Based On The Ubiquitin Proteasome Pathway, The Effect Of Qianzheng Powder And Dabuyin Pills On The In Vitro Parkinson's Model Was Studied

Objective:PC12 cells were induced by(Proteasome inhibitor,PSI)to establish Parkinson's disease model in vitro.The cell proliferation cycle,apoptosis,SOD activity,MDA level and ubiquitin proteasome pathway were studied.To investigate the effects of Qianzheng Powder and Dabuyin Pill on cell proliferation cycle,apoptosis,SOD activity and MDA level,as well as the expression of TBP1-19 and PA28a in ubiquitin proteasome pathway.The effect of Qianzheng Sanhe Dabuyin Pill on PC12 cells was clarified at the cellular and molecular levels,so as to determine the effect of Qianzheng Sanhe Dabuyin Pill on Parkinson's disease.Methods:MTT method was used to detect the cytotoxicity of Qianzheng Powder and Dabuyin Pill on PC12 cells.The appropriate dose of Parkinson's disease cell model induced by PSI was determined by inverted fluorescence microscopy with DAPI staining and eosin staining. The eosinophilic bodies of PC12 cells induced by PSI in vitro were observed by HE staining.MTT method was used to determine the effects of Qianzheng Powder Decoction and Dabuyin Pill Decoction on the proliferation of PC12 cells induced by PSI in vitro.Flow cytometry was used to detect the cell cycle changes and cell death of PC12 cell model induced by PSI after 48 hours of intervention with Qianzheng Powder Decoction and Dabuyin Pill decoction.Detection of apoptotic rate by annexin V/fitc flow cytometry.The effects of Qianzheng Powder and Dabuyin Pill Decoction on SOD and MDA in PC12 cell model induced by PSI were determined by SOD and MDA.Western Blot technique was used to determine the effects of the decoction of Qianzheng Powder and Dabuyin Pill on the expression of intracellular related factors TBP1-19 and PA28a in mice with or without PSI induction.Results:MTT results showed that different concentrations of Qianzheng Powder and Dabuyin Pill had different effects on cell proliferation.At 48h,the decoction of Qianzheng Powder showed inhibitory effect on cell proliferation from 6 mg/ml concentration.The inhibitory rates of 12 and 24 mg/ml concentration were 61%and 91%,respectively.The water decoction of Dabuyin Pills showed inhibitory effect on cell proliferation from 0.125 mg/ml.The inhibitory rates of 0.25,0.5,1 and 2 mg/ml were 14%,9.8%,0.64%and 26.46%respectively.Different concentrations of Qianzheng Powder Decoction and Dabuyin Pill decoction can improve the inhibition of PSI on PC12 cell proliferation.ICO,IC25 and IC50 were calculated by Using SPSS Statistics 20 Software.The ICO,IC25,IC50 and IC50 of Qianzheng Powder Decoction were 0.2mg/ml,12.292 mg/ml and 22.652 mg/ml respectively after 48 hours of action on rat pheochromocytoma PC12 cells.The ICO,IC25 and IC50 of Dabuyin Pill were 0.814 mg/ml,4.485 mg/ml and 9.004 mg/ml respectively after 48 hours of action on rat pheochromocytoma PC12 cells.MTT assay in vitro showed that the decoction of Qianzheng Powder at 12.292 mg/ml(IC25)concentration and Dabuyin Pill at 4.485mg/ml(IC25)concentration could significantly alleviate the damage of PC12 cell model induced by PSI.DAPI and eosin staining showed that the morphological changes of PC12 cells and eosinophils in the cytoplasm were most typical when PSI concentration was 6 micromol/L.After HE staining,the morphology and quantity of PC12 changed under the action of PSI under inverted microscope.HE staining showed that the number of cells decreased,aggregation tendency,cell volume increased,Parkinson's characteristic pathological changes appeared in the cytoplasm of Lewis corpuscles similar to the structure of eosinophilic corpuscles.Flow cytometry showed that PSI-induced PC12 cells in rat pheochromocytoma could affect cell cycle.However,Qianzheng Powder and Dabuyin Pill did not significantly improve cell cycle.Annexin V/FITC flow cytometry showed that the apoptotic rate increased in PSI group,and decreased in different degrees in drug-added group.The results of OD and MDA assay showed that compared with the blank control group,the SOD activity of PC12 cells in PSI group decreased,while the MDA content of lipid peroxidation product increased.After treatment,the SOD activity and MDA content of Qianzheng San Decoction and Dabuyin Pill Decoction in each dose group were higher than those in PSI group.Western Blot results showed that PSI could increase the expression of TBP1-19 and PA28a in PC12 cells.Compared with the PSI group,the water decoction of Qianzheng Powder in each dose group could decrease the expression of TBP1-19 and PA28a protein.The water decoction of Dabuyin Pills in each dose group could reduce the expression of TBP1-19 and PA28a protein.Conclusions:PSI-induced Parkinson's model of PC12 in vitro is of scientific significance.The Decoction of Qianzheng Powder and Dabuyin Pill can alleviate the inhibition of PSI on PC12 Parkinson's model in vitro and improve the survival rate of PC12 cells induced by PSI.The mechanism is to regulate the apoptosis of PC12 cells induced by PSI and the content of SOD and MDA in cells.In addition,the metabolism of PC12 cells is affected by regulating the expression of TBP1-19 and PA28alpha proteins in ubiquitin proteasome pathway,which can alleviate Parkinson's disease(trembling syndrome).