高脂喂养加干燥气体损伤构建颈动脉狭窄动物模型

目的探讨建立经济实用、稳定可靠的适合颈动脉粥样硬化性狭窄外科治疗研究的动物模型的条件。方法日本大耳白兔40只,采用血管内膜气体干燥损伤法在动物的颈总动脉上造成特定条件的损伤(160ml/min×15min),然后以特定高胆固醇饲料(饲料中含2%的胆固醇和6%的花生油)喂养动物不同时间(30天,60天和90天)。通过血脂测定、DSA、HE染色方法评价各组动物血管狭窄程度,再观察其病理改变特点。结果病理检查证实:随着喂养时间的延长,动脉粥样硬化程度在加重,高脂喂养加干燥气体损伤90天时,血管狭窄率达71.53±3.65%,动脉的粥样硬化病理改变已属十较成熟的粥样斑块期。结论按本实验方法,90天时颈动脉狭窄程度和病理改变程度符合颈动脉粥样硬化性狭窄外科治疗之实验研究需要。     

颈动脉狭窄动物模型的构建及核因子-κB和胰岛素样生长因子-1的表达

目的 探讨建立经济实用、稳定可靠的适合颈动脉粥样硬化性狭窄外科治疗研究的动物模型的条件.通过测量核因子(NF)-κB、胰岛素样生长因子(IGF)-1的表达,探讨它们在颈动脉粥样硬化性狭窄发生、发展中的作用.方法 日本大耳白兔40只,采用血管内膜气体干燥损伤法在动物的颈总动脉上造成特定条件的损伤(160 ml/min×15 min),然后以特定高脂饲料(饲料中含2%的胆固醇和6%的花牛油)喂养动物不同时间(30、60和90 d).通过血脂测定、DSA、HE染色评价各组动物血管狭窄程度,再观察其病珲改变特点.应用免疫组织化学方法观察NF-KB和IGF-1在动脉粥样硬化斑块中的表达特点.结果 病理检查证实:随着喂养时间的延长,动脉粥样硬化程度加重,高脂喂养加干燥气体损伤90 d时,血管狭窄率达71.53%±3.65%(q=568.417,P=0.000),动脉的粥样硬化病理改变已属较成熟的粥样斑块期.正常血管的内膜和中膜内均未见明显的NF-κB和IGF-1的表达,模型组血管斑块内NF-κB、IGF-1表达呈强阳性,表达面积分别为59.66%±12.04%(t=22.98,P＜0.01)、54.48%±12.92%(t=5.76,P＜0.01),明显高于对照组(分别为8.34%±2.66%和16.38%±8.22%).结论 按本实验方法,90 d时颈动脉狭窄程度和病理改变程度符合颈动脉粥样硬化性狭窄外科治疗的实验研究需要.NF-κB及其靶基因IGF-1的激活与动脉粥样硬化病变的发生和发展密切相关,在颈动脉粥样硬化斑块形成过程中发挥重要作用.     

颈动脉粥样硬化性狭窄动物模型的建立

目的 探讨建立经济实用、稳定可靠的适于颈动脉粥样硬化性狭窄(CASS)外科治疗研究的CASS动物模型的条件。方法 新西兰白兔25只，采用血管内膜气体干燥损伤法在动物的颈总动脉上造成特定条件的损伤，然后以特定高脂饲料喂养动物不同时间。评价各组动物血管狭窄程度和病理改变特点。结果 1月组、2月组和3月组各组中重度狭窄均达到80％。病理检查证实高脂喂养2个月时，动脉的粥样硬化病理改变已属于较成熟的纤维斑块期。结论 按本实验方法，损伤后喂养2个月的动物颈动脉狭窄程度和病理改变程度皆符合CASS外科治疗之实验研究需要。     

高脂饲料喂养与动脉内膜球囊损伤结合建立兔腹主动脉粥样硬化模型

背景：以往研究多采用单纯饲喂高脂饲料、正常或高脂血症动物动脉内膜损伤致动脉粥样硬化狭窄模型。 目的：拟采用喂养高脂饲料与动脉内膜球囊损伤相结合的方法建立腹主动脉粥样硬化的模型。 设计、时间及地点：对比观察实验,于2007-01/03在解放军总医院动物实验中心完成。 材料：新西兰白兔20只,随机分为单纯饲喂高脂饲料组、高脂饲料与动脉内膜损伤结合组,每组10只。高脂饲料由普通颗粒饲料+4%胆固醇+10%猪油组成+10%蛋黄粉组成。 方法：单纯饲喂高脂饲料组单纯喂养高脂饲料,高脂饲料与动脉内膜损伤结合组喂养高脂饲料4周后,行腹主动脉内膜球囊损伤术。 主要观察指标：12周后取主动脉行病理检查,计算机计算脂纹脂斑厚度,内、中膜厚度,内膜厚度比值,并进行血清总胆固醇、三酰甘油和高密度脂蛋白胆固醇、低密度脂蛋白胆固醇的测定。 结果：12周后,两组兔血脂血清总胆固醇、三酰甘油、高密度脂蛋白胆固醇、低密度脂蛋白胆固醇均升高,高脂饲料与动脉内膜损伤结合组升高更明显(P < 0.05)。喂养高脂饲料的两组动物均出现动脉粥样硬化斑块,横段面纤维斑块切片出现钙化。与单纯饲喂高脂饲料组比较,高脂饲料与动脉内膜损伤术结合组内膜明显增厚, 形成了明显的斑块及纤维帽结构,斑块厚度与内中膜厚度比值增加明显(P < 0.01)。 结论：所诱导产生的斑块与人类斑块成分具有一定相似性,包括斑块的纤维帽、钙化、脂核等,提示采用动脉内膜损伤与高脂饮食结合方式来制作动脉粥样硬化斑块的模型是可行的、实用的。     

改良硅橡胶圈加高胆固醇喂养诱导颈动脉狭窄兔模型的建立

目的探讨粥样硬化性颈动脉狭窄兔模型的建立。方法40只雄性新西兰大白兔随机分为4组:空白对照组4只(A组)、高胆固醇喂养组4只(无硅橡胶圈干预,B组)、硅橡胶圈干预组16只(C组,分C1,C2、C3、C4亚组),高胆固醇 硅橡胶圈干预组(D组,分D1、D2、D3、D4亚组)。硅橡胶圈置于右颈动脉外膜,对侧颈动脉行伪手术,术后次日给予1%胆固醇饲料喂养2周,过量麻醉处死动物前,动物模型行DSA证实颈动脉狭窄程度,切除硅橡胶圈下方狭窄的动脉及对侧相应位置的颈动脉标本,光学显微镜观察病理变化。结果置入改良的硅橡胶圈加高胆固醇喂养2周后,病理证实D组的硅橡胶圈诱导的颈动脉内膜面增厚、管腔狭窄;中膜平滑肌细胞排列紊乱。病理和DSA证实模型颈动脉管腔狭窄程度为12%～88%之间,其中以D4组的颈动脉粥样硬化斑块和狭窄程度较为显著。结论应用改良硅橡胶圈加高胆固醇喂养的方法成功地建立了与人有相似病理表现的粥样硬化性颈动脉狭窄模型兔,颈动脉狭窄程度与硅橡胶圈的形状、内壁直径及弹性回缩力相关,模型制作方法简便易行,便于重复。     

硼替佐米对LDLR-/-小鼠动脉粥样硬化血管平滑肌细胞凋亡的影响

目的观察泛素-蛋白酶体抑制剂(UPI)硼替佐米对LDLR-/-小鼠动脉粥样硬化血管平滑肌细胞(VSMC)凋亡的影响。方法取12只周龄为8w的雄性LDLR-/-小鼠,适应性喂养1w,随机将12只小鼠分为高脂饮食+硼替佐米组(6只)和高脂饮食+生理盐水组(6只)。将上述2组小鼠分别给予硼替佐米(50μg·kg~(-1))和同等剂量生理盐水进行腹腔注射,1w2次,共8w。处死小鼠,取主动脉根部,观察主动脉根部的病理变化;利用免疫组化方法检测VSMC中激活型caspase-3的表达水平。利用Tunel技术检测VSMC凋亡,计算凋亡指数。结果光镜下苏木精-伊红染色显示盐水组小鼠主动脉根部内膜出现较多泡沫细胞、内膜广泛增厚,硼替佐米组主动脉根部内膜局部增厚,泡沫细胞的数量较少。硼替佐米组小鼠主动脉根部激活型caspase-3表达高于生理盐水组(0.048±0.0133 vs 0.021±0.008,P <0.05)。硼替佐米组小鼠主动脉根部凋亡率较生理盐水组明显增高(0.302±0.105 vs 0.164±0.039,P <0.05)。结论蛋白酶体抑制剂硼替佐米可通过诱导VSMC凋亡从而延缓LDLR-/-小鼠主动脉动脉粥样硬化的进展,其机制可能与调控激活型caspase-3表达有关。     

小型猪颈动脉粥样硬化性狭窄模型的初步建立

目的探索建立适合于神经介入研究的动脉粥样硬化性颈动脉狭窄动物模型的方法。方法以12头广西巴马小型猪为实验对象,以高脂饲养和动脉球囊损伤为模型建立的主要手段。应用体外超声、数字减影血管造影(DSA)和血管内超声(IVUS)评价颈动脉狭窄的形成情况,并对颈动脉的病理改变特点进行评价,最后分析总结出创建动脉粥样硬化性颈动脉狭窄动物模型的理想方案。结果16周时的DSA发现：高脂饲养和动脉球囊损伤组平均狭窄率达33．32%±12．84%；病理检查证实有明显的粥样硬化斑块形成。结论高脂饲养加动脉球囊损伤,16周可以初步建立广西巴马小型猪颈动脉粥样硬化性狭窄模型。     

小檗碱对家兔颈动脉粥样硬化干预的实验研究

目的　探讨小檗碱对家兔颈动脉粥样硬化形成的干预作用。方法　2 4只日本大耳白家兔随机分成正常组、对照组和小檗碱干预组,正常组8只,普通饲料喂养5周;对照组8只,高脂喂养一周后行颈总动脉内膜空气干燥术,术后继续喂养4周;小檗碱干预组8只,高脂喂养一周后行颈总动脉内膜空气干燥术,术后继续高脂喂养并每日肌肉注射小檗碱,剂量为5 mg/ kg,时间为4周。实验满5周后,行右侧脑血管造影(DSA)术并观察正常组、对照组和小檗碱干预组的颈动脉的病理变化和计算内膜中膜比(I/ M)。结果　正常组的颈动脉的内膜围一层单皮细胞,中膜为规则的平滑肌肌层;对照组内膜明显增厚,中膜变薄,HE染色可见大量泡沫细胞堆积和坏死的物质;小檗碱干预组内膜也有增厚的现象,增厚程度明显小于对照组,HE染色见有泡沫细胞形成,堆积程度小于对照组。DSA发现对照组的颈动脉有不同程度的狭窄,而小檗碱预防组的家兔没有发现明显的颈动脉狭窄。结论　小檗碱可以干预家兔颈动脉粥样硬化的形成过程,延缓粥样硬化的发生。     

银杏黄酮苷元干预兔颈总动脉损伤后内膜增生及血凝素样氧化低密度脂蛋白受体1的表达

背景：银杏叶提取物中的黄酮苷元具有较强的抗氧化活性,课题组前期实验证实可抑制氧化低密度脂蛋白诱导内皮细胞表达血凝素样氧化低密度脂蛋白受体1。 目的：进一步探讨银杏黄酮苷元对兔颈总动脉内皮损伤后内膜增生和血凝素样氧化低密度脂蛋白受体1表达的影响。 方法：将雄性新西兰大白兔随机分为对照组、假手术组、模型组和治疗组。对照组予普通饲料,其余各组予高脂饮食,假手术组仅作颈外动脉结扎,模型组和治疗组均球囊损伤右颈总动脉,治疗组损伤后用银杏黄酮苷元灌胃。4周后检测各组血脂水平,观察各组右颈总动脉形态,用免疫组织化学法和RT-PCR检测血凝素样氧化低密度脂蛋白受体1蛋白和mRNA表达。 结果与结论：模型组术后4周内膜增生明显,有粥样斑块形成,血凝素样氧化低密度脂蛋白受体1表达明显增加(P < 0.05)。治疗组银杏黄酮苷元灌胃4周后内膜增生较轻,内膜面积,血凝素样氧化低密度脂蛋白受体1阳性细胞数和其基因表达水平均低于模型组(P < 0.05),但血脂与模型组比较无差异。提示银杏黄酮苷元可减轻新生内膜增生及动脉粥样硬化病粥样斑块的形成,这种作用可能与抑制兔颈总动脉内皮损伤后血凝素样氧化低密度脂蛋白受体1的表达有关。     

经股动脉入路建立球囊损伤兔颈动脉狭窄模型

目的经股动脉入路制作一种实用、可靠且稳定的颈动脉狭窄模型,用于探讨颈动脉再狭窄的防治。方法新西兰大白兔20只,随机分为对照组、实验1周组、实验2周组和实验4周组,每组5只,常规喂养。所有动物采用经右侧股动脉入路置入HyperForm球囊,实验组行右颈总动脉定比扩张,对照组行单纯右颈动脉路图显示,术后以普通饮食喂养。实验1、2、4周组实验兔分别于手术后1周、2周和4周处死,并在第4周同时处死对照组兔。右颈动脉分叉近心端1～2cm节段取材送光镜和电镜检查,分别观察血管壁增生情况并计算血管内膜和中膜厚度。结果与对照组比,实验组新生内膜厚度呈持续性显著增加(P<0.01),实验2周组与实验1周组比较及实验4周组与实验2周组相比,内膜厚度均显著增加(P<0.01)。各组中膜厚度未见明显统计学差异(P>0.05)。结论经股动脉入路用Hyper Form球囊行颈动脉定比扩张能成功地制作一种稳定、简便可靠的颈动脉狭窄动物实验模型。血管平滑肌细胞的过度增生是造成PTA术后管腔再狭窄的主要因素。     

Prevention of muscle disuse atrophy by MG132 proteasome inhibitor

Introduction: Our goal was to determine whether in vivo administration of the proteasome inhibitor MG132 can prevent muscle atrophy caused by hindlimb unloading (HU). Methods: Twenty‐seven NMRI mice were assigned to a weight‐bearing control, a 6‐day HU, or a HU+MG132 (1 mg/kg/48 h) treatment group. Results: Gastrocnemius wasting was significantly less in HU+MG132 mice (?6.7 ± 2.0%) compared with HU animals (?12.6 ± 1.1%, P = 0.011). HU was also associated with an increased expression of MuRF‐1 (P = 0.006), MAFbx (P = 0.001), and USP28 (P = 0.027) mRNA, whereas Nedd4, E3α, USP19, and UBP45 mRNA did not change significantly. Increases in MuRF‐1, MAFbx, and USP28 mRNA were largely repressed after MG132 administration. β5 proteasome activity tended to increase in HU (+16.7 ± 6.1%, P = 0.086). Neither β1 and β2 proteasome activities nor ubiquitin‐conjugated proteins were changed by HU. Conclusions: Our results indicate that in vivo administration of MG132 partially prevents muscle atrophy associated with disuse and highlight an unexpected regulation of MG132 proteasome inhibitor on ubiquitin‐ligases. Muscle Nerve, 2011     

Differentiation-dependent sensitivity to cell death induced in the developing retina by inhibitors of the ubiquitin-proteasome proteolytic pathway

The effects of inhibitors of proteasome function were studied in the retina of developing rats. Explants from the retina of neonatal rats at postnatal day (P) 3 or P6 were incubated with various combinations of the proteasome inhibitor carbobenzoxyl-leucinyl-leucinyl-leucinal (MG132), the protein synthesis inhibitor anisomycin, or the adenylyl cyclase activator forskolin. MG132 induced cell death in a subset of cells within the neuroblastic (proliferative) layer of the retinal tissue. The cells sensitive to degeneration induced by either MG132 or anisomycin, were birthdated by bromodeoxyuridine injections. This showed that the MG132-sensitive population includes both proliferating cells most likely in their last round of cell division, and postmitotic undifferentiated cells, at a slightly earlier stage than the population, sensitive to anisomycin-induced cell death. The results show that sensitivity to cell death induced by proteasome inhibitors defines a window of development in the transition from the cell cycle to the differentiated state in retinal cells.     

Differential effect of calmodulin antagonists on MG132-induced mitochondrial dysfunction and cell death in PC12 cells

Defects in proteasome function have been suggested to be involved in the pathogenesis of neurodegenerative diseases. We examined the effect of calmodulin antagonists on proteasome inhibitor-induced mitochondrial dysfunction and cell viability loss in undifferentiated PC12 cells. Caspase inhibitors (z-IETD.fmk, z-LEHD.fmk and z-DQMD.fmk) and antioxidants attenuated cell death and decrease in GSH contents in PC12 cells treated with 20 microM MG132, a proteasome inhibitor. Calmodulin antagonists (trifluoperazine, W-7 and calmidazolium) had a differential inhibitory effect on the MG132-induced cell death and GSH depletion depending on concentration with a maximal inhibitory effect at 0.5-1 microM. Addition of trifluoperazine and W-7 reduced the MG132-induced nuclear damage, loss of the mitochondrial transmembrane potential followed by cytochrome c release, formation of reactive oxygen species and elevation of intracellular Ca(2+) levels in PC12 cells. Calmodulin antagonists at 5 microM exhibited a cytotoxic effect on PC12 cells but attenuated the cytotoxicity of MG132. The results suggest that the toxicity of MG132 on PC12 cells is mediated by activation of caspase-8, -9 and -3. Trifluoperazine and W-7 at the concentrations of 0.5-1 microM may attenuate the MG132-induced viability loss in PC12 cells by suppressing change in the mitochondrial membrane permeability and by lowering of the intracellular Ca(2+) levels as well as calmodulin inhibition.     

Inhibition of MG132-induced mitochondrial dysfunction and cell death in PC12 cells by 3-morpholinosydnonimine

The effect of 3-morpholinosydnonimine (SIN-1) against the cytotoxicity of MG132, a proteasome inhibitor, in differentiated PC12 cells was assessed by measuring the effect on the mitochondrial membrane permeability. Treatment of PC12 cells with MG132 resulted in the nuclear damage, decrease in the mitochondrial transmembrane potential, cytosolic accumulation of cytochrome c, activation of caspase-3, increase in the formation of reactive oxygen species (ROS), and depletion of GSH. Addition of SIN-1, a producer of nitric oxide (NO) and superoxide, differentially reduced the MG132-induced cell death and GSH depletion concentration dependently with a maximal inhibitory effect at 150 microM. Carboxy-PTIO, superoxide dismutase, Mn-TBAP, and ascorbate prevented the inhibitory effect of SIN-1 on the cytotoxicity of MG132. SIN-1 inhibited the MG132-induced change in the mitochondrial membrane permeability, ROS formation and decrease in GSH contents in PC12 cells. S-nitroso-N-acetyl-DL-penicillamine reduced the MG132-induced cell death in PC12 cells, whereas peroxynitrite and H2O2 did not affect the cytotoxicity of MG132. The results suggest that NO and superoxide liberated from SIN-1 exert an inhibitory effect against the cytotoxicity of MG132. SIN-1 may inhibit the MG132-induced viability loss in PC12 cells by suppressing change in the mitochondrial membrane permeability that is associated with oxidative damage.     

Elevation of heme oxygenase‐1 by proteasome inhibition affords dopaminergic neuroprotection

Postmortem studies have shown that heme oxygenase‐1 (HO‐1) immunoreactivity is increased in patients with Parkinson disease. HO‐1 expression is highly upregulated by a variety of stress. Since the proteasome activity is decreased in patients with Parkinson disease, we investigated whether proteasome activity regulates HO‐1 content. MG‐132, a proteasome inhibitor, increased the amount of HO‐1 protein mainly in astrocytes of primary mesencephalic cultures. Quantitative RT‐PCR analysis revealed that lactacystin upregulated HO‐1 mRNA expression. Proteasome inhibition with MG132 also increased the cytomegalovirus promoter‐driven expression of Flag‐HO‐1 protein and resulted in an accumulation of ubiquitinated Flag‐HO‐1 in Flag‐HO‐1‐overexpressing PC12 cells. In addition, a cycloheximide chase assay demonstrated that the degradation of Flag‐HO‐1 protein was slowed by MG‐132. Next, the function of HO‐1 which was upregulated by proteasome inhibitors was examined. Proteasome inhibitors protected dopaminergic neurons from 6‐hydroxydopamine (6‐OHDA)‐induced toxicity and this neuroprotection was abrogated by co‐treatment with zinc protoporphyrin IX, a HO‐1 inhibitor. Furthermore, 6‐OHDA‐induced toxicity was blocked by bilirubin and carbon monoxide, products of the HO‐1‐catalyzed degradation of heme. These results suggest that mesencephalic HO‐1 protein level is regulated by proteasome activity and the elevation by proteasome inhibition affords neuroprotection. © 2010 Wiley‐Liss, Inc.     

Proteasome inhibition modulates kinase activation in neural cells: Relevence to ubiquitination,ribosomes, and survival

In this study we examined whether established signal transduction cascades, p44/42 mitogen‐activated protein kinase (ERK1/2) and Jun N‐terminal kinases (JNK) pathways, are altered in N2a neural cells in response to proteasome inhibition. Additionally, we sought to elucidate the relative contribution of these signal transduction pathways to the multiple downstream effects of proteasome inhibition. Our data indicate that ERK1/2 and JNK are activated in response to proteasome inhibition. Washout of proteasome inhibitor (MG132) results in an enhancement of ERK1/2 activation and amelioration of JNK activation. Treatment with an established MAPK inhibitor resulted in an increase in proteasome inhibitor toxicity, and incubation with JNK inhibitor was observed to attenuate proteasome inhibitor toxicity significantly. Subsequent studies demonstrated that inhibition of ERK1/2 and JNK activity does not alter the gross increase in ubiquitinated protein following proteasome inhibitor administration. Similarly, ERK1/2 and JNK activity do not appear to play a role in the disruption of polysomes following proteasome inhibitor administration in neural cells. Together these data indicate that ERK1/2 and JNK activation may play differential roles in modulating neurochemical disturbances and neurotoxicity induced by proteasome inhibition. © 2009 Wiley‐Liss, Inc.     

Impairment of proteasome and anti-oxidative pathways in the induced pluripotent stem cell model for sporadic Parkinson's disease

BackgroundParkinson's disease (PD) is associated with the progressive degeneration of dopaminergic neurons with abnormal accumulation of α-synuclein mainly in the ventral midbrain. However, the lack of live human neurons from PD patients and their heterogeneous pathogenic nature limit mechanistic studies and therefore the development of drugs to modify the disease progression of PD. The evolution of induced pluripotent stem cell (iPSC) technology makes it possible to generate patient-specific neurons to explore the pathogenesis in individual PD patients.MethodsWe generated PD-iPSCs from a sporadic early onset PD patient carrying a heterozygous deletion of exon 5 (Ex5del) in PARK2. The expression of α-synuclein and proteasome and anti-oxidative functions were examined in differentiated iPSC-derived neurons.ResultsThe neurons derived from our PD-iPSCs demonstrated abnormal α-synuclein accumulation and down-regulation of the proteasome and anti-oxidative pathways. Environmental triggers such as proteasome inhibitor MG132 and H₂O₂ markedly induced cell death, while the proteasome enhancer benzamil and anti-oxidative compound genipin significantly rescued these increased susceptibilities.ConclusionsThese results demonstrate that unique genetic–environmental interactions are involved in neuronal death in PD patients. Our findings also provide a new model to identify potential disease-modifying strategies and an insight into personalized medicine for patients with PD.     

LRRK2 interferes with aggresome formation for autophagic clearance

Autosomal-dominant mutations in the gene encoding leucine-rich repeat kinase 2 (LRRK2) account for the most common monogenic form of Parkinson's disease (PD). A link between autophagy dysregulation and LRRK2 has consistently been reported, but it remains poorly defined which step is targeted by LRRK2. Here, we sought to examine the effect of LRRK2 on the sequestration and degradation of aggregated protein complexes for autophagic clearance. Because two major intracellular protein degradation systems, the ubiquitin proteasome system and the autophagy, are functionally coupled, proteasome inhibition is suggested to activate autophagy. So, we induced protein quality control-associated autophagy using the proteasome inhibitor MG132 in differentiated SH-SY5Y cells and mice expressing G2019S mutant LRRK2 to uncover how the autophagy pathway is affected by LRRK2. We found that LRRK2 disrupted aggresome formation for autophagic clearance of accumulated protein aggregates. Specifically, we observed the following in differentiated SH-SY5Y cells with overexpressed wild-type and G2019S LRRK2: 1) large, clear, perinuclear aggresomes were not detected under MG132, instead, much smaller aggregates were broadly distributed in the cytosol; 2) enhanced accumulation of LC3-II and p62/ubiquitin-positive protein inclusions were noted; and 3) protein aggregates were not cleared even after a recovery period, which exacerbated the MG132-induced cytotoxicity. Notably, higher protein accumulation was detected in the brains of G2019S transgenic mice than in the brains of littermate control mice under proteasome inhibition. Our present findings provide insight into the precise mechanisms that underlie autophagy dysregulation in the brains of patients with PD with LRRK2 mutations.     

蛋白酶体抑制剂MG-132作用下失神经骨骼肌myf-5的表达

背景：MG-132是蛋白酶体抑制剂的一种，有报道显示其对失神经骨骼肌萎缩有延缓作用。 目的：进一步验证蛋白酶体抑制剂MG-132对大鼠骨骼肌成肌调节因子myf-5基因表达的影响。 方法：SD大鼠随机被分成3组，空白对照组不切断坐骨神经，仅做假手术，去神经组和去神经MG-132干预组切断坐骨神经1 cm以上，制作失神经骨骼肌动物模型，去神经MG-132干预组肌肉内注射MG-132。分别于去神经第2，7，28 d处死大鼠。用反转录聚合酶链式反应技术检测myf-5 mRNA表达情况，Western-blot检测myf-5蛋白表达的变化。 结果及结论：在去神经支配后第2，7，28天，与空白对照组比较，去神经组、去神经MG-132干预组myf-5mRNA和蛋白质均表达上调(P < 0.01)；去神经MG-132干预组myf-5mRNA和蛋白表达较去神经组均明显上调(P < 0.01)。结果提示蛋白酶体抑制剂MG-132可以通过抑制泛素-蛋白酶体途径来上调myf-5的表达，从而起到延缓骨骼肌萎缩的作用。     

Regulation of the content of progesterone and estrogen receptors, and their cofactors SRC-1 and SMRT by the 26S proteasome in the rat brain during the estrous cycle

In this work we have determined the role of the 26S proteasome in the regulation of the content of progesterone receptors (PR-A and PR-B), estrogen receptors (ER-alpha and ER-beta), the coactivator SRC-1 and the corepressor SMRT in the rat brain during the estrous cycle. The 26S proteasome inhibitor MG132 was injected once into the lateral ventricle on proestrous day; and 24h later, on estrous day we evaluated the content of PR and ER isoforms, SRC-1 and SMRT in the hypothalamus, the preoptic area and the hippocampus by Western blot. A significant increase in the content of both PR isoforms, ER-beta and SRC-1 was observed after the administration of MG132 in the three studied cerebral regions. SMRT content was increased in the hypothalamus and the preoptic area and a significant increase in ER-alpha content was only observed in the preoptic area. These results suggest that essential proteins that participate in progesterone and estrogen actions in the brain should be regulated by the 26S proteasome in a tissue-specific manner in physiological conditions.