Effectiveness Of Magnolia Extract On Endoplasmic Reticulum Stress And The Ubiquitin-proteasome System In PD Model

BackgroundParkinson disease (PD) is a neurodegenerative disease common in the elderly. With the advent of an aging society, the PD prevalence is worldwide increased year by year, according to the World Parkinson Disease Association recent data show that the PD disease patients around the world have more than4million. China ranks first in the world. Parkinson’s disease has become a major medical and social problems for our country and even around the world.The exact cause of this disease is unknown, modern medicine lacks the desired effect of therapy, Chinese medicine Magnolia extract have accumulated a lot of treatment of experience in the PD clinical studies but so far Chinese medicine Magnolia extract has not cleared at the molecular level the mechanisms of effecting endoplasmic reticulum stress and the ubiquitin-proteasome system in the PD model. Therefore, in order to play to the characteristics and advantages of Chinese medicine, and actively explore the Chinese medicine treatment way for the PD disease and research and development Chinese medicine new drugs for the treatment of Parkinson’s disease have practical significance.According to the latest progress and developments of the international study of Parkinson’s disease, by continuous injection of MPTP-induced simulate the animal model that has human chronic course of the Parkinson’s disease and Lewy body formation pathological features. research the impact of Magnolia extract for endoplasmic reticulum stress and the ubiquitin-the proteasome system and its pharmacodynamic mechanism of action, further clear the relationship of treatment of PD efficacy and provide a scientific basis for traditional Chinese medicine new drug development.The study shows that:the midbrain’ s substantia nigra dopamine (DA) neuron degeneration missing with intracytoplasmic Lewy body formation is the main pathological changes of Parkinson’s disease Lewy body gathered a number of proteins, including lipid, neurofilament and related proteins a-synuclein and ubiquitin and ubiquitin pathway enzymes. Now people generally agreed that neuronal cell death due to the a-synuclein protein conformational change, the formation of amyloid filaments, abnormal accumulation. The present main studys lie in that the toxic protein biosynthetic pathway, few studies its degradation and metabolic pathways. The ubiquitin-proteasome system (UPS) is an important intracellular not lysosomal degradation system, which can find and destruct these improper assembly and misfolded protein, so that timely degradation of them. UPS dysfunction in PD is a very important link.Continuous abnormal protein aggregation is closely related UPS function defect. The latest study found that:the endoplasmic reticulum stress is a vital link in this process. When severe or prolonged endoplasmic reticulum stress damages the function of the endoplasmic reticulum it can lead to apoptosis. There are three ways in the cell apoptosis induced by the endoplasmic reticulum stress: the activation transcription of the CHOP/GADD153gene; JNK’s activation pathway:Endoplasmic reticulum-specific cysteine protease caspase-12activation. In addition, the study also showed that the endoplasmic reticulum stress response iconic protein is GRP78/Bip upward in early the disease expression. GRP78/Bip upward is a sign of the ERS, and long-term stress start the apoptosis pathway, damage cells.Today, ERS and UPS has become a research abroad hot, but it has not yet developed drugs that can effective interventions for ERS and to improve UPS function. Domestic Chinese medicine is still blank about the intervention of the ERS and UPS yetModern medical therapy for this PD disease, dopamine and dopamine receptor agonists can get significant early results, but there are the side effect serious, expensive, long-term application inducting gradual resistance. Therefore, actively explore the Chinese medicine treatment is important signifiant.This PD disease belongs the category of fibrillation syndrome in traditional Chinese medicine the areas. Tutor professor Wu Zhengzhi have a new view about the pathogenesis of this disease. That is this diseases happen to root he muscles, the four main symptoms; tremor, muscle rigidity, slow movement, gait abnormalities and these symptoms are relate closely to the muscle.Spleen in charge of muscles according to Chinese medicine theory, the disease treatment should be to start from Spleen. The happen of this disease, it’ s basic cause lie in the spleen. Spleen weak can not metabolism wet and moist. Moist properties is viscous. and they are easy to choke Spleen funtions. Wet stops in muscles, gas actve is chocked so the body weight, slow movement; spleen weak not product enough of qi and blood and induct that blood not raised muscle ficiency. Blood deficiency must induct the wind so the tremors, muscle rigidity, gait abnormalities. The famous Chinese medicine encephalopathy experts also think the spleen is the root about this disease. Liver is the external performance ". Therefore, the key to the pathogenesis of this disease is wet trapped spleen and spleen metabolism function is resistanced, muscle loses its functions. Treatment key is to improve spleen active; remove wet and moisture in spleen; to eliminated spleen qi chock. Supplemented by promoting blood actives and raise Liver to reach stoping spasm Magnolia extract is the screening optimization out of the natural monomer of Chinese medicine. which is choiced by tutor Professor Wu Zhengzhi during he participates and bears the15national scientific and technological key project "Comprehensive treatment optimization scheme research in Parkinson’s disease treatment with combining traditional Chinese and western medicine" of work time, and upon bases the whole system of ancient and modern literature related to prescription, combined with years of research and clinical experience, and according to new proposed traditional Chinese medicine pathogenesis hypothesis. In years of clinical application Magnolia extract curative effect is distnnct for the early or mid-Parkinson’s disease patient. and also has certain curative effect for the late Parkinson’s disease patients.Research purpose The subject Combined with a latest new dynamic and the latest new progress of Parkinson’s disease research. continuous injection of MPTP can induce animal model which have simulate the human Parkinson’s disease chronic process and Lewy body formation pathological features so as to investigate the role of ERS, UPS in Parkinson’s disease occurrence and development. sometimes observed Magnolia extract’s impact for the ERS, UPS and Lewy body formation in the model of Parkinson’s disease, to reveal its treatment of PD disease intervention targets and molecular mechanism and to provide experimental basis for the further developing treating PD Traditional Chinese new Drug and open up new ideas for the clinical treatment.Methods(1) Establishment of PD model mouse and experimental groupsSelect the health of48male C57BL/5J mice (9weeks old) were randomly divided into three groups, the PD model group of16; normal control group of16; traditional Chinese medicine group16.(2) Observation of animal behaviorAfter injection of7d,14d and21d, by0.25mg/kg intraperitoneal injection apomorphine we observed the changes in the rotational behavior that were average recorded30min. at the same time we observe the tremors、slow activity、grip、sniffing and other abnormal behavior.(3) Immunohistochemistry detection of the nigral TH、GFAP、ox-42After21days injection, under2.5%sodium pentobarbital anesthesia the4%formaldehyde is pour into the left ventricle intubation.After perfusion, mice brains were removed. Through a series of biochemical fixed.by near substantia nigra continuous coronal sections, picked up the symmetry of the substantia nigra slice we do the TH and GFAP immunohistochemistry and observed the changs of tyrosine hydroxylase(TH)-positive cells, astrocytes (GFAP) and OX-42in the substantia nigra as to understand the DA neuron damage and whether selective damage to dopaminergic neurons and GFAP, OX-42whether that have activation. And then, by a series of biochemical immune markers, slices is set microscope, counting both sides of the substantia nigra TH-positive cell number.(4) Immunohistochemistry detection of the Lewy body、a-synuclein Parkin and ubiquitinExpression of aggregated protein of Lewy body and its main component of a-synuclein, Parkin. And ubiquitin are observed by immunohistochemistry staining in DA neurons. Through the series of biochemical immune markers again, slice is put under fluorescence microscopy, dimensional microscopy image analysis system and do quantitative detection. Model group normal group and Chinese Medicines group of the processing time and dose are the same.(5) substantia nigra organization Caspase-12mRNA expression by RT-PCR detection:GenBank retrieval mice Caspase-12sequence, self-designed primers were synthesized by Shanghai Sangon Company. Caspase-12upstream primers (506bp):5’-the AGGC AACTCTCATGCAGTTC-3’(1319-1338bp), downstream:5’-AACCATGTATGCCAGAG AC C-3’(1847-1866bp). Taking the total RNA of4ul do series of biochemical treatment, Taking PCR amplification product10μ1line do agarose gel electrophoresis, by gel imaging system scan Caspase-12gray value. Compare the caspase-12mRNA expression of the substantia nigra in each group (6) Western blot detective the substantia nigra organizations Caspase-12, CHOP and Bip/GRP78protein expression:80μg black mass organization do the series biochemical enzyme mark after Chemiluminescence reagent reaction X film exposure, after development and upper fuser processing observing the results. Touching2002gel imaging system scan images analysis the gray degrees. Comparison of the substantia nigra zone of Caspase-12, Bip/GRP78’s expression(7) Quantitative analysis of microscopic images:The Laboratory of Intelligent automatic microscopic image quantitative analysis system does quantitative analysis.(8) Statistics:Statistical data using the mean (x)±standard deviation (s), using SAS software package do single factor analysis of Variance analysis and inspection, to P<0.05says there are significant differences. Results1. The animal behaviorAbdominal cavity continuous injection of MPTP has been successful got chronic course of PD mouse model, results in mice behavioral experiments tip:Magnolia extract could improve limb motor coordination on the MPTP-induced PD mouse model PD motor signs has significantly improved.2. Nigral TH、GFAP、OX-42immune detectionThe results showed:Compared with the control group, the substantia nigra TH expression in PD model mice was significantly lower (P<0.01), indicating that TH-positive neurons have a significant loss, Tip DP neurons apoptosis obvious; Magnolia extract group’s mice TH positive cells have not significantly reduced. and tip Magnolia extract group DP neurons is protect by the Chinses medicine to not obvious injury. GFAP was not significantly changed (P>0.05)、OX-42expression increases tip MTPT has selectively damaged DP neurons.3. Lewy small body, a-synuelein, Parkin and ubiquitin immunofluorescence double-labeled detectionThe results show that:Compared with model group, Magnolia extract group substantia nigra cell apoptosis have not significantly reduced (P<0.01),Magnolia extract group of a-synuclein, the ubiquitin-positive cell counts were reduced (P <0.01) showed that the toxicity protein a-synuclein that is generated by the endoplasmic reticulum reduced. and toxic protein’ s UPS degradative pathway is normal. these responses Show that the ERS function get protective interventions and the functional activity of UPS. is good, and also tip that Magnolia can inhibit apoptosis the substantia nigra cells of PD mouse model, and protect and improve the ERS and UPS function.4. RT-PCR detect the substantia nigra organizations Caspase-12mRNA expressionRT-PCR results:(1) PD model Group:Caspase-12mRMA expression levels are significantly higher than the control group.(2)Magnolia extract group:the expression of Caspase-12mRMA expression levels compared with the model is significantly reduced. The level of Caspase expression-12mRMA tips endoplasmic reticulum stress response of the level. Tiping intraperitoneal injection of MPTP can induced the endoplasmic reticulum stress response of substantia nigra neurons.Magnolia extract can ease endoplasmic reticulum stress.5. Western blot protein expression of caspase-12, CHOP, Bip/GRP78in substantia nigra zoneThe results showed that Western blot analysis of model mice substantia nigra Caspase-12, and CHOP protein expression levels that compared with normal control group was that expression of Caspase-12significantly rise(P<0.01). CHOP protein levels upward tips the endoplasmic reticulum stress response in the neurons of the substantia nigra is improved. Magnolia extract group Caspase-12and CHOP protein levels that compared with the model group was significantly lower (P<0.05),which tip magnolia extract has apparent ease for the endoplasmic reticulum stress response. In the substantia nigra organization of model mice, GRP78/Bip protein expression compared with normal control group increase, GRP78/Bip is the iconic protein of the endoplasmic reticulum stress:GRP78/Bip of increase is ERS ease a sign; long-term stress to start the apoptosis pathway, damage cells. GRP78/Bip protein expression of Magnolia extract group was significantly up-regulated compared with model group (P<0.05). The above results suggested that intraperitoneal injection of MPTP induced substantia nigra neurons’s endoplasmic reticulum stress response and the Magnolia extract has obvious ease for this stress response.Conclusions Mice behavior showed that the mode by MPTP injecting induced was successful; Magnolia extract can improve limb motor coordination of mouse model induced by MPTP-induced with PD movement signs. Endoplasmic reticulum stress and the ubiquitin-proteasome system may be the PD dopaminergic neuronal apoptosis key factors. Endoplasmic reticulum stress adjustment in the early has contained activity of the ubiquitin-proteasome system degradation to abnormal proteins. ERS and UPS promote action each other in order to achieve the dynamic balance of the endoplasmic reticulum protein synthesis and degradation, and ensure the stability of the internal environment. Thought interventing to substantia nigra organization Caspase-12mRNA expression and caspase-12, CHOP, Bip/GRP78protein of target, Chinese medicine Magnolia achieves to impact and improvement for the Endoplasmic reticulum stress response (ESR) in order to reduce the cytotoxicity of protein synthesis. By the a-synuelein, and ubiquitin protein targets of intervention Chinese medicine Magnolia achieves to impact and improvement for the ubiquitin-proteasome system (UPS) and improve the ability ahout detecting and destructing the inproper assembly and misfolded proteins, so that they degradate and eliminate of the cytotoxic proteins in a timely manner. therefore they improve the protection for the nigral dopamine (DA) neurons nerve cells, reduced apoptosis. Thus, Chinese medicine Magnolia has a significant protective effection on dopaminergic neurons.