Clinical Observation And Experimental Study Of Zhichan Decoction For Parkinson’s Disease And Its Movement Complications

Objective:To explore the effect of Zhichan Decoction on improving the symptoms of Parkinson’s disease rats and the effect on autophagy-related proteins.By using network pharmacology,use database,establish a network,analyze the network,screen the most potential targets of Zhichan Decoction.To study the regulation mechanism of Zhichan Decoction on cell chain induced by rotenone on autophagy-related and apoptosis-related proteins;To observe the therapeutic efficacy of Zhichan Decoction Granules on Levodopa-Induced Dyskinesias(LID).Methods:Study 1: We selected thirty healthy adult SD rats and divided them into 3 groups:a control group,a model group,and a traditional Chinese medicine(TCM)group randomly.Each group had10 rats in it.Through injecting 6-OHDA stereotactic,we established the PD rat model and studied the behavioral improvement and neuroprotective effects of Zhichan Decoction on the PD rat model.We observed the change in the expression of the abnormal protein a-Syn and autophagy-related protein p62 and LC3 in the brain of the PD rat after the intervention of Zhichan Decoction.Study 2: We used the research method of network pharmacology and relevant databases to search for PD-related target proteins.We tested the effective active ingredients of Huangqi,Danshen,Gouteng,Zhimu,Baishao,Zhidahuang,and Shenma in the prescription of Zhichan Decoction based on the database and mapped the network with drug-related targets and the disease-related targets,screened the core targets of the drug acting on PD and verified the continuous experiment.Study 3: We used the cell chain model induced by rotenone to study the effect of the intervention serum contained of Zhichan Decoction on the chain model.We explored the cytoprotective effect of Zhichan Decoction on PD cell model by CCK8 detection and studied the best time of action.We observed the improvement effect of Zhichan Decoction on cell apoptosis by flow double staining detection.We explored the effect of Zhichan Decoction on the key protein BCL2 in the balance of autophagy and apoptosis by the WB method.Study 4: A randomized,double-blind,placebo-controlled method was conduced to evaluate the effectiveness of Zhichan Decoction in treating 100 LID patients.There were 50 patients in the treatment group and 50 patients in the control group.In addition to the original anti-Parkinson’s drug,patients took Zhichan Decoction granules in the treatment group,while patients took placebo granules in the control group for 12 weeks.The curative effect was performed at the time of enrollment,the4 th week,the 8th week,and the 12 th week.Indicator observation: Unified Parkinson’s Disease Rating Scale(UPDRS score),Abnormal Involuntary Movement Scale(AIMS)score,TCM syndrome score and Efficacy Comparison.Results:1.Zhichan Decoction can improve the central activity time,central activity distance,defecation and total distance in PD rats(P<0.001);electron microscopy and TH staining indicate that Zhichan Tang has a protective effect on the neurons of PD rats;TH staining suggests that,the expression of TH in the model group significantly decreased(P<0.001),and the expression of TH in TCM group increased(P<0.05);The results in WB suggested that,the expression of p62 and LC3 in model group increased(P<0.05,P<0.05),the expression of that in TCM group decreased(P<0.05,P<0.05);the expression of LC3 in model group was lower than that of drug group(P<0.05),and the expression of that in TCM group increased(P<0.05).2.Search the effective active ingredients of seven herbs included Huangqi,Danshen,Gouteng,Zhimu,Baishao,Zhidahuang,and Shenma from TCMSP,TCMID and other databases,and removed the common ingredients and non-targeted ingredients.We get a total of 343 effective active ingredients.1038 PD-related targets were retrieved from the Gene Expression Omnibus(GEO)database.Through PPI network analysis,a network of 57 key targets acting on PPD was obtained after mapping with drug targets.GO analysis and enrichment analysis were performed on the drug targets acting on PD,and a target path network including 20 pathways and 35 candidate targets was established.We found that BCL2 protein was the most critical target of Zhichan Decoction(P<0.05).As a protein that regulates the balance of autophagy and apoptosis,BCL2 will be one of the key proteins in subsequent experiments.3.After Zhichan Decoction interfered with rotenone-induced cell model,CCK8analysis indicated that Zhichan Decoction reduced cytotoxicity and improved cell viability after 24 h,48h,72 h intervention with TCM(P <0.01,P<0.05,P<0.001),and the TCM low-dose-group and the 24h-intervention-time were the best;The flow cytometric double staining analysis showed that the apoptosis of the model group increased compared with the control group,and declined after being interfered.When the BCL2 inhibitor was added to reduce the anti-apoptotic effect,the cell apoptosis significantly increased in the model group.When the autophagy agonist was added,the cell apoptosis decreased;WB suggested that after 24 h intervention with the low-dose decoction group,the expression of BCL2 phosphorylation increased,the expression of BAD and CASP9 decreased,which means Zhichan Decoction can inhibite apoptosis.In the process of autophagy,the expression of SQSTM1(P62)in the model group was higher,and it was lower in the drug group.,while the expression of LC3 was higher in the drug group.Zhichan Decoction promotes autophagy and inhibits apoptosis,thereby protecting cells and improving PD.4.We used Zhichan Decoction granules to treat LID patients.We found that UPDRSIII scale scores in the treatment group decreased at the 8th and 12 th week of treatment(P<0.01,P<0.01),and UPDRS IV-A scale scores in the treatment group decreased at the 8th and 12 th week of treatment(P<0.05,P<0.01);the AIMS limb score was lower at the 12 th week of treatment(P<0.01).The AIMS total standard score in the treatment group was lower at the 8th and 12 th week during the treatment(P<0.01,P<0.01).The total AIMS score in the treatment group decreased at the 8th and 12 th week during the treatment(P<0.05,P<0.01).The TCM syndrome score in the treatment group decreased significantly at the 8th and 12 th week during the treatment(P<0.01,P<0.01).The total clinical efficacy of all scales and TCM syndrome in the treatment group was better(P <0.01).Conclusions:1.Zhichan Decoction can improve the behavior of PD rats and have a protective effect on the neurons of PD rats.Its mechanism of action may be achieved by promoting autophagy,clearing PD characteristic abnormal protein a-Syn,and maintaining the stability of internal environment..2.It is found that BCL2 protein is the the key target of Zhichan Decoction on the treatment of PD.3.Zhichan Decoction reduces the cytotoxicity of rotenone-induced cell models,improves cell viability,inhibits apoptosis,promotes autophagy,thereby protects cells and treats PD.4.Zhichan Decoction granules can improve the LID clinical symptom,and play a role on the effectiveness from the 8th week,especially on the symptoms of limbs;and as the medication time is extended,the improvement is more obvious.