藏红花素诱导人胶质瘤U251细胞内质网应激性凋亡的研究

目的：研究藏红花素对人胶质瘤U251细胞的促凋亡作用和可能的机制。方法：不同浓度藏红花素处理U251细胞后,MTT法检测细胞活力,TUNEL染色观察细胞凋亡情况。结果：①藏红花素显著抑制U251细胞的增殖,并诱导其发生凋亡。②藏红花素增加了U251细胞胞浆内钙离子的含量,并上调了内质网分子伴侣GRP78的表达。③藏红花素处理后的U251细胞内质网相关凋亡分子CHOP,Caspase-4,JNK活性明显增高。结论：藏红花素通过诱导内质网应激性凋亡抑制人胶质瘤U251细胞的增殖。     

藏红花素诱导人胶质瘤U251细胞内质网应激性凋亡的研究

目的:研究藏红花素对人胶质瘤U251细胞的促凋亡作用和可能的机制。方法:不同浓度藏红花素处理U251细胞后,MTT法检测细胞活力,TUNEL染色观察细胞凋亡情况。结果:①藏红花素显著抑制U251细胞的增殖,并诱导其发生凋亡。②藏红花素增加了U251细胞胞浆内钙离子的含量,并上调了内质网分子伴侣GRP78的表达。③藏红花素处理后的U251细胞内质网相关凋亡分子CHOP,Caspase-4,JNK活性明显增高。结论:藏红花素通过诱导内质网应激性凋亡抑制人胶质瘤U251细胞的增殖。     

Melatonin Inhibits Endoplasmic Reticulum Stress and Epithelial-Mesenchymal Transition during Bleomycin-Induced Pulmonary Fibrosis in Mice

Several reports indicate that melatonin alleviates bleomycin (BLM)-induced pulmonary fibrosis in rodent animals. Nevertheless, the exact mechanism remains obscure. The present study investigated the effects of melatonin on endoplasmic reticulum (ER) stress and epithelial-mesenchymal transition (EMT) during BLM-induced lung fibrosis. For the induction of pulmonary fibrosis, mice were intratracheally injected with a single dose of BLM (5.0 mg/kg). Some mice were intraperitoneally injected with melatonin (5 mg/kg) daily for a period of 3 wk. Twenty-one days after BLM injection, lung fibrosis was evaluated. As expected, melatonin significantly alleviated BLM-induced pulmonary fibrosis, as evidenced by Sirius red staining. Moreover, melatonin significantly attenuated BLM-induced EMT to myofibroblasts, as determined by its repression of α-SMA expression. Further analysis showed that melatonin markedly attenuated BLM-induced GRP78 up-regulation and elevation of the cleaved ATF6 in the lungs. Moreover, melatonin obviously attenuated BLM-induced activation of pulmonary eIF2α, a downstream target of the PERK pathway. Finally, melatonin repressed BLM-induced pulmonary IRE1α phosphorylation. Correspondingly, melatonin inhibited BLM-induced activation of XBP-1 and JNK, two downstream targets of the IRE1 pathway. Taken together, these results suggest that melatonin alleviates ER stress and ER stress-mediated EMT in the process of BLM-induced pulmonary fibrosis.     

Respiratory Syncytial Virus Reverses Airway Hyperresponsiveness to Methacholine in Ovalbumin-Sensitized Mice

Each year, approximately 20% of asthmatics in the United States experience acute symptom exacerbations, which commonly result from pulmonary viral infections. The majority of asthma exacerbations in very young children follow infection with respiratory syncytial virus (RSV). However, pathogenic mechanisms underlying induction of asthma exacerbations by RSV are not well understood. We therefore investigated the effect of post-sensitization RSV infection on lung function in ovalbumin (OVA)-sensitized BALB/c mice as a model of RSV asthma exacerbations. OVA sensitization of uninfected female BALB/c mice increased bronchoalveolar lavage fluid (BALF) eosinophil levels and induced airway hyperresponsiveness to the muscarinic agonist methacholine, as measured by the forced-oscillation technique. In contrast, intranasal infection with replication-competent RSV strain A2 for 2–8 days reduced BALF eosinophil counts and reversed airway hyperresponsiveness in a pertussis toxin-sensitive manner. BALF levels of the chemokine keratinocyte cytokine (KC; a murine homolog of interleukin-8) were elevated in OVA-sensitized, RSV-infected mice and reversal of methacholine hyperresponsiveness in these animals was rapidly inhibited by KC neutralization. Hyporesponsiveness could be induced in OVA-sensitized, uninfected mice by recombinant KC or the Gαi agonist melittin. These data suggest that respiratory syncytial virus induces KC-mediated activation of Gαi, resulting in cross-inhibition of Gαq-mediated M₃-muscarinic receptor signaling and reversal of airway hyperresponsiveness. As in unsensitized mice, KC therefore appears to play a significant role in induction of airway dysfunction by respiratory syncytial virus. Hence, interleukin-8 may be a promising therapeutic target to normalize lung function in both asthmatics and non-asthmatics with bronchiolitis. However, the OVA-sensitized, RSV-infected mouse may not be an appropriate model for investigating the pathogenesis of viral asthma exacerbations.     

Visualization of Endoplasmic Reticulum Subdomains in Cultured Cells

The lipids and proteins in eukaryotic cells are continuously exchanged between cell compartments, although these retain their distinctive composition and functions despite the intense interorganelle molecular traffic. The techniques described in this paper are powerful means of studying protein and lipid mobility and trafficking in vivo and in their physiological environment. Fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP) are widely used live-cell imaging techniques for studying intracellular trafficking through the exo-endocytic pathway, the continuity between organelles or subcompartments, the formation of protein complexes, and protein localization in lipid microdomains, all of which can be observed under physiological and pathological conditions. The limitations of these approaches are mainly due to the use of fluorescent fusion proteins, and their potential drawbacks include artifactual over-expression in cells and the possibility of differences in the folding and localization of tagged and native proteins. Finally, as the limit of resolution of optical microscopy (about 200 nm) does not allow investigation of the fine structure of the ER or the specific subcompartments that can originate in cells under stress (i.e. hypoxia, drug administration, the over-expression of transmembrane ER resident proteins) or under pathological conditions, we combine live-cell imaging of cultured transfected cells with ultrastructural analyses based on transmission electron microscopy.     

Normal CFTR Inhibits Epidermal Growth Factor Receptor-Dependent Pro-Inflammatory Chemokine Production in Human Airway Epithelial Cells

Mutations in cystic fibrosis transmembrane conductance regulator (CFTR) protein cause cystic fibrosis, a disease characterized by exaggerated airway epithelial production of the neutrophil chemokine interleukin (IL)-8, which results in exuberant neutrophilic inflammation. Because activation of an epidermal growth factor receptor (EGFR) signaling cascade induces airway epithelial IL-8 production, we hypothesized that normal CFTR suppresses EGFR-dependent IL-8 production and that loss of CFTR at the surface exaggerates IL-8 production via activation of a pro-inflammatory EGFR cascade. We examined this hypothesis in human airway epithelial (NCI-H292) cells and in normal human bronchial epithelial (NHBE) cells containing normal CFTR treated with a CFTR-selective inhibitor (CFTR-172), and in human airway epithelial (IB3) cells containing mutant CFTR versus isogenic (C38) cells containing wild-type CFTR. In NCI-H292 cells, CFTR-172 induced IL-8 production EGFR-dependently. Pretreatment with an EGFR neutralizing antibody or the metalloprotease TACE inhibitor TAPI-1, or TACE siRNA knockdown prevented CFTR-172-induced EGFR phosphorylation (EGFR-P) and IL-8 production, implicating TACE-dependent EGFR pro-ligand cleavage in these responses. Pretreatment with neutralizing antibodies to IL-1R or to IL-1alpha, but not to IL-1beta, markedly suppressed CFTR-172-induced EGFR-P and IL-8 production, suggesting that binding of IL-1alpha to IL-1R stimulates a TACE-EGFR-IL-8 cascade. Similarly, in NHBE cells, CFTR-172 increased IL-8 production EGFR-, TACE-, and IL-1alpha/IL-1R-dependently. In IB3 cells, constitutive IL-8 production was markedly increased compared to C38 cells. EGFR-P was increased in IB3 cells compared to C38 cells, and exaggerated IL-8 production in the IB3 cells was EGFR-dependent. Activation of TACE and binding of IL-1alpha to IL-1R contributed to EGFR-P and IL-8 production in IB3 cells but not in C38 cells. Thus, we conclude that normal CFTR suppresses airway epithelial IL-8 production that occurs via a stimulatory EGFR cascade, and that loss of normal CFTR activity exaggerates IL-8 production via activation of a pro-inflammatory EGFR cascade.     

Administration of DHA Reduces Endoplasmic Reticulum Stress-Associated Inflammation and Alters Microglial or Macrophage Activation in Traumatic Brain Injury

We investigated the effects of the administration of docosahexaenoic acid (DHA) post-traumatic brain injury (TBI) on reducing neuroinflammation. TBI was induced by cortical contusion injury in Sprague Dawley rats. Either DHA (16 mg/kg in dimethyl sulfoxide) or vehicle dimethyl sulfoxide (1 ml/kg) was administered intraperitonially at 5 min after TBI, followed by a daily dose for 3 to 21 days. TBI triggered activation of microglia or macrophages, detected by an increase of Iba1 positively stained microglia or macrophages in peri-lesion cortical tissues at 3, 7, and 21 days post-TBI. The inflammatory response was further characterized by expression of the proinflammatory marker CD16/32 and the anti-inflammatory marker CD206 in Iba1⁺ microglia or macrophages. DHA-treated brains showed significantly fewer CD16/32⁺ microglia or macrophages, but an increased CD206⁺ phagocytic microglial or macrophage population. Additionally, DHA treatment revealed a shift in microglial or macrophage morphology from the activated, amoeboid-like state into the more permissive, surveillant state. Furthermore, activated Iba1⁺ microglial or macrophages were associated with neurons expressing the endoplasmic reticulum (ER) stress marker CHOP at 3 days post-TBI, and the administration of DHA post-TBI concurrently reduced ER stress and the associated activation of Iba1⁺ microglial or macrophages. There was a decrease in nuclear translocation of activated nuclear factor kappa-light-chain-enhancer of activated B cells protein at 3 days in DHA-treated tissue and reduced neuronal degeneration in DHA-treated brains at 3, 7, and 21 days after TBI. In summary, our study demonstrated that TBI mediated inflammatory responses are associated with increased neuronal ER stress and subsequent activation of microglia or macrophages. DHA administration reduced neuronal ER stress and subsequent association with microglial or macrophage polarization after TBI, demonstrating its therapeutic potential to ameliorate TBI-induced cellular pathology.     

The Endoplasmic Reticulum: A Social Network in Plant Cells

The endoplasmic reticulum (ER) is an interconnected network comprised of ribosome-studded sheets and smooth tubules. The ER plays crucial roles in the biosynthesis and transport of proteins and lipids, and in calcium (Ca²⁺) regulation in compartmentalized eukaryotic cells including plant cells. To support its well-segregated functions, the shape of the ER undergoes notable changes in response to both developmental cues and outside influences. In this review, we will discuss recent findings on molecular mechanisms underlying the unique morphology and dynamics of the ER, and the importance of the interconnected ER network in cell polarity. In animal and yeast cells, two family proteins, the reticulons and DP1/Yop1, are required for shaping high-curvature ER tubules, while members of the atlastin family of dynamin-like GTPases are involved in the fusion of ER tubules to make an interconnected ER network. In plant cells, recent data also indicate that the reticulons are involved in shaping ER tubules, while RHD3, a plant member of the atlastin GTPases, is required for the generation of an interconnected ER network. We will also summarize the current knowledge on how the ER interacts with other membrane-bound organelles, with a focus on how the ER and Golgi interplay in plant cells.     

Titanium Dioxide Nanoparticles Induce Endoplasmic Reticulum Stress-Mediated Autophagic Cell Death via Mitochondria-Associated Endoplasmic Reticulum Membrane Disruption in Normal Lung Cells

Nanomaterials are used in diverse fields including food, cosmetic, and medical industries. Titanium dioxide nanoparticles (TiO₂-NP) are widely used, but their effects on biological systems and mechanism of toxicity have not been elucidated fully. Here, we report the toxicological mechanism of TiO₂-NP in cell organelles. Human bronchial epithelial cells (16HBE14o-) were exposed to 50 and 100 μg/mL TiO₂-NP for 24 and 48 h. Our results showed that TiO₂-NP induced endoplasmic reticulum (ER) stress in the cells and disrupted the mitochondria-associated endoplasmic reticulum membranes (MAMs) and calcium ion balance, thereby increasing autophagy. In contrast, an inhibitor of ER stress, tauroursodeoxycholic acid (TUDCA), mitigated the cellular toxic response, suggesting that TiO₂-NP promoted toxicity via ER stress. This novel mechanism of TiO₂-NP toxicity in human bronchial epithelial cells suggests that further exhaustive research on the harmful effects of these nanoparticles in relevant organisms is needed for their safe application.     

Staphylococcus aureus Inhibits IL-8 Responses Induced by Pseudomonas aeruginosa in Airway Epithelial Cells

Pseudomonas aeruginosa (PA) and Staphylococcus aureus (SA) are major respiratory pathogens and can concurrently colonize the airways of patients with chronic obstructive diseases, such as cystic fibrosis (CF). Airway epithelial cell signalling is critical to the activation of innate immune responses. In the setting of polymicrobial colonization or infection of the respiratory tract, how epithelial cells integrate different bacterial stimuli remains unknown. Our study examined the inflammatory responses to PA and SA co-stimulations. Immortalised airway epithelial cells (Beas-2B) exposed to bacteria-free filtrates from PA (PAF) induced a robust production of the neutrophil chemoattractant IL-8 while bacteria-free filtrates from SA (SAF) had a minimal effect. Surprisingly, co-stimulation with PAF+SAF demonstrated that SAF strongly inhibited the PAF-driven IL-8 production, showing that SAF has potent anti-inflammatory effects. Similarly SAF decreased IL-8 production induced by the TLR1/TLR2 ligand Pam₃CysSK₄ but not the TLR4 ligand LPS nor TLR5 ligand flagellin in Beas-2B cells. Moreover, SAF greatly dampened TLR1/TLR2-mediated activation of the NF-κB pathway, but not the p38 MAPK pathway. We observed this SAF-dependent anti-inflammatory activity in several SA clinical strains, as well as in the CF epithelial cell line CFBE41o-. These findings show a novel direct anti-inflammatory effect of SA on airway epithelial cells, highlighting its potential to modulate inflammatory responses in the setting of polymicrobial infections.     

Dexamethasone Inhibits Repair of Human Airway Epithelial Cells Mediated by Glucocorticoid-Induced Leucine Zipper (GILZ)

Background

Glucocorticoids (GCs) are a first-line treatment for asthma for their anti-inflammatory effects, but they also hinder the repair of airway epithelial injury. The anti-inflammatory protein GC-induced leucine zipper (GILZ) is reported to inhibit the activation of the mitogen-activated protein kinase (MAPK)-extracellular-signal-regulated kinase (ERK) signaling pathway, which promotes the repair of airway epithelial cells around the damaged areas. We investigated whether the inhibition of airway epithelial repair imposed by the GC dexamethasone (DEX) is mediated by GILZ.

Methods

We tested the effect of DEX on the expressions of GILZ mRNA and GILZ protein and the MAPK-ERK signaling pathway in human airway epithelial cells, via RT-PCR and Western blot. We further evaluated the role of GILZ in mediating the effect of DEX on the MAPK-ERK signaling pathway and in airway epithelium repair by utilizing small-interfering RNAs, MTT, CFSE labeling, wound-healing and cell migration assays.

Results

DEX increased GILZ mRNA and GILZ protein levels in a human airway epithelial cell line. Furthermore, DEX inhibited the phosphorylation of Raf-1, Mek1/2, Erk1/2 (components of the MAPK-ERK signaling pathway), proliferation and migration. However, the inhibitory effect of DEX was mitigated in cells when the GILZ gene was silenced.

Conclusions

The inhibition of epithelial injury repair by DEX is mediated in part by activation of GILZ, which suppressed activation of the MAPK-ERK signaling pathway, proliferation and migration. Our study implicates the involvement of DEX in this process, and furthers our understanding of the dual role of GCs.     

气道上皮细胞在哮喘中的作用

随着现代医学的发展,人们对支气管哮喘发病机制的研究有了进一步发展.支气管哮喘(简称哮喘)是一种由多种细胞,多种细胞因子参与形成的慢性气道炎症性疾病.支气管上皮细胞是气道结构细胞,它是抵抗外界损伤因素的第一道防线,当吸人性刺激物质时,首先激化支气管上皮细胞并破坏支气管上皮细胞的正常结构和生理功能,在应激状态下的上皮细胞通过分泌炎性介质与自身细胞或其他气道结构细胞、炎性细胞、抗原递呈细胞等相互作用,积极参与哮喘的气道慢性炎症发生与发展进程.因此气道上皮损伤是影响哮喘发生发展的重要因素,阐明维持气道上皮正常结构和功能的分子机制是目前防治哮喘的重要课题.本文综述气道上皮在哮喘发生发展中的作用及相关机制研究进展.     

The Endoplasmic Reticulum and the Golgi Structures in Maize Root Cells

Maize root tips were fixed in potassium permanganate, embedded in epoxy resin, sectioned to show silver interference color, and studied with the electron microscope. All the cells were seen to contain an endoplasmic reticulum and apparently independent Golgi structures. The endoplasmic reticulum is demonstrated as a membrane-bounded, vesicular structure comparable in many aspects to that of several types of animal cells. With the treatment used here the membranes appear smooth surfaced. The endoplasmic reticulum is continuous with the nuclear envelope and, by contact at least, with structures passing through the cell wall. The nuclear envelope is characterized by discontinuities, as previously reported for animal cells. The reticula of adjacent cells seem to be in contact at or through the plasmodesmata. Because of these contacts the endoplasmic reticulum of a given cell appears to be part of an intercellular system. The Golgi structures appear as stacks of platelet-vesicles which apparently may, under certain conditions, produce small vesicles around their edges. Their form changes markedly with development of the cell.     

Ivermectin Inhibits Growth of Chlamydia trachomatis in Epithelial Cells

Ivermectin is currently approved for treatment of both clinical and veterinary infections by nematodes, including Onchocerca cervicalis in horses and Onchocerca volvulus in humans. However, ivermectin has never been shown to be effective against bacterial pathogens. Here we show that ivermectin also inhibits infection of epithelial cells by the bacterial pathogen, Chlamydia trachomatis, at doses that could be envisioned clinically for sexually-transmitted or ocular infections by Chlamydia.     

CHOP Mediates Endoplasmic Reticulum Stress-Induced Apoptosis in Gimap5-Deficient T Cells

Gimap5 (GTPase of the immunity-associated protein 5) has been linked to the regulation of T cell survival, and polymorphisms in the human GIMAP5 gene associate with autoimmune disorders. The BioBreeding diabetes-prone (BBDP) rat has a mutation in the Gimap5 gene that leads to spontaneous apoptosis of peripheral T cells by an unknown mechanism. Because Gimap5 localizes to the endoplasmic reticulum (ER), we hypothesized that absence of functional Gimap5 protein initiates T cell death through disruptions in ER homeostasis. We observed increases in ER stress-associated chaperones in T cells but not thymocytes or B cells from Gimap5^−/− BBDP rats. We then discovered that ER stress-induced apoptotic signaling through C/EBP-homologous protein (CHOP) occurs in Gimap5^−/− T cells. Knockdown of CHOP by siRNA protected Gimap5^−/− T cells from ER stress-induced apoptosis, thereby identifying a role for this cellular pathway in the T cell lymphopenia of the BBDP rat. These findings indicate a direct relationship between Gimap5 and the maintenance of ER homeostasis in the survival of T cells.     

NELL2 Function in the Protection of Cells against Endoplasmic Reticulum Stress

Continuous intra- and extracellular stresses induce disorder of Ca²⁺ homeostasis and accumulation of unfolded protein in the endoplasmic reticulum (ER), which results in ER stress. Severe long-term ER stress triggers apoptosis signaling pathways, resulting in cell death. Neural epidermal growth factor-like like protein 2 (NELL2) has been reported to be important in protection of cells from cell death-inducing environments. In this study, we investigated the cytoprotective effect of NELL2 in the context of ER stress induced by thapsigargin, a strong ER stress inducer, in Cos7 cells. Overexpression of NELL2 prevented ER stress-mediated apoptosis by decreasing expression of ER stress-induced C/EBP homologous protein (CHOP) and increasing ER chaperones. In this context, expression of anti-apoptotic Bcl-xL was increased by NELL2, whereas NELL2 decreased expression of pro-apoptotic proteins, such as cleaved caspases 3 and 7. This anti-apoptotic effect of NELL2 is likely mediated by extracellular signal-regulated kinase (ERK) signaling, because its inhibitor, U0126, inhibited effects of NELL2 on the expression of anti- and pro-apoptotic proteins and on the protection from ER stress-induced cell death.     

白藜芦醇对高糖条件下大鼠晶状体上皮细胞线粒体活性氧产生及内质网的影响

目的：探讨白藜芦醇（resveratrol,Res）对高糖条件下大鼠晶状体上皮细胞（LECs）凋亡、线粒体活性氧产生以及内质网表达的影响。方法：用含30 mmol·L-1葡萄糖浓度的培养基体外培养LECs,随后加入25 mg·L-1Res共培养48 h。流式细胞术检测LECs细胞凋亡情况和线粒体膜电位的变化。激光共聚焦显微镜观察线粒体活性氧变化情况,并用免疫组化法检测内质网表达。结果：在高糖培养条件下,与对照组相比,LECs死亡率明显增高,线粒体膜电位降低,活性氧增多。内质网阳性率明显下降。经Rev干预后,细胞凋亡率显著降低,线粒体膜电位和内质网阳性率均升高,活性氧产生明显减少（P〈0.05）。结论：白藜芦醇能在一定程度上减轻糖尿病性白内障大鼠晶状体凋亡的发生并维持正常细胞器功能,从而延缓白内障的发生和发展。     

CFTR Delivery to 25% of Surface Epithelial Cells Restores Normal Rates of Mucus Transport to Human Cystic Fibrosis Airway Epithelium

Dysfunction of CFTR in cystic fibrosis (CF) airway epithelium perturbs the normal regulation of ion transport, leading to a reduced volume of airway surface liquid (ASL), mucus dehydration, decreased mucus transport, and mucus plugging of the airways. CFTR is normally expressed in ciliated epithelial cells of the surface and submucosal gland ductal epithelium and submucosal gland acinar cells. Critical questions for the development of gene transfer strategies for CF airway disease are what airway regions require CFTR function and how many epithelial cells require CFTR expression to restore normal ASL volume regulation and mucus transport to CF airway epithelium? An in vitro model of human CF ciliated surface airway epithelium (CF HAE) was used to test whether a human parainfluenza virus (PIV) vector engineered to express CFTR (PIVCFTR) could deliver sufficient CFTR to CF HAE to restore mucus transport, thus correcting the CF phenotype. PIVCFTR delivered CFTR to >60% of airway surface epithelial cells and expressed CFTR protein in CF HAE approximately 100-fold over endogenous levels in non-CF HAE. This efficiency of CFTR delivery fully corrected the basic bioelectric defects of Cl⁻ and Na⁺ epithelial ion transport and restored ASL volume regulation and mucus transport to levels approaching those of non-CF HAE. To determine the numbers of CF HAE surface epithelial cells required to express CFTR for restoration of mucus transport to normal levels, different amounts of PIVCFTR were used to express CFTR in 3%–65% of the surface epithelial cells of CF HAE and correlated to increasing ASL volumes and mucus transport rates. These data demonstrate for the first time, to our knowledge, that restoration of normal mucus transport rates in CF HAE was achieved after CFTR delivery to 25% of surface epithelial cells. In vivo experimentation in appropriate models will be required to determine what level of mucus transport will afford clinical benefit to CF patients, but we predict that a future goal for corrective gene transfer to the CF human airways in vivo would attempt to target at least 25% of surface epithelial cells to achieve mucus transport rates comparable to those in non-CF airways.