缺血再灌注损伤脑微血管内皮细胞Necroptosis模型的建立

目的:采用拟缺血再灌注损伤结合z-VAD-FMK(Benzyloxyearbonyl-Val-Ala-Asp-fluoromethylketone,z-VAD-FMK)干预,探索一种脑微血管内皮细胞(Brain Microvascular Endothelial Cells,BMECs)Necroptosis模型.方法:首先利用原代大鼠脑微血管内皮细胞,采用氧糖剥夺及复氧复糖方法,筛选出拟缺血再灌注损伤时间点.在拟缺血再灌注模型基础上,予Casepase抑制剂z-VAD-FMK 20 μmol/L干预,采用CCK-8检测细胞活性;透射电镜观察细胞超微结构;Annexin V-FITC/PI(Propidium Iodide)双染色法检测细胞死亡方式.结果:确定氧糖剥夺2 h复氧复糖8 h,作为拟缺血再灌注时间点;z-VAD-FMK作用于拟缺血再灌注损伤BMECs后,细胞活性无统计学意义;z-VAD-FMK干预组在电镜下呈现明显的Necroptosis特征;流式检测显示,各象限细胞比率无明显变化,但Necroptosis特异性抑制剂Nec-1可显著降低Q2象限细胞比率,提示z-VAD-FMK干预抑制了细胞晚期凋亡,诱导了Necroptosis的发生.结论:z-VAD-FMK可诱导拟缺血再灌注脑微血管内皮细胞发生Necroptosis,为以后研究缺血性脑中风necroptosis机制提供了细胞实验模型.

Necroptosis在中枢神经系统损伤修复中作用的研究进展

Necroptosis是一种新发现的程序性细胞死亡方式，由死亡受体与其配体的结合所启动，通过特定的信号通路执行。Necroptosis已被证实参与了多种疾病的病理进程，包括肿瘤、免疫性疾病、脑外伤及脑部缺血再灌注损伤等。

Neurodegenerative disorders affect more than 30 million individuals throughout the world and lead to signiifcant disability as well as death. These statistics will increase almost exponentially as the lifespan and age of individuals increase globally and individuals become more susceptible to acute disorders such as stroke as well as chronic diseases that involve cognitive loss, Alzheimer’s disease, and Parkinson’s disease. Current therapies for such disorders are effective only for a small subset of individuals or provide symptomatic relief but do not alter disease progression. One exciting therapeutic approach that may turn the tide for addressing neurodegenerative disorders involves the mammalian target of rapamycin (mTOR). mTOR is a component of the protein complexes mTOR Complex 1 (mTORC1) and mTOR Complex 2 (mTORC2) that are ubiquitous throughout the body and control multiple functions such as gene transcription, metabolism, cell survival, and cell senescence. mTOR through its relationship with phosphoinositide 3-kinas e (PI 3-K) and protein kinase B (Akt) and multiple downstream signaling pathways such as p70 ribosomal S6 kinase (p70S6K) and proline rich Akt substrate 40 kDa (PRAS40) promotes neuro-nal cell regeneration through stem cell renewal and oversees critical pathways such as apoptosis, autophagy, and necroptosis to foster protection against neurodegenerative disorders. Targeting by mTOR of speciifc pathways that drive long-term potentiation, synaptic plasticity, andβ-amyl oid toxicity may offer new strategies for disorders such as stroke and Alzheimer’s disease. Overall, mTOR is an essential neuroprotective pathway but must be carefully targeted to maximize clini-cal efifcacy and eliminate any clinical toxic side effects.

Necroptosis is characterized by programmed necrotic cell death and autophagic activation and might be involved in the death process of dopaminergic neurons in Parkinson’s disease. We hypothesized that necrostatin-1 could block necroptosis and give protection to dopaminergic neurons. There is likely to be crosstalk between necroptosis and other cell death pathways, such as apoptosis and autophagy. PC12 cells were pretreated with necroststin-1 1 hour before expo-sure to 6-hydroxydopamine. We examined cell viability, mitochondrial membrane potential and expression patterns of apoptotic and necroptotic death signaling proteins. The results showed that the autophagy/lysosomal pathway is involved in the 6-hydroxydopamine-induced death pro-cess of PC12 cells. Mitochondrial disability induced overactive autophagy, increased cathepsin B expression, and diminished Bcl-2 expression. Necrostatin-1 within a certain concentration range (5–30 μM) elevated the viability of PC12 cells, stabilized mitochondrial membrane potential, inhibited excessive autophagy, reduced the expression of LC3-II and cathepsin B, and increased Bcl-2 expression. These findings suggest that necrostatin-1 exerted a protective effect against injury on dopaminergic neurons. Necrostatin-1 interacts with the apoptosis signaling pathway during this process. This pathway could be a new neuroprotective and therapeutic target in Par-kinson’s disease.

脑缺血后 necroptosis的机制及神经保护

细胞程序性坏死（ necroptosis）是一种新型的细胞死亡方式。目前研究的多的机制为死亡受体（ death re-ceptors，DRs）介导的信号转导途径。受体交互作用蛋白（receptor interaction protein ，RIP）1和3是necroptosis信号通路中极为重要的调节蛋白。并且necroptosis能诱导相关的炎症反应，加重组织损伤。新近研究表明脑缺血后脑组织的损伤与necroptosis密切相关。本文就necroptosis的机制及有关神经保护方面的分子靶向研究进展进行综述。

活性氧自由基在肾小管上皮细胞necroptosis中的作用

目的:探讨活性氧自由基(ROS)在肾小管上皮细胞necroptosis中的作用.方法:构建肾小管上皮细胞HK-2细胞necroptosis模型,检测其ROS升高程度.并使用NADPH酶抑制剂Apocynin 抑制HK-2细胞necroptosis模型中ROS的生成,通过流式细胞计数及检测necroptosis的关键蛋白观察HK-2细胞necroptosis的变化.结果:使用肿瘤坏死因子α、苄氧羰酰-缬氨酰-丙氨酰-天冬氨酰-氟甲基酮及抗霉素A成功建立了HK-2细胞necroptosis模型,并观察到HK-2细胞发生necroptosis时ROS显著升高(43.29±2.49 vs 25.90±1.27,P<0.001),而使用necrostatin-1抑制necroptosis后ROS生成受到抑制(35.58±1.08 vs 43.29±2.49,P=0.002).当对necroptosis模型使用Apocynin干预时,HK-2细胞ROS明显下降(30.71±2.82 vs 43.29±2.49,P<0.001),并且流式细胞计数结果显示坏死细胞比例减少(2.00%±0.30% vs 6.99%±2.79%,P<0.001),同时受体相关蛋白3和混合系列蛋白激酶样结构域的磷酸化水平降低.结论:ROS参与了HK-2细胞的necroptosis,并且通过抑制ROS的生成可减少necroptosis发生,提高损伤状态下HK-2细胞存活率,减轻急性肾小管坏死.

HIF-1α在缺糖缺氧诱导的原代皮质神经元necroptosis中的表达

目的 探讨低氧诱导因子-1α(hypoxia-inducible factor-1α,HIF-1α)是否参与缺糖缺氧诱导的原代皮质神经元necroptosis.方法 原代皮质神经元培养14天,予caspase抑制剂Z-VAD-FMK(20 μmol/L)预保护30 min,缺糖缺氧2 h,再灌注0、2、6、12、24、48 h,进行LDH测定;RT-PCR测HIF-1α RNA表达;Western blot检测总HIF-1α蛋白表达情况;分别提取细胞质和细胞核蛋白,Western blot分别检测胞质、胞核内HIF-1α蛋白表达情况.结果 caspase抑制剂Z-VAD-FMK预作用30 min、缺糖缺氧2 h、再灌注2 h后培养基中LDH增加(P＜0.05),再灌注12 h达高峰;缺糖缺氧后HIF-1α RNA表达无变化(P＞0.05);HIF-1α总蛋白表达增加(P＜0.05),再灌注12 h达高峰;再灌注6 h时细胞质HIF-1α蛋白表达达高峰(P＜0.05),随后降低;再灌注12 h时,细胞核HIF-1α蛋白表达达高峰(P＜0.05),随后降低.结论 HIF-1α参与缺糖缺氧诱导的原代皮质神经元necroptosis.

RIP3在necroptosis信号通路中的胞内定位

目的 研究necroptosis信号通路中受体相互作用蛋白3(RIP3)的胞内定位.方法 SD大鼠原代皮质神经元细胞培养12天,zVAD 20 μmol/L预处理30 min,TNFα 30 ng/ml处理不同时间,Western blot检测胞质、胞核中RIP3蛋白水平,结合免疫荧光观察RIP3的胞内定位.结果 随着TNFα作用时间的延长,胞质、胞核中RIP3蛋白水平均呈上升趋势,胞质中RIP3蛋白水平在8 h达高峰,随后下降,胞核中RIP3蛋白水平在12 h达高峰,即在12 h出现核转位的高峰(P＜0.05).结论 正常细胞中RIP3存在于胞质,necroptosis时发生核转位.

Necrostatin-1对输尿管梗阻小鼠肾脏炎症的影响

目的 探讨necroptosis(坏死性凋亡)抑制剂Necrostatin-1(Nec-1)对单侧输尿管梗阻(unilateral ureter obstruction,UUO)小鼠肾脏炎症的影响.方法 雄性C57BL/6J小鼠随机分为假手术组(Sham组)、左侧输尿管结扎组(UUO组)、UUO+ Nec-1治疗组(UN组),术后7天处死小鼠取左肾.收集血清测定肌酐(Scr)、尿素氮(BUN)水平;HE染色观察肾脏形态学改变;免疫组化法和Western blot法分别检测肾脏组织中坏死性凋亡相关指标RIP1、RIP3、MLKL以及炎症相关因子TNF-α、IL-1β、MCP-1的表达.结果 与Sham组相比,UUO组小鼠RIP1、RIP3和MLKL蛋白表达明显升高,同时伴有TNF-α、IL-1β、MCP-1表达增多.Nec-1治疗显著降低了上述蛋白的表达水平,同时HE染色显示肾间质炎性反应和肾小管损伤程度有所减轻,Scr和BUN水平提示肾功能有所改善.结论 Nec-1通过抑制necroptosis减轻了单侧输尿管梗阻小鼠肾脏的炎症反应,这可能成为治疗肾脏继发炎症的新靶点.

Necroptosis的分子机制及其在心血管疾病中的研究进展

Necroptosis作为一种新的半胱氨酸天冬酰胺特异蛋白酶非依赖性的细胞死亡方式,在细胞内环境稳态中扮演重要的角色.necroptosis通过肿瘤坏死因子受体(TNFR)激活启动,活化受体相互作用蛋白激酶1 (RIP1)-受体相互作用蛋白激酶3(RIP3)-混合系列蛋白激酶样结构域(MLKL)信号通路诱导其发生.研究发现necroptosis与凋亡和自噬具有十分密切的关系,因此探讨这3种信号机制间的相互作用关系对研究疾病进展及治疗方向具有十分重要的价值.本文以necroptosis分子调控机制及与凋亡、自噬三者间的关系进行综述,同时探讨其在心血管疾病心肌损伤中的作用,为临床认识、治疗心血管疾病提供方向.

Cell death plays an important role in the regulation of inflammation and may be the result of inflammation. The maintenance of tissue homeostasis necessitates both the recognition and removal of invading microbial pathogens as well as the clearance of dying cells. In the past few decades, emerging knowledge on cell death and inflammation has enriched our molecular understanding of the signaling pathways that mediate various programs of cell death and multiple types of inflammatory responses. This review provides an overview of the major types of cell death related to inflammation. Modification of cell death pathways is likely to be a logical therapeutic target for inflammatory diseases.