| Objective:Study 1:To evaluate the possibility and reliability of phosphorylated α-synuclein(p-α-syn)in blood a biomarker of multiple system atrophy(MSA),so as to provide peripheral biological basis for the differential diagnosis and prognosis assessment of MSA.Study 2:By observing the differences between structural magnetic resonance imaging(MRI),resting state functional MRI(rs-fMRI)in MSA and the control group,and the correlation between multi-modal MRI parameters and clinical data,the pathological characteristics of MSA were understood from both structural and functional levels,and at the same time,the multi-modal MRI reference was provided for the differential diagnosis and prognosis observation of MSA.Study 3:Combined with blood biomarkers and multimodal MRI that are valuable for the diagnosis and prognosis of MSA,the possible mechanism of the treatment of MSA by Wenshen Jiannao formula was explored in multiple dimensions by comparing the differences of symptom assessment scale,blood biomarkers and multimodal MRI before and after treatment with Chinese medicine.Method:Study 1:Study on blood biomarkers of MSAEnzyme linked immunosorbent assay(ELISA)was used to detect p-α-syn in plasma and erythrocyte p-α-syn of MSA and Parkinson’s Disease(PD),Healthy Control(HC),and BCA method was used to detect total protein concentration in samples,levels of.Plasma and erythrocyte p-α-syn levels and their ratio to total protein were compared between MSA with predominant parkinsonism(MSA-P)subtype and MSA with predominant cerebellar ataxia(MSA-C)subtype.The performance of p-α-syn in differentiating MSA from control groups was verified by receiver operating characteristic(ROC)curves.The relationship between p-α-syn level and patient age,disease course and clinical rating scale was analyzed.Study 2:Study on multimodal MRI of MSAThe 3.0T superconducting MRI scanner was used for multimodal MRI image acquisition of the subjects.Differences in multimodal MRI were compared between MSA patients and controls.In terms of structural MRI,voxel-based morphometry(VBM)analysis was used to compare the differences of gray matter density and gray matter volume in different brain regions of MSA and control group,so as to understand the characteristics of brain atrophy in MSA.Gray matter density was used to construct the brain structure network,and the structural covariant network(SCN)analysis was used to understand the change characteristics of MSA brain structure network.In the aspect of rs-fMRI,functional connectivity(FC)was compared between the MSA patients and the control group to understand the changing characteristics of the brain functional network of MSA.At the same time,the differences between the MSA-C subtype and the MSA-P subtype in structural MRI and fMRI were compared,and the correlations between MRI parameters and age,disease course,and clinical rating scale were analyzed.Study 3:Study on the effect and mechanism of Wenshen Jiannao formula in treating MSAPatients with MSA were included and treated with Wenshen Jiannao formula for 3 months on the basis of the patient’s original medication.Motor and non-motor symptom rating scale and TCM syndrome scale of kidney deficiency and pulp reduction were evaluated before and after treatment.Blood p-α-syn levels were measured in some patients before and after treatment,as well as multimodal MRI scans.Symptom scales,p-α-syn and multimodal MRI were compared before and after treatment.Results:Study 1:A total of 46 patients with MSA,25 patients with PD,and 12 HC were included.There were no significant differences in gender distribution and age between MSA and the two control groups(P>0.05),indicating comparability.(1)Comparison between MSA and control group:Compared with HC,p-α-syn and its ratio to total protein levels in plasma and erythrocytes of MSA were significantly increased(all P<0.001).Compared with PD,there was no significant difference in plasma p-α-syn(P>0.05),but the plasma p-α-syn/total protein level in MSA was significantly lower than that in PD(P<0.01).Erythrocyte p-α-syn level was lower in MSA than in PD,and the difference was statistically significant(P<0.05),but there was no significant difference in erythrocyte p-α-syn/total protein level compared with PD level(P>0.05).ROC curve analysis showed,the Area Under Curve(AUC)of erythrocyte p-α-syn was 0.998(95%CI:99.2-100.0%),when the critical value was 38.19 ng/mL,the sensitivity,specificity,positive predictive value,and negative predictive value were 100%,97.8%,100%,and 92.3%,respectively.When plasma and erythrocyte p-α-syn levels were combined to distinguish between MSA and HC,AUC reached 1(95%CI:100-100%),sensitivity was 100%,and specificity was 100%.When erythrocyte p-α-syn and erythrocyte p-α-syn/total protein were combined to distinguish MSA from PD,the AUC was 0.691(95%CI:55.6-81.6%),the sensitivity was 72.0%,and the specificity was 65.2%.(2)Comparison between the two subtypes of MSA:There were no significant differences in plasma and erythrocyte p-α-syn and its ratio to total protein between MSA-C subtype and MSA-P subtype(P>0.05).(3)The correlation between p-α-syn and MSA clinical data:Plasma p-α-syn level was weakly negatively correlated with Montreal Cognitive Assessment Scale(MoCA)score of patients,and the higher the level of p-α-syn,the lower the MoCA score of patients,with statistical significance(P<0.05).The erythrocyte p-α-syn level was negatively correlated with Unified Multiple System Atrophy Rating Scale(UMSARS),Unified Parkinson’s Disease Rating Scale(UPDRS),Hoehn-Yahr(H-Y)grades,MSA non-motor Symptom Scale(MSA-NMSS),non-motor symptoms scale(NMSS),mini-mental state examination(MMSE)and MoCA scales,and the UPDRS-Ⅲ and MSA-NMSS scales were negatively correlated with the erythrocyte p-α-syn level.The results were statistically significant(P<0.05).Study 2:A total of 93 patients with MSA,22 patients with PD,and 33 patients with HC were included.There was no significant difference in age and sex distribution between the MSA group and the control group(P>0.05),indicating comparability.(1)Structural MRI:①Difference of gray matter volume between MSA and control group:Compared with HC,the gray matter volume of MSA decreased significantly in cerebellar hemisphere and cerebellar vermis,and increased in the right amygdala,inferior occipital gyrus,anterior ventral thalamus,internal thalamic nucleus and red nucleus,with statistical significance(P<0.05 or P<0.01).In addition,the volume of cortex gray matter in partial frontal and temporal regions of MSA showed a decreasing trend,but the difference was not statistically significant(P>0.05).Compared with PD,MSA reduced the gray matter volume in the broad cerebellar hemispheres and the vermis,and the differences were statistically significant(P<0.05 or P<0.01).②Difference of gray matter density between MSA and control group:Compared with HC,the gray matter density of MSA decreased in cerebellar hemispheres and broad regions of cerebellar vermis,and slightly increased in the right amygdala,ventral anterior thalamus,internal thalamic nucleus,ventral tegmental area of midbrain,and dorsal raphe nucleus,with statistical significance(P<0.05 or P<0.01).In addition,the decrease trend of MSA gray matter density was observed in some frontal and insular regions,but the difference was not statistically significant(P>0.05).Compared with PD,the gray matter density of MSA decreased in cerebellar hemispheres,extensive areas of cerebellar vermis and locus cyanulus,with statistical significance(all P<0.05).③Differences of structural networks between MSA and control group:structural networks were constructed based on cortical gray matter density.Compared with HC,extensive cortical and subcortical connections were significantly weakened in MSA(P<0.01).Compared with PD,the connections between the cerebellum and some cortical regions and subcortical areas in MSA were significantly weakened(P<0.01).④Comparison of structural networks between the two subtypes of MSA:Compared with MSA-P,the connections between the cerebellum and the vermis of the cerebellum and part of the cortex and subcortex were weakened in MSA-C,with statistical significance(P<0.05).⑤Correlation between structural MRI parameters and clinical variables in MSA patients:Correlation analysis was conducted between the volume and density of gray matter in MSA patients and the clinical scale of MSA.The results showed that gray matter volume in part of cerebellar hemispheres was negatively correlated with the course of the disease,while gray matter volume in posterior thalamus,inferior frontal gyrus insula and anterior central gyrus was positively correlated with the course of the disease.There was a positive correlation between the orbital gray matter volume of the inferior frontal gyrus and the total score of the UMSARS The gray matter volume of the orbital gyrus was positively correlated with the UMSARS total score and UMSARS-Ⅳ.The gray matter density in cerebellar hemispheres was negatively correlated with the course of disease.The density of large cells in the medial thalamus was positively correlated with UMSARS-Ⅳ.The correlation was statistically significant(P<0.05 or P<0.01).(2)rs-fMRI:①Difference of global functional network between MSA and control group:The whole brain functional network was constructed based on the Automated Anatomical Labeling.The results showed that compared with HC,the connections between cerebellar hemisphere,cerebellar vermis,thalamus and extensive cortex,subcortex and basal ganglia were weakened,and the connections between cortex and subcortex were weakened in MSA,with statistical significance(P<0.05 or P<0.01).The brain tissue was divided into 11 subnetworks to construct the whole brain functional network.The results showed that,compared with HC,MSA had extensive cortical and subcortical FC network abnormalities,mainly in the insula,occipital,temporal,hippocampus and brainstem nuclei and other brain regions or interbrain connectivity weakened.Compared with MSA,PD had weaker connections between hippocampus and parietal lobe,with statistical significance(P<0.05 or P<0.01).②Comparison of functional networks between the two subtypes of MSA:Compared with MSA-C type,MSA-P type had weaker average network connections in the basal ganglia region,and the connections between the basal ganglia region and insula lobe were weaker,with statistical significance(P<0.05).③Correlation between fMRI and clinical variables in MSA patients:mean insular intranetwork connection was negatively correlated with the total score of UMSARS and the score of MSA-NMSS,and the correlation was statistically significant(P<0.05).Study 3:A total of 87 patients with MSA were included,all of whom completed the efficacy related scale assessment before and after treatment.30 patients completed multimodal MRI scanning before and after treatment,and 11 patients completed blood P-alpha-SYN detection before and after treatment.(1)Improvement of patients’ condition:①Changes of main therapeutic indexes:After 3 months of treatment with Wenshen Jiannao formula,the exercise symptom evaluation scale UMSARS-Ⅲ was slightly improved.Though UMSARS-Ⅰ,UMSARS-Ⅱ,and the total score of UMSARS were slightly improved,the differences were not statistically significant(P>0.05).Compared with 3 months ago,UMSARS-Ⅲ were improved,with statistical significance(P<0.05).MSA-NMSS was significantly lower than 3 months ago(P<0.001).Further detailed analysis of 12 kinds of symptoms of MSA-NMSS showed that sleep symptoms and psychosomatic symptoms were improved after treatment,and the differences were significant(P<0.001).Defecation function,digestive function,and sweating function were better than before,with statistical significance(P<0.05).Systemic symptoms showed a trend of improvement compared with before,while head symptoms,urinary function,sexual function,cold/heat sense adaptive regulation,and cognitive function were slightly improved compared with before,but the differences did not reach statistical significance(all P>0.05).②Changes of secondary efficacy indexes:after treatment,H-Y grade and UPDRS-I were decreased compared with 3 months ago,while UPDRS-Ⅱ and Ⅲ were slightly increased,but the differences were not statistically significant(P>0.05).The NMSS of the patients was decreased,and the difference was statistically significant(P<0.05).After treatment,the principal syndrome integral part of TCM scale of kidney deficiency and pulp reduction decreased significantly(P<0.05),the concurrent syndrome integral part decreased significantly(P<0.001),and the total score of TCM scale of kidney deficiency and pulp reduction also decreased significantly(P<0.001).(2)Changes of blood markers in patients:there were no significant changes in plasma p-α-syn and its ratio to total protein after treatment compared with before treatment(P>0.05).The erythrocyte p-α-syn level was higher than that before treatment,the difference was significant(P<0.01),and the erythrocyte p-α-syn/total protein level was also higher than that after treatment,the difference was statistically significant(P<0.05).(3)Multimodal MRI changes in patients:①Structural MRI:After treatment,there were no significant changes in the volume and density of gray matter in each brain area compared with before treatment(P>0.05);The structural network comparison based on SCN showed that the connections in the cerebellum and between the cerebellum and brainstem nuclei were weakened after treatment compared with those before treatment,and the difference was statistically significant(P<0.05).②rs-fMRI:The whole brain functional network connectivity analysis showed that after treatment,the cortical and subcortical connections,mainly in the frontal lobe,tended to be enhanced compared with the anterior ones.Specifically,the average intra-network connections in the frontal and occipital lobes were enhanced compared with the anterior ones,and the connections between the frontal lobe and the insula,limbic lobes,hippocampus,basal ganglia,occipital lobe,parietal lobe,temporal lobe,and brain stem nucleus were enhanced compared with the anterior ones.The connections between occipital lobe and insula,temporal lobe and parietal lobe were enhanced,but the differences were not statistically significant(P>0.05).Conclusion:Study 1:The detection of p-α-syn in peripheral blood,especially in erythrocytes,has great potential in MSA,and is expected to become a potential liquid biomarker of MSA,which is of great value in the differential diagnosis,prognosis assessment and treatment of MSA.Study 2:MSA has extensive and complex structural and functional abnormalities in the brain.The combined application of structural and functional MRI will contribute to the study of the pathogenesis,differential diagnosis and longitudinal observation of MSA.Study 3:Wenshen Jiannao formula can delay the progression of motor symptoms and improve non-motor symptoms in MSA patients,especially in sleep and psychosomatic symptoms.The mechanism may be through enhancing the brain’s ability to clear toxic α-syn,increasing the peripheral transport of α-syn,and thus increasing the uptake of α-syn by peripheral red cells.At the same time,the therapeutic effect of Wenshen Jiannao formula on MSA may be closely related to the enhancement of functional network connections in the brain,especially in the frontal cortex and subcortical functional network connections. |
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