人参皂苷Rb1在Aβ损伤的PC12细胞中激活线粒体自噬的机制研究＊

目的 基于Aβ损伤的PC12模型，观察人参皂苷Rb1对线粒体自噬的调节作用。方法 用Aβ损伤PC12细胞建立体外AD模型，采用人参皂苷Rb1进行干预后，利用透射电镜观察自噬情况，Western blot检测LC3Ⅱ/Ⅰ、p62、PINK1、parkin、NDP52和OPTN蛋白的表达，荧光实时定量PCR检测LC3、p62、PINK1、parkin、NDP52、OPTN mRNA水平。结果 人参皂苷Rb1高剂量促进细胞中自噬小体包裹受损线粒体，升高LC3Ⅱ/Ⅰ比值（P < 0.05），减少p62蛋白水平（P < 0.05）；同时，人参皂苷Rb1显著增加了PINK1蛋白表达（P < 0.01），同时减少OPTN及NDP52蛋白水平（P < 0.05）；此外，人参皂苷Rb1高剂量组parkin、OPTN mRNA表达显著上调（P < 0.01），低剂量组PINK1、NDP52 mRNA表达也显著上调（P < 0.05）。结论 人参皂苷Rb1神经保护作用与调节线粒体自噬相关，通过激活PINK1/parkin通路促进线粒体自噬而改善体外AD模型损伤情况。     

构建SD大鼠鱼藤酮中毒模型及其线粒体DNA突变的初步探索

目的构建SD大鼠鱼藤酮慢性中毒模型,同时对SD大鼠组织标本(脑组织和肝脏)进行线粒体DNA突变的初步探索,为后续实验提供线索。方法将75只雄性SD鼠随机分为三组,实验组:0.5 mg/(kg·d)鱼藤酮皮下注射4~8周,共45只;空白组:不予任何特殊处理4~8周,共15只;对照组:相同体积的二甲基亚砜(DMSO)皮下注射4~8周,共15只。其中实验组随机分为1、2、3小组,每小组15只;空白组和对照组也分别分为1、2、3小组,每小组5只。每组内相应的1、2、3小组分别在实验的第4、6、8周处死老鼠并留取相应的组织标本(中脑组织和肝脏),存储于-80℃冰箱中待用。在实验组的每个小组内分别随机选取3对组织标本,共9对;在空白组和对照组的每个小组内分别随机选取1对组织标本,共6对。随后进行组织DNA提取,采用2对引物进行长PCR并将PCR产物进行凝胶电泳分析,同时设计并利用线粒体DNA引物,用一代测序的方法进行线粒体全基因组测序。实验中观察各组大鼠的一般情况并进行行为学测试。结果1)实验组大鼠在实验中表现出较为明显的中毒症状。在体重增长速度上与对照组和空白组存在明显差异(P0.05)。在行为学上,实验组大鼠亦表现出了较为明显的行为学异常(P0.05)。2)大鼠脑以及肝脏组织的长PCR凝胶电泳结果未发现明显的异常条带;所有测序突变(缺失、插入、点突变)均为大鼠固有突变。结论 0.5 mg/(kg·d)鱼藤酮皮下注射4~8周可导致SD大鼠鱼藤酮慢性中毒。SD幼鼠的选择、鱼藤酮剂量及作用时间、一代测序等多种因素可能是导致本次实验未检测到有意义的线粒体DNA突变的原因。因此,后续实验应做相应改进。     

Dependency of 2-methoxyestradiol-induced mitochondrial apoptosis on mitotic spindle network impairment and prometaphase arrest in human Jurkat T cells

The present study sought to determine the correlation between 2-methoxyestradiol (2-MeO-E₂)-induced cell cycle arrest and 2-MeO-E₂-induced apoptosis. Exposure of Jurkat T cell clone (JT/Neo) to 2-MeO-E₂ (0.5–1.0 μM) caused G₂/M arrest, Bak activation, Δψm loss, caspase-9 and -3 activation, PARP cleavage, intracellular ROS accumulation, and apoptotic DNA fragmentation, whereas none of these events except for G₂/M arrest were induced in Jurkat T cells overexpressing Bcl-2 (JT/Bcl-2). Under these conditions, Cdk1 phosphorylation at Thr-161 and dephosphorylation at Tyr-15, up-regulation of cyclin B1 expression, histone H1 phosphorylation, Cdc25C phosphorylation at Thr-48, Bcl-2 phosphorylation at Thr-56 and Ser-70, Mcl-1 phosphorylation at Ser-159/Thr-163, and Bim phosphorylation were detected irrespective of Bcl-2 overexpression. Concomitant treatment of JT/Neo cells with 2-MeO-E₂ and the G₁/S blocking agent aphidicolin resulted in G₁/S arrest and abrogation of all apoptotic events, including Cdk1 activation, phosphorylation of Bcl-2, Mcl-1 and Bim, and ROS accumulation. The 2-MeO-E₂-induced phosphorylation of Bcl-2 family proteins and mitochondrial apoptotic events were suppressed by a Cdk1 inhibitor, but not by an Aurora A kinase (AURKA), Aurora B kinase (AURKB), JNK, or p38 MAPK inhibitor. Immunofluorescence microscopic analysis revealed that 2-MeO-E₂-induced mitotic arrest was caused by mitotic spindle network impairment and prometaphase arrest. Whereas 10–20 μM 2-MeO-E₂ reduced the proportion of intracellular polymeric tubulin to monomeric tubulin, 0.5–5.0 μM 2-MeO-E₂ increased it. These results demonstrate that the apoptogenic effect of 2-MeO-E₂ (0.5–1.0 μM) was attributable to mitotic spindle defect-mediated prometaphase arrest, Cdk1 activation, phosphorylation of Bcl-2, Mcl-1, and Bim, and activation of Bak and mitochondria-dependent caspase cascade.     

Luteolin promotes mitochondrial protection during acute and chronic periods of isoproterenol induced myocardial infarction in rats

ObjectiveAn attempt has been made to evaluate the mitochondrial protection in acute and chronic periods after isoproterenol (ISO)-induced myocardial-infarction (MI) in male Wistar rats.Materials and methodsLuteolin was supplemented by intra-gastric intubation at a daily dose of 0.3 mg/kg body weight for 30 days. In the acute MI model, luteolin had been administered once per day to rat groups during 30 days. On 29th and 30th days, the rats of the acute MI control groups were administered 85 mg/kg body weight, isoproterenol, intra-peritoneally at an interval of 24 h. In the chronic MI model luteolin was supplemented to the rat group during 30 days. On the 1st and 2nd days, the rats of the chronic MI control and luteolin treatment groups were administered ISO by the same way.ResultsThe isoproterenol-treated rats both in acute and chronic models showed an increase in the level of TBARS and a decrease in the activities of mitochondrial antioxidants in MI rats, an increase in levels of mitochondrial lipid profile except phospholipids and the activities of mitochondrial enzymes were decreased in isoproterenol-treated rats. Oral treatments with luteolin in both acute and chronic models showed a significant decrease in the levels of mitochondrial lipid peroxidation, increase in the mitochondrial antioxidant levels and also decrease in the mitochondrial enzymes.ConclusionThus the present study revealed that luteolin ameliorates mitochondrial damage in isoproterenol induced myocardial infarction by maintaining lipid peroxidation metabolism due to its free radical scavenging, mitochondrial lipids, antioxidants and mitochondrial enzymes. Histopathological observations were also in correlation with the biochemical parameters.     

Hepatotoxicity reports in the FDA adverse event reporting system database: A comparison of drugs that cause injury via mitochondrial or other mechanisms

Drug-induced liver injury (DILI) is a leading reason for preclinical safety attrition and post-market drug withdrawals. Drug-induced mitochondrial toxicity has been shown to play an essential role in various forms of DILI, especially in idiosyncratic liver injury. This study examined liver injury reports submitted to the Food and Drug Administration (FDA) Adverse Event Reporting System (FAERS) for drugs associated with hepatotoxicity via mitochondrial mechanisms compared with non-mitochondrial mechanisms of toxicity. The frequency of hepatotoxicity was determined at a group level and individual drug level. A reporting odds ratio (ROR) was calculated as the measure of effect. Between the two DILI groups, reports for DILI involving mitochondrial mechanisms of toxicity had a 1.43 (95% CI 1.42–1.45; P < 0.0001) times higher odds compared to drugs associated with non-mitochondrial mechanisms of toxicity. Antineoplastic, antiviral, analgesic, antibiotic, and antimycobacterial drugs were the top five drug classes with the highest ROR values. Although the top 20 drugs with the highest ROR values included drugs with both mitochondrial and non-mitochondrial injury mechanisms, the top four drugs (ROR values > 18: benzbromarone, troglitazone, isoniazid, rifampin) were associated with mitochondrial mechanisms of toxicity. The major demographic influence for DILI risk was also examined. There was a higher mean patient age among reports for drugs that were associated with mitochondrial mechanisms of toxicity [56.1 ± 18.33 (SD)] compared to non-mitochondrial mechanisms [48 ± 19.53 (SD)] (P < 0.0001), suggesting that age may play a role in susceptibility to DILI via mitochondrial mechanisms of toxicity. Univariate logistic regression analysis showed that reports of liver injury were 2.2 (odds ratio: 2.2, 95% CI 2.12–2.26) times more likely to be associated with older patient age, as compared with reports involving patients less than 65 years of age. Compared to males, female patients were 37% less likely (odds ratio: 0.63, 95% CI 0.61–0.64) to be subjects of liver injury reports for drugs associated with mitochondrial toxicity mechanisms. Given the higher proportion of severe liver injury reports among drugs associated with mitochondrial mechanisms of toxicity, it is essential to understand if a drug causes mitochondrial toxicity during preclinical drug development when drug design alternatives, more clinically relevant animal models, and better clinical biomarkers may provide a better translation of drug-induced mitochondrial toxicity risk assessment from animals to humans. Our findings from this study align with mitochondrial mechanisms of toxicity being an important cause of DILI, and this should be further investigated in real-world studies with robust designs.     

创伤弧菌诱导树突状细胞凋亡的过程观察

目的 实验观察创伤弧菌(Vibrio vulnificus,Vv)诱导鼠树突状细胞(dendritic cell,DC)凋亡的过程.方法 建立小鼠树突状细胞(DC2.4株)与创伤弧菌(Vv1.1758株)混合培养模型,DAPI荧光染色分析细胞凋亡的形态特征,DNA Ladder检测凋亡细胞的DNA片段化水平分析,Annexin V FITC/PI染色分析DC2.4细胞凋亡率,分光光度法测定caspase-3、caspase-8活性,JC-1荧光标记检测线粒体膜电位(△Ψm)变化.结果 Vv1.1758株与DC2.4细胞混合培养4h时DAPI荧光染色出现典型的凋亡特征——染色质浓缩及边缘化；DNA琼脂糖凝胶电泳出现凋亡条带；2、4、6h细胞凋亡率分别为(37.8±9.8)％、(54.3±12.7)％和(68.2±14.6)％；1、2、4h线粒体膜电位(△ψm)分别下降了7.1％、16.1％与46.7％；caspase-8活性在1.5h增高,2h达高峰(2.48±0.19) U/μg,而caspase-3活性于3h开始增高,4h达高峰(1.91±0.16) U/μg.结论 创伤弧菌诱导树突状细胞可通过线粒体膜电位下降及激活caspase-8启动子两条途径,最终活化效应因子caspase-3,促使细胞凋亡发生.     

线粒体介导的细胞凋亡与男性不育的研究进展

随着世界工业化进程的加快,环境污染逐渐加重,男性不育的发病率呈全球性的增高趋势。疾病、毒物接触、电磁辐射、环境雌激素都可能是造成生育能力下降的诱因,但造成男性不育的机制尚不明确。精子质量低下是男性不育的根本原因,主要包括精子DNA损伤、精子活动力下降、精子密度降低等。线粒体损伤与精子细胞凋亡率升高常伴随以上原因出现。该文对线粒体介导的细胞凋亡与男性不育之间的关系进行简要综述。