| Background and objectiveParkinson’s disease(PD)is a worldwide common central nervous system neurodegenerative disease,and its incidence and prevalence are only second to Alzheimer’s disease.The incidence of Parkinson’s disease is related to age,and is more common in the elderly.Patients before the age of 50 are rare,and the prevalence and incidence of Parkinson’s disease are rapidly increasing in people over the age of 60.Studies have shown that the prevalence of Parkinson’s disease in people aged 50-59 is 107/100,000,and the prevalence of Parkinson’s disease in people aged 70-79 can reach 1087/100,000.The pathogenesis of Parkinson’s disease is a chronic process.The symptoms of early Parkinson’s disease are mainly non-motor symptoms,which are characterized by olfactory dysfunction,sleep disorders,pain symptoms,fatigue,mental symptoms,cognitive disorders,and autonomic dysfunction.As the disease progresses,Parkinson’s disease patients have typical motor symptoms,including instability of walking gait,increased muscle tone,bradykinesia,and resting tremor.Parkinson’s disease causes serious economic losses to the society,and it also causes a decline in the living quality of patients with Parkinson’s disease.Therefore,Parkinson’s disease has become one of the major diseases that endanger human health.The progression of Parkinson’s disease is a long-term phase.It can last for several years from the appearance of early non-motor symptoms to the late typical motor dysfunction.We call this period the prodromal stage of motor symptoms.Some scholars have shown that the prodromal stage of motor symptoms is also belong to the early pathological progress of Parkinson’s disease.The typical motor symptoms are not obvious in the early stage since the dopaminergic neuron cells in the nigrostriatal system are less affected and he body has a corresponding compensatory mechanism.When the substantia nigra dopaminergic neurons are reduced by more than 50%and the striatal dopamine levels are reduced by more than 70%,typical motor symptomsand systemic symptoms begin to appear which is the late stage of Parkinson disease.The prodromal stage of motor symptoms provides a valuable opportunity for the prevention and treatment of Parkinson’s disease.Effective prevention and treatment can delay or even reverse the progression of Parkinson’s disease.Parkinson’s disease is a complex neurological disease,and its specific pathogenesis is still unclear.Several factors such as genetic factors,environmental factors,age factors,and history of traumatic brain injury are associated with Parkinson’s disease.A large number of epidemiological and toxicological studies have shown that environmental factors,especially various neurotoxins in the environment,are closely related to the pathogenesis and progression of Parkinson’s disease.Two factors are mainly involved in the progression of Parkinson’s disease.First,dopaminergic neurons loss spontaneously with the increase of age.The other is the neurotoxins in the environment accelerate the aging process of dopaminergic neurons,and the rate of loss of dopaminergic neurons is significantly accelerated.MPTP is the first neurotoxin found to induce Parkinson’s disease.The discovery of MPTP caused people to focus on the correlation between environmental factors and Parkinson’s disease.MPTP itself is not neurotoxic.MPTP is metabolized to MPP+ in vivo,and the main target of MPP+destruction in the body is dopaminergic neurons.MPTP inhibits the function of the mitochondrial complex I of the oxidative respiratory chain,thereby impairing mitochondrial function of dopaminergic neurons and causing oxidative stress.Since the discovery of MPTP,more and more MPTP analogs have been discovered,including herbicides,alkaloids,and beta-carbolines.In this study,TaClo,which has a similar structure to MPP+ belongs to β-carboiines.TaClo is a metabolite of trichloroethylene in the body.Since trichloroethylene and TaClo are more soluble in fat,they are easier to penetrate the blood-brain barrier than MPTP.The neurotoxicity of TaClo is stronger than MPP+.In 1995,Bringmann G.et al first discovered that TaClo can cause symptoms of Parkinson’s disease.TaClo is spontaneously synthesized by biogenic amines(Ta)and trichloroacetaldehyde(Clo)in vivo.Trichloroacetaldehyde is metabolized by trichloroethylene in the body.Trichloroethylene is a commonly used industrial organic solvent,and is often used in pesticides,extractants,detergents,etc.The main sources of trichloroethylene in natural environment are industrial wastewater pollution and air pollution.Long-term exposure to trichloroethylene can lead to spontaneous synthesis of TaClo in humans.Kochen W.et al tracked three patients with Parkinson’s disease who had been exposed to trichloroethylene for a long time and found that the blood levels of TaClo in the three patients reached the nanogram level.Gash DW.also reported that a cluster of 30 industrial co-workers exposed to trichloroethylene for 8-33years developed Parkinsion’s disease.The content of TaClo in the body will increase with the prolonged exposure time since TaClo is difficult to be metabolized out of the body after spontaneous synthesis.When the neurotoxic effects of TaClo accumulate to a certain extent,it will cause typical motor symptoms of Parkinson’s disease.Bringmann G.et al further studied of TaClo in vivo and showed that TaClo induced disruption of dopaminergic neurons is associated with disruption of mitochondrial complex I on oxidative respiratory chain.Since the discovery of the neurotoxicity of TaClo,it has quickly attracted widespread attention.Many in vitro and in vivo studies have demonstrated that TaClo can disrupt the nigrostriatal system,lead to a decrease of dopaminergic neurons and induce symptoms of Parkinson’s disease.At present,researches on the mechanism of TaClo induced Parkinson’s disease mainly showed that TaClo inhibits mitochondrial complex 1,induces the production of reactive oxygen species and inhibits mitochondrial function.Bing G.et al studies C57BL/6 mice which were given trichloroethylene by intragastric administration per day for 8 months and found that there was a decrease in dopaminergic neurons in the substantia nigra which was accompanied by activation of microglia in the substantia nigra.These pathological changes are caused by TaClo,not directly caused by TCE.However,how TaClo destroys the nigrostriatal system,the molecular biological mechanisms under TaClo induced loss of dopaminergic neurons,and TaClo-induced pathological changes are still lacking further research.This study conducts a preliminary study on how TaClo causes dopaminergic neuron reduction and how it induces the pathological changes of Parkinson’s disease by in vitro and in vivo experiments.It aims to provide new ideas for the prevention and treatment of Parkinson’s disease.ContentPart 1:We investigated the cytotoxic effect of TaClo on the dopaminergic cell line SH-SH5Y and the pathway of apoptosis induced by TaClo was by in vitro experiments.Part 2:By using stereotactic injection technique,we constructed a TaClo rats Parkinson’s disease model to explore the toxic effects of TaClo on the nigrostriatal system and the pathological changes induced by TaClo.MethodsPart 11.Cytotoxicity of TaClo on SH-SH5Y cell line was detected by CCK-8 and cell viability assay.Then,the mitochondrial oxidative stress in SH-SH5Y cell line stimulated by different concentrations of TaClo was detected by mitochondrial ROS fluorescent staining kit and mitochondrial membrane potential JC-1 staining kit.2.The apoptosis of SH-SH5Y induced by TaClo was detected by TUNEL staining and flow cytometry.3.Western Blot was used to explore the pathways and mechanisms of TaClo-induced apoptosis in SH-SH5Y.Part 2:1.The relationship between morbidity and oxidative stress and inflammatory response-related gene expression in patients with Parkinson’s disease was analyzed by a data set(GSE7621-GPL570)on the gene expression of the substantia nigra in patients with Parkinson s disease in the GEO public database.2.By using stereotactic injection technique,TaClo was injected into the striatum of male Wistar rats and the rats in the control group were injected with the solvent.Then,Open field experiment wae used to test behavioral of Wistar rat model.On the 28th day,the rat striatum dopamine neurotransmitter levels were detected by liquid chromatography-mass spectrometry to determine whether the Wistar rat Parkinson’s disease model was successfully modeled.3.The reduction of tyrosine hydroxylase-positive cells in the substantia nigra of the experimental group and the control group was detected by Western blot,immunofluorescence staining andimmumohistochemical staining and the damage of nerve was observed by Nissl staining.4.Flow cytometry was used to detect TH+ cell ROS levels in the nigrostriatal system of experimental and control groups.Meanwhile,JC-1 staining was performed on the substantia nigra to observe the effect of TaClo on mitochondrial membrane potential.5.Immunohistochemical staining,RT-PCR and western blot were used to detect the activation of microglia in the substantia nigra and the expression of inflammatory factors,including IL-6,TNF-α,iNOS and Cox-2,and the expression of apoptosis protein Caspase-3 and activated Caspase-3 of experimental and control groups.6.By using the diffusion tensor imaging technique(DTI),the nerve fibers of the nigrostriatal pathway system were subjected to nerve fiber tracing,and the ADC and FA values of the nigrostriatal system were compared.The nerve fiber myelin sheath was stained by LFB to observe the changes of neuromyelin.7.After the intervention of antioxidant NAC in the successfully constructed Wistar rat Parkinson’s disease model,the level of inflammatory factors and apoptotic proteins in the substantia nigra were analyzed by western blot.ResultsPart 1:1.TaClo inhibited the proliferation and cell viability of SH-SH5Y cell line and induced mitochondrial oxidative stress in SH-SH5Y cell line which increased mitochondrial ROS level and decreased mitochondrial membrane potential by concentration gradient.2.Flow cytometry showed that TaClo induced an increase in the proportion of apoptosis in SH-SH5Y cells,and the results of TUNEL staining also showed that TaClo induced apoptosis in SH-SH5Y cells.3.Western blot analysis showed that TaClo induced an increase in the ratio of Bax/Bcl-2 in SH-SH5Y cells,and induced Caspase-9 and Caspase-3 activation.After the inhibition of NAC,the effect of TaClo was inhibited Those results indicated that TaClo induced mitochondrial apoptosis in SH-SH5Y cells.Part 2:1.By analyzing the dataset(GSE7621-GPL570)on the gene expression of the substantia nigra in patients with Parkinson’s disease in the GEO public database,the expression of some inflammatory factors in the substantia nigra of patients with Parkinson’s disease was elevated including IL-6、iNOS、Cox-2 and NF-κB.The expression levels of some genes related to oxidative stress also increased including GABPA and Keap 1.These results indicate that both oxidative stress and inflammatory responses are associated with Parkinson’s disease.2.The behavioral test results showed that the trajectories of the experimental group were significantly less than those of the control group on the 21st and 28th day after the modeling operation.And the rats in the experimental group showed symptoms of bradykinesia,and the range of motion gradually narrowed.The results of striatum dopamine levels showed that the dopamine level in the experimental group was significantly lower than that in the control group.These results show that the rat Parkinson’s disease model was successfully modeled.3.Western blot,immunofluorescence staining and immumohistochemical staining showed that the TH+ positive cells in the substantia nigra of rats in the experimentalgroup were significantly reduced.At the same time,the results of Nissl staining showed that the Nissl body was reduced in the substantia nigra of the experimental group.These results indicate that TaClo causes nerve damage and a decrease of dopaminergic neurons in the substantia nigra.4.Flow cytometry showed that the ROS level of TH+ cells in the substantia nigra of the experimental group was significantly higher than that of the control group,and JC-1 staining showed that the mitochondrial membrane potential decreased in the experimental group.The results indicate that TaClo induces mitochondrial oxidative stress in dopaminergic neurons and causes early apoptosis in the substantia nigra.5.Iba-1 Immunohistochemical staining results showed that TaClo induced activation of microglia in the substantia nigra.At the same time,RT-PCR and western blot showed that the expression levels of inflammatory factors IL-6,TNF-α,iNOS and Cox-2 in the substantia nigra of rats in the experimental group were higher than those in the control group.The results indicated that TaClo induced an inflammatory reaction in the substantia nigra of rats.6.The diffusion tensor imaging technique showed that the neurofibrillary of the nigrostriatal pathway was apparently reduced in the nigrostriatal pathway.The parametric analysis of the nigrostriatal pathway nerve fibers showed that the ADC value of the experimental group was significantly increased,indicating that the lateral water molecule dispersion of the nerve fibers increased which was related to the demyelination of nerve fibers.The results of LFB neuromyelin staining showed demyelination in the substantia nigra of rats in the experimental group.These results suggest that TaClo-induced Parkinson’s disease is associated with pathological changes of demyelination of neurons in the nigrostriatal system.7.Western blot showed that NAC intervention can reverse the neurotoxic effects of TaClo,reduce the level of inflammatory factors and the expression of apoptosis-related proteins.Preliminary experiments confirmed the therapeutic effect of antioxidants against TaClo neurotoxicity.Conclusion1.In vitro,TaClo activates the endogenous mitochondrial apoptotic pathway to induce apoptosis in SH-SH5Y cells.ROS inhibitors can attenuate TaClo-induced apoptosis and inhibit TaClo-induced activation of the mitochondrial apoptotic pathway.2.In vivo,the neurotoxin TaClo participates in the progression of Parkinson’s disease by mediating mitochondrial oxidative stress in dopaminergic neurons and abnormal activation of microglia,which promotes apoptosis of dopaminergic neurons in the nigrostriatal system.ROS inhibitor NAC can attenuate TaClo-induced inflammatory responses and apoptosis3.TaClo induces demyelinating pathology of never fiber in the nigrostriatal pathway,which leads to a decrease in dopamine levels in the striatum and induces typical motor symptoms of Parkinson’s disease. |
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