The Protective Effects And Its Mechnisms Of Baicalin On Dopaminergic Neurons

Parkinson’s disease (PD) also named Paralysis agitans, is the second chronic neurodegenerative disease after Alzheimer’s Disease (AD). The main clinical features of PD include resting tremor, bradykinesia, postural instability and rigidity. As the population of the elders is growing, the incidence of PD increases in China.Though PD is aneurodegenerative disease with effective symptomatic treatment, modern medicine has not found an effective neuroprotective treatment or strategy to prevent the loss of dopaminergic neurons in PD. Therefore, development of new drugs with better curative effect and fewer side effects for PD is urgently needed.Animal models are invaluable tools in the identification of pathogenic mechanisms and testing new therapeutic strategies for PD.The1-methyl4-pheny1-1,2,3,6-tetrahydropyridine (MPTP)-induced PD mice model is the most commonly used animal model in PD research, neuropathology and motor deficits induced by MPTP is strikingly similar to PD patients. Establishment of a stable and sensitive evaluation platform for the animal model is very important for the treatment screening in PD.Scutellaria baicalensis Georgi, an important medicinalherb, has been widely used in China to treat the inflammatory diseases and ischemic stroke for thousands of years.Baicalin, a flavonoid compound isolated from S. baicalensisGeorgi, possesses anti-oxidant, anti-inflammatory and anti-apoptotic properties.In present study, we made the PD mice model by intraperitoneal administration of MPTP to the C57/BL6mice. Then, we investigated the gait and postural changes in PD mice using the computer-assisted CatWalk system, and screened the sensitive parameters by correlating the gait and postural parameters with the TH protein levels in the substantial nigra. The gait and postural readouts, includingstride length,diagonal dual support, three-point supports,variation of walking speed, base of support between hind limbs, duty cycle in the hind limbs and initial dual stance of the forelimbswere proved to be the satble and sensitive parameters for evaluating the movement disorders in PD mice. We have screened a lot of drugs for PD, and found that Bacalin can effectively alleviate the functional disorder and the degeneration of dopaminergic neurons. Some questions arise:1. Through which pathway Bacalin prevent dopaminergic neurons degeneration?2. Which protein is the key one during Baicalin’s neuroprotective role?To answer these questions, we profile the quantitative proteomic differences of substantial nigra in PD mice with or without Bacaline treatment by two-dimensional gel electrophoresis (2-DE) and Mass Spectrometry (MS) analysis. Integration of the quantitative proteomics with global protein interaction data using the String software leads to the identification of several novel proteins, which may be important in find new targets for PD therapy.This study was divided into4parts:PART I ESTABLISHMENTOF THE MPTP-INDUCED PD MICE MODEL AND ITS EVALUATION SYSTEMObjective:To establish the MPTP-induced PD mice model and screen the sensitive and stable gait parameters by correlating the CatWalk test with TH protein levels in substantial nigra of PD mice.Methods:1. The C57/BL6mice received one injection of MPTP-HClintraperitoneally at the dose of40mg/kg for5consecutive days, the control mice were treated with the same volume of normal saline.2. On the21day after the last MPTP injection, brain of the C57/BL6mice in both control and PD groups were dissected for TH immunohistochemistry analysis3. On the4,7,12,16,21day after the last MPTP injection, C57/BL6mice in both control and PD groups performed the open field test. 4. On the2day before MPTP injection, and2,4,7,12,16,21day after the last MPTP injection, the substantial nigra and striatum of mice in both control and PD groups were dissected for detecting the TH protein level by Western Blotting analysis.5. On the4,7,12,16,21day after the last MPTP injection, C57/BL6mice in both control and PD groups performed the CatWalk test. We will screen the sensitive and stable gait parameters by correlating the gait parameters and TH level in substantial nigra.Results:1. The dopaminergic neurons subjecting to this regimen of MPTP injection induced a significant loss to30%of that of control group after the last injection of MPTP. The challenge with MPTP also resulted in a great depletion of TH-ir in striatum.2. In the open field test, the walking distance of PD mice decreased significantlyon the4th day after the last MPTP injection, increased slightly on the7th day. There is no different between PD mice and control mice on the12and16day. On the21day, the distance of PD mice decreased stably and significantly.3. The gait parameters including stride length,diagonal dual support, three-point supports,variation of walking speed, base of support between hind limbs, duty cycle in the hind limbs and initial dual stance of the forelimbscorrelated well with the TH protein levels in substantial nigra of PD mice. They are served as the stable and sensitive parameters for analysis gait in MPTP-induced PD mice.4. A significant fall in TH was evident in both substantianigra andstriatum of mice starting on day2of MPTP administration, followed by a slight rebound at twoweeks. TH then remained at the lowest levels on three weeks post-MPTP injection. It was noted that the down regulationof TH by MPTP in the striatum was more profound than that in the substantia nigra.5. The correlation analysis between CatWalk test and TH level shows significant positive correlations of the levels of TH in the substantia nigra of MPTP-treatedmice with readouts derived from CatWalk tests of diagonaldual support, stride length in all limbs, and swing speed inthe forelimbs three weeks post MPTP administration. Substantially negative correlations of TH levels with stancein the hind limbs, step cycle, duty cycle, initial dual stance, terminal dual stance, three-point support, four-point support,walking speed variation, cadence,and base of supportbetween hind limbs were noted. On the other hand, thelevels of TH correlated poorly with the trajectories in theopen field tests.Conclusion:The MPTP-induced subacute PD mice model can effectively mimic the pathological and behavioral feature in PD patients. The computer-assisted CatWalk system can evaluate the motor deficits in PD mice in a friendly, automated,fast and objective way, and is an important part of the evaluation system in PD model.PART II THE PROTECTIVE EFFECT OF BACAILIN ON THE MPTP PD MICEObjective:To screen the therapeutic drugs on the MPTP-induced PD mice using the evaluation system established in the first part.Method:1. Animals were divided into four groups:Control, PD, PD+Baicalin,Baicalin. Mice in PD and PD+Baicalin group were injected with MPTP, mice in PD+Baicalin and Bacalin group were treated with Bacalin, and mice in the Control group were injected with normal saline.2. The open field test was performed on the7th day after MPTP/Saline injection.3. The CatWalk test was performed on the8th day after MPTP/Saline injection.4. After the baicalin/saline treatment, mice in different groups were perfused and fixed transcardially. The brain were dissected and embedded in Tissue-Tek O.C.T compound, after immunostaining with TH antibody, total number of TH-positive neurons inSNpc and the TH positive terminal in striatum in all groups was estimated.5. After the baicalin/saline treatment, the substantial nigra and striatum of mice in all groups were dissected for detecting the TH and other proteins level by Western Blotting analysis.Results:1. After Baicalin treatment, the traveling distance in10minutes and time of mice in PD+Baicalin group increased while rest time decreased greatly than mice in PD group.2. After MPTP treatment, the stride length,diagonal dual support, duty cycle in the hind limbs and swing speed decreased significantly, and the three-point supports,variation of walking speed, base of support between hind limbs, and initial dual stance of the forelimbs increased significantly. Bacalin treatment can effectively improve the gait and postural disorders in PD mice.3. After the subacute MPTP treatment, TH-irpositive neurons decreased greatly in the pars compacta. Baicalin treatment can effectively prevent the neuron loss in PD mice.4. After the subacute MPTP treatment, the protein levels of TH and cleaved-Caspase-3decreased, while Bax and iNOS increased in the substantial nigra pars compactagreatly. Baicalin treatment can effectively reverse the change in substantial nigra in PD mice.Conclusion:Baicalin can effectively inhibit the damage in DA neurons and alleviate the movement disorders in PD mice. The protective mechanism of Baicalin may related with its anti-apoptosis and anti-inflammation effects.PARTⅢ QUANTITATIVE PROTEOMIC ANALYSIS OF THE ANTIPARKINSONIAN ROLE OF BAICALIN Objective:To identify and analyze the key proteins associated with the antiparkinsonian effects of baicalin. Comparativeanalyse of2-DE protein patterns between the PD and PD+Baiclin groups were done using computerized image analysis. Differentially expressed proteins (at least two-fold) were identified by MASCOT software. RT-PCR was carried out to confirm the gene expression patterns of those screened proteins.Methods:1. After the baicalin treatment, mice brain of PD and PD+Baicalin group were removed, and the substantial nigra was dissected and lysis.2. The total proteins from substantial nigra were separated by2-DE. Comparativeanalyses of2-DE protein patterns between the PD and PD+Baiclin groups were done using computerized image analysis. Differentially expressed proteins (at least two-fold) were identified by MASCOT software.3. RT-PCR was carried out to confirm the gene expression patterns of those screened proteins.Results:1. A total of36differentially expressed proteinswere identified by the MASCOT software.2. The RT-PCR results domistrated that the gene expression patterns of the screened proteins were almost in accordance with the protein expressing pattern.Conclusion:Baicalin may play neuroprotective role by regulating the aggregation of protein and energy metabolizing, inhibiting the oxidative stress and modulating some sigal pathways.PART IV BIOINFORMATIC ANALYSIS UNDERLYING THE ANTIPARKINSONIAN EFFECTS OF BAICALINObjective:To investigate the protein-protein interaction and biological pathways associated with antiparkinsonian effects of baicalin using String, KEGG and Pathwaylinkersoftware. To confirm the role of proteins and pathways by Western Blotting to provide new thoughts for therapeutic study of PD.Methods:1. Predict the key proteins and pathways involved in the antiparkinsonian effects of baicalin using KEGG and cytoscape software.2. Confirm the role of the key proteins and pathways using Western Blotting analysis.Results:1. All the differentially expressed proteins screened using the2-D method involve in about14biological pathways, including the Parkinson’s disease pathway, the Alzermer’s disease pathway, the actin regulating pathway, the axon guidance pathway and the Coli infection pathway and soon on.2. There are22interacting proteins in the differentially expressed proteins. All the interacting proteins can be devided into5sub-groups including the actin-related protein group, CRMP/DPYL family proteins and Septin family proteins.3. Integrating the information of the proteomic and bioinformatic results, we predict that the proteins including GFAP, GAPDH and Stipl and the MAPK/ERK pathway may play important role in the protective effects of Baicalin.4. Western Blotting results showed that the expressing of GFAP and ERK1/2increased after MPTP injection, while HSP70and LC-Ⅱdecreased. After treated with Baicalin, expression of GFAP and ERK1/2decreased while HSP70and LC-Ⅱincreased significantly.Conclusion:The protective effects of Baicalin may mainly relating with the protein processing and energy metabolism. The antiparkinsonian effects of baicalin may involve multipleproteins including GFAP, GAPDH and Stipl. The MAPK/ERK pathway may also play important role in the antiparkinsonian effects of baicalin.Summary:We found that the computer-assisted CatWalk system can provide sensitive and stable parameters for analysising the motor disorders in PD mice. Baicalin can effectively inhibit the damage in DA neurons and alleviate the movement disorders in PD mice. Baicalin may play neuroprotective role through the anti-inflammatory, anti-apoptosis, anti-aggregation and regulating the energy metabolizing and some signal pathways. The antiparkinsonian effects of baicalin involve multiple proteins including GFAP, GAPDH and Stipl. The MAPK/ERK pathway may play important role in the antiparkinsonian effects of baicalin.