| Objective Firstly,the effects and mechanisms of early acupuncture intervention on the pathogenesis of Parkinson's disease were investigated from the prodrome gastro intestinal dysfunction of the model mice.Secondly,to explore the projection relatio nship between the substantia nigra and the motor dorsal nucleus vagus,and to furth er explore the neuro-anatomical mechanism of Parkinson's gastrointestinal dysfuncti on.Finally,Parkinson's cell model was established in vitro to investigate the effect and molecular mechanism of rotenone-induced microglial cell activation in promotin g dopaminergic neuron injury.Methods 1.Long-term low-dose subcutaneous injection of rotenone induces PD mo use model to reproduce Parkinson's typical neuropathy and gastrointestinal symptoms.Through continuous observation of symptoms and signs of PD model mice,as wel l as tests of fecal water content,intestinal propulsion rate and neurobehavior at diff erent time points,the correlation between constipation and movement disorders in P D model mice was explored.Second,start acupuncture intervention on building day,observed the curative early intervention of signs and symptoms of the PD mice in duced by rotenone,neuroethology,intestinal inflammation,colon neurotransmitters,s ubstantia nigra and the vagus nerve movement back nuclear TH,Iba-1 and the infl uence of characteristic of Parkinson's pathology protein expression,from the behavio r,the enteric nervous system,including the central nervous system comprehensive st udy intervention in Parkinson's disease mouse model to the mechanism of action,to explore brain gut axis theory under the guidance of whether can choose acupunctu re point on the central nervous degeneration disease process retard the biological m echanism;2.Nerve tracer and immunofluorescence techniques were used to study the projecti on relationship between the substantia nigra and the dorsal motor nucleus of the va gus nerve,so as to further explore the neuroanatomical mechanism of Parkinson's g astrointestinal dysfunction.3.Choose rotenone as the neurotoxin to treat BV-2 cells,take the supernatant and co-culture with MN9 D cells to establish a PD cell model.Through the inhibition of gene silencing NLRP3 inflammasome,with a determined by MTT method to det ect BV-2,MN9 D cells activity;ELISA to detect inflammatory factor,IL-1?,TNF-?,IL-6,IL-8 levels;Weste-blot method detects protein levels such as NLRP3,pro-Caspase-1,and caspase-1;The morphological changes of microglia cells were analyzed by i mmunofluorescence staining and the changes of neuronal apoptosis and membrane p otential were detected by flow cytometry.To explore the role and molecular mecha nism of activation of NLRP3 inflammasome in rotenone-induced activation of micro glia cells to promote DA-induced neuronal injury.Results 1.The study found that rotenone successfully reproduced the brain patholog y and non-motor gastrointestinal dysfunction of PD.And electroacupuncture interven tion significantly increased the spontaneous activity of PD mice,played a role in i mproving motor disorders.Electroacupuncture significantly increased the expression of tyrosine hydroxylase(TH),reduced the expression of Iba-1 in the brain substanti a nigra(SN),showed the effect of alleviating neuropathological damage.In addition,electroacupuncture significantly reduced the deposition of ?-synuclein(?-syn)both in colon and SN,increased the expression of choline acetyltransferase(Ch AT),TH,n NOS,ZO1 tight junction protein(ZO-1)and Occludin in colon,reduced intestinal inflammation,and had a protective effect on the enteric nervous system and intestin al barrier in PD mice model.In conclusion,electroacupuncture has a good effect o n gastrointestinal and motor functions of PD mice and can alleviate neuroinflammati on and neuropathic injury of PD by alleviating intestinal inflammation,promoting i ntestinal barrier repair,and reducing the deposition of ?-syn in colon.2.The anterograde tracer VSV-EGFP was micro-injected into SNC.After 6 days,antero-grade tracer VSV-EGFP labeled the fibers of the whole DVC head and tail.The retrograde tracer cholera toxin subunit B(CTB)was injected into the bilateral DVC.Under fluoresce-nce microscopy,section staining showed that the concentrate d area of CTB was limited to the bilateral DVC,including DMV and NTS.The e nds of CTB markers appeared in the red fluorescent particles,and the fibers labele d across the ganglia terminated at the tail of the lateral NTS.After 14 days,CTB p ositive neurons were found scattered in SNC.In addition,co-localization of TH-IR n eurons at SNC and DMV and anterograde tracer VSV-EGFP showed that these VS V-positive neurons had TH immunoreactivity(IR),indicating their catecholaminergic and dopaminergic neurochemical phenotypes.3.After the treatment of BV-2 microglia with 5 nmol/L rotenone at different time p eriods(0h,6h,12 h,24h),the BV2 cells had obvious morphological changes.BV2 microglia exposed to rotenone could induce the production of a large number of pr oinflammatory factors,in which the levels of IL-1?,IL-8,IL-6 and TNF-? increase d significantly with the prolongation of exposure time,and reached the peak value at 24 h.Rotenone can directly activate NLRP3 inflammasome in BV-2 microglia,and the expression level of activated NLRP3 inflammasome not only increased with th e prolongation of rotenone incubation time,but also increased the expression of oth er components of inflammasome multi-prote in complex;NLRP3 silentation can slo w down the activation of BV-2 cells,inhibit the secretion of IL-1?,IL-8,TNF-? in flammatory factors,and reduce the activation of caspase-1 induced by rotenone;Si R NA-NLRP3 alleviates the damage of rotenone to the activity of MN9 D neurons to a certain extent,and protects the morphological and structural integrity of MN9 D n eurons.In addition,si RNA-NLRP3 significantly inhibited the expression of ?-synucl ein in MN9 D neurons and increased the expression of TH and Neurofilament.Conclusion 1.This study found that electroacupuncture improved the motor and ga strointestinal dysfunction in PD model mice,mainly manifested as increased spontan eous activity and reduced fecal moisture.In addition,electroacupuncture relieved int estinal inflammation,promoted intestinal barrier repair,reduced intestinal?-syn deposition,thus slowed down neuroinflammation and improved neuropathy of PD.Electroacupuncture plays an important role in improving neuroinflammation and motor dysfunction of PD mice.2.In this study,a single synaptic substantia nigra-vagus pathway was identified i n C57 mice using neural tracer techniques,demonstrating that the vagal nerve com plex,including the motor dorsal and solitary tract nuclei,receives direct projection from substantia nigra neurons.3.The BV2 and MN9 D cell lines simulation principle of Parkinson's disease,in vitro experiments confirmed that rotenone could be induced by activation of NLRP3 inflammasome microglia activation,the release of proinflammatory factor,and fu rther lead to dopamine neurons apoptosis,and found that silence NLRP3 inflammat ory corpuscle can suppress the MN9 D cells apoptosis induced by rotenone and infla mmatory cascade reaction. |
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