively.Abeta 25-35 was screened out.-The optimum time,dosage and concentration of drug-containing serum for PC12 cell injury induced by 35.After screening the conditions,continue the experiment according to the scheme.Cell morphology was observed under a microscope.Cell supernatants and cells were collected and preserved.The contents of SOD and MDA in the supernatants of each group were detected by Elisa method,the expressions of ERK1/2 and P-ERK1/2 were detected by Western blot,and the expression of erk1/2 was detected by RT-PCR.To explore the protective mechanism of Bushen Jiannao Decoction serum on PC12 cells damaged by A beta 25-35 and its relationship with MAPK/erk1/2 signal transduction pathway.Results:Part I: Selection of Modeling Conditions by MTT Method1.Selection of modeling time,under the same conditions of drug serum concentration and drug concentration,the different time for the longitudinal coordinates were made into a column statistical map to compare,and the results showed that 24 hours was the best choice.2.Under the same serum concentration and 24-hour action time,the best concentration of20umol/L A beta 25-35 was selected by comparing the columnar statistical charts of different concentration of modelling drugs in longitudinal coordinates.3.Selection of drug-containing serum concentration.Under the condition of 20 umol/L of model drug and 24 hours of culture,column diagram was made with drug-containing serum concentration as ordinate.By comparison,it was found that medium containing 15%drug-containing serum was the most suitable condition for experimental selection.Part II: Protective effect of serum containing Bushen Jiannao Decoction on PC12 cell model1.Effect of Serum Containing Bushen Jiannao Decoction on the Morphology of PC12 Cells Damaged by Abeta 25-35Under the inverted microscope,the cells in the blank control group were densely distributed in the field of vision,with spindle shape and full inclusions;the number of living cells in the model group decreased,with shrinkage and floating phenomenon;the cell viability in the Bushen Jiannao Decoction group was stronger than that in the model group,and the field of vision was re-densely distributed,the pseudopods extended,the shrinkage gradually recovered,and the growth condition became better.2.Effect of serum containing Bushen Jiannao Decoction on MDA content in PC12 cells injured by A beta 25-35Compared with the model group,the content of MDA in the cells of the normal control group was lower(P < 0.05);compared with the blank control group,the content of MDA in the cells of the model group increased significantly(P < 0.05);compared with the model group,the content of MDA in the middle and high dose groups of Bushen Jiannao Decoction decreased significantly(P < 0.05);compared with the model group,the content of MDA in the low dose group of Bushen Jiannao Decoction decreased,but there was no significant difference.(P < 0.05).3.Effect of serum containing Bushen Jiannao Decoction on SOD content in PC12 cellsinjured by A beta 25-35Compared with the model group,the expression of SOD in the cells of the normal control group was higher(P < 0.05);compared with the model group,the expression of SOD in the cells of the low,middle and high dose groups of Bushen Jiannao Decoction increased significantly(P < 0.05).4.Effect of serum containing Bushen Jiannao Decoction on ERK1/2 mRNA expression in PC12 cells injured by A beta 25-35Compared with the normal control group,the expression of ERK1/2 in the model group was significantly lower(P<0.05).Compared with the model group,the expression of ERK1/2was increased in the middle and high dose groups of Bushen Jiannao Decoction and the high dose group of Bushen Jiannao Decoction plus erk1/2 inhibitor(P < 0.05);compared with the high dose group of Bushen Jiannao Decoction,the expression of erk1/2 in the high dose group of Bushen Jiannao Decoction plus erk1/2 inhibitor was significantly decreased(P < 0.05).5.Effect of serum containing Bushen Jiannao Decoction on ERK1/2 protein expression in PC12 cells injured by A beta 25-35Compared with the normal control group,the expression of ERK1/2 and P-ERK1/2protein in the model group was significantly lower(P < 0.05);compared with the model group,the expression of ERK1/2 protein in the middle and high dose groups of Bushen Jiannao Decoction increased,and the difference was statistically significant(P < 0.05).Compared with the high dose group of Bushen Jiannao Decoction,the expression of ERK1/2protein in the high dose + inhibitor group of Bushen Jiannao Decoction decreased significantly(P < 0.05).Conclusion:1.The serum containing Bushen Jiannao Decoction can protect PC12 cells damaged by A beta 25-35.Among them,15% of the serum containing Bushen Jiannao Decoction and 20umol/LA beta 25-35 intervene for 24 hours are the most suitable conditions for the experiment.2.The serum containing Bushen Jiannao Decoction has protective effect on PC12 cells damaged by Abeta 25-35.Its mechanism may be to maintain the integrity of the membrane structure of PC12 cells,maintain the morphological rules of PC12 cells,reduce the MDAcontent of PC12 cells,increase SOD activity,enhance the antioxidant capacity of cells,and reduce the damage of cells caused by oxidative stress.43.The serum containing Bushen Jiannao Decoction can significantly up-regulate the expression of ERK1/2 mRNA,increase the phosphorylation level of ER1/2 protein,and exert its protective effect on AD cells in many ways.It is suggested that Bushen Jiannao Decoction may have preventive and therapeutic effects on Alzheimer’s disease from many aspects.