Enhanced release of thromboxane A(2) after exposure of human airway epithelial cells to meconium.

Meconium aspiration syndrome (MAS) is a cause of significant morbidity and mortality in the perinatal period. Despite the clinical relevance of MAS, its pathogenesis is poorly understood. Epithelial cell-derived prostanoids are involved in the regulation of several cellular functions within the lung, including the control of tone and reactivity of airway and vascular smooth muscle. In this study, we evaluated whether exposure to meconium affects the metabolic function of human airway epithelial cells. Monolayers of A549 cells, a transformed human epithelial cell line, were incubated with various concentrations of meconium. Control cells were incubated with serum-free medium in a similar manner. The supernatant fluid was removed at various time points and assayed for thromboxane A(2) (TXA(2)) production. The latter was accomplished by measuring its immediate and stable metabolite thromboxane B(2), using an enzyme-linked immunosorbent assay (ELISA). In selected experiments, the modulatory effects of indomethacin (10(-6) M), dexamethasone (10(-6) M), and L-nitroarginine methyl ester (L-NAME, 10(-6) M) on TXA(2) production were evaluated. Results were expressed in terms of pg/mg protein (mean +/- SE). We found that exposure to meconium produced a significant release of TXA(2) from A549 cells. Indomethacin, dexamethasone, and in part, L-NAME inhibited meconium-induced release of TXA(2). Our findings demonstrate that meconium enhances the production of thromboxanes from A549 cells, suggesting that airway epithelial cells and their metabolic products may play an important role in the pathogenesis of MAS.     

Involvement of p38 mitogen‐activated protein kinase pathway in honokiol‐induced apoptosis in a human hepatoma cell line (hepG2)

Background: Honokiol has been known to have antitumour activity. This study was conducted to evaluate the antiproliferative potential of honokiol against the hepG2 heptocellular cell line and its mechanism of action. Methods: hepG2 cells were treated with honokiol of 0–40 μg/ml concentration. The cytotoxic effect of honokiol was determined by a 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) assay. The apoptosis was evaluated by flow cytometry. Western blots were used to analyse the expression of various proteins (procaspase‐9, procaspase‐3, cleaved caspase‐3, cytochrome c, Bcl‐2, Bax, Bad, Bcl‐X_L and p38). Results: Honokiol induced apoptosis with a decreased expression of procaspase‐3 and ‐9 and an increased expression of active caspase‐3. Exposure of hepG2 cells to honokiol resulted in the downregulation of Bcl‐X_L and Bcl‐2 expression and the release of mitochondrial cytochrome c to the cytosol. In addition, honokiol activated the p38 mitogen‐activated protein kinase (MAPK) pathway, and the inhibition of this pathway by SB203580 reduced honokiol‐induced apoptosis and activation of caspase‐3. Conclusion: Honokiol induces apoptosis of hepG2 human hepatocellular carcinoma cells through activation of the p38 MAPK pathway, and, in turn, activation of caspase‐3.     

Melatonin enhances hyperthermia‐induced apoptotic cell death in human leukemia cells

Melatonin is an endogenous indoleamine with a wide range of biological functions. In addition to modulating circadian rhythms, it plays important roles in the health as an antioxidant. Melatonin has also the ability to induce apoptosis in cancer cells and to enhance the antitumoral activity of chemotherapeutic agents. In this study, the effect of melatonin on hyperthermia‐induced apoptosis was explored using human leukemia cells. The results demonstrate that melatonin greatly improved the cytotoxicity of hyperthermia in U937 cells. The potentiation of cell death was achieved with 1 mmol/L concentrations of the indoleamine but not with concentrations close to physiological levels in blood (1 nmol/L). This effect was associated to an enhancement of the apoptotic response, revealed by an increase in cells with hypodiploid DNA content and activation of multiple caspases (caspase‐2, caspase‐3, caspase‐8, and caspase‐9). Melatonin also increased hyperthermia‐induced Bid activation as well as translocation of Bax from the cytosol to mitochondria and cytochrome c release. Hyperthermia‐provoked apoptosis and potentiation by melatonin were abrogated by a broad‐spectrum caspase inhibitor (z‐VAD‐fmk) as well as by specific inhibitors against caspase‐8 or caspase‐3. In contrast, blocking of the mitochondrial pathway of apoptosis either with a caspase‐9 inhibitor or overexpressing the anti‐apoptotic protein Bcl‐2 (U937/Bcl‐2) reduced the number of apoptotic cells in response to hyperthermia but it was unable to suppress melatonin enhancement. Melatonin appears to modulate the apoptotic response triggered by hyperthermia in a cell type‐specific manner as similar results were observed in HL‐60 but not in K562 or MOLT‐3 cells.     

Modular effects of estradiol on ethanol-induced apoptosis in human intestinal epithelial cells

OBJECTIVE: Epidemiological data indicate that females develop alcohol-induced liver disease (ALD) more rapidly and more severely than males. Though the contribution of gut-derived endotoxin to the onset and development of ALD suggests the loss of epithelial cell viability that results in impaired intestinal function due to alcohol exposure, the additional effects of female sex hormones on intestinal cell viability is not known. The aim of this study was to examine the influence of estradiol on the intestinal cell death induced by acute and low concentrations of ethanol in an in vitro system. MATERIAL AND METHODS: Human intestinal epithelial Caco-2 cells were incubated with 0, 5, and 10% ethanol for 3 h. Estradiol stimulation, concentration of 3, 30, and 300 pg/ml occurred in the presence or absence of 10% ethanol for 2 h. Phosphatidylserine (PS) externalization, caspase-mediated cytokeratin 18 (CK18) cleavage, and DNA fragmentation were quantified using flow cytometry. RESULTS: Treatment with 10% ethanol markedly induced PS externalization, caspase activation, and DNA fragmentation after 2 h incubation. Whereas estradiol itself did not affect cell viability, physiological concentrations of estradiol enhanced PS externalization and DNA fragmentation induced by 10% ethanol, and these were remarkable at 300 pg/ml estradiol. CONCLUSIONS: Ethanol-induced apoptosis was potentiated by physiological concentrations of estradiol, especially at the higher level which is found only in females. Our data suggest that enhanced ethanol-induced intestinal epithelial cell apoptosis in the presence of estradiol could cause greater intestinal permeability, which allows endotoxin to enter the circulation and eventually results in more severe ALD in females.     

The novel proteasome inhibitor carfilzomib induces cell cycle arrest,apoptosis and potentiates the anti‐tumour activity of chemotherapy in rituximab‐resistant lymphoma

Targeting the proteasome system with bortezomib (BTZ) results in anti‐tumour activity and potentiates the effects of chemotherapy/biological agents in multiple myeloma and B‐cell lymphoma. Carfilzomib (CFZ) is a more selective proteasome inhibitor that is structurally distinct from BTZ. In an attempt to characterize its biological activity, we evaluated CFZ in several lymphoma pre‐clinical models. Rituximab‐sensitive cell lines (RSCL), rituximab‐resistant cell lines (RRCL), and primary tumour cells derived from B‐cell lymphoma patients were exposed to CFZ or BTZ. Cell viability and changes in cell cycle were determined. Western blots were performed to detect PARP‐cleavage and/or changes in Bcl‐2 (BCL2) family members. CFZ was 10 times more active than BTZ and exhibited dose‐ and time‐dependent cytotoxicity. CFZ exposure induced apoptosis by upregulation of Bak (BAK1) and subsequent PARP cleavage in RSCL and RRCL; it was also partially caspase‐dependent. CFZ induced G2/M phase cell cycle arrest in RSCL. CFZ demonstrated the ability to overcome resistance to chemotherapy in RRCL and potentiated the anti‐tumour activity of chemotherapy agents. Our data suggest that CFZ is able to overcome resistance to chemotherapeutic agents, upregulate pro‐apoptotic proteins to promote apoptosis, and induce G2/M cell cycle arrest in lymphoma cells. Our pre‐clinical data supports future clinical evaluation of CFZ in B‐cell lymphoma.     

Carbocisteine inhibits oxidant-induced apoptosis in cultured human airway epithelial cells

Background and objective: Increased oxidant levels have been associated with exacerbations of COPD, and L‐carbocisteine, a mucolytic agent, reduces the frequency of exacerbations. The mechanisms underlying the inhibitory effects of L‐carbocisteine on oxidant‐induced COPD exacerbations were examined in an in vitro study of human airway epithelial cells. Methods: In order to examine the antioxidant effects of L‐carbocisteine, human tracheal epithelial cells were treated with L‐carbocisteine and exposed to hydrogen peroxide (H₂O₂). Cell apoptosis was assessed using a cell death detection ELISA, and the pathways leading to cell apoptosis were examined by measurement of caspase‐3 and caspase‐9 by western blot analysis with fluorescent detection. Results: The proportion of apoptotic cells in human tracheal epithelium was increased in a concentration‐ and time‐dependent manner, following exposure to H₂O₂. Treatment with L‐carbocisteine reduced the proportion of apoptotic cells. In contrast, H₂O₂ did not increase the concentration of LDH in supernatants of epithelial cells. Exposure to H₂O₂ activated caspase‐3 and caspase‐9, and L‐carbocisteine inhibited the H₂O₂‐induced activation of these caspases. L‐carbocisteine activated Akt phosphorylation, which modulates caspase activation, and the inhibitors of Akt, LY294002 and wortmannin, significantly reversed the inhibitory effects of L‐carbocisteine on H₂O₂‐induced cell apoptosis. Conclusions: These findings suggest that in human airway epithelium, L‐carbocisteine may inhibit cell damage induced by H₂O₂ through the activation of Akt phosphorylation. L‐carbocisteine may have antioxidant effects, as well as mucolytic activity, in inflamed airways.     

Melatonin inhibits apoptosis and improves the developmental potential of vitrified bovine oocytes

Vitrification of oocytes has been shown to be closely associated with increased levels of reactive oxygen species (ROS) and apoptotic events. However, little information is available the effect of melatonin on the ROS levels and apoptotic events in vitrified oocytes. Therefore, we studied the effect of melatonin on ROS and apoptotic events in vitrified bovine oocytes by supplementing vitrification solution or in vitro maturation (IVM) and vitrification solution with 10^?9 m melatonin. We analyzed the ROS, mitochondrial Ca²⁺ (mCa²⁺) and membrane potential (ΔΨm), externalization of phosphatidylserine (PS), caspase‐3 activation, DNA fragmentation, mRNA expression levels of Bax and Bcl2 l1, and developmental potential of vitrified bovine oocytes. Vitrified bovine oocytes exhibited increased levels of ROS, mCa²⁺, Bax mRNA, and caspase‐3 protein and higher rates of PS externalization and DNA fragmentation, and decreased ΔΨm and Bcl2 l1 mRNA expression level. However, melatonin supplementation in vitrification solution or IVM and vitrification solution significantly decreased the levels of ROS, mCa²⁺, Bax mRNA expression, and caspase‐3 protein, and PS externalization and DNA fragmentation rates, and increased the ΔΨm and Bcl2 l1 mRNA expression level in vitrified oocytes, resulting in an increased developmental ability of vitrified bovine oocytes after parthenogenetic activation. The developmental ability of vitrified oocytes with melatonin supplementation in IVM and vitrification solution was similar to that of fresh ones. This study showed that supplementing the IVM and vitrification medium or vitrification medium with 10^?9 m melatonin significantly decreased the ROS level and inhibited apoptotic events of vitrified bovine oocytes, consequently increasing their developmental potential.     

Ghrelin inhibits 5-fluorouracil-induced apoptosis in colonic cancer cells

Background and Aim: The aim of the present study was to investigate if ghrelin inhibits apoptosis in colonic cancer cells. Methods: Cell viability in HT‐29 cells was measured using 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide assay. Apoptosis was measured using 4′,6‐diamidino‐2‐phenylindole staining and flow cytometry. The protein expression of Bcl‐2, Bax, and caspase‐3 activation was examined using Western blotting. Results: Ghrelin dose dependently decreased the growth inhibition of HT‐29 cells induced by 5‐fluorouracil (5‐FU). Cells treated with 5‐FU displayed chromatin condensation and nuclear fragmentation, which are typical changes of apoptosis. However, co‐treatment with ghrelin reduced these changes. Flow cytometry after staining with Annexin V and propidium iodide showed that ghrelin decreased the apoptotic rate of HT‐29 cells induced by 5‐FU. Caspase‐3 activation was significantly lower in the co‐treated group than in the group treated with 5‐FU alone. In addition, ghrelin reversed the 5‐FU‐induced Bcl‐2/Bax protein ratio. Conclusion: Ghrelin inhibits 5‐FU‐induced apoptosis in colon cancer cells through the regulation of the Bcl‐2/Bax protein ratio.     

Low concentrations of ethanol induce apoptosis in human intestinal cells

Background: Increased systemic levels of endotoxin have been detected in human alcoholics and are thought to be derived from the gut. Although a ‘leaky gut’ is considered to be a necessary factor for alcohol‐induced endotoxemia followed by chronic liver injury, the effects of low concentrations of ethanol on intestinal epithelial cells have not been fully understood. The aim of this study was to evaluate intestinal epithelial cell death induced by acute, low concentrations of ethanol in an in vitro system. Methods: The human intestinal Caco‐2 cell line was incubated with 0%, 5%, 10% ethanol for up to 3?h. Phosphatidylserine (PS) externalization, caspase‐mediated cytokeratin 18 (CK18) cleavage, and DNA fragmentation were evaluated using flow cytometry. The caspase inhibitor zVAD‐fmk was used to test the role of caspases in ethanol‐induced cell death. Results: Treatment with 5% and 10% ethanol for 3?h led to a gradual increase in PS externalization. Caspase‐mediated CK18 was significantly enhanced as early as 1?h after 10% ethanol incubation, while DNA fragmentation was detected from 2?h onwards. Not only caspase activation but also both PS externalization and DNA fragmentation were completely prevented by pretreatment with the caspase inhibitor. Conclusions: Apoptotic cell death in confluent Caco‐2 cells was induced by acute and low concentrations of ethanol. These results suggest that clinically achievable doses of ethanol impair intestinal barrier function by induction of apoptosis in intestinal epithelial cells. This impairment of the barrier function would allow endotoxin to enter the circulation and evoke hepatic inflammation.     

Ethanol enhances susceptibility to apoptotic cell death via down-regulation of autophagy-related proteins

Background: Alcohol induces cellular stress and promotes cell death in immune cells. Molecular mechanisms by which ethanol impairs the function of immune cells are largely unknown. Autophagy is a degradation pathway, acting either as a pro‐survival or pro‐death mechanism activated during stress conditions. We examined whether ethanol influences autophagy in monocytic human U937, CD4 Jurkat, and MCF‐7 cells. Methods: Effects of ethanol during starvation‐induced autophagy were investigated, treating cells with ethanol alone and in combination with activation of autophagy by rapamycin or inhibition by wortmannin. Apoptotic and necrotic cell death features such as the breakdown of the mitochondrial membrane potential, DNA fragmentation, and cell permeability were assessed using FACS analyses. Expression level of Beclin‐1, LC3‐II, Bcl‐2, and the activation of caspase‐3, and PARP‐1 were determined using Western blot analyses. Influence of ethanol on formation of LC3‐II complexes was assessed using fluorescence microscopy in MCF‐7 cells stable transfected with a GFP‐LC3‐II‐expression vector. Results: Ethanol down regulated autophagy proteins such as Beclin‐1 and LC3‐II. Apoptosis was enhanced as shown by breakdown of mitochondrial potential, up‐regulation of cleaved caspase‐3 and PARP‐1 and down‐regulation of anti‐apoptotic protein Bcl‐2. Formation of LC3‐II complexes was inhibited by ethanol in caspase‐3 deficient MCF‐7 cells. Stimulation of autophagy by rapamycin prevented ethanol‐induced apoptotic cell death. Inhibition of autophagy by wortmannin aggravated ethanol‐mediated necrotic cell death. Conclusion: Inhibition of autophagy via ethanol enhances susceptibility to cell death.     

Increased leucocyte apoptosis in transfused β‐thalassaemia patients

This exploratory study assessed apoptosis in peripheral blood leucocytes (PBL) from β‐thalassaemia patients receiving chronic transfusions and chelation therapy (deferasirox or deferoxamine) at baseline, 1, 6, and 12 months. At baseline, thalassaemic PBLs presented 50% greater levels of Bax (BAX), 75% higher caspase‐3/7, 48% higher caspase‐8 and 88% higher caspase‐9 activities and 428% more nucleosomal DNA fragmentation than control subjects. Only neutrophils correlated significantly with apoptotic markers. Previously, we showed that over the treatment year, hepatic iron declined; we now show that the ratio of Bax/Bcl‐2 (BCL2), (−27·3%/year), and caspase‐9 activity (−13·3%/year) declined in both treatment groups, suggesting that chelation decreases body iron and indicators of PBL apoptosis.     

Bcl‐2 overexpression in hepatic stellate cell line CFSC‐2G,induces a pro‐fibrotic state

Background and Aim: Development of hepatic fibrosis is a complex process that involves oxidative stress (OS) and an altered balance between pro‐ and anti‐apoptotic molecules. Since Bcl‐2 overexpression preserves viability against OS, our objective was to address the effect of Bcl‐2 overexpression in the hepatic stellate cells (HSC) cell‐line CFSC‐2G under acetaldehyde and H₂O₂ challenge, and explore if it protects these cells against OS, induces replicative senescence and/or modify extracellular matrix (ECM) remodeling potential. Methods: To induce Bcl‐2 overexpression, HSC cell line CFSC‐2G was transfected by lipofection technique. Green fluorescent protein‐only CFSC‐2G cells were used as a control. Cell survival after H₂O₂ treatment and total protein oxidation were assessed. To determine cell cycle arrest, proliferation‐rate, DNA synthesis and senescence were assessed. Matrix metalloproteinases (MMP), tissue‐inhibitor of MMP (TIMP), transglutaminases (TG) and smooth muscle a‐actin (α‐SMA) were evaluated by western blot in response to acetaldehyde treatment as markers of ECM remodeling capacity in addition to transforming growth factor‐β (TGF‐β) mRNA. Results: Cells overexpressing Bcl‐2 survived ≈ 20% more than control cells when exposed to H₂O₂ and ≈ 35% proteins were protected from oxidation, but Bcl‐2 did not slow proliferation or induced senescence. Bcl‐2 overexpression did not change α‐SMA levels, but it increased TIMP‐1 (55%), tissue transglutaminases (tTG) (25%) and TGF‐β mRNA (49%), when exposed to acetaldehyde, while MMP‐13 content decreased (47%). Conclusions: Bcl‐2 overexpression protected HSC against oxidative stress but it did not induce replicative senescence. It increased TIMP‐1, tTG and TGF‐β mRNA levels and decreased MMP‐13 content, suggesting that Bcl‐2 overexpression may play a key role in the progression of fibrosis in chronic liver diseases.     

Analysis of cellular senescence induced by lipopolysaccharide in pulmonary alveolar epithelial cells

In this work, it was examined the possibility of lipopolysaccharide (LPS) causing cellular senescence in lung alveolar epithelial cells. Then, it was clarified how this cellular senescence phenomenon is associated with oxidative stress effect induced by LPS and whether antioxidants could inhibit reduced cellular viability by oxidant stress effect of LPS. In cell viability using cell counting kit-8, exposure to LPS decreased cellular viability and induced growth arrest in a concentration-dependent manner. The pre-apoptotic concentration of LPS was determined by caspase activation using a Caspase-Glo 3/7 luminescence assay kit. This concentration of LPS caused morphologic characteristics shown in senescent cells and elevated senescence-associated β-galactosidase activity. In addition, lysosomal content associated with senescence was increased by LPS at the pre-apoptotic concentration. However, this concentration of LPS did not shorten the telomere length. Exposure to LPS resulted in the formation of hydrogen peroxide in a concentration-dependent manner. The ability of LPS to reduce cellular viability was inhibited by the presence of glutathione. This study revealed that LPS could induce cellular senescence in lung alveloar epithelial cells, and these phenomena were closely associated with hydrogen peroxide production by LPS. Taken together, it is suggested that LPS-induced cellular senescence may play an important role in limiting the tissue repair response after sepsis.     

Thioredoxin protects fetal type II epithelial cells from hyperoxia‐induced injury

Oxygen toxicity is known to be one of the major contributors to bronchopulmonary dysplasia, a chronic lung disease in premature infants. Thioredoxin (Trx) is an antioxidant that prevents oxidative stress‐induced cell death, suggesting a potential therapeutic role in bronchopulmonary dysplasia. The aim of this study was to determine the role of Trx in the pathogenesis of hyperoxia‐induced alveolar epithelial cell injury. Alveolar type II epithelial cells from fetal rat lung were exposed to hyperoxia in vitro in the presence or absence of recombinant human Trx (rhTrx 2 µg/ml). Cell viability was assessed by 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl tetrazolium bromide assay. Apoptosis and levels of reactive oxygen species (ROS) were measured by flow cytometry. Activation of mitogen‐activated protein kinase (MAPK) and phosphatidylinositol 3‐kinase‐Akt (PI3K‐Akt) pathways were detected by Western blotting. We also investigated the effects of rhTrx on the following antioxidants (superoxide dismutase, catalase, and glutathione peroxidase). Trx significantly reduced hyperoxia‐induced cell death and increased cell viability. In addition, ROS generation in type II cells was inhibited by rhTrx under hyperoxic conditions. We demonstrated that rhTrx protected type II cells against hyperoxic injury via sustaining the extracellular signal regulated kinase and PI3K activation, and decreasing of c‐Jun N‐terminal protein kinase and p38 activation. The results also showed manganese superoxide dismutase and glutathione peroxidase activities were increased by rhTrx in type II cells exposed to hyperoxia.Taken together, these results demonstrate that rhTrx administration markedly attenuates hyperoxia‐induced type II cell injury through reduction of ROS generation, elevation of antioxidant activities and regulation of both MAPK and PI3K‐Akt signaling pathways. Pediatr Pulmonol. 2010;45:1192–1200. © 2010 Wiley‐Liss, Inc.     

Preservation of the Characteristics of the Cultured Human Type II Alveolar Epithelial Cells

The human type II alveolar epithelial cells lost their specific characteristics during cultivation. We examined the ultrastructural and biochemical nature of the human type II cells cultured by two culture systems. To make a physiological alveoli model, the epithelial cells were seeded onto the cell culture insert and allowed contact with the air directly. The cells exposed to the air expressed polarity and immature lamellar bodies in their cytoplasm. Separately, the alveolar epithelial cells were cultured as spheroids to construct the three-dimensional condition. These cells expressed mature morphological characteristics as epithelial cells and lamellar bodies. The expression of the surfactant apoprotein-A (SP-A) and -C (SP-C) mRNA was compared in the cells cultured as a monolayer, the air exposed and the spheroids. SP-A mRNA was detected in all the cultured epithelial cells, but SP-C mRNA, a specific protein for the type II cells, was expressed only in the cells forming spheroids. The expression of uPA, one of the fibrinolytic enzymes, its receptor (uPAR) and its inhibitor-1 (PAI-1) were also examined. The epithelial cells exposed to the air and formed spheroids expressed a larger amount of uPA mRNA than the monolayer, although the amount of uPAR mRNA were comparable in these cells. The amount of PAI-1 mRNA significantly increased when the epithelial cells were exposed to the air. These results indicate that the type II alveolar epithelial cells induced and preserved their specific characteristics by taking the physiological three-dimensional structure, and these characteristics were partially restored by exposure to the air. Those findings suggest that the alveolar epithelial cells should be cultivated in three-dimensional form with contact to the air to regenerate an appropriate alveolar tissue.     

Infant formula alters surfactant protein A (SP-A) and SP-B expression in pulmonary epithelial cells

Surfactant proteins A (SP-A) and SP-B are critical in the ability of pulmonary surfactant to reduce alveolar surface tension and provide innate immunity. Aspiration of infant milk formula can lead to lung dysfunction, but direct effects of aspirated formula on surfactant protein expression in pulmonary cells have not been described. The hypothesis that infant formula alters surfactant protein homeostasis was tested in vitro by assessing surfactant protein gene expression in cultured pulmonary epithelial cell lines expressing SP-A and SP-B that were transiently exposed (6 hr) to infant formula. Steady-state levels of SP-A protein and mRNA and SP-B mRNA in human bronchiolar (NCI-H441) and mouse alveolar (MLE15) epithelial cells were reduced in a dose-dependent manner 18 hr after exposure to infant formula. SP-A mRNA levels remained reduced 42 hr after exposure, but SP-B mRNA levels increased 10-fold. Neither soy formula nor non-fat dry milk affected steady-state SP-A and SP-B mRNA levels; suggesting a role of a component of infant formula derived from cow milk. These results indicate that infant formula has a direct, dose-dependent effect to reduce surfactant protein gene expression. Ultimately, milk aspiration may potentially result in a reduced capacity of the lung to defend against environmental insults.     

Intracellular zinc depletion induces caspase activation and p21 Waf1/Cip1 cleavage in human epithelial cell lines

To better understand the mechanisms by which zinc deficiency induces epithelial cell death, studies were done of the effects of intracellular zinc depletion induced by the zinc chelator TPEN on apoptosis-related events in human malignant epithelial cell lines LIM1215 (colonic), NCI-H292 (bronchial), and A549 (alveolar type II). In TPEN-treated cells, depletion of zinc was followed by activation of caspase-3 (as demonstrated by enzymatic assay and Western blotting), DNA fragmentation, and morphologic changes. Increase in caspase-3 activity began 12 h after addition of TPEN, suggesting that zinc may suppress a step just before the activation of this caspase. Caspase-6, a mediator of caspase-3 processing, also increased, but later than caspase-3. Effects of TPEN on apoptosis were completely prevented by exogenous ZnSO4 and partially prevented by peptide caspase inhibitors. A critical substrate of caspase-3 may be the cell cycle regulator p21Waf1/Cip1, which was rapidly cleaved in TPEN-treated cells to a 15-kDa fragment before further degradation.     

泡状棘球蚴原头节与肝癌细胞共培养的相互作用研究

目的 探究泡状棘球蚴原头节与肝癌细胞共培养后,原头节的活性、成囊以及肝癌细胞的增殖与凋亡情况。方法 构建泡状棘球蚴原头节与肝癌细胞共培养模型,以存在肝癌细胞共培养的泡球蚴原头节为实验组,无肝癌细胞共培养的泡球蚴原头节为对照组,光镜下观察泡状棘球蚴原头节存活数量、活性以及成囊情况;以存在原头节共培养的肝癌细胞为实验组,无原头节共培养的肝癌细胞为对照组,利用CCK-8检测肝癌细胞增殖情况,Annexin V-FITC/PI 流式细胞术检测肝癌细胞凋亡情况,Real-Time PCR检测凋亡相关分子caspase3、caspase9、Bax、Bcl-2、P21、P53的mRNA表达情况,Western-blot方法检测caspase3、caspase9、Bax、Bcl-2的蛋白表达情况。结果共培养后,实验组泡球蚴原头节的存活率[(91.42±2.47)%]显著高于无饲养细胞组[(73.10±4.68)%,t=-7.738,P<0.05],且活性较高;利用肝癌细胞作为饲养细胞时原头节的成囊率[(66.72±2.02)%]显著高于无饲养细胞组的成囊率[(30.21±2.25)%,t=11.228,P<0.01];CCK-8结果显示随着原头节的比例增高,肝癌细胞的增殖能力逐渐增强,且显著高于对照组;Real-Time PCR显示实验组的Bcl-2表达量显著高于对照组,而caspase3、caspase9、Bax、P21、P53的表达量低于对照组。Western blot结果与实时荧光定量PCR结果相一致,显示实验组Bcl-2表达量显著高于对照组,而caspase3、caspase9、Bax的表达量低于对照组。结论 泡球蚴原头节与肝癌细胞共培养时肝癌细胞可以促进原头节的存活,且会使原头节保持高活性状态,促进其成囊;反之原头节也会促进肝癌细胞的增殖,抑制肝癌细胞的凋亡。     

Ambient particulate matter affects occludin distribution and increases alveolar transepithelial electrical conductance

Background and objective: Inhaled particulate matter (PM) causes lung inflammation and epithelial dysfunction. However, the direct effect of PM on alveolar epithelial barrier integrity is not well understood. Our aim is to determine whether PM exposure affects the alveolar epithelial cells (AEC) transepithelial electrical conductance (Gt) and tight junction (TJ) proteins. Methods: Human AEC (A549) and primary rat AEC were exposed to PM of <10 µm in size (PM₁₀) and diesel exhaust particles (DEP), using titanium dioxide (TiO₂) as a control for particle size effects. Gt and permeability to fluorescein isothiocyanate‐dextran (FITC‐Dextran) were measured to assess barrier integrity. TJ integrity was evaluated by analysing penetration of Lanthanum nitrate (La³⁺) under transmission electron microscopy. Surface proteins were labelled with biotin and analysed by western blot. Immunofluorescence was performed to assess colocalization of TJ proteins including occludin and zonula occludens‐1 (ZO‐1). PM induced dissociation of occludin‐ZO‐1 was evaluated by co‐immunoprecipitation. Results: PM₁₀ and DEP increased Gt and disrupted TJ after 3 h of treatment. PM₁₀ and DEP induced occludin internalization from the plasma membrane into endosomal compartments and dissociation of occludin from ZO‐1. Overexpression of antioxidant enzymes manganese superoxide dismutase (MnSOD) and catalase, prevented PM‐induced Gt increase, occludin reduction from the plasma membrane and its dissociation from ZO‐1. Conclusions: PM induces alveolar epithelial dysfunction in part via occludin reduction at the plasma membrane and ZO‐1 dissociation in AEC. Furthermore, these effects are prevented by overexpression of two different antioxidant enzymes.