| BackgroundParkinson’s Disease(PD) is one of the common movement disorders. PD severelyaffects the life qualities of the patients, but there isn’t any breakthrough for therapy up tillnow. The pathogenesis of PD is still unclear. Now it is considered that PD is not a kind ofdisease caused by a single factor, but by the interactions of multiple factors, leading toneuronal degeneration and loss of numerous dopaminergic(DA) neurons in substantia nigra(SN). The net effect of dopamine depletion is to produce hypokinesia, an overall reductionin motor output. Studies on the molecules that are possibly involved in the occurrence andsurvival of DA neurons also attract considerable attention.The normal expressionEngrailed1(EN1), a homeodomain transcription factor,is very important for thedevelopment, survival and maintenance of function of the DA neurons of midbrain. What’smore, it probably has special neuroprotective effect. Researches confirm thatKeap1-Nrf2-ARE pathway changed during PD. Keap1-Nrf2-ARE signalling pathway playsan important role in the modulating of oxidative stress and toxicant metabolism。Nrf2incytoplasm is transferred to the nucleus through a specific signal,then it activates theexpressions of detoxification enzymes and antioxida nt enzyme containing ARE,and protectcells from oxidative stress injuries.After being transferred from the cytoplasm to thenucleus, Nrf2activates the expressions of detoxification enzymes and antioxidant enzymescontaining ARE, and then protects cells from oxidative stress injuries. a-SYN is the maincomponent of LB. The abnormal aggregation of a-SYN within neurons of PD patients maybe associated with death of DA neurons in PD. At present it is believed that thetranscription factors Nrf2,EN1and a-SYN are closely associated with occurrence andprogress of PD. However, there are few reports on characters of morphological expression ofEN1,Nrf2and a-SYN in PD, as well as the relationship between them. ObjectiveTo investigate the spontaneous activities of PD mice models, we used the open-fieldtest. Considering the transcription factors which contribute to the development and survival ofthe DA neurons, we researched the possiblely effective factors in pathogenesis ofPD-EN1,Nrf2and α-SYN by characterizing their expressions and the sequence of abnormalexpressions.We explored the underlying relationships among them and potentialmechanisms in the injuries of DA neurons, hoping to find theoretical basis for thedevelopment and clinical therapy of PD.Methods9week-old C57BL/6mice with both sexes were used in our study. PD mice modelswere induced by unilateral(the right striatum) stereotaxic intra-striatal6-OHDA injections.In the first place, to examine the spontaneous behaviors of PD mice models.The mice incontrol group(n=10) received no treatment while in the model group(n=10), they acceptedPD surgery and were managed to survive. Then the5minute open-field test was used toassess behaviors of two groups in the following7days,and electronic scale were applied todetect their wights. In the second place, to feature the expressions of molecules-EN1,Nrf2and α-SYN,which were probably involved in the development of PD.Expressions ofEN1,Nrf2,α-SYN and tyrosine hydroxylase (TH) in the midbrains of PD mice models wereexamined in specific time point-3/6/9/12/15/18/21/24hours(n=6/time point) byimmunohistochemistry and immunofluorescence. The aim is to learn about their changingcharacteristics and whether they colocalized in DA neurons.The target proteins expressionsin specific time point-18h/24h/3d (n=8/time point) were measured through Western Blot.Results1. Alterations of Spontaneous behaviors of PD mice models in open-field test①Compared with the control group, wights of mice in the model group transientlydecreased after6-OHDA injection and recovered rapidly.②Mice in the model groupshowed persistent rotation to the experimental side(the right side), but their motor functionrecovered to normal gradually in7days. Their trails were also abnormal.Their totaldistance travelled following the operation descended on first day and showed a gradualrecovering trend,but there was no statistically differences compared with control group(P＞0.05). The time that they spent in the centre-zone gradually reduced and was significantlydifferent from control group7days later(P<0.05). 2. Characteristics of expressions of EN1,Nrf2,α-SYN in midbrains of acute PD micemodels⑴morphological results:①Compared with the control side, TH-positive DA neuronsin the front midbrain of experimental side were progressively decreased with the time afterinjection of6-OHDA. In6h, the number of TH positive cells was similar to that in thecontrol side, and in12h, the difference has reached statistical significance.②In theexperimental side, expression of α-SYN in DA neurons in SN pars compacta wassignificantly increased in6and12hs after injection of6-OHDA,with visible aggregation inthe cytoplasm.③EN1positive cells in the experimental side were continuously decreasedafter surgery. The expression of EN1were initially decreased in3h, and severely decreasedin6h. In the midbrain,EN1were mainly localized in the nucleus and sometimes in thecytoplasm.EN1positive cells were largely distributed in the DA neurons in mesencephalicSN and VTA,and occasionally in some neurons in other areas.④The alteration ofexpressions of target proteins-TH、α-SYN、EN1in mesencephalic SN pars compacta andVTA began from front(near the striatum) to back.(2)WB: Compared with the control side,in the acute period after injection (18h/24h/3d),WB testing of target protein TH in total experimental side of the midbrain showed nostatistical changes,while the expression levels of Nrf2/α-SYN were significantly higher.Conclusion1. The behavioural impairment and changes in emotion of mice models confirmsuccess of models.2.⑴D ecrease of expression of EN1is followed by the change of α-SYN,TH.Thismay show that gathering of α-SYN is related to the death of DA neurons, and that the downregulation of EN1may lead to the high expression of α-SYN.(2)We verified the expressionof EN1in cytoplasm in the midbrain from the morphological point of view.⑶The alterationof expressions of target proteins-TH,α-SYN,EN1in mesencephalic SN pars compacta andVTA began from front(near the striatum) to back.⑷Increasing in Nrf2expression indicatesthe presence of oxidative stress and alteration in Nrf2-ARE pathway.Our study is the first to determine the changes of expressions of EN1,Nrf2,α-SYand TH in the midbrain and the sequence of these changes. Also, we discuss the relationshipbetween the four factors and their possible roles in DA neurons injury and PD. Our results confirm the expression of EN1in cytoplasm in the midbrain, and may show the possibilityof EN1as peptide signals between cells.We hope that this could provide theoretical basisfor clinical treatment of PD. |
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