软骨细胞凋亡与内质网应激

背景：软骨细胞的凋亡最终会导致骨关节炎的发生。目前发现的软骨细胞凋亡机制有很多,但内质网应激引起其凋亡的机制尚处于初始阶段。目的：综述分析内质网应激如何引起软骨细胞凋亡,并探寻骨关节炎治疗的新方法。方法：第一作者通过计算机检索,检索了PubMed、CNKI、万方数据库2000年1月至2015年1月的相关文献。使用PubMed检索时,输入检索词“endoplasmic reticulum stress,chondrocyte,apoptosis”之间以“AND”形式连接,使用CNKI、万方数据库检索时,输入主题词“内质网应激,软骨细胞,凋亡”之间以“并且/与”连接,选择模糊查询进行检索。选择文章内容涉及骨关节炎的文献,较多采用近期发表在权威杂志上的相关文献。结果与结论：初检得到中英文文献共计62篇,其中57篇符合纳入标准,对其进行综述。PERK、IRE1和ATF6三条信号通路对软骨细胞同样有重要作用。内质网应激介导的细胞凋亡机制有未折叠蛋白质反应和Ca²⁺起始信号2种,但具体机制及凋亡途径间的交叉作用还未明了。若从上述信号通路中探索软骨细胞凋亡效应的抑制分子,并将其作为治疗骨关节炎的靶点,可能会为治疗骨关节炎提供新的方法。中国组织工程研究杂志出版内容重点：组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程     

Involvement of endoplasmic reticulum stress in myofibroblastic differentiation of lung fibroblasts

Stress that impairs endoplasmic reticulum (ER) function leads to an accumulation of unfolded or misfolded proteins in the ER (ER stress) and triggers the unfolded protein response (UPR). Recent studies suggest that ER stress is involved in idiopathic pulmonary fibrosis (IPF). The present study was undertaken to determine the role of ER stress on myofibroblastic differentiation of fibroblasts. Fibroblasts in fibroblastic foci of IPF showed immunoreactivity for GRP78. To determine the role of ER stress on α-smooth muscle actin (α-SMA) and collagen type I expression in fibroblasts, mouse and human lung fibroblasts were treated with TGF-β1, and expression of ER stress-related proteins, α-SMA, and collagen type I was analyzed by Western blotting. TGF-β1 significantly increased expression of GRP78, XBP-1, and ATF6α, which was accompanied by increases in α-SMA and collagen type I expression in mouse and human fibroblasts. A chemical chaperone, 4-PBA, suppressed TGF-β1-induced UPR and α-SMA and collagen type I induction. We also showed that TGF-β1-induced UPR was mediated through the reactive oxygen species generation. Our study provides the first evidence implicating the UPR in myofibroblastic differentiation during fibrosis. These findings of the role of ER stress and chemical chaperones in pulmonary fibrosis may improve our understanding of the pathogenesis of IPF.     

动脉粥样硬化中巨噬细胞凋亡与内质网应激机制

巨噬细胞是机体免疫系统的重要细胞成份,具有多种生理功能,在动脉粥样硬化等血管疾病的发生和发展中具有重要作用.巨噬细胞凋亡是造成动脉粥样硬化斑块不稳定的重要因素,在晚期动脉粥样硬化中,内质网应激与巨噬细胞凋亡密切相关.目前已知的巨噬细胞凋亡途径包括外源性的死亡受体途径、内源性的线粒体途径及内质网应激凋亡途径,其中内质网应...     

Involvement of endoplasmic reticulum stress in a novel Classic Galactosemia model

Inherited deficiency of galactose-1-phosphate uridyltransferase (GALT) activity in humans leads to a potentially lethal disorder called Classic Galactosemia. It is well known that patients often accumulate high levels of galactose metabolites such as galactose-1-phosphate (gal-1-p) in their tissues. However, specific targets of gal-1-p and other accumulated metabolites remain uncertain. In this study, we developed a new model system to study this toxicity using primary fibroblasts derived from galactosemic patients. GALT activity was reconstituted in these primary cells through lentivirus-mediated gene transfer. Gene expression profiling showed that GALT-deficient cells, but not normal cells, responded to galactose challenge by activating a set of genes characteristic of endoplasmic reticulum (ER) stress. Western blot analysis showed that the master regulator of ER stress, BiP, was up-regulated at least threefold in these cells upon galactose challenge. We also found that treatment of these cells with galactose, but not glucose or hexose-free media reduced Ca2+ mobilization in response to activation of Gq-coupled receptors. To explore whether the muted Ca2+ mobilization is related to reduced inositol turnover, we discovered that gal-1-p competitively inhibited human inositol monophosphatase (hIMPase1). We hypothesize that galactose intoxication under GALT-deficiency resulted from accumulation of toxic galactose metabolite products, which led to the accumulation of unfolded proteins, altered calcium homeostasis, and subsequently ER stress.     

钙联蛋白调控内质网应激诱导的细胞凋亡

内质网应激(ERS)可诱导多种细胞凋亡,与疾病发生发展密切相关.钙联蛋白(CNX)是内质网中重要的类凝集素分子伴侣,通过参与未折叠蛋白反应募集凋亡相关因子、激活IRE1-JNK激酶等途径,调控ERS诱导的凋亡.本文就其研究现状进行阐述.     

Ischemia induces endoplasmic reticulum stress and cell apoptosis in human brain

In animal models, endoplasmic reticulum (ER) stress and apoptosis take place around cerebral infarction areas during ischemia, which presumably protect tissues from necroses-induced injury as well as promote cells toward death. We examined whether these pathological changes, especially temporal occurrence, were present in patients who suffered from cerebral ischemia. The studies by immunohistochemistry show that ER chaperone glucose-regulated protein (GRP78) and caspase-9 elevate around infarction areas. The experiments by terminal deoxynucleotidy transferase-mediated 2′-deoxyuridine 5′-triphosphate-biotin nick-end labeling (TUNEL) illustrate that TUNEL-positive cells are higher around infarction tissues than controls. Moreover, GRP78, caspase-9 and TUNEL cells emerge one after another during ischemia. In conclusion, ER stress, apoptosis initiation and DNA fragment develop sequentially in ischemic human brain. ER stress during excessive ischemia stimulates apoptotic cell death beyond activating a defense for nerve cells being away from injury.     

内质网应激在Bim介导缺氧致心肌细胞凋亡中的作用

 目的：探讨内质网应激在Bim介导缺氧致心肌细胞凋亡中的作用。方法：在体外原代培养出生1~3 d大鼠心肌细胞,并用抗α-横纹肌肌动蛋白免疫组化法进行鉴定。设计并化学合成3对靶向bim的siRNA,用脂质体法将siRNA转染心肌细胞,筛选沉默效率最高的siRNA。实验分组：（1）空白对照组;（2）缺氧组;（3）缺氧+脂质体组;(4)缺氧+阴性对照siRNA组;(5)缺氧+Bim-siRNA组。MTT法观察细胞活性;流式细胞术检测细胞凋亡率及细胞内钙离子浓度变化情况;Western blotting检测内质网应激标志分子caspase-12和三磷酸肌醇（IP3）的表达情况。结果：免疫组化鉴定证实大鼠心肌细胞原代培养成功。在荧光显微镜下,转染了阴性对照siRNA组的细胞中观察到绿色荧光,即转染成功;Western blotting 结果显示,Bim-siRNA转染均能有效降低Bim蛋白的表达,其中第2对沉默效率最高,达到86.73%。缺氧损伤导致心肌细胞活性明显下降（P<005）,转染Bim-siRNA后细胞活性较阴性对照组升高。缺氧细胞凋亡率较对照组明显增加（P<0.01）,细胞内钙离子浓度明显增高,而沉默bim的表达能降低细胞凋亡率和细胞内钙离子浓度。缺氧导致内质网应激标志分子caspase-12和IP3表达较空白对照组明显上调（均P<005）,而抑制Bim表达后caspase-12和IP3表达明显降低。结论：沉默bim的表达能有效抑制缺氧导致心肌细胞凋亡的作用,内质网应激标志分子caspase-12和IP3可能参与了Bim介导缺氧致心肌细胞凋亡的过程。这有望为临床心肌缺血缺氧损伤的治疗提供新思路。     

Serpinopathy and endoplasmic reticulum stress

We have recently identified a novel human gene, megsin, which is a new serine protease inhibitor (serpin) predominantly expressed in the kidney. Our previous studies suggested a role of megsin in the pathogenesis of human renal diseases, but its exact biopathological significance remained unknown. During the analysis of experimental animals overexpresssing the human megsin gene, we unexpectedly generated a serpinopathy model involving the kidney and pancreas and discovered a novel mechanism of renal injury, that is, cellular damage by endoplasmic reticulum (ER) stress caused by conformational disorder of protein tertiary structure within the ER. In vitro induction of ER stress may play a role in renal cell injury by various stimuli, but the involvement of ER stress in human renal disease remains elusive. Further research for ER structure and function may open new exciting prospects in the pathology of human renal diseases.     

Involvement of endoplasmic reticulum in hepatitis B virus replication

The mitochondrial calcium and downstream proline-rich tyrosine kinase-2 (PyK2) signaling pathway are critical to hepatitis B virus (HBV) replication, and the endoplasmic reticulum (ER) plays an important role in intracellular calcium regulation. To investigate the role of ER in HBV replication, the HBV genome transfected HepG2.2.15 cells were treated by cyclosporine A (CsA), cyclopiazonic acid (CPA), ryanodine and U73122, which are all specific blockers of calcium channels located in either ER or mitochondria. The HBV replication level was evaluated by two methods: slot blot hybridization analysis of intracellular HBV DNA and real-time polymerase chain reaction (PCR) analysis of secreted HBV DNA in supernatant; the activation of PyK2 kinase was detected by Western blot analysis. Results indicated that the HBV replication was inhibited when mitochondrial permeability transition pore, ER Ca2+ -ATPase and ER inositol 1,4,5-trisphosphate receptor (IP3R) were blocked by CsA, CPA and U73122, respectively; but not inhibited when ER ryanodine receptor was blocked by ryanodine. The PyK2 phosphorylation level declined after treatment of 2 microg/ml CsA, 5 microM CPA and 25 microM U73122, but not changed apparently after 50 microM ryanodine treatment. Compared with monotreatment, a more powerful inhibitory effect was achieved when the CsA, CPA and U73122 were combined used in twosome or triple manner, while the HBV replication level did not change apparently when ryanodine combined with CsA, CPA or U73122. In conclusion, besides the mitochondria, the ER also participates in the HBV replication through calcium-PyK2 signaling pathway; the calcium channels of ER Ca2+ -ATPase and ER IP3R are responsible for this role; during this complicated process, an interaction between ER and mitochondria maybe involved.     

西洋参茎叶总皂苷通过抑制内质网应激减轻毒胡萝卜素诱导的心肌细胞凋亡

 目的:研究西洋参茎叶总皂苷 (PQS) 减轻毒胡萝卜素(TG) 诱导的心肌细胞凋亡的分子机制。方法:原代培养的心肌细胞分为：control组、TG组、PQS (40 mg/L、80 mg/L及160 mg/L)+TG组、牛磺酸 (40 mmol/L)+TG组、蛋白激酶R样内质网激酶 (PERK)敲低+TG组及随机双链RNA转染对照 (mock)+TG组。通过向培养液中加入1 μmol/L TG作用24 h诱导离体培养的乳大鼠心肌细胞凋亡。以RNAi方法敲低心肌细胞PERK基因。CCK-8法和流式细胞术检测细胞活性及凋亡率,Western blotting方法检测内质网应激(ERS)标志分子葡萄糖调节蛋白78 (GRP 78)、钙网蛋白 (CRT)、PERK、p-PERK、真核起始因子2α (eIF2α)、p- eIF2α、活化转录因子4 (ATF4)、C/EBP同源蛋白 (CHOP) 及凋亡相关蛋白Bcl-2、Bax的表达。结果:与对照组比较,TG孵育明显诱导细胞凋亡,降低细胞活力,上调PERK和eIF2α磷酸化以及GRP78、CRT、ATF4、CHOP及促凋亡蛋白Bax表达,降低抗凋亡蛋白Bcl-2表达 (P<0.05);与TG组比较,PQS 160 mg/L及敲低PERK均可显著降低细胞凋亡率,升高细胞活力 (P<0.05);3种不同浓度的PQS可呈剂量依赖性降低Bax蛋白表达,升高Bcl-2蛋白表达 (P<0.05),敲低PERK基因及PQS (160 mg/L) 预处理均可降低ERS分子GRP78、CRT、ATF4及CHOP表达,降低PERK及eIF2α的磷酸化水平 (P<0.05)。结论: PQS 160 mg/L 减轻ERS诱导剂TG诱导的心肌细胞凋亡,PQS预处理可以模拟PERK敲低减轻TG致心肌细胞凋亡的效应,提示PERK-eIF2α-ATF4-CHOP途径参与PQS减轻ERS相关凋亡的作用。     

Bevacizumab induces A549 cell apoptosis through the mechanism of endoplasmic reticulum stress in vitro

Aims: To observe the effect of bevacizumab on human A549 cells and explore its mechanism. Methods: After different concentrations (0 μM, 1 μM, 5 μM, 25 μM) of bevacizumab treating in A549 cells, CCK8 assay detect the impact of bevacizumab on A549 cell proliferation and flow cytometry determine the effect of bevacizumab on human A549 cells apoptosis. Real-time PCR and Western blotting detect the changing expression of the target gene (CHOP, caspase-4, IRE1, XBP-1) on mRNA and Protein level. Results: Treatment with bevacizumab for 24-hr have induced cell death in a does-dependent manner dramatically (P<0.05). In terms of the mRNA level, expression of XBP-1 has increased obviously in each group (1 μM, 5 μM, 25 μM) (P<0.01); the expression of CHOP (25 μM) and caspase-4 (5 μM) have increased slightly (P<0.05). In terms of the protein level, the expression of CHOP has increased obviously in each group (1 μM, 5 μM, 25 μM) when compared with the control group (0 μM) (P<0.05). As for caspase-4 (5 μM, 25 μM), the expression have increased slightly when compared with the control group (0 μM) (P<0.05). Conclusion: Bevacizumab can induce A549 cell apoptosis through the mechanism of endoplasmic reticulum stress.     

Involvement of endoplasmic reticulum stress-associated apoptosis in a heart failure model induced by chronic myocardial ischemia

Apoptosis plays a critical role in the pathogenesis of chronic myocardial ischemia (CMI) and heart failure (HF). Endoplasmic reticulum stress (ERS) is one of the newly defined signaling pathways which initiate apoptosis. Previous studies have shown that ERS-associated apoptosis is involved in the pathogenesis of HF induced by pressure-overload and acute myocardial infarction. Also, in?vitro experiments have proved that ischemia is a strong stimulus of ERS. This study aimed to demonstrate whether ERS-associated apoptosis is involved in the pathogenesis of CMI-induced HF. We established a HF model induced by CMI in mini pigs via placement of an ameroid constrictor around the proximal anterior descending branch of the left coronary artery (LAD). Furthermore, we used myocardial perfusion imaging, echocardiographic and hemodynamic measurements, hematoxylin-eosin staining, and terminal deoxynucleotidyl transferase-mediated DNA nick-end labeling staining to identify the existence of myocardial ischemia and cardiac dysfunction and of enhanced apoptosis in the ischemic heart. We performed immunohistochemistry, Western blot, and real-time PCR to analyze the hallmark of ERS glucose-regulated protein 78 (GRP78). The ERS-associated apoptotic pathways, CCAAT/enhancer-binding protein homologous protein (CHOP), caspase-12, and c-Jun NH2-terminal kinase?1 (JNK1) were also examined. We found that all three of these pathways were activated and that GRP78 protein and mRNA levels were significantly enhanced in the myocardium of HF mini pigs induced by CMI. These results suggest that ERS is present in the CMI-induced HF pig model, and that ERS-associated apoptosis is involved in the pathophysiology of HF induced by CMI.     

Role of Herp in the endoplasmic reticulum stress response

Application of differential display to cultured rat astrocytes allowed cloning of Herp cDNA. Although Herp was strongly induced by endoplasmic reticulum (ER) stress, it decayed rapidly consequent to proteasome-mediated degradation. To investigate the role of this molecule in terms of the stress response, Herp knockout cells were developed using F9 embryonic carcinoma cells. F9 Herp null cells were more vulnerable to ER stress compared with F9 wild-type cells. In the early period of ER stress (0-8 h after tunicamycin treatment), Herp null cells displayed enhanced ER stress signalling and stabilization of an endogenous ERAD substrate, compared with wild-type cells. In the intermediate period (8-20 h after tunicamycin treatment), Herp null cells displayed reduced ER stress signalling, whereas in the late period (20-40 h after tunicamycin treatment), Herp null cells manifested irreversible cellular changes that lead to apoptotic cell death. Transfection analysis revealed that the N-terminal region, including the ubiquitin-like domain of Herp, was required for the survival of F9 cells under ER stress. These results indicate that Herp is a short-lived Ub-like protein improving the balance of folding capacity and protein loads in the ER and plays crucial roles for the ER stress resistance in F9 cells.     

一氧化氮和内质网应激

一氧化氮(nitric oxide,NO)是一种重要的多功能内源性气体分子,能舒张血管、参与免疫反应和作为一种神经递质在神经元的信息传递中发挥重要作用,因而广泛参与机体心血管系统、神经系统和免疫系统等的生理和病理调节[1].     

松香酸通过诱导自噬减轻AGEs诱导的心肌H9c2细胞凋亡和内质网应激反应

目的:本文旨在探索松香酸(abietic acid,AA)对晚期糖基化终末产物(advanced glycosylation end products,AGEs)诱导的心肌H9c2细胞凋亡和内质网应激的影响及其相关机制。方法:H9c2细胞分为5组:对照组加入生理盐水处理24 h;AGEs处理组加入AGEs(100 mg/L)处理24 h;AGEs+AA(10、25和50μmol/L)组同时加入AGEs(100 mg/L)和AA(10、25和50μmol/L)处理24 h。MTT法检测H9c2细胞活力。Western blot分析肌红蛋白(myoglobin,Mb)、肌酸激酶MB同工酶(creatine kinase MB isoenzyme,CK-MB)、心肌肌钙蛋白I(cardiac troponin I,c Tn I)、C/EBP同源蛋白(C/EBP homologous protein,CHOP)、cleaved caspase-12、GADD34、Bi P、LC3、P62和beclin 1的蛋白水平。ELISA检测乳酸脱氢酶(lactate dehydrogenase,LDH)活性。流式细胞术分析细胞凋亡。结果:低浓度(低于50μmol/L)的松香酸对H9c2细胞活力无明显影响,高浓度(高于50μmol/L)的松香酸会降低H9c2细胞活力。AGEs组Mb、CK-MB和c Tn I蛋白表达及LDH的水平明显高于对照组(P0.05);与AGEs组相比,AGEs+AA(10、25和50μmol/L)组Mb、CK-MB和c Tn I的蛋白表达及LDH的表达水平明显降低(P0.05)。松香酸(10、25和50μmol/L)可抑制AGEs诱导的心肌细胞凋亡、CHOP和cleaved caspase-12蛋白水平的升高及GADD34和Bi P表达的降低(P0.05)。而且,松香酸(10、25和50μmol/L)可抑制AGEs诱导的心肌细胞LC3-Ⅱ/LC3-Ⅰ比值和beclin 1表达的降低及P62表达的升高(P0.05)。自噬抑制剂3-甲基腺嘌呤可逆转松香酸对心肌细胞LC3、Mb、cleaved caspase-12和Bi P蛋白水平的影响(P0.05)。结论:松香酸通过诱导自噬减轻AGEs诱导的心肌H9c2细胞凋亡和内质网应激反应。     

栀子苷元通过抑制内质网应激改善脾细胞凋亡治疗脓毒症的机制研究

目的 评估栀子苷元 (Genipin) 对盲肠结扎穿孔术 (Cecal Ligation and Puncture,CLP) 诱导的脓毒症小鼠模型的治疗效果,并探讨其潜在的作用机制。方法 将雄性 C57BL/6 小鼠分为假手术组 (Sham组)、脓毒症模型组 (CLP组)、栀子苷元治疗脓毒症组 (Genipin+CLP组)。栀子苷元治疗组在建模后0 h和20 h经尾静脉注射2.5 mg/kg栀子苷元,而Sham组和CLP组注射等体积生理盐水。实验1,观察栀子苷元对脓毒症小鼠120 h生存率的影响;实验2,基于生存率的结果选取建模后36 h为采样时间点,并行以下方法检测：利用酶联免疫吸附法 (Enzym-linked Immunosorbent Assay,ELISA) 检测肿瘤坏死因子α (Tumor Necrosis Factor α,TNF-α) 和白细胞介素6(Interleukin 6,IL-6) 含量;常规苏木精-伊红 (Hematoxylineosin,HE) 染色法检测肺和肝组织病理损害;Western Blot法检测脾脏内质网应激相关蛋白的表达变化,包括葡萄糖调节蛋白 78 (Glucose Regulated Protein 78,GRP78)、蛋白激酶 R 样内质网激酶 (Protein Kinase R-like Endoplasmic Reticulum Ki‐ nase,PERK)、磷酸化真核翻译起始因子2α (p-eukaryotic Initation Factor 2α,p-eIF2α) 和C/EBP同源蛋白 (C/EBP Homolo‐ gous Protein,CHOP);脱氧核糖核苷酸末端转移酶介导的缺口末端标记法 (Terminal-deoxynucleotidyl Transferase-mediated dUTP Nick End-labeling,TUNEL) 检测脾细胞凋亡变化。结果 与 CLP组相比,栀子苷元治疗提高了脓毒症小鼠 120 h生存率 (P＜0.05),降低了血清 TNF-α和 IL-6含量 (P＜0.05),并减轻肺和肝脏组织病理学损伤。另外,栀子苷元显著下调了脾脏GRP78、PERK、p-eIF2α和CHOP蛋白的表达水平 (P＜0.05),并进而减少内质网应激诱导的脾细胞凋亡数量 (P＜0.05)。 结论 栀子苷元能够明显减轻脓毒症所导致的脏器损害和过度炎症反应,改善脓毒症预后。该效应的作用机制可能与栀子苷元减轻脾脏内质网应激导致其凋亡机制的启动被阻断,进而减轻脾细胞凋亡有关。     

内质网应激在肺肿瘤中的作用

内质网是细胞的蛋白质加工厂,主要负责蛋白质的合成、折叠和装配。各种生理和病理条件（如缺氧、氧化还原状态的变化）可能会干扰内质网的功能,并导致未折叠蛋白在内质网中积累,导致内质网应激（endoplasmic reticulum stress,ERS）。ERS是细胞抵抗外来不良刺激的一种重要保护机制,也是决定细胞命运的关键。适度的ERS能够促进肺肿瘤细胞生存和转移,过度的ERS则促进肺肿瘤细胞凋亡。多种抗肿瘤药物都可通过加重ERS而促进肺肿瘤细胞凋亡。