曲格列酮诱导人心肌细胞氧化应激和自噬的作用

目的研究曲格列酮的心肌细胞毒性特征,并从线粒体氧化应激和自噬角度探讨其潜在的毒作用机制。方法人源心肌细胞AC16给予不同浓度曲格列酮0~40μmol·L^-1孵育24 h。倒置显微镜观察细胞形态,CCK-8法检测细胞存活率,漏出法检测乳酸脱氢酶(LDH)释放量;荧光探针TMRM检测线粒体膜电位和CM-H2DCFDA检测全细胞活性氧的含量;Western印迹法检测微管相关蛋白Ⅱ/Ⅰ轻链3(LC3-Ⅱ/LC3-Ⅰ)比值和P62蛋白表达水平。结果与细胞对照组相比,曲格列酮可浓度依赖性地引起细胞质皱缩、细胞存活率下降(r=-0.928,P<0.05)和LDH释放量增加(r=0.746,P<0.05);曲格列酮10和20μmol·L^-1可明显降低细胞线粒体膜电位(P<0.05),增加全细胞活性氧含量(P<0.05);显著增加LC3-Ⅱ/LC3-Ⅰ比值,上调P62蛋白表达(P<0.05)。结论曲格列酮可引起心肌细胞损伤和线粒体功能障碍,其机制与线粒体氧化应激和自噬体降解受阻密切相关。     

Inhibition of autophagy increases apoptosis during re‐warming after cold storage in renal tubular epithelial cells

Prolonged cold storage and re‐warming (CS/REW) of kidneys are risk factors for delayed graft function (DGF). Studies in renal tubular epithelial cells (RTECs) have determined apoptosis and autophagy in models of either cold storage (CS) or re‐warming alone. The effect of both cold storage and re‐warming on apoptosis and autophagy, in RTECS is not known and is relevant to DGF as the kidney is subjected to both CS and re‐warming. We hypothesized that CS/REW of RTECs would induce autophagy that protects against apoptosis. In CS/REW, there was increased autophagic flux of RTECs. Autophagy inhibition using an Atg5 siRNA resulted in increased cleaved caspase‐3 and increased apoptotic cells (on both morphology and annexin V staining) during CS/REW. The effect of autophagy inhibition on necrosis in RTECs is unknown. There were increased necrosis and caspase‐1, a mediator of necrosis, during CS/REW, and the Atg5 siRNA had no effect on necrosis and caspase‐1. In a kidney transplant model, there was an increase in LC3 II, a marker of autophagy, in kidneys transplanted after cold storage. In summary, autophagic flux is increased during CS/REW. Autophagy inhibition resulted in increased cleaved caspase‐3 and increased apoptosis during CS/REW without an effect on necrosis or caspase‐1. In conclusion, autophagy inhibition in RTECs after CS/REW induces apoptotic cell death and may be deleterious as a therapy to decrease DGF.     

Effect of high volume hemofiltration on CHOP protein from a canine model of multiple organ dysfunction syndrome

Objective To observe the effect of high volume hemofiltration (HVHF) on the expression of CCAAT enhancer binding protein(CHOP) during the treatment of multiple organ dysfunction syndrome (MODS). To investigate the role of CHOP protein act in apoptosis pathway mediated by the endoplasmic reticulum stress. Methods Twelve Beagle dogs were subjected to hemorrhagic shock plus resuscltation and endotixemia to establish MODS model, then they were randomly divided into two groups: HVHF group (n=6) and MODS group (n=6). After endotoxin injection completed, the HVHF group received HVHF treatment for 24 hours; MODS group did not receive. Vivo experiments: Blood samples were obtained at different time points(before operation, 0 h, 6 h, 12 h, 24 h after the injection of endotoxin). The dogs were killed and the tissue samples from lung, liver and kidney were took, then the expression of CHOP mRNA was determined. Vitro experiments: human umbilical vein endothelial cells (HUVECs) were induced by two groups’ blood samples to establish the apoptosis model. Gene expression, protein quantification and cell apoptosis rate were determined before and after the interference. Results Vivo experiments: The levels of CHOP mRNA from lung, liver and kidney had no significant difference between the two groups (P＞0.05). Vitro experiments: (1)The expression of CHOP mRNA: Compared with MODS group, the expression levels of CHOP mRNA were significantly decreased in HVHF group at 6 h, 24 h after the injection of endotoxin (P＜0.05). Compared with before, the expression levels of CHOP mRNA in the two groups were both significantly decreased after CHOP siRNA interference (P＜0.05). (2)The expression of CHOP protein: Compared with MODS group, the expression levels of CHOP protein were significantly decreased in HVHF group at each time points (P＜0.05). Compared with before, the expression levels of CHOP protein in the two groups were both significantly decreased after CHOP siRNA interference(P＜0.05). (3)Endothelial cell apoptosis rate: Compared with the preoperative rate, the two group’s endothelial cell apoptosis rate was decreased significantly at each time points(P＜0.05). Compared with MODS group, the endothelial cell apoptosis rate was significantly decreased in HVHF group at each time points(P＜0.05). Compared with before, the endothelial cell apoptosis rate in the two groups was both significantly decreased after CHOP siRNA interference(P＜0.05). Conclusion In the treatment of MODS process, HVHF can reduce endothelial cell apoptosis which may be related to the inhibition of CHOP mRNA expression and protein synthesis.     

内质网应激相关蛋白CHOP/GADD153在结肠癌组织中的表达及意义

目的：探讨结肠癌组织内质网应激相关蛋白CHOP/GADD153的表达,并分析其与临床病理特征的关系。方法： 选择82例结肠癌患者的结肠癌组织与相应的癌旁正常结肠组织（距癌组织>5 cm）制作蜡块后构建组织芯片,分别用免疫组化法及Western blot法检测CHOP/GADD153蛋白的表达,并分析其表达与患者临床病理特征分关系。结果：免疫组化与Western blot结果均显示,结肠癌组织CHOP/GADD153蛋白的表达水平明显癌旁正常结肠组织（P<0.05）;与临床病理特征的关系分析显示,结肠癌组织中CHOP/GADD153蛋白的表达水平随结肠癌组织分化程度的降低而增强（P<0.05）,而与患者的年龄、性别、肿瘤的大小、肿瘤的浸润深度和淋巴结转移等因素无关（均P>0.05）。结论：结肠癌组织中CHOP/GADD153蛋白的表达增高,并与结肠癌织的分化程度密切相关,提示内质网应激可能参与了肿瘤分化的调控。

     

The role of Ca2+ release from sarcoplasmic reticulum in the regulation of sinoatrial node automaticity

Summary The role of Ca²⁺ release channels in the sarcoplasmic reticulum in modulating physiological automaticity of the sinoatrial (SA) node was studied by recording transmembrane action potentials and membrane ionic currents in small preparations of the rabbit SA node. Ryanodine, which modifies the conductance and gating behavior of the Ca²⁺ release channels, was used to block Ca²⁺ release from the sarcoplasmic reticulum. Superfusion of 1-mM ryanodine decreased the spontaneous firing frequency as well as the maximal rate of depolarization of the SA, and these reductions reached a steady state within approximately 5min. The action potential recordings revealed that the latter part of diastolic depolarization was depressed and that the take-off potential became less negative. This suggested that the negative chronotropic effect of ryanodine resulted from the blockade of physiological Ca²⁺ release from the sarcoplasmic reticulum. In voltage clamp experiments, using double-microelectrode techniques, ryanodine did not markedly reduce the Ca²⁺ current (I_Ca) but decreased the delayed rectifying K+ current (I_K), the steady-state inward current (I_ss), and the hyperpolarization-activated inward current (I_h). These observations suggest that, even when the function of Ca²⁺ channels in the cell membrane is normally maintained, depression of Ca²⁺ release channels in the sarcoplasmic reticulum would prevent sufficient elevation of the Ca²⁺ concentration in SA node cells for the activation of various ionic currents, and, thus adversely affect the physiological automaticity of this primary cardiac pacemaker.     

自噬抑制剂在内质网应激状态下对肝癌HepG2细胞和正常肝细胞L-02作用的差异

目的：研究自噬在内质网应激( ERS)状态下对肝癌HepG2细胞和正常肝细胞L-02作用的差异。方法体外常规培养的人肝癌HepG2细胞和正常肝细胞L-02,分别给予衣霉素( TM)单药和TM联合自噬抑制剂3-甲基腺嘌呤( TM+3-MA)或氯喹( TM+CQ)作用12、24、48 h后,采用噻唑蓝( MTT)法检测细胞活力变化,流式细胞术检测细胞凋亡率, Western blot法检测自噬蛋白LC3的变化。结果 TM可引起HepG2细胞和L-02细胞死亡并呈时间依赖关系,3-MA或CQ均可增加TM对HepG2细胞的生长抑制作用,24 h细胞存活率分别为TM+3-MA组(60%)、TM+CQ组(72%)、TM组(86%),差异有统计学意义(P<0．01);但对于L-02细胞,其存活率分别为83%、84%、83%,活力没有明显差异;流式细胞术显示TM+3-MA、TM+CQ和TM组对HepG2细胞的凋亡率分别为15%、11%、7%,差异有统计学意义( P<0．01),但对L-02细胞,凋亡率分别为16%、17%、16%,未见明显差异;Western blot法结果显示TM作用引起两种细胞自噬增加,自噬抑制剂3-MA与CQ可引起两种细胞自噬作用减弱。结论自噬抑制剂(3-MA 或 CQ)均可显著增加TM对肝癌HepG2细胞的生长抑制作用,但对正常肝细胞L-02的生长抑制作用差异无统计学意义。自噬在ERS状态下可对肝癌细胞的生存提供保护,但对正常肝细胞无保护作用。     

MANF蛋白表达水平与肝纤维化程度的相关性研究

目的：探讨中脑星形胶质细胞源性神经营养因子(MANF)蛋白在慢性乙型肝炎病毒(HBV)感染患者肝纤维化发生发展中的作用及其与临床特点的相关性。方法采用相对和绝对定量同位素标记( ITRAQ)蛋白质组学和免疫组化法检测肝脏穿刺组织中MANF蛋白的表达,分析其表达水平与肝脏炎症纤维化分期的关系;采用实时荧光定量PCR技术检测正常对照( NC)、乙肝病毒携带者( ASC)、慢性乙型肝炎患者( CHB)及乙肝后肝硬化患者( LC)外周血白细胞中MANF mRNA的表达水平,分析其与不同阶段慢性HBV感染者的临床病毒学和生化学指标的相关性。结果 MANF蛋白主要在肝细胞质中表达,其表达水平随着肝脏炎症及纤维化的程度加重而升高。 NC 组、ASC 组和 CHB 组分别与LC组比较,MANF mRNA的表达差异均有统计学意义( P<0．01)。 MANF mRNA 的表达水平在乙肝病毒表面抗原(HBsAg)<1．5×106 IU/L、(1．5×106~2．0×107) IU/L和>2．0×107 IU/L 3组间差异有统计学意义(P<0．05);在乙肝病毒e抗原( HBeAg)阳性组与阴性组之间差异有统计学意义(P<0．01);在胆红素正常组与异常组之间差异有统计学意义( P <0．01)。结论 MANF 蛋白可能参与了慢性HBV感染者肝纤维化的发生发展,并与其临床特点相关。     

内质网应激与相关眼科疾病

内质网是一个非常重要的细胞器.当内质网应激发生,细胞内信号分子活化,启动未折叠蛋白反应,细胞最终得以适应性存活或者启动凋亡.诸如白内障、糖尿病性视网膜病变(diabetic retinopathy,DR)、青光眼、色素性视网膜炎(retinitis pigmentosa,RP)等眼科疾病的发生和发展都与内质网应激过程密切相关,就此研究动态予以综述.