环境毒性物MPTP帕金森病模型中硫氧还蛋白抑制内质网应激的分子机理

发布时间：2018-02-03 09:13

本文关键词： 帕金森病 MPP~+/MPTP 内质网应激硫氧还蛋白分子机理　出处：《昆明理工大学》2014年博士论文　论文类型：学位论文

【摘要】：帕金森病(Parkinson's disease, PD)是一种常见的神经退行性疾病,其病理特征是中脑黑质区(substantia nigra pars compacta, SNpc)多巴胺能神经元进行性变性坏死,导致多巴胺水平降低。PD的分子机制已经被广泛的研究,然而仍不清楚。大量的研究表明氧化应激(oxidative stress)和内质网应激(endoplasmic reticulum stress, ER stress)与PD的发病机理有关。未折叠蛋白在内质网的积累激活了通路蛋白的转录即未折叠蛋白应答(unfolded protein response, UPR)。内质网应激引发两个细胞保护应答反应,一方面是减少蛋白质的合成,另一方面是促进蛋白质折叠分子伴侣基因的表达上调。UPR的主要信号通路包括：(1)肌醇需求酶1(inositol-requiring enzyme1, IRE1)活化拼接X-box结合蛋白1(X-box binding protein1, XBP1)的mRNA;(2)活化转录因子(activating transcription factor6, ATF6)从内质网释放,活化后进入细胞核上调内质网应答相关基因转录水平；(3)蛋白激酶RNA样内质网激酶(protein kinase RNA-like ER kinase, PERK)活化使真核生物翻译起始因子2a(eukaryotic translation initiation factor2a, eIF2a)发生磷酸化,导致翻译起始抑制。如果内质网应激持续时间过长,UPR就会引发末期事件,如内质网Ca2+释放以及促凋亡通路的活化,包括肿瘤坏死因子受体相关因子2(tumor necrosis factor receptor associated factor2,TRAF2)、凋亡信号调节激酶1(apoptosis signal regulating kinase1, ASK1)和c-jun N-末端激酶(c-jun N-terminal kinase, JNK),最终导致细胞死亡。硫氧还蛋白-1(Thioredoxin-1, Trx-1)是一个12kDa的多功能蛋白,含有一个保守的活性位点序列：-Cys-Gly-Pro-Cys-。越来越多的研究表明Trx-1可以抵抗细胞损伤和环境应激而起细胞保护作用。Trx-1能够清除单氧自由基和羟自由基。Trx-1可以保护细胞免于过氧化氢(hydrogen peroxide, H2O2)、紫外线照射和局部脑缺血引起的损伤。我们以前的研究表明MPP+处理大鼠肾上腺嗜铬细胞瘤PC12细胞显著降低Trx-1的表达,并且Trx-1高表达减轻了MPP+诱导的神经毒性,Trx-1高表达抑制内质网凋亡分子caspase12的表达,然而它们的分子机制仍不清楚。帕金森病毒性化合物1-甲基-4-苯基-1,2,3,6-四氢吡啶(1-Methy-4-phenyl-1,2,3,6-tetrahydropyridine, MPTP)及其活性代谢物1-甲基-4-苯基吡啶离子(1-methyl-4-phenylpyridinium ion, MPP+)常被用来构建PD体内和体外的实验模型。在本论文中,我们研究了MPP+处理PC12细胞对葡萄糖调节蛋白78(Glucose Regulated Protein78, GRP78)、IRE1α、TRAF2、C/EBP同源蛋白(C/EBP homologous protein, CHOP)、含半胱氨酸的天冬氨酸蛋白水解酶原12(pro-cysteinyl aspartate specific proteinase-12, procaspase-12)和JNK表达的影响。我们还进一步研究了Trx-1对GRP78、IRE1α、TRAF2、 CHOP、procaspase-12和JNK的调节作用。本论文的主要研究结果如下： (1)MPP+诱导PC12细胞发生内质网应激。0.3mM的MPP+处理PC12细胞不同的时间(0、2、6、12和24h或0、15、30、45和60min)后,用Western Blot检测MPP+对GRP78、 IRE1α、TRAF2、CHOP、procaspase-12和JNK表达的影响。结果显示MPP+以时间依赖的方式诱导GRP78、IRE1α、TRAF2和CHOP的表达和procaspase-12的切割活化,MPP+处理在很短的时间内就活化了JNK。为了确认ER凋亡途径的执行者procaspase-12是否是被JNK活化,用JNK的抑制剂SP600125预处理PC12细胞30min,再用MPP+刺激24h,结果显示procaspase-12的活化被SP600125抑制。 (2)Trx-1调节MPP+诱导的细胞毒性。MPP+处理显著降低了PC12细胞的存活率,MPP+以时间依赖的方式降低PC12细胞中Trx-1的表达,且MPTP降低野生型C57BL/6小鼠SNpc区Trx-1的表达。为阐明Trx-1是否在抑制MPP+毒性中起重要作用,转染siRNA降低Trx-1表达或转染GFP-Trx-1质粒使Trx-1高表达,用MTT分析检测Trx-1低表达或高表达对MPP+毒性作用的影响。转染Trx-1siRNA或GFP-human Trx-1质粒对PC12细胞无明显的细胞毒性。Trx-1表达下调增加了PC12细胞对MPP+诱导毒性的敏感性。相反,Trx-1高表达抑制了MPP+诱导的细胞毒性。这些结果表明Trx-1在MPP+诱导的细胞毒性中起重要的调节作用。 (3)Trx-1低表达加重MPP+诱导的内质网应激。PC12细胞转染Trx-1siRNA24h, MPP+刺激24h或15min,我们研究了Trx-1表达下调对MPP+诱导的内质网应激的影响。Trx-1敲低后MPP+诱导的GRP78表达进一步增加,表明Trx-1siRNA加重了MPP+诱导的内质网应激。我们也检测了Trx-1siRNA对IRE1α、TRAF2、CHOP、JNK和procaspase-12的影响。Trx-1siRNA进一步增加了MPP+诱导的IRE1α、TRAF2和CHOP的表达以及JNK和procaspase-12的活化。这些结果表明Trx-1siRNA通过增加IRE1α、TRAF2和CHOP的表达以及活化JNK和procaspase-12，，加重MPP+诱导的内质网应激。 (4)Trx-1高表达减轻MPP+诱导的内质网应激。PC12细胞转染GFP-human Trx-1质粒高表达Trx-1,然后检测Trx-1高表达对MPP+诱导的内质网应激的影响。结果显示Trx-1高表达抑制了MPP+诱导的GRP78表达增加。而且MPP+诱导的IRE1α、TRAF2和CHOP的表达以及JNK和procaspase-12的活化也被Trx-1高表达抑制。这些数据表明Trx-1高表达通过抑制IRE1α、TRAF2和CHOP的表达,以及减少JNK和procaspase-12的活化,从而减轻了MPP+诱导的内质网应激。 (5)Trx-1抑制MPTP诱导的内质网应激。我们检测了Trx-1是否能在小鼠模型抑制MPTP诱导的毒性。野生型MPTP组和human Trx-1转基因MPTP组小鼠腹腔注射MPTP(20mg/kg,每天2次,注射7天),野生型对照组和human Trx-1转基因对照组小鼠注射等体积的生理盐水。野生型MPTP组小鼠与野生型对照组相比运动能力显著降低,而human Trx-1转基因MPTP组小鼠与hTrx-1转基因对照组相比运动能力没有降低。在本论文中,我们还检测了Trx-1对MPTP引起的肢体运动损伤的影响。实验结果显示Trx-1高表达改善了MPTP诱导的肢体损伤。为评估多巴胺能神经元的丢失,我们用免疫组织化学和Western Blot分析检测了小鼠SNpc区酪氨酸羟化酶(tyrosine hydroxylase, TH)的表达情况。MPTP处理后,野生型小鼠黑质区TH表达显著降低,而转基因小鼠中Trx-1高表达回复了TH表达。这些数据表明Trx-1高表达保护多巴胺能神经元免于MPTP诱导的神经毒性损伤。与PC12细胞内结果一致,在转基因小鼠中Trx-1高表达显著抑制了黑质区GRP78表达的增加,表明Trx-1高表达减轻了MPTP诱导的内质网应激。并且Trx-1高表达抑制了MPTP诱导的IRE1α、TRAF2和CHOP的表达以及JNK和procaspase-12的活化。这些数据表明Trx-1高表达通过抑制IRE1α、 TRAF2和CHOP的表达,以及JNK和procaspase-12的活化,从而抑制了MPTP的神经毒性。在本论文中,我们系统地在体外和体内研究了Trx-1抑制MPP+/MPTP诱导的内质网应激的分子机制。从实验结果我们得出结论：Trx-1在帕金森病中通过抑制IRE1α、TRAF2、CHOP、JNK和procaspase-12的活化,抑制了内质网应激,从而发挥神经保护功能。
[Abstract]:Parkinson's disease (Parkinson's disease PD) is a common neurodegenerative disease, the pathological feature of midbrain substantia nigra (substantia nigra pars compacta, SNpc) of dopaminergic neurons degeneration and necrosis, resulting in decreased levels of dopamine.PD molecular mechanism has been extensively studied, however, is still not clear.
A large number of studies show that oxidative stress (oxidative stress) and endoplasmic reticulum stress (endoplasmic reticulum stress, ER stress) associated with the pathogenesis of PD. The unfolded protein in the endoplasmic reticulum activates transcription pathway protein accumulation of unfolded protein response (unfolded protein, response, UPR). The endoplasmic reticulum stress triggered two cell protection in response, one is to reduce the protein synthesis, on the other hand is the main pathway to promote the expression of.UPR protein folding molecular chaperone genes include: (1) 1 (inositol-requiring enzyme1 inositol requiring enzyme, IRE1) activated splicing X-box binding protein 1 (X-box binding protein1, XBP1 mRNA); (2) activation transcription factors (activating transcription factor6, ATF6) release from the endoplasmic reticulum, endoplasmic reticulum increased into the nucleus after activation response related gene transcription level; (3) like protein kinase RNA Er kinase (protein kinase RNA-like ER kinase, PERK) activation of the eukaryotic translation initiation factor 2A (eukaryotic translation initiation factor2a, eIF2a) phosphorylation, resulting in inhibition of translation initiation. If ER stress lasts for a long time, UPR will lead to the end of the event, such as the activation of endoplasmic reticulum Ca2+ release and apoptosis pathway, including tumor necrosis factor receptor associated factor 2 (tumor necrosis factor receptor associated FACTOR2, TRAF2), apoptosis signal regulating kinase 1 (apoptosis signal regulating kinase1, ASK1) and c-jun (c-Jun N-terminal kinase N- terminal kinase, JNK), eventually leading to cell death.
Thioredoxin -1 (Thioredoxin-1, Trx-1) is a multifunctional protein 12kDa, containing a conserved active site sequence: -Cys-Gly-Pro-Cys-. an increasing number of studies show that Trx-1 can resist cell damage and environmental stress and cell protective effect of.Trx-1 can remove singlet oxygen and hydroxyl radical.Trx-1 can protect cells from hydrogen peroxide (hydrogen peroxide, H2O2), caused by ultraviolet irradiation and local cerebral ischemia injury. Our previous study showed that MPP+ treatment of rat pheochromocytoma PC12 cells significantly decreased Trx-1 expression and high expression of Trx-1 reduces MPP+ induced neurotoxicity, high expression of Trx-1 inhibits the expression of endoplasmic reticulum apoptosis caspase12, however, their molecular mechanisms is still not clear.
Parkinson viral compounds 1- methyl -4- phenyl -1,2,3,6- tetrahydropyridine four (1-Methy-4-phenyl-1,2,3,6-tetrahydropyridine, MPTP) and its active metabolite 1- methyl -4- phenyl pyridine ion (1-methyl-4-phenylpyridinium ion MPP+) is often used to construct the experimental model of PD in vivo and in vitro.
In this paper, we study the MPP+ treatment of PC12 cells to glucose regulated protein 78 (Glucose Regulated, Protein78, GRP78), IRE1 alpha, TRAF2, C/EBP (C/EBP homologous protein homologous protein, CHOP), containing cysteine aspartic acid proteolytic zymogens 12 (pro-cysteinyl aspartate specific proteinase-12, procaspase-12 and JNK) on the expression of we further studied the Trx-1 of GRP78, TRAF2, IRE1 alpha, CHOP, regulation of procaspase-12 and JNK. The main results are as follows:
(1) PC12 cells to endoplasmic reticulum stress induced by.0.3mM MPP+ MPP+ in PC12 cells treated with different time (0,2,6,12 and 24h or 0,15,30,45 and 60min), Western Blot detection of MPP+ on GRP78, TRAF2, CHOP, IRE1 alpha, procaspase-12 expression and JNK. The results showed that MPP+ induced by GRP78 in a time-dependent manner. IRE1 alpha, procaspase-12 and the expression of TRAF2 and cleavage of CHOP activation, MPP+ treatment in a very short period of time in order to confirm the activation of JNK. ER apoptosis pathway executive procaspase-12 is activated by JNK, with SP600125 JNK inhibitor pretreatment PC12 30min cell stimulation with MPP+ 24h, the results showed that procaspase-12 activation is the inhibition of SP600125.
(2) Trx-1 in the regulation of cell toxicity of.MPP+ induced by MPP+ treatment significantly reduced the survival rate of PC12 cells in a time-dependent manner, MPP+ reduced the expression of Trx-1 in PC12 cells, and MPTP reduced Trx-1 expression in wild-type C57BL/6 mice SNpc. To clarify whether Trx-1 plays an important role in inhibiting the toxicity of MPP+, transfection of siRNA decreased the expression of Trx-1 or GFP-Trx-1 plasmid was transfected into the high expression of Trx-1, Trx-1 expression analysis of low or high toxicity effect on the expression of MPP+ with MTT Trx-1siRNA or GFP-human Trx-1 plasmid. Transfection of PC12 cells without cytotoxicity.Trx-1 expression significantly increased the sensitivity of PC12 cells to MPP+ induced toxicity. On the contrary, the high expression of Trx-1 inhibits cell the toxicity induced by MPP+. These results suggest that Trx-1 may play an important role in MPP+ induced cytotoxicity.
(3) the low expression of Trx-1 increased MPP+ induced endoplasmic reticulum stress in.PC12 cells transfected with Trx-1siRNA24h, 24h or 15min MPP+ stimulation, we studied the effect of down-regulation of Trx-1 expression of endoplasmic reticulum stress induced by MPP+.Trx-1 on MPP+ induced by low GRP78 expression after further increase showed that Trx-1siRNA increased the endoplasmic reticulum stress induced by MPP+. We also detection of Trx-1siRNA on IRE1 alpha, TRAF2, CHOP, JNK and procaspase-12.Trx-1siRNA further increased MPP+ induced IRE1 alpha, TRAF2 and the expression of CHOP and JNK and the activation of procaspase-12. That Trx-1siRNA by increasing the IRE1 alpha of these results, the expression of TRAF2 and CHOP and activation of JNK and procaspase-12, increased endoplasmic reticulum stress MPP+ induced.
(4) the high expression of Trx-1 reduced MPP+ induced endoplasmic reticulum stress in.PC12 cells transfected with GFP-human plasmid Trx-1 with high expression of Trx-1, then detect the effect of Trx-1 overexpression on the endoplasmic reticulum stress induced by MPP+. The results showed that high expression of Trx-1 inhibited MPP+ induced increased expression of GRP78. MPP+ and IRE1 induced by alpha, TRAF2 and CHOP expression and the activation of JNK and procaspase-12 were high expression of Trx-1 inhibited. These data suggest that high expression of Trx-1 by inhibiting IRE1 alpha, expression of TRAF2 and CHOP, and reduce the activation of JNK and procaspase-12, thus reducing endoplasmic reticulum stress induced by MPP+.
(5) Trx-1 inhibition of endoplasmic reticulum stress induced by MPTP. We examined whether Trx-1 can inhibit MPTP induced toxicity in mice. Wild type MPTP group and human Trx-1 transgenic mice were intraperitoneally injected with MPTP MPTP (20mg/kg, 2 times daily injections for 7 days), the wild type control saline group and human transgenic Trx-1 mice injected with wild-type MPTP. Mice and wild type compared to the control group exercise capacity decreased significantly, while the human Trx-1 transgenic mice in group MPTP and hTrx-1 transgenic compared to the control group, exercise capacity did not decrease. In this thesis, we also examined the effect of Trx-1 on MPTP induced limb injury. Experimental results show that the high expression of Trx-1 improved limb injury induced by MPTP. In order to evaluate the loss of dopaminergic neurons, we examined mice SNpc tyrosine hydroxylase immunohistochemistry and Western Blot analysis (TY Rosine hydroxylase, TH) expression after.MPTP treatment, the expression of wild type mice substantia nigra TH decreased significantly, and the high expression of Trx-1 in transgenic mice responded to TH expression. These data suggest that overexpression of Trx-1 protects dopaminergic neurons from MPTP induced neurotoxicity in PC12 cells. And the results are consistent, high Trx-1 in transgenic mice the expression increased significantly inhibited the expression of GRP78 in substantia nigra, showed high expression of Trx-1 reduces endoplasmic reticulum stress induced by MPTP. And the high expression of Trx-1 inhibited MPTP induced IRE1 alpha, TRAF2 and the expression of CHOP and JNK and the activation of procaspase-12. These data suggest that the high expression of Trx-1 by inhibiting the expression of TRAF2 and IRE1 alpha. CHOP, and the activation of JNK and procaspase-12, thus inhibiting the neurotoxicity of MPTP.
In this paper, the molecular mechanism of endoplasmic reticulum stress we systematically investigated in vitro and in vivo inhibition of Trx-1 induced by MPP+/MPTP. From the experimental results, we can draw a conclusion: Trx-1 by inhibiting IRE1 alpha, in Parkinson's disease TRAF2, CHOP, JNK and procaspase-12 activation, inhibition of endoplasmic reticulum stress, and thus play a neuroprotective function.

【学位授予单位】：昆明理工大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R742.5;R-332

【共引文献】