Nickel oxide nanoparticles induce hepatocyte apoptosis via activating endoplasmic reticulum stress pathways in rats

Nickel oxide nanoparticles (nano NiO) could induce hepatocyte apoptosis, while its potential mechanisms are unclear. This study aimed to explore the role of endoplasmic reticulum (ER) stress pathways in hepatocyte apoptosis induced by nano NiO. Male Wistar rats were administrated with nano NiO (0.015, 0.06, and 0.24 mg/kg b.w.) and micro NiO (0.24 mg/kg b.w.) by intratracheal instillation twice a week for 6 weeks. We measured the hepatocyte apoptosis levels by TdT‐mediated dUTP nick‐end labeling (TUNEL) staining, ER stress related gene and protein expression levels in rat liver. The results showed that the TUNEL positive cells increased after exposure nano NiO, hinting hepatocyte apoptosis. The up‐regulated gene and protein levels of 78 kD glucose regulated protein and CCAAT/enhancer binding protein homologous protein suggested that nano NiO triggered ER stress. Nano NiO exposure contributed to the increased protein contents of inositol‐requiring enzyme 1 (IRE‐1)α, p‐IRE‐1α, X box protein‐1S, pancreatic ER kinase (PERK), p‐PERK, eukaryotic initiation factor‐2 alpha (eIF‐2α), p‐eIF‐2α, caspase‐12, ?9, and ?3, implicating that nano NiO can activate the pathways of ER stress‐mediated apoptosis. These findings indicate that the ER stress pathways may play an important role in hepatocyte apoptosis induced by nano NiO.     

Cantharidin-induced LO2 cell autophagy and apoptosis via endoplasmic reticulum stress pathway in vitro

Cantharidin (CTD), an important active compound derived from the traditional Chinese medicine Mylabris (also called Banmao), has been used in the treatment of diseases such as tumors and dermatosis. However, Mylabris has been shown to induce hepatotoxicity in clinical practice and animal experiments, limiting its use. Further, a detailed mechanism underlying CTD-induced hepatotoxicity has not been determined. In the present study, we aimed to explore the effect of endoplasmic reticulum stress (ERS), autophagy, and apoptosis on CTD-induced hepatotoxicity. We found that CTD could inhibit the proliferation of LO2 cells; increase alanine aminotransferase, aspartate aminotransferase, lactate dehydrogenase, and malondialdehyde levels; and reduce glutathione peroxidase and superoxide dismutase activities. Western blotting showed that low concentrations of CTD induced the expressions of ERS-related proteins [GRP78, ATF4, PERK, p-PERK, XBP1–1 s, and CHOP], but high concentrations of CTD inhibited their expressions. Furthermore, high concentrations of CTD activated autophagy (LC3, Beclin-1, Atg3, Atg4A, Atg4B, and Atg7), induced the expressions of apoptotic proteins (Bax/Bcl-2 and caspase-3), and increased LO2 toxicity. Taken together, these results indicated that CTD can induce LO2 cytotoxicity by inhibiting ERS and inducing autophagy and apoptosis, which provides a scientific basis for CTD-induced hepatotoxicity.     

毒胡萝卜素诱导大鼠皮层神经元内质网应激及丹红注射液的干预作用

目的：探讨毒胡萝卜紊诱导大鼠皮层神经元内质网应激凋亡的机制及丹红注射液的干预作用。方法：体外培养SD乳鼠皮层神经元，免疫组织化学、免疫荧光染色鉴定神经元纯度。流式细胞术Annexin V、PI双标检测凋亡率及活性caspase-3、caspase-8、caspase-7、caspase-9表达，Western Noting免疫印迹分析caspase-12、GRP78、Bcl-2、细胞色素C蛋白表达，Fura-2／AM法荧光分光光度计检测细胞内钙浓度（[Ca^2＋]i）。结果：SD乳鼠皮层神经元可纯化体外培养。2μmol／L毒胡萝卜素作用神经元24、48h细胞凋亡率分别是17.88％、21.38％，丹红治疗组分别是6．30％、6．11％，两组比较差异有统计学意义（P〈0．05）。毒胡萝卜素诱导神经元GRP78表达上调，剪切活化caspase-3、caspase-8、caspase-9、caspase-12，使细胞色素C表达增加，Bcl-2表达减少。丹红注射液促进细胞Bcl-2表达，抑制细胞色素C释放，减少活化的caspase-3、caspase-8、caspase-9含量，稳定游离钙浓度。结论：毒胡萝卜素诱导神经元内质网应激反应性凋亡。丹红注射液能抑制体外培养神经元内质网应激所致凋亡。     

辛伐他汀通过内质网应激途径诱导K562细胞凋亡

探讨内质网应激在辛伐他汀诱导K562细胞凋亡中的作用。采用荧光显微镜观察凋亡细胞的形态变化，AnnexinV-FITC/PI双染法检测细胞凋亡率，激光扫描共聚焦显微镜检测细胞内Ｃa²⁺浓度，RT-PCR检测葡萄糖调节蛋白78(glucose regulated protein 78，GRP78)、钙蛋白酶(calpain)基因mRNA表达水平，Western blotting检测GRP78、 calpain、 caspase-3， -6， -7， -9， -12蛋白水平。结果显示，10、 20、 30 μmol·L^-1辛伐他汀(simvastatin，Sim)作用K562细胞72 h后，细胞出现典型的凋亡形态，凋亡率分别为12.41%、 19.08%和23.41%；细胞内Ca²⁺浓度增加，荧光强度分别为43、54和64；GRP78、calpain基因mRNA表达上调；calpain、 caspase-3， -6， -7， -9， -12蛋白剪切活化、GRP78蛋白表达增强。以上结果表明，内质网作为细胞凋亡的重要途径参与了辛伐他汀诱导K562细胞的凋亡。辛伐他汀将可能被用于临床治疗白血病。     

活性氧与内质网应激

内质网(endoplasmic reticulum,ER)是细胞加工蛋白质和贮存Ca2+的主要场所,对应激极为敏感,其功能紊乱时出现错误折叠与未折叠蛋白在腔内聚集以及Ca2+平衡紊乱的状态,称为内质网应激(endoplasmic reticulum stress,ERS)。活性氧(reactive oxygen species,ROS)作为第二信使,在细胞生物学功能的调节中起着重要作用。细胞内氧化还原状态的改变促进了ROS的产生和凋亡诱导因子的激活,致使细胞凋亡的同时又加剧了细胞内氧化还原状态的改变。研究发现细胞内氧化还原水平的改变在ERS介导的细胞凋亡过程中承担重要的角色,推测ROS可能是ERS介导的凋亡通路的上游信号分子,该文就ROS与ERS之间的关系作一综述。     

内质网应激与心房颤动研究进展

心房颤动（AF）是临床常见的持续性心律失常,是卒中和心力衰竭的独立危险因素,然而其具体发病机制尚不完全清楚。内质网是调控蛋白质合成、细胞内Ca2+浓度、氧化应激水平、诱导细胞凋亡信号通路的主要细胞器,在心律失常发生和发展中的作用日益受到重视。多种致病因素可导致内质网应激（ERS）,其主要通过未折叠蛋白反应（UPR）来恢复内质网稳态。ERS的过度激活可导致心房肌细胞Ca2+超载、氧化应激失衡和细胞凋亡,在AF的发病机制中发挥重要作用。本文对ERS和AF研究进展进行了综述。     

Nickel sulfate induced apoptosis via activating ROS‐dependent mitochondria and endoplasmic reticulum stress pathways in rat Leydig cells

Nickel can induce apoptosis of testicular Leydig cells in mice, whereas the mechanisms remain unclear. In this study, we investigated the role of nickel‐induced reactive oxygen species (ROS) generation in mitochondria and endoplasmic reticulum stress (ERS) mediated apoptosis pathways in rat Leydig cells. Fluorescent DCF and Annexin‐V FITC/PI staining were performed to measure the production of ROS and apoptosis in Leydig cells. RT‐qPCR and Western blot were conducted to analyze the key genes and proteins involved in mitochondria and ERS apoptotic pathways. The results showed that nickel sulfate induced ROS generation, consequently resulted in nucleolus deformation and apoptosis in testicular Leydig cells, which were then attenuated by ROS inhibitors of N‐acetylcysteine (NAC) and 2,2,6,6‐tetramethyl‐1‐piperidinyloxy (TEMPO). Nickel sulfate‐triggered Leydig cells apoptosis via mitochondria and ERS pathways was characterized by the upregulated mRNA and proteins expression of Bak, cytochrome c, caspase 9, caspase 3, GRP78, GADD153, and caspase 12, which were inhibited by NAC and TEMPO respectively. The findings indicated that nickel‐induced ROS generation was involved in apoptosis via mitochondria and ERS pathways in rat Leydig cells.     

Zonisamide,an antiepileptic drug,alleviates diabetic cardiomyopathy by inhibiting endoplasmic reticulum stress

Endoplasmic reticulum stress(ER stress)plays a key role in the development of cardiac hypertrophy and diabetic cardiomyopathy(DCM).Zonisamide(ZNS)was originally developed as an antiepileptic drug.Studies have shown that ZNS suppresses ER stress-induced neuronal cell damage in the experimental models of Parkinson’s disease.Herein,we investigated whether ZNS improved DCM by attenuating ER stress-induced apoptosis.C57BL/6J mice were fed with high-fat diet(HFD)and intraperitoneally injected with low-dose streptozotocin(STZ)to induce type 2 diabetes mellitus(T2DM),and then treated with ZNS(40 mg·kg^?1·d^?1,i.g.)for 16 weeks.We showed that ZNS administration slightly ameliorated the blood glucose levels,but significantly alleviated diabetes-induced cardiac dysfunction and hypertrophy.Furthermore,ZNS administration significantly inhibited the Bax and caspase-3 activity,upregulated Bcl-2 activity,and decreased the proportion of TUNEL-positive cells in heart tissues.We analyzed the hallmarks of ER stress in heart tissues,and revealed that ZNS administration significantly decreased the protein levels of GRP78,XBP-1s,ATF6,PERK,ATF4,and CHOP,and elevated Hrd1 protein.In high glucose(HG)-treated primary cardiomyocytes,application of ZNS(3μM)significantly alleviated HG-induced cardiomyocyte hypertrophy and apoptosis.ZNS application also suppressed activated ER stress in HG-treated cardiomyocytes.Moreover,preapplication of the specific ER stress inducer tunicamycin(10 ng/mL)eliminated the protective effects of ZNS against HG-induced cardiac hypertrophy and ER stress-mediated apoptosis.Our findings suggest that ZNS improves the cardiac diastolic function in diabetic mice and prevents T2DM-induced cardiac hypertrophy by attenuating ER stress-mediated apoptosis.     

缺血性脑中风中与内质网应激相关炎症反应研究进展

内质网是机体微环境的重要组成结构和功能单位,内质网内稳态失衡导致的内质网应激成为近年来的一个研究热点。该文主要针对内质网应激在缺血性脑中风中的作用及相关信号通路的研究进展进行综述,特别对内质网应激触发脑中风炎症反应的机制进行了系统的整理和总结,以期为缺血性中风的防治提供新的研究思路。     

腺苷诱导人食管癌EC109细胞凋亡及其内质网分子机制

目的探讨内质网应激途径在腺苷诱导食管癌EC109细胞凋亡中的作用。方法腺苷2 mmol.L-1作用于EC109细胞24～72 h或腺苷0.5～4 mmol.L-1作用于EC109细胞36 h,MTT法观察腺苷对EC109细胞存活率的时效和量效关系;免疫荧光法检测葡萄糖调节蛋白78(GRP78),胱天蛋白酶4,胱天蛋白酶3,转录因子CHOP和核因子κB(NF-κB)的表达及亚细胞定位;原位末端转移酶标记技术检测细胞凋亡;Western蛋白印迹法检测内质网应激相关蛋白表达。结果腺苷对EC109细胞生长具有明显的抑制作用。腺苷2 mmol.L-1与EC109细胞作用24,36,48和72 h,细胞存活率分别为(57.7±15.0)%,(56.5±11.1)%,(43.8±5.7)%和(28.8±4.1)%,呈时间依赖性下降(r=0.9192,P<0.01);腺苷0.5,1,2和4 mmol.L-1与EC109细胞作用36 h,随药物浓度增加,细胞存活率依次为(83.1±11.2)%,(67.9±6.7)%,(55.3±5.0)%和(45.4±5.4)%,呈浓度依赖性降低(r=0.8252,P<0.01)。腺苷0.5～4mmol.L-1与EC109细胞作用36 h,细胞凋亡率分别为(15.5±1.1)%,(28.2±0.8)%,(40.1±2.2)%和(50.6±1.3)%,与对照组(2.1±0.3)%相比均明显增加(P<0.05)。腺苷2 mmol.L-1与EC109细胞作用36 h,与正常对照组相比,GRP78、胱天蛋白酶4、胱天蛋白酶3和CHOP表达明显增强(P<0.05,P<0.01),胱天蛋白酶4,胱天蛋白酶3和CHOP发生核易位。GRP78、胱天蛋白酶4、胱天蛋白酶3、CHOP和NF-κB表达呈浓度依赖性增加(rGRP78=0.9471,r胱天蛋白酶4=0.8977,r胱天蛋白酶3=0.968,rCHOP=0.9762,rNF-κB=0.9471,P<0.05)。结论腺苷可诱导人食管癌EC109细胞凋亡,其分子机制可能与内质网应激凋亡途径的启动有关。     

Palmitate enhanced the cytotoxicity of ZnO nanomaterials possibly by promoting endoplasmic reticulum stress

We recently synthesized ZnO nanomaterials (denoted as ZnO nanorods [NRs] and Mini‐NRs) and suggested that their cytotoxicity could be related with the activation of endoplasmic reticulum (ER) stress apoptosis. However, in a complex biological microenvironment, the ER stress‐apoptosis pathway could also be modulated by biological molecules, such as free fatty acids, leading to unpredicted biological effects. In this study, we investigated the combined toxicity of ZnO NRs/Mini‐NRs and palmitate (PA) to THP‐1 macrophages. PA influenced the zeta potential and solubility of ZnO NRs and ZnO Mini‐NRs in water, which indicated a change of colloidal stability. Exposure to ZnO NRs and Mini‐NRs dose‐dependent decreased cellular viability and release of soluble monocyte chemotactic protein 1 (sMCP‐1), and these effects were significantly promoted with the presence of PA. However, ZnO NR‐ and Mini‐NR‐induced intracellular Zn ions or reactive oxygen species were not significantly affected by PA. ZnO NRs and ZnO Mini‐NRs significantly promoted the expression of ER stress genes HSPA5, DDIT3, XBP‐1s and apoptotic gene CASP3, whereas PA also modestly promoted the expression of HSPA5, DDIT3 and CASP3. Interestingly, the ER stress inducer thapsigargin showed a similar effect as PA to promote the cytotoxicity of ZnO NRs and ZnO Mini‐NRs. It is suggested that PA might promote the cytotoxicity of ZnO NRs and ZnO Mini‐NRs possibly by promoting ER stress.     

Wogonin ameliorates lipotoxicity-induced apoptosis of cultured vascular smooth muscle cells via interfering with DAG-PKC pathway

Aim:

To investigate the effects of wogonin (5,7-dihydroxy-8-methoxyflavone) extracted from Scutellaria baicalensis Georgi (S baicalensis) on lipotoxicity-induced apoptosis of vascular smooth muscle cells (VSMCs) and the underlying mechanisms.

Methods:

Cultured VSMCs were used. Apoptosis of VSMCs was induced by palmitate (0.75 mmol/L), and detected using TUNEL assay. The expression levels of protein and phosphorylated protein were measured using Western blot analysis.

Results:

Treatment of VSMCs with wogonin (10, 25 and 50 μmol/L) significantly attenuated the apoptosis and endoplasmic reticulum (ER) stress induced by palmitate in concentration- and time-dependent manners. Wogonin (50 μmol/L) decreased palmitate-induced reactive oxygen species (ROS) generation. The ER stress inhibitor 4-phenyl butyric acid (5 mmol/L) significantly decreased palmitate-induced apoptotic cells, and occluded the anti-apoptotic effect of wogonin (25 μmol/L). Wogonin (10, 25 and 50 μmol/L) significantly reduced the intracellular diacylglycerol (DAG) accumulation and expression levels of phosphorylated PKCs in palmitate-treated VSMCs.

Conclusion:

Our results suggest that wogonin inhibits lipotoxicity-induced apoptosis of VSMCs via suppressing the intracellular DAG accumulation and subsequent inhibition of PKC phosphorylation. Wogonin has therapeutic potential for the prevention and treatment of atherosclerosis.     

高糖对肾小管上皮细胞内质网应激的影响及冬虫夏草的干预作用

目的：观察高糖对肾小管上皮细胞凋亡和内质网应激标志物表达的影响,研究冬虫夏草提取液对高糖诱导的肾小管上皮细胞内质网应激的可能保护机制。方法：体外培养大鼠肾小管上皮细胞,分为正常低糖组、低糖+冬虫夏草组、高渗组、高渗+冬虫夏草组、高糖组、高糖+冬虫夏草组和高糖+高渗+冬虫夏草组,用Realtime-PCR法检测各组细胞葡萄糖调解蛋白78（Grp78）、半胱氨酸蛋白酶蛋白12（caspase-12）及I型胶原mRNA的表达,采用酶联免疫吸附法测定细胞上清液中I型胶原的分泌,Western-blotting检测Grp78及caspase-12蛋白表达。结果：高糖组与低糖组相比,Grp78、Caspase-12及Ⅰ型胶原mRNA和蛋白水平明显上升,差异均有统计学意义（P<0.05）;低糖+冬虫夏草组、高渗组及高渗+冬虫夏草组与低糖组相比,Grp78、Caspase-12 mRNA及Ⅰ型胶原mRNA和蛋白水平无明显差别（P>0.05）;与高糖组相比,高糖+冬虫夏草干预组Grp78、Caspase-12及Ⅰ型胶原mRNA和蛋白表达下降,差异均有统计学意义（P<0.05）。结论：冬虫夏草对高糖诱导下肾小管上皮细胞凋亡及肾脏纤维化具有一定抑制作用,其机制可能与抑制内质网应激反应有关。     

Atrazine induces endoplasmic reticulum stress‐mediated apoptosis of T lymphocytes via the caspase‐8‐dependent pathway

Atrazine (ATR) is one of the most commonly applied broad‐spectrum herbicides. Although ATR is well known to be a biologically hazardous molecule with potential toxicity in the immune system, the molecular mechanisms responsible for ATR‐induced immunotoxicity remain unclear. In this study, we found that the immunotoxic properties of ATR were mediated through the induction of apoptotic changes in T lymphocytes. Mice exposed to ATR for 4 weeks exhibited a significant decrease in the number of spleen CD3⁺ T lymphocytes, while CD19⁺ B lymphocytes and nonlymphoid cells were unaffected. ATR exposure also led to inhibition of cell growth and induction of apoptosis in human Jurkat T‐cells. Importantly, ATR triggered the activation of caspase‐3 and the cleavage of caspase‐8 and PARP, whereas it did not affect the release of cytochrome c from the mitochondria in Jurkat T‐cells. In addition, ATR activated the unfolded protein response signaling pathway, as indicated by eIF2α phosphorylation and CHOP induction. Our results demonstrate that ATR elicited an immunotoxic effect by inducing ER stress‐induced apoptosis in T‐cells, therefore providing evidence for the molecular mechanism by which ATR induces dysregulation of the immune system. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 998–1008, 2016.     

Thymoquinone protects rat liver after partial hepatectomy under ischaemia/reperfusion through oxidative stress and endoplasmic reticulum stress prevention

Ischaemia reperfusion (I/R) is associated with liver injury and impaired regeneration during partial hepatectomy (PH). The aim of this study was to investigate the effect of thymoquinone (TQ), the active compound of essential oil obtained from Nigella sativa seeds, on rat liver after PH. Male Wistar rats were divided equally into four groups (n = 6) receiving an oral administration of either vehicle solution (sham and PH groups) or TQ at 30 mg/kg (TQ and TQ + PH groups) for 10 consecutive days. Then, rats underwent PH (70%) with 60 minutes of ischaemia followed by 24 hours of reperfusion (PH and TQ + PH groups). Alanine aminotransferase (ALT) activity and histopathological damage were determined. Also, antioxidant parameters, liver regeneration index, hepatic adenosine triphosphate (ATP) content, endoplasmic reticulum (ER) stress and apoptosis were assessed. In response to PH under I/R, liver damage was significantly alleviated by TQ treatment as evidenced by the decrease in ALT activity (P < .01) and histological findings (P < .001). In parallel, TQ preconditioning increased hepatic antioxidant capacities. Moreover, TQ improved mitochondrial function (ATP, P < .05), attenuated ER stress parameters and repressed the expression of apoptotic effectors. Taken together, our results suggest that TQ preconditioning could be an effective strategy to reduce liver injury after PH under I/R. The protective effects were mediated by the increase of antioxidant capacities and the decrease of ER stress and apoptosis.     

内质网应激与脑缺血/再灌注损伤

内质网是蛋白质合成、加工和转运的主要场所。内质网应激是指缺血缺氧、葡萄糖或营养物质缺乏、药物、毒素等因素破坏内质网的稳态,出现错误折叠与未折叠蛋白在腔内聚集以及Ca2+平衡紊乱的状态。内质网早期或未折叠蛋白反应(unfolded protein response,UPR)能提高细胞在有害因素下的生存能力。但剧烈或者持久的ERS,能诱发凋亡机制。研究表明,脑缺血/再灌注后发生ERS,导致神经细胞凋亡和坏死,是引起再灌注损伤的重要原因。本文对近年来ERS在细胞凋亡调控中的重要作用及脑缺血/再灌注损伤与ERS的关系进行综述,这对脑卒中的理论及临床研究具有重要意义,也给治疗脑卒中的药物发现提供新思路。     

Berberine reduces endoplasmic reticulum stress and improves insulin signal transduction in Hep G2 cells

Aim:

Endoplasmic reticulum (ER) stress plays an important role in the pathogenesis of insulin resistance and pancreatic β-cell dysfunction. The aim of this study is to investigate whether the insulin-sensitizing action of berberine is related to reducing ER stress.

Methods:

ER stress in cultured Hep G2 cells was induced with tunicamycin. Cells were pretreated with berberine in combination with or without insulin. The concentration of glucose was measured by glucose oxidase method. The molecular markers of ER stress, including ORP150, PERK, and eIF2α were analyzed by Western blot or real time PCR. The activity of JNK was also evaluated. Moreover, the insulin signaling proteins such as IRS-1 and AKT were determined by Western blot.

Results:

The production of glucose stimulated with insulin was reduced. The expressions of ORP150 was decreased both in gene and protein levels when cells were pretreated with berberine, while the activation of JNK was blocked. The levels of phosphorylation both on PERK and eIF2α were inhibited in cells pretreated with berberine. The level of IRS-1 ser³⁰⁷ phosphorylation was decreased, whereas IRS-1 tyr phosphorylation was increased notablely. AKT ser⁴⁷³ phosphorylation was also enhanced significantly in the presence of berberine.

Conclusion:

The antidiabetic effect of berberine in Hep G2 cells maybe related to attenuation of ER stress and improvement of insulin signal transduction.     

Dithiothreitol enhanced arsenic‐trioxide‐induced cell apoptosis in cultured oral cancer cells via mitochondrial dysfunction and endoplasmic reticulum stress

Arsenic is naturally occurring toxic metalloid and drinking As₂O₃ containing water are recognized to be related to increased risk of neurotoxicity, liver injury, blackfoot disease, hypertension, and cancer. On the contrary, As₂O₃ has been an ancient drug used in traditional Chinese medicine with substantial anticancer activities, especially in the treatment of acute promyelocytic leukemia as well as chronic wound healing. However, the cytotoxicity and detail mechanisms of As₂O₃ action in solid cancer cells, such as oral cancer cells, are largely unknown. In this study, we have primarily cultured four pairs of tumor and nontumor cells from the oral cancer patients and treated the cells with As₂O₃ alone or combined with dithiothreitol ( DTT). The results showed that 0.5 μM As₂O₃ plus 20 μM DTT caused a significant cell death of oral cancer cells but not the nontumor cells. Also As₂O₃ plus DTT upregulated Bax and Bak, downregulated Bcl‐2 and p53, caused a loss of mitochondria membrane potential in oral cancer cells. On the other way, As₂O₃ also triggered endoplasmic reticulum stress and increased the levels of glucose‐regulated protein 78, calpain 1 and 2. Our results suggest that DTT could synergistically enhance the effects of As₂O₃ on killing oral cancer cells while nontoxic to the nontumor cells. The combination is promising for clinical practice in oral cancer therapy and worth further investigations. © 2015 Wiley Periodicals, Inc. Environ Toxicol 32: 17–27, 2017.