动力相关蛋白1的小泛素化相关修饰物化修饰在脑缺血/再灌注损伤中的作用

背景 近年研究发现动力相关蛋白1(dynamin-related protein 1,Drp1)介导的线粒体分裂在脑缺血/再灌注损伤(cerebral ischemia/reperfusion injury,CI/RI)中发挥了重要作用,Drp1的小泛素化相关修饰物(small ubiquitin-related modifier,SUMO)化修饰能影响自身生物学效应.目的 阐述Drp1的SUMO化修饰在CI/RI中的作用.内容 分别描述Drp1蛋白、SUMO蛋白、SUMO相关酶(SUMO化酶/去SUMO化酶)及SUMO化修饰对Drp1生物学效应的影响.趋向 研究SUMO化修饰影响Drp1的生物学效应的机制,为减轻或治疗CI/RI找到新的靶点或提供新的思路.     

HECT型E3泛素连接酶在肿瘤中作用的研究现状和进展

肿瘤的发生和发展是多因素、多步骤的复杂过程。而泛素化是多步级联的蛋白质修饰过程,是维持真核细胞内稳态的重要机制。其中E3泛素连接酶家族是泛素-蛋白酶体系统的重要成分,可催化多种蛋白底物的泛素化,促进其被蛋白酶体系统降解。迄今为止,E3泛素连接酶在多种肿瘤细胞生物学过程中发挥着重要作用,包括细胞增殖、凋亡及周期调控。而HECT型E3泛素连接酶作为E3泛素连接酶最早被研究的一种,其主要参与蛋白质翻译后转录调控的泛素化修饰过程。本文就HECT型E3泛素连接酶及其在肿瘤中作用的现状和最新研究进展进行综述。     

靶向Neddylation修饰通路抗结直肠癌作用及其机制研究进展

蛋白类泛素化修饰Neddylation是一种重要的蛋白质翻译后修饰。Neddylation修饰在结直肠癌中过度活化,并且与结直肠癌患者的预后不良密切相关。小分子抑制剂MLN4924通过阻断Neddylation修饰通路诱导结直肠癌细胞凋亡、自噬、DNA损伤及细胞衰老发挥抗肿瘤效应。此外,MLN4924还可作为化学增敏剂...     

PIAS在前列腺癌发生发展中的作用

PIAS(protein inhibitors of activated STAT)是信号转导与转录激活因子(STAT)的特异性抑制蛋白,能够作用于雄激素受体(AR)以及核因子-kappa B(NF-κB)信号通路,且具有小泛素蛋白修饰分子(SUMO)连接酶E3的活性,参与转录因子SUMO化修饰,调控肿瘤细胞周期进程。近年来诸多研究已经证实PIAS与前列腺癌的发生与演变具有密切的关系。本文主要阐述PIAS在前列腺癌发生、发展以及转移等方面的研究进展,为前列腺癌的治疗提供新的理论基础与研究思路。     

泛素-蛋白酶体系统与精子DNA损伤修复的研究进展

泛素-蛋白酶体系统(UPS)是体内广泛存在的一种蛋白酶体系统,由泛素(Ub)、泛素激活酶(E1)、泛素结合酶(E2)、泛素连接酶(E3)、26S蛋白酶体和去泛素化酶(DUBs)组成,能够调控体内蛋白降解。研究发现UPS除了蛋白降解功能以外,还参与细胞周期调控、免疫应答、信号传导以及DNA损伤修复等过程。精子发生要经历染色体联会、同源重组等过程,精子DNA在这些过程中易受干扰而出现损伤。近年来研究发现,UPS与精子发生当中的DNA损伤修复有关。UPS参与DNA损伤的修复机制包括:泛素化调节DNA损伤修复相关酶类、协助识别DNA损伤位点、募集损伤修复相关蛋白、启动DNA损伤修复途径、维持染色体稳定,从而保证精子发生正常进行。     

小泛素样修饰蛋白通路在骨肉瘤中异常激活与靶向治疗的研究

目的运用分子生物学方法观察小泛素样修饰蛋白(small ubiquitin-related modifier,SUMO)通路成员在骨肉瘤中的表达特点,为基于蛋白质SUMO化修饰的靶向治疗提供理论依据。方法收集2017年1月至2019年6月于我院就诊并手术的新鲜骨肉瘤组织样本18例,经Western blot及免疫组织化学方法检测癌灶及癌旁SUMO通路核心成员SUMO1、SAE1、Ubc9、SENP1的蛋白表达水平。分别干涉SUMO1、干涉UBC9及过表达SENP1,进行如下分组:空白对照组、对照组、siR-SUMO1组、siR-Ubc9组和SENP1组。Western blot验证基因转染效率,细胞增殖检测试剂盒检测EdU的阳性表达率,划痕实验及Transwell侵袭实验检测细胞迁移和侵袭能力,流式细胞术检测细胞凋亡率。以骨髓间充质干细胞(BMSCs)为研究对象,评估三种治疗方案的副作用。结果 Western blot及免疫组织化学结果均显示,骨肉瘤组织中SUMO1、SAE1、Ubc9的蛋白表达水平均明显高于癌旁组织(P<0.05),但SENP1明显低于癌旁组织。基于143B骨肉...     

烧伤中的氧化应激与DNA损伤

氧化物在正常细胞的生物反应中起着重要的作用 ,也参与许多人类疾病的病理过程。一旦氧化物与抗氧化物的平衡破坏 ,即氧化应激 (ｏｘｉｄａｔｉｖｅｓｔｒｅｓｓ)产生 ,氧负荷增加和 (或 )抗氧化物防卫能力下降 ,氧化物则修饰靶分子的结构和功能导致细胞损伤。损伤广泛时 ,可产生组织水平和器官功能的表现。一、烧伤与氧化应激研究发现 ,氧自由基 (ｒｅａｃｔｉｖｅｏｘｙｇｅｎｓｐｅｃｉｅｓ ,ＲＯＳ)在烧伤局部创面损伤和进一步的水肿形成、休克的发生、全身炎症反应和远隔器官损伤等过程中有重要作用[1- 4] ,同时也参与了烧伤后免疫功能…     

缺血预处理调节内质网应激及促进自噬机制的研究进展

缺血预处理(IPC)对于缺血再灌注损伤(IRI)发生后更迅速的恢复生理功能和组织结构和减少损伤具有重要的作用。细胞受到外界刺激后,内质网应激(ERS)启动,可以通过减少未折叠蛋白的累积并增加未折叠蛋白的降解,保护内质网稳态,泛素化降解系统也参与其中,但当细胞受到外界刺激持续或者过强时,ERS和泛素化降解系统无法消除未折...     

piRNA通路在生精过程中的作用

piRNA是一类特异性与Argonuat蛋白家族中的PIWI蛋白相互结合形成piRNA沉默复合体(piRNA-induced silencing complex,piRISC)而在生物体内发挥重要作用的非编码小RNA,主要作用于生殖系统。piRNA通路不仅通过抑制转座子,还通过泛素化降解、调控mRNA表达、基因甲基化修饰等方式来发挥在生精过程中的特定作用。本文拟对piRNA通路在生精过程中的作用进行综述。     

SUMO-1对p53基因诱导HepG2细胞凋亡的增强作用

目的研究小分子泛素样修饰体1(smallubiquitin likemodifier1,SUMO1)在野生型p53基因诱导HepG2细胞凋亡中的作用。方法用含人野生型p53基因质粒pcDNA3wtp53(pwtp53)、含人双微粒体基因2〔(humandoubleminutegene2(HDM2);鼠同源基因为MDM2〕质粒pCMV HDM1B(pMDM2)、含人SUMO1基因质粒pcDNA3His6SUMO1(pSUMO1)和空质粒pcDNA3分别或同时转染HepG2细胞,获得各转染细胞系,应用Westernblot检测转染后细胞中质粒蛋白的表达及流式细胞技术检测细胞凋亡比例。结果转染pwtp53和pMDM2质粒的HepG2细胞均可见p53及MDM2蛋白条带,同时转染pSUMO1质粒的细胞分别可见被SUMO1修饰的相对分子量较大的p53和MDM2蛋白条带,在未转染任何质粒、仅转染空质粒和pSUMO1质粒的细胞中只检测到少量p53蛋白表达。转染pwtp53及pwtp53+pSUMO1质粒的HepG2细胞凋亡比例分别为(16.79±1.62)%和(18.15±1.36)%,转染pwtp53+pMDM2+pSUMO1质粒的细胞凋亡比例为(14.06±1.84)%,转染pwtp53+pMDM2质粒的细胞凋亡比例则下降至(5.17±1.23)%,与前三者比较差异有显著性意义(P<0.01);其他转染系细胞中的细胞凋亡比例均≤2%,差异无显著性意义。结论SUMO1通过与p53蛋白的结合或翻译后修饰,抑制MDM2等癌基因蛋白对p53蛋白的降解,可增强p53抑癌基因诱导的细胞凋亡。     

microRNAs在脊髓损伤中的研究进展

脊髓损伤(spinal cord injury,SCI)的继发性病变如氧化应激、凋亡等会进一步加重神经功能损害.microRNAs(miRNAs)是一类新型的小非编码RNAs,其通过沉默翻译或干扰靶mRNAs的表达调控蛋白质的产生和细胞功能.SCI改变了氧化应激、炎症和凋亡等许多继发性损伤相关的miRNAs的表达.本综...     

Increased protein oxidation and decreased creatine kinase BB expression and activity after spinal cord contusion injury

Traumatic injury to the spinal cord triggers several secondary effects, including oxidative stress and compromised energy metabolism, which play a major role in biochemical and pathological changes in spinal cord tissue. Free radical generation and lipid peroxidation have been shown to be early events subsequent to spinal cord injury. In the present study, we demonstrated that protein oxidation increases in rat spinal cord tissue after experimental injury. As early as h after injury, the level of protein carbonyls at the injury epicenter was significantly higher than in control (169%, p < 0.05) and increased gradually over the next 4 weeks to 1260% of control level. Both caudal and rostral parts of the injured spinal cord demonstrated a mild increase of protein carbonyls by 4 weeks postinjury (135-138%, p < 0.05). Immunocytochemical analysis of protein carbonyls in the spinal cord cross-sections showed increased protein carbonyl immunoreactivity in the epicenter section compared to rostral and caudal sections of the same animal or control laminectomy animals. Increased protein carbonyl formation in damaged spinal cord tissue was associated with changes in activity and expression of an oxidative sensitive enzyme, creatine kinase BB, which plays an important role in the maintenance of ATP level in the CNS tissue. Damage to CK function in the CNS may severely aggravate the impairment of energy metabolism. The results of our study indicate that events associated with oxidative damage are triggered immediately after spinal cord trauma but continue to occur over the subsequent 4 weeks. These results suggest that antioxidant therapeutic strategies may be beneficial to lessen the consequences of the injury and potentially improve the restoration of neurological function.     

Current role of methylprednisolone in the treatment of acute spinal cord injury

High doses of methylprednisolone (MPSS) came into use as part of a therapeutic protocol for acute spinal cord injuries following the published results from the NASCIS II study in 1992; they soon became a standard of care around the world. However, the results of this study have been critically reviewed and questioned by many authors since the beginning. The major argument is based on the fact that its effectiveness in reducing post-injury neurological damage has not been conclusively proved; in addition, there has been increasing evidence of serious side effects of steroids administered at high doses. In the Czech Republic, as part of pre-hospital care, MPSS according to the NASCIS II (or NASCIS III) protocol is used in all regional centres of emergency medical service. In the Czech spinal surgery centres involved in treating acute spinal cord injuries, there are 19 of them, attitudes towards the use of MPSS vary. In 16% of the centres a certainty of its beneficial effect is still maintained, faith in its effect together with fear of a "non-lege artis" procedure is the reason for MSPP use in 21%, and the fear of sanctions only leads to its use in 63% of the centres. There is no standard practice in application of the NASCIS II and NASCIS III protocols and no standard exclusion criteria exist. The two protocols are used equally, and one institution has its own modification. The recommended MPSS dose is administered with no exception in 63% of the centres; dose adjustment is employed according to the form of spinal cord lesion in 11%, the level of spinal cord injury in 5%, associated diseases in 16% and patient age in 11% of the spinal surgery centres. After the results of studies on MPSS administration in acute spinal cord injury have been analysed, many medical societies have changed their recommendations. In view of later relevant publications it is no longer possible to regard MPSS administration as a standard of cure for acute spinal cord injury. Current evidence suggests that MPSS administration in a 24-hour regimen after an initial dose given within 8 hours of injury is the therapeutic procedure that needs individual consideration in each patient according to their state of health and potential complications. MPSS administration at an interval longer than 8 hours after injury and for more than 24 hours is not justified, nor is it justified to use a high MPSS dose at the place of injury by an emergency ambulance crew. Key words: corticosteroids, methylprednisolone, spinal cord trauma, neurological damage.     

Polyethylene glycol improves function and reduces oxidative stress in synaptosomal preparations following spinal cord injury

Spinal cord injury (SCI) results in rapid and significant oxidative stress. We have previously demonstrated that administration of polyethylene glycol (PEG) inhibits oxidative stress using an in vitro model of SCI. In this study we tested the effects of PEG in vivo, to elucidate the mechanism of PEG-mediated neuroprotection. We show that a compression injury at T10-11 induced diffusive oxidative stress in crude synaptosomal preparations, correlated with synaptosomal dysfunction and increased intrasynaptosomal calcium. Administration of PEG immediately post-injury produced a marked decrease in synaptosomal oxidative stress and calcium, associated with an increase in synaptosomal function. Confocal microscopy using fluorescein conjugated PEG revealed that PEG entered the cells of the injured spinal cord, placing the polymer in a position to directly interact with cellular organelles. PEG attenuates calcium-induced functional compromise of normal spinal cord synaptosomes and mitochondria in vitro. These results indicate that PEG may exert its neuroprotective effect through direct interaction with mitochondria, besides its known ability to rescue neurons and their axons by repairing the plasma membranes. We submit that PEG is likely to interfere with the cascade of secondary injury by several mechanisms of action that in concert reduce oxidative stress.     

脊髓损伤与男性不育(英文)

脊髓损伤 (SCI)通常会对育龄男性造成一定的影响。脊髓受损后的男性大多会在以下方面发生问题 :如勃起和射精功能障碍 ,精子发生受损 ,精子存活力、活率及形态异常 ,泌尿生殖系统感染以及内分泌异常。本文将从病理生理、评价和治疗等方面对脊髓损伤引起的男性不育症进行论述。脊髓损伤可对精浆产生影响并加速精液氧化 ,从而使得SCI男性的精液质量下降。本文还将对用于SCI男性的精子复苏技术和辅助生育技术所取得的进展进行讨论。     

Beta2-adrenoreceptor agonist-enhanced recovery of locomotor function after spinal cord injury is glutathione dependent

The beta2-adrenoreceptor agonist, clenbuterol, has been shown to spare spinal cord tissue and enhance locomotor recovery in an experimental model of spinal cord contusion injury. A likely mechanism of neurodegeneration following spinal cord injury involves generation of toxic levels of reactive oxygen species (ROS), e.g., O2-*, H2O2 and OH*, which overwhelm endogenous antioxidants. Agents, such as clenbuterol, that oppose neurodegeneration and improve recovery of locomotor function may possibly act by improving redox status. Consistent with reduced oxidative stress by beta2-agonist treatment following injury, prior blockade of synthesis of the antioxidant tripeptide, glutathione, with buthionine sulfoximine completely inhibited the ability of clenbuterol to enhance locomotor recovery and spare spinal cord tissue. Moreover, at 8 h postinjury, clenbuterol caused an increase in glutathione reductase activity, an indicator of cellular redox status, at the injury site that was also blocked by buthionine sulfoximine. Although clenbuterol improved locomotor recovery only when administered within a therapeutic window of several days postinjury, the accumulation of protein carbonyls in the spinal cord at 1 week postinjury, a consequence of ongoing ROS-mediated neurodegeneration, was also decreased by clenbuterol in a glutathione-dependent manner. Together, these results suggest that activation of beta2-adrenoreceptors during the acute phase of injury stimulates glutathione-dependent antioxidative processes, that lead to reduced oxidative damage and greater locomotor function as the injury evolves during the subacute and chronic phases.     

C-reactive protein, oxidative stress, homocysteine, and troponin as inflammatory and metabolic predictors of atherosclerosis in ESRD

Mortality in patients with end-stage renal disease remains high, with cardiovascular disease accounting for half of these deaths. Novel risk factors such as inflammation, oxidative stress, hyperhomocysteinemia, and high troponin levels are associated with cardiovascular risk in the general population. While there are substantial epidemiologic data confirming that these novel risk factors are associated with cardiovascular risk in end-stage renal disease patients, a causal relationship has not been established. Inflammation is readily identified by the presence of high levels of C-reactive protein, while studies of oxidative stress are hampered by the lack of a standardized test. The cause of both is unknown. Hyperhomocysteinemia results from decreased remethylation to methionine, although vitamin supplementation only partially corrects the defect, suggesting that uremic inhibition of the enzymatic process may be important. The most promising strategies for correcting oxidative stress and hyperhomocysteinemia are vitamin E and folinic acid therapy, respectively. Troponin I appears to be a more specific marker of myocardial injury than Troponin T, but troponin T retains its ability to predict cardiovascular mortality as well as all-cause mortality. Sorting out the role of each of these risk factors may be difficult since the factors may influence each other, may increase oxidative stress, and may mediate atherosclerosis through oxidative modification of lipids.     

Resveratrol, a red wine polyphenol, protects spinal cord from ischemia-reperfusion injury

OBJECTIVE: The cardioprotective effect of red wine has been attributed to resveratrol. The resveratrol-induced protection against ischemia-reperfusion (I/R) injury has been documented in heart, kidney, and brain. Resveratrol scavenges free O(2) radicals and upregulates nitric oxide (NO). However, the presence of resveratrol-induced spinal cord protection against I/R injury has not been reported in the literature. The objective of this study was to evaluate the effects of resveratrol on neurologic functions, histopathologic changes, and NO metabolism following temporary spinal cord ischemia (SCI) in rabbits.Material and methods SCI was induced with occlusion of the infrarenal aorta in rabbits. In addition to the sham group (group S, n = 7), group C (n = 7) received vehicle 30 minutes before ischemia. Group R1 (n = 7) and R10 (n = 7) received 1 mg/kg and 10 mg/kg resveratrol instead of vehicle, respectively. Blood samples were taken to obtain nitrite/nitrate levels during the surgical procedure. After neurologic evaluation at the 48th hour of reperfusion, lumbar spinal cords were removed for histopathologic examination and malondialdehyde measurement as a marker of oxidative stress. RESULTS: Five animals in group C had paraplegia while 5 in group R10 had normal neurologic functions. The average Tarlov score of group R10 was significantly higher than that the score of group C (4.1 +/- 1.2, vs 1.2 +/- 2.2; P =.014). Histopathologic examination revealed higher neuronal viability index in group R10 compared with that of group C (0.82 +/- 0.24 vs. 0.46 +/- 0.34; P =.018). Nitrite/nitrate levels decreased in group C (from 357 +/- 20.15 micromol/L to 281 +/- 47.9 micromol/L; P <.01) whereas they increased both in group R1 and group R10 (from 287+/-28 micromol/L to 310 +/- 33.9 micromol/L and from 296 +/- 106 micromol/L to 339 +/- 87 micromol/L, respectively) during SCI. Malondialdehyde levels of group R10 was lower than those of group C (55 +/- 12.9 nmol/mg protein vs 83.9 +/- 15.1 nmol/mg protein; P =.001, respectively). CONCLUSIONS: In this model of SCI, resveratrol decreased oxidative stress, increased NO release, and protected spinal cord from I/R injury. Resveratrol-induced neuroprotection is probably mediated by its antioxidant and NO promoting properties. Before considering the clinical use of this natural antioxidant, further research is warranted about its mechanism of effects, timing, and optimum dose. CLINICAL RELEVANCE: Paraplegia that results from spinal cord ischemia is a catastrophic complication of thoracic and thoracoabdominal aorta surgical procedures. Despite several surgical modifications and pharmacologic approaches, paraplegia has not been totally eliminated. On clinical grounds, the efficiency of currently used pharmacologic agents to prevent spinal cord injury during thoracic and thoracoabdominal aorta surgery is very limited and their benefit is controversial. Preischemic infusion of resveratrol protects the spinal cord from ischemia reperfusion injury in rabbits. Following clarification of the underlying protective mechanism, optimal dose, and timing, resveratrol may used in humans as an adjunct to eliminate this catastrophic complication.     

Persistent phosphorylation of NKCC1 and WNK1 in the epicenter of the spinal cord following contusion injury

Background contextNKCC1 regulates neuronal homeostasis of chloride ions and mediates GABAergic activities in nociceptive processing. WNK1 is an upstream regulator of NKCC1 and acts via SPAK (STE20/SPS1-related proline/alanine-rich kinase) and oxidative stress-responsive kinase 1. NKCC1 activity has been shown to be important in edema formation and nociception following spinal cord injury (SCI).PurposeTo determine the role of NKCC1 and WNK1 in spinal cord tissues in the acute and chronic phases following contusional SCI.Study designAn experimental study investigating the phosphorylation profile of an important Cl-regulatory protein Na+-K+-Cl? cotransporter 1 (NKCC1) and its regulatory-kinase WNK1 (kinase with-no-lysine).MethodsSprague-Dawley rats underwent a contusive SCI at T9. The epicenter spinal cord tissues were harvested at Days 1, 3, and 7 for acute phase of injury or Days 35 and 42 in the chronic phase of injury. Western blot was used to compare phosphorylated levels of both NKCC1 and WNK1 in injured tissues compared with those of sham.ResultsA sustained increase in phosphorylation of NKCC1 and WNK1 was detected in the lesion epicenter in spinal cord during both acute and chronic phases following SCI.ConclusionsThese results suggest that persistent activation of NKCC1 and WNK1 may play an important role in SCI.