Study On The Characteristics Of The Syndromes Of Parkinson's Disease And The Regulation Of The Mechanism Of The Degradation Of α-synuclein By Bushen Pinggan Recipe

OBJECTIVE:To study the characteristics of traditional Chinese medicine(TCM)syndromes elements of Parkinson’s disease(PD),to explore the mechanism of the formula of nourish the kidney and soft the liver in the treatment of PD based on network pharmacology,and to study the degradation of a-synuclein by the formula of nourish the kidney and soft the liver with PD A53T transgenic model,so as to provide a scientific basis for the treatment of PD with TCM.METHODS:(1)A multi-center cross-sectional survey was conducted to collect 254 patients(PD group)who were clinically diagnosed as PD from Dongzhimen Hospital,Beijing Hospital and the Third Hospital of Peking University.The distribution of TCM syndromes in the PD group was investigated by using the PD-PES scale as the evaluation criterion,and 63 non-PD subjects(group non-PD)matched with their age were compared with PD group.Statistical methods of chi-square test were used to summarize the characteristics of common TCM syndromes elements in PD group.(2)On the basis of network pharmacology,TCMSP,ETCM and BATMAN-TCM databases are used to collect the active ingredients and corresponding targets of drugs in the formula of nourish the kidney and soft the liver,and CTD and Genecards databases are used to collect disease targets in PD’.According to the theory of "JunchenZuoshi" in TCM,the interaction between them is explored,and the core components and targets of each drug in the formula of nourish the kidney and soft the liver are analyzed.To summarize the core targets of the formula of nourish the kidney and soft the liver drugs and PD diseases.PPI network was constructed by STRING database.GO enrichment analysis and KEGG Pathways enrichment analysis were carried out by GO and KEGG Pathways.Combined with the network structure chart of"drug-core component-core target-related pathway",the mechanism of the formula of nourish the kidney and soft the liver drug treatment for PD was comprehensively analyzed.(3)PD A53T transgenic rats were randomly divided into model group(n=22),Chinese and Western medicine combined group(n=22)and herb group(n=22),and compared with normal control group(n=16).The intervention measures of distilled water,Madopa combined with the formula of nourish the kidney and soft the liver drug,the formula of nourish the kidney and soft the liver drug,distilled water were adopted respectively.After 24 weeks,behavioral Rotarod test and 24-hour autonomous activity experiment were used to explore the effect of the formula of nourish the kidney and soft the liver drug on motor function of PD rats.The positive expression of alpha-synuclein and TH was quantitatively analyzed by immunohistochemistry(IHC)test.The correlation of alpha-synuclein,tyrosine hydroxylase(TH)and lysosome was detected by Western Blot(WB)test.The expression of relative protein content of protein-2A(Lysossomal associated protein 2A,LAMP-2A),microtubule-associated protein 1 light chain 3Ⅱ/Ⅰ(Microtubule-associated protein 1 light chain3 Ⅱ/Ⅰ,LC3 Ⅱ/Ⅰ),62 proteasome(P62)was studied.After the experiment,one-way ANOVA or non-parametric test were selected to analyze the regulation effect of the formula of nourish the kidney and soft the liver drug on a-synuclein.RESULT:(1)The distribution of TCM syndrome elements in PD group showed that marrow deficiency(87.80%)was the highest,followed by liver wind(72.05%)and Qi deficiency(58.66%).Compared with the age-matched PD-PES in non-PD group,the results showed that there were significant differences in syndrome elements of marrow deficiency(p=0.000),liver wind(p=0.000),kidney deficiency(p=0.029)and yin deficiency(p=0.013).(2)According to the theory of"emperor-minister assistant envoy",the most effective targets of Cornus officinalis,Lycium barbarum,Gastrodia elata and Rehmannia Rehmanniae were found by network pharmacological method.There were 1146 targets for the emperor-minister drugs and 741 targets for the emperor-minister drugs and PD,accounting for 64.4%of the core targets.Through the study of core target and core components of single drug,58 core components of Cornus officinalis and 1054 core targets were found in the formula of nourish the kidney and soft the liver,91 core components of Lycium barbarum and 746 core targets were found to be core drugs in the formula of nourish the kidney and soft the liver.Among the core targets,SNCA,TH,LAMP2,HSPA1L,PSMC5,HSPB1,HSPA5,UBE2B and other core targets with research significance are found.The core target is to construct PPI network interaction by STRING for GO enrichment and KEGG Pathway s enrichment analysis.A total of 258 Pathway s related to Kidney-Tonifying and Liver-Calming therapy for PD are summarized.According to the top 20 Pathway s ranked by PValue,relevant literature verifies that they are closely related to Autophagy.At the same time,KEGG Pathway is enriched to Parkinson’s disease(4.59E-14),Autophagy-animal(6.09E-12)and Mitopani.Mal(2.03E-09).(3)In the study of the mechanism of PD a-synuclein degradation by the formula of nourish the kidney and soft the liver,the duration of the Rotarod test was significantly longer in the herb group of the formula of nourish the kidney and soft the liver group than in the model group(p=0.028).There was no statistical difference between the herb group of the formula of nourish the kidney and soft the liver and the model group(p>0.05),but there was an increasing trend in the 24-hour autonomous activity experiment.In the immunohistochemistry(IHC)test,the herb group of the formula of nourish the kidney and soft the liver was compared with the model group,the positive expression of TH could be increased(p=0.005)and the positive expression of alpha-synuclein could be reduced(p=0.001).Compared with the model group,the herb group of the formula of nourish the kidney and soft the liver could improve the expression of TH(p=0.000),LAMP-2A(p=0.000)and LC3 in the substantia nigra of PD A53T transgenic rats through regulation and control.The relative protein content of Ⅱ/Ⅰ(p>0.05)and P62(p>0.05)decreased the expression of a-synuclein(p=0.042).CONCLUSION:Marrow deficiency and liver wind are the common TCM syndromes elements of PD.The core targets of the formula of nourish the kidney and soft the liver and PD include SNCA,TH and LAMP-2,which may regulate Autophagy lysosomal pathway(ALP)related biological processes through the target of LAMP-2to improve the degradation of a-synuclein in substantia nigra of rat midbrain.