Clinical Observation Of Shuimu Hening Prescription In The Treatment Of Autonomic Dysfunction Symptoms Of Parkinson’s Disease And Its Effect On Ubiquitin-proteasome Pathway In PC12 Cells

Objective:The clinical study collected the cases of Parkinson’s Disease(PD)with autonomic dysfunction to observe the clinical efficacy of Shuimu hening prescription in the treatment of Parkinson’s disease with autonomic dysfunction.Additionally,by constructing an in vitro cell model of Parkinson’s disease,we plan to explore the mechanism of Shuimu hening prescription in the treatment of Parkinson’s Disease based on ubiquitin-proteasome pathway(UPS).Methods:1.We collected one hundred clinical patients in Parkinson’s Disease with autonomic dysfunction in our hospital.Then we divided them into a treatment group and a control group randomly,fifty patients in each group.The treatment group was treated with traditional Chinese medicine Shuimu hening prescription combined with Madopar,while the control group used Madopar alone for eight weeks.The Parkinson’s Disease Rating Scale Part III(UPDRS-III)and the Autonomic Symptom Scale(The Scale for Outcomes in Parkinson’s Disease for Autonomic Symptoms,SCOPA-OUT),constipation scoring system(CSS),Parkinson’s disease TCM syndrome scores,TCM syndrome efficacy evaluation were evaluated after treatment,and SPSS 26.0 was used for statistical analysis to assess clinical efficacy.During the course of treatment,safety assessment was performed,and adverse reactions of patients were recorded.2.MTT assay was used to select optimal drug concentration for modeling drug 1-Methyl-4-phenylpyridinium(MPP~+),Shuimu hening prescription and Madopar in PC12 cells.The in vitro cell model of Parkinson’s Disease induced by MPP~+was used to intervene PC12 cells with the optimal concentrations of Shuimu hening prescription and Medopa respectively.After24 hours,the OD value of the cells was measured to calculate the cell viability.3.The in vitro cell model of Parkinson’s Disease was induced by MPP~+,and the Chinese medicines Shuimu hening prescription and Medopa were given drug intervention respectively.After 24 hours of drug action,We used Real-time PCR method to detect the m RNA expression ofα-syn,TH and ubiquitin,UCH-L1,Parkin and UBE1 related to the ubiquitin proteasome system.Western blot was used to detect the expressions ofα-syn,TH,and ubiquitin,UCH-L1,Parkin,and UBE1 proteins related to the ubiquitin-proteasome system.Results:1.Clinical research:(1)Baseline analysis:There were no significant differences in gender,age,course of disease,educational level,co-morbidities,and H-Y classification between the two groups,indicating that the baselines were consistent and comparable(P>0.05)(2)Assessment of motor function in Parkinson’s disease:Both Shuimu hening prescription and Medopa can reduce the UPDRS-III score and improve the motor symptoms of Parkinson’s disease patients,and the former is better than the latter(P<0.05);(2)Parkinson’s disease autonomic dysfunction assessment:Shuimu hening prescription combined with Madopar can significantly reduce the patient’s SCOPA-OUT score,improve the patient’s autonomic nervous function symptoms such as digestion,urinary,cardiovascular,thermoregulation,and reproductive system,and reduce the severity of constipation.degree,and the effect was better than that of Medopa alone(P<0.05);(3)Evaluation of the efficacy of TCM syndromes:Both Shuimu hening prescription and Medopa can improve the TCM syndrome scores of the patients,and the overall efficacy of the formmer(84%)is better than the latter(68%)(P<0.05);(4)Drug safety assessment:There were no obvious adverse reactions between in Shuimu hening prescription during the medication period,and the drug safety was good.2.With the increase of MPP~+concentration,the viability of the cells showed a downward trend.When the concentration of MPP~+was 1 mmol/L,the cell viability of PC12 cells was50.57%.Therefore,we chose 1 mmol/L as MPP~+optimum concentration to build Parkinson’s disease patients cell model.Cell experiments showed that within a certain concentration range,Shuimu hening prescription and Madopa were not toxic to PC12cells,and with the increase of drug concentration,the cell viability decreased correspondingly.The optimal drug concentration of Shuimu hening prescription acting on PC12 cells was 6mg/m L by MTT assay and the optimal drug concentration of Madopar was 3.125μg/m L.Both Shuimu hening prescription and Medopa can improve the cell viability of PC12 cells,inhibit the damage of MPP~+to PC12 cells,so they have a protective effect on PC12 cells,and the protective effect of Shuimu hening prescription group on PC12 cells is better than that of Medopa group(P<0.05).3.The results of Real-time PCR showed that compared with the control group,the expression ofα-syn m RNA in the MPP~+group was significantly up-regulated,while TH,UBE1,Parkin,UCH-L1 and ubiquitin were down-regulated significantly(P<0.05).Compared with MPP~+group,the expression ofα-syn m RNA in Shuimu hening prescription group was down-regulated significantly,and TH,UBE1,Parkin,UCH-L1 and ubiquitin was up-regulated significantly(P<0.05).Compared with the Medopa group,the expression ofα-syn m RNA in the Shuimu hening prescription groups was significantly down-regulated while TH,UCH-L1 and ubiquitin were significantly up-regulated(P<0.05).Western blot results showed that compared with the control group,the expression ofα-syn in the MPP~+group was up-regulated,and TH,UBE1,Parkin,UCH-L1,and ubiquitin were down-regulated obviously(P<0.05).Compared with MPP~+group,the expression ofα-syn was down-regulated,and the expressions of TH,UBE1,Parkin,UCH-L1 and ubiquitin were up-regulated evidently(P<0.05).Compared with the Medopa group,theα-syn protein in the Shuimu hening prescription group was down-regulated while TH,UCH-L1 and ubiquitin proteins were up-regulated significantly(P<0.05).Conclusion:1.Shuimu hening prescription combined with Madopar can not only improve the motor symptoms of Parkinson’s disease patients with liver-kidney deficiency and phlegm and blood stasis blocking collaterals,but also improve the patients’autonomic dysfunction and TCM syndromes,additionally the clinical efficacy is better than that of Medopa alone,with reliable clinical safety.2.Shuimu hening prescription can inhibit the damage of MPP~+to PC12 cells,and has a protective effect on PC12 cells,and its protective effect is better than that of Madopar alone.