香烟烟雾提取物抑制肺泡上皮细胞的增殖并诱导其凋亡

香烟烟雾提取物（cigarette smoke extract,CSE）中含有丰富的氧化剂和自由基,由它所引起的氧化应激可导致肺泡壁的损伤进而发展为肺气肿.近年来,围绕CSE损伤肺泡壁作用机制的研究较为活跃,但其结果却一直存在着分歧.本实验的目的是观察CSE对肺泡Ⅱ型上皮细胞的损伤作用并探讨与其相关的分子机制.MTT比色法的结果显示,CSE以时间和剂量依赖性的方式降低细胞的增殖活力,流式细胞术的分析结果表明细胞增殖周期被阻滞在G1/S期.Hoechst 33258染色以及透射电镜观察从形态上确认CSE诱导细胞凋亡的发生,DNA梯的出现和Annexin V-FITC/碘化丙啶双染色的结果从分子水平得到进一步的证实.同时,运用流式细胞术检测到CSE诱导的凋亡伴随着Fas受体的高表达和caspase-3的显著活化.另外,使用H2DCFDA染色,经激光共聚焦显微镜术测得细胞内氧自由基在细胞受到CSE刺激以后大量快速积累.结果表明CSE能够抑制肺泡Ⅱ型上皮细胞来源的A549细胞的生长和增殖,并诱导细胞凋亡,由Fas受体所介导的死亡受体途径参与此凋亡过程,而CSE所引起的氧化应激则可能是阻止肺泡上皮细胞生长增殖并诱导其凋亡的始动因素.     

Vasorin/ATIA Promotes Cigarette Smoke–Induced Transformation of Human Bronchial Epithelial Cells by Suppressing Autophagy-Mediated Apoptosis

BACKGROUND: Escaping cell death pathways is an important event during carcinogenesis. We previously identified anti-TNFα-induced apoptosis (ATIA, also known as vasorin) as an antiapoptotic factor that suppresses reactive oxygen species (ROS) production. However, the role of vasorin in lung carcinogenesis has not been investigated. METHODS: Vasorin expression was examined in human lung cancer tissues with immunohistochemistry and database analysis. Genetic and pharmacological approaches were used to manipulate protein expression and autophagy activity in human bronchial epithelial cells (HBECs). ROS generation was measured with fluorescent indicator, apoptosis with release of lactate dehydrogenase, and cell transformation was assessed with colony formation in soft agar. RESULTS: Vasorin expression was increased in human lung cancer tissues and cell lines, which was inversely associated with lung cancer patient survival. Cigarette smoke extract (CSE) and benzo[a]pyrene diol epoxide (BPDE)–induced vasorin expression in HBECs. Vasorin knockdown in HBECs significantly suppressed CSE-induced transformation in association with enhanced ROS accumulation and autophagy. Scavenging ROS attenuated autophagy and cytotoxicity in vasorin knockdown cells, suggesting that vasorin potentiates transformation by impeding ROS-mediated CSE cytotoxicity and improving survival of the premalignant cells. Suppression of autophagy effectively inhibited CSE-induced apoptosis, suggesting that autophagy was pro-apoptotic in CSE-treated cells. Importantly, blocking autophagy strongly potentiated CSE-induced transformation. CONCLUSION: These results suggest that vasorin is a potential lung cancer–promoting factor that facilitates cigarette smoke–induced bronchial epithelial cell transformation by suppressing autophagy-mediated apoptosis, which could be exploited for lung cancer prevention.     

Activation of the UPR Protects against Cigarette Smoke-induced RPE Apoptosis through Up-Regulation of Nrf2

Recent studies have revealed a role of endoplasmic reticulum (ER) stress-induced unfolded protein response (UPR) in the regulation of RPE cell activity and survival. Herein, we examined the mechanisms by which the UPR modulates apoptotic signaling in human RPE cells challenged with cigarette smoking extract (CSE). Our results show that CSE exposure induced a dose- and time-dependent increase in ER stress markers, enhanced reactive oxygen species (ROS), mitochondrial fragmentation, and apoptosis of RPE cells. These changes were prevented by the anti-oxidant NAC or chemical chaperone TMAO, suggesting a close interaction between oxidative and ER stress in CSE-induced apoptosis. To decipher the role of the UPR, overexpression or down-regulation of XBP1 and CHOP genes was manipulated by adenovirus or siRNA. Overexpressing XBP1 protected against CSE-induced apoptosis by reducing CHOP, p-p38, and caspase-3 activation. In contrast, XBP1 knockdown sensitized the cells to CSE-induced apoptosis, which is likely through a CHOP-independent pathway. Surprisingly, knockdown of CHOP reduced p-eIF2α and Nrf2 resulting in a marked increase in caspase-3 activation and apoptosis. Furthermore, Nrf2 inhibition increased ER stress and exacerbated cell apoptosis, while Nrf2 overexpression reduced CHOP and protected RPE cells. Our data suggest that although CHOP may function as a pro-apoptotic gene during ER stress, it is also required for Nrf2 up-regulation and RPE cell survival. In addition, enhancing Nrf2 and XBP1 activity may help reduce oxidative and ER stress and protect RPE cells from cigarette smoke-induced damage.     

Tobacco smoke reduces viability in human lung fibroblasts: protective effect of glutathione S-transferase P1

Cigarette smoking is thought to be a major risk factor in various lung diseases including lung cancer and emphysema. However, the direct effect of cigarette smoke on the viability of lung-derived cells has not been fully elucidated. In this study, we investigated the viability of human lung fibroblast-derived (HFL1) cells to different concentrations of cigarette smoke extract (CSE). CSE induced apoptosis at lower concentrations (10-25%) and necrosis at higher concentrations (50-100%). We also examined the effects of glutathione S-transferase P1 (GSTP1), one of the xenobiotic metabolizing and antioxidant enzymes in the lung, against the cytotoxicity of CSE. Our results indicated that the level of HFL1 cell death was decreased by transfection with a GSTP1 expression vector and was increased by GSTP1 antisense vector transfection. Therefore, transient overexpression and underexpression of GSTP1 appeared to inhibit and enhance the cytotoxic effects of CSE on HFL1 cells, suggesting that GSTP1 may have protective effects against cigarette smoke in the airway cells.     

Cigarette smoke induces Akt protein degradation by the ubiquitin-proteasome system

Emphysema is one of the characteristic features of chronic obstructive pulmonary disease, which is caused mainly by cigarette smoking. Recent data have suggested that apoptosis and cell cycle arrest may contribute to the development of emphysema. In this study, we addressed the question of whether and how cigarette smoke affected Akt, which plays a critical role in cell survival and proliferation. In normal human lung fibroblasts, cigarette smoke extract (CSE) caused cell death, accompanying degradation of total and phosphorylated Akt (p-Akt), which was inhibited by MG132. CSE exposure resulted in preferential ubiquitination of the active Akt (myristoylated), rather than the inactive (T308A/S473A double mutant) Akt. Consistent with cytotoxicity, CSE induced a progressive decrease of phosphorylated human homolog of mouse double minute homolog 2 (p-HDM2) and phosphorylated apoptosis signal regulating kinase 1 (p-ASK1) with concomitant elevation of p53, p21, and phosphorylated p38 MAPK. Forced expression of the active Akt reduced both CSE-induced cytotoxicity and alteration in HDM2/p53/p21 and ASK1/p38 MAPK, compared with the inactive Akt. Of note, CSE induced expression of the tetratrico-peptide repeat domain 3 (TTC3), known as a ubiquitin ligase for active Akt. TTC3 siRNAs suppressed not only CSE-induced Akt degradation but also CSE-induced cytotoxicity. Accordingly, rat lungs exposed to cigarette smoke for 3 months showed elevated TTC3 expression and reduced Akt and p-Akt. Taken together, these data suggest that cigarette smoke induces cytotoxicity, partly through Akt degradation via the ubiquitin-proteasome system, in which TTC3 acts as a ubiquitin ligase for active Akt.     

Vasoactive intestinal peptide and pituitary adenylate cyclase-activating polypeptide attenuate the cigarette smoke extract-induced apoptotic death of rat alveolar L2 cells.

Chronic obstructive pulmonary disease is a major clinical disorder usually associated with cigarette smoking. A central feature of chronic obstructive pulmonary disease is inflammation coexisting with an abnormal protease/antiprotease balance, leading to apoptosis and elastolysis. In an in vitro study of rat lung alveolar L2 cells, cigarette smoke extract (CSE) induced apoptotic cell death. Exposure of L2 cells to CSE at a concentration of 0.25% resulted in a 50% increase of caspase-3 and matrix metalloproteinase (MMP) activities. Specific inhibitors for caspases and MMPs attenuated the cytotoxicity of CSE. RT-PCR amplification identified VPAC2 receptors in L2 cells. A radioligand-binding assay with (125)I-labeled vasoactive intestinal peptide (VIP) found high affinity and saturable (125)I-labeled VIP-binding sites in L2 cells. VIP and pituitary adenylate cyclase-activating polypeptide (PACAP27) were approximately equipotent for both VIP receptor binding and stimulation of cAMP production in L2 cells. Both neuropeptides, at concentrations higher than 10(-13) m, produced a concentration-dependent inhibition of CSE-induced cell death in L2 cells. VIP, at 10(-7) m, reduced CSE-stimulated MMP activity and caspase-3 activation. The present study has shown that VIP and PACAP27 significantly attenuate the cytotoxicity of CSE through the activation of VPAC2 receptor, and the protective effect of VIP may partly be the result of a reduction in the CSE-induced stimulation of MMPs and caspases.     

STAT3 activation inhibits human bronchial epithelial cell apoptosis in response to cigarette smoke exposure

We have previously reported that cigarette smoke can induce DNA damage in human lung cells without leading to apoptosis or necrosis. In this study, we report that STAT3 is required for the survival of human bronchial epithelial cells (HBECs) following cigarette smoke-induced DNA damage. Cigarette smoke extract (CSE) exposure increases STAT3 phosphorylation (Tyr 705) and DNA binding activity in HBECs. CSE also stimulates IL-6 release and mRNA expression. Anti-IL-6 neutralizing antibody partially blocks STAT3 activation and renders the cells sensitive to CSE-induced DNA damage. Suppression of STAT3 by siRNA results in severe DNA damage and cell death in response to CSE exposure. These findings suggest that STAT3 mediates HBEC survival in response to CSE-induced DNA damage, at least in part, through the IL-6/STAT3 signaling pathway.     

新型鬼臼毒素衍生物诱导人肺腺癌A549细胞凋亡及其机制研究

目的探讨新型鬼臼毒素衍生物10Ⅲg诱导人肺腺癌细胞A549细胞凋亡及其调控机制。方法采用四甲基偶氮唑蓝（MTr）比色法、流式细胞术测定细胞周期细胞凋亡率,DNA琼脂糖凝胶电泳和微管蛋白组化染色,Western印迹法检测凋亡蛋白Bax、Caspase-3的表达。结果新型鬼臼毒素衍生物10m。对A549细胞增殖具有明显的剂量和时间依赖性抑制作用,细胞周期分析显示S期细胞数明显增多,出现G2／M期阻滞;10Ⅲg作用48h后DNA电泳可见明显的梯状条带;10Ⅲg能破坏A549细胞的细胞骨架,与依托泊苷相比有明显促进微管解聚现象;10Ⅲg浓度为10^-6mol/L时能显著促进Bax、Caspase-3蛋白的表达。结论新型鬼臼毒素衍生物10Ⅲg通过诱导A549细胞发生G2／M期阻滞而抑制其增殖,其机制可能与抑制细胞微管解聚及诱导细胞凋亡有关。     

Cigarette smoke adversely affects functions and cell membrane integrity in c-kit+ cardiac stem cells

Cigarette smoking is a major risk factor for numerous diseases including cardiovascular diseases. Exposure to cigarette smoke (CS) leads to increased cardiovascular risk, myocardial injury, and mortality. Stem cell therapy is one of the promising therapeutic options available to treat myocardial injuries. Understanding the impact of cigarette smoke extract (CSE) on stem cell function would be valuable in determining the risk passed on during transplant. In this study, the impact of CSE on cardiac stem cell (CSC) functions was investigated using c-kit+ rat cardiac stem cells as the experimental model. Here, we hypothesized that CSE attenuates CSC membrane integrity, causes cytotoxicity, and affects many CSC functions via multiple mechanisms including modulation of extracellular stress-regulated kinase (ERK) (44/42) signaling and oxidative stress. The effects of CSE on CSCs were examined in vitro. Based on a published method, CSE was prepared. CSE-induced ERK signaling was detected by western blotting. CSE-induced modulation of catalase activity was also measured. Functional modulations due to CSE were examined via several methods including Apostain, BrdU, and LDH assays. In agreement with the CSE-induced activation of ERK, CSE-induced reduction in viability, migration, and increase in both cytotoxicity and para-cellular permeability were observed in CSCs. These results suggest that CSE impaired CSC responses that contribute to decreased ability of CSC to respond to stress or injury leading to exacerbation of the damage. Our findings will contribute to the understanding of the discipline and might contribute to the development of stem cell therapy approaches in the future.     

Cigarette Smoke Causes Caspase-Independent Apoptosis of Bronchial Epithelial Cells from Asthmatic Donors

Background

Epidemiologic studies have demonstrated important links between air pollution and asthma. Amongst these pollutants, environmental cigarette smoke is a risk factor both for asthma pathogenesis and exacerbation. As the barrier to the inhaled environment, the bronchial epithelium is a key structure that is exposed to cigarette smoke.

Objectives

Since primary bronchial epithelial cells (PBECs) from asthmatic donors are more susceptible to oxidant-induced apoptosis, we hypothesized that they would be susceptible to cigarette smoke-induced cell death.

Methods

PBECs from normal and asthmatic donors were exposed to cigarette smoke extract (CSE); cell survival and apoptosis were assessed by fluorescence-activated cell sorting, and protective effects of antioxidants evaluated. The mechanism of cell death was evaluated using caspase inhibitors and immunofluorescent staining for apoptosis-inducing factor (AIF).

Results

Exposure of PBEC cultures to CSE resulted in a dose-dependent increase in cell death. At 20% CSE, PBECs from asthmatic donors exhibited significantly more apoptosis than cells from non-asthmatic controls. Reduced glutathione (GSH), but not ascorbic acid (AA), protected against CSE-induced apoptosis. To investigate mechanisms of CSE-induced apoptosis, caspase-3 or -9 inhibitors were tested, but these failed to prevent apoptosis; in contrast, CSE promoted nuclear translocation of AIF from the mitochondria. GSH reduced the number of nuclear-AIF positive cells whereas AA was ineffective.

Conclusion

Our results show that PBECs from asthmatic donors are more susceptible to CSE-induced apoptosis. This response involves AIF, which has been implicated in DNA damage and ROS-mediated cell-death. Epithelial susceptibility to CSE may contribute to the impact of environmental tobacco smoke in asthma.     

Autophagic proteins regulate cigarette smoke-induced apoptosis: protective role of heme oxygenase-1

Cigarette smoke-induced cell death contributes to the pathogenesis of chronic obstructive pulmonary disease, though the relative roles of apoptosis and autophagy remain unclear. The inducible stress protein heme oxygenase-1 (HO-1) confers cytoprotection against oxidative stress. We examined the relationships between these processes in human bronchial epithelial cells (Beas-2b) exposed to cigarette smoke extract (CSE). CSE induced morphological and biochemical markers of autophagy in Beas-2b cells and induced autophagosome formation as evidenced by formation of GFP-LC3 puncta and electron microscopic analysis. Furthermore, CSE increased the processing of microtubule-associated protein-1 light chain-3 (LC3B-I) to LC3B-II, within 1 hr of exposure. Increased LC3B-II was associated with increased autophagy, since inhibitors of lysosomal proteases and of autophagosome-lysosome fusion further increased LC3B-II levels during CSE exposure. CSE concurrently induced extrinsic apoptosis in Beas-2b cells involving early activation of death-inducing-signaling-complex (DISC) formation and downstream activation of caspases (-8,-9,-3). The induction of extrinsic apoptosis by CSE was dependent in part on autophagic proteins. Reduction of Beclin 1 levels with beclin 1 siRNA inhibited DISC formation and caspase-3/8 activation in response to CSE. LC3B siRNA also inhibited caspase-3/8 activation. The stress protein HO-1 protected against CSE-induced cell death by concurrently downregulating apoptosis and autophagy-related signaling. Adenoviral mediated expression of HO-1 inhibited DISC formation and caspase-3/9 activation in CSE-treated epithelial cells, diminished the expression of Beclin 1, and partially inhibited the processing of LC3B-I to LC3B-II. Conversely, transfection of Beas-2b with ho-1 siRNA augmented CSE-induced DISC formation and increased intracellular reactive oxygen species formation. HO-1 expression augmented CSE-induced phosphorylation of NFkappaB p65 in Beas-2b cells. Consistently, expression of IkappaB, the inhibitor of NFkappaB, increased CSE-induced DISC formation. LC3B siRNA also enhanced p65 phosphorylation. In fibroblasts from beclin 1 heterozygous knockout mice, p65 phosphorylation was dramatically upregulated, while CSE-induced DISC formation was inhibited, consistent with an anti-apoptotic role for NFkappaB and a pro-apoptotic role for Beclin 1. These studies demonstrated an interdependence of autophagic and apoptogenic signaling in CSE-induced cell death, and their coordinated downregulation by HO-1. An understanding of the regulation of cell death pathways during smoke exposure may provide therapeutic strategies in smoke-related illness.     

Pro-apoptotic effect and cytotoxicity of genistein and genistin in human ovarian cancer SK-OV-3 cells

We investigated the effects of genistein and genistin on proliferation and apoptosis of human ovarian SK-OV-3 cells and explored the mechanism for these effects. SK-OV-3 cells were treated with genistein and genistin at various concentrations (ranging from 1 to 100 muM) either alone or in combination for 24 and 48 h. Cell proliferation was estimated using an MTT assay, and cell cycle arrest was evaluated using FACS. Caspase-3 activity and annexin-based cell cycle analysis were used as measures of apoptosis. In addition, genistein- and genistin-induced cytotoxicity was determined by measuring release of LDH. Genistein treatment for 24 or 48 h substantially inhibited SK-OV-3 cell proliferation in a dose-dependent manner, and genistin treatment for 48 h also inhibited cell proliferation. Genistein caused cell cycle arrest at G2/M phase in dose- and time-dependent manner, and genistin caused cell cycle arrest not only at G2/M phase but also at G1 phase. Genistein markedly induced apoptosis and significantly increased LDH release, whereas genistin did not affect LDH release. Moreover, exposure to both genistein and genistin in combination for 48 h induced apoptosis without increasing LDH release. Genistein and genistin inhibit cell proliferation by disrupting the cell cycle, which is strongly associated with the arrest induction of either G1 or G2/M phase and may induce apoptosis. Based on our findings, we speculate that both genistein and genistin may prove useful as anticancer drugs and that the combination of genistein and genistin may have further anticancer activity.     

Long non-coding RNA MEG3 regulates CSE-induced apoptosis and inflammation via regulating miR-218 in 16HBE cells

Chronic obstructive pulmonary disease (COPD) is a prevalent disease worldwide, mainly caused by cigarette smoking. Maternally expressed gene 3 (MEG3) functions as the lncRNA and is upregulated in COPD patients and human bronchial epithelial cells after fine particulate matter (PM2.5) treatment. However, the molecular mechanism of MEG3 in COPD remains unknown. The expression of MEG3 and miR-218 in COPD tissues and cigarette smoke extract (CSE)-treated 16HBE cells was detected by RT-qPCR. The effects of MEG3 and miR-218 on proliferation and apoptosis in (CSE)-treated 16HBE cells were analyzed by CCK-8 and flow cytometry assay, respectively. The protein levels of inflammatory cytokines (IL-1β IL-6 and TNF-α) were detected in 16HBE cells by ELISA. MEG3 and miR-218 binding interaction was predicted by LncBase Predicted v.2 and further confirmed by dual luciferase reporter assay and RNA Immunoprecipitation (RIP) assay. MEG3 was upregulated in COPD tissues and inversely related to FEV1%. MEG3 was upregulated in (CSE)-treated 16HBE cells, and knockdown of MEG3 mitigated CSE-repressed proliferation and CSE-triggered apoptosis or inflammation. MiR-218 was demonstrated as a target miRNA of MEG3. MiR-218 was downregulated in COPD tissues and (CSE)-treated or MEG3 overexpressed 16HBE cells. MiR-218 overexpression attenuated CSE-blocked proliferation and CSE-induced apoptosis or inflammation. Deficiency of MEG3 counteracted CSE-blocked proliferation CSE-induced apoptotic rate and inflammatory cytokine (IL-1β IL-6 and TNF-α) levels, while introduction of anti-miR-218 reversed these effects. MEG3 regulated CSE-inhibited proliferation and CSE-induced apoptosis or inflammation by targeting miR-218, providing a possible therapeutic target for treatment of CSE-induced COPD.     

Protein kinase C alpha and zeta differentially regulate death-inducing signaling complex formation in cigarette smoke extract-induced apoptosis

Cigarette smoke, a major risk factor in emphysema, causes cell death by incompletely understood mechanisms. Death-inducing signaling complex (DISC) formation is an initial event in Fas-mediated apoptosis. We demonstrate that cigarette smoke extract (CSE) induces DISC formation in human lung fibroblasts (MRC-5) and promotes DISC trafficking from the Golgi complex to membrane lipid rafts. We demonstrate a novel role of protein kinase C (PKC) in the regulation of DISC formation and trafficking. The PKC isoforms, PKCalpha, zeta, epsilon, and eta, were activated by CSE exposure. Overexpression of wild-type PKCalpha inhibited, while PKCzeta promoted, CSE-induced cell death. Dominant-negative (dn)PKCzeta protected against CSE-induced cell death by suppressing DISC formation and caspase-3 activation, while dnPKCalpha enhanced cell death by promoting these events. DISC formation was augmented by wortmannin, an inhibitor of PI3K. CSE-induced Akt phosphorylation was reduced by dnPKCalpha, but it was increased by dnPKCzeta. Expression of PKCalpha in vivo inhibited DISC formation, caspase-3/8 activation, lung injury, and cell death after prolonged cigarette smoke exposure, whereas expression of PKCzeta promoted caspase-3 activation. In conclusion, CSE-induced DISC formation is differentially regulated by PKCalpha and PKCzeta via the PI3K/Akt pathway. These results suggest that modulation of PKC may have therapeutic potential in the prevention of smoke-related lung injury.     

MAPK pathway mediates EGR-1-HSP70-dependent cigarette smoke-induced chemokine production

Cigarette smoking, a major risk factor for chronic obstructive pulmonary disease, can cause airway inflammation, airway narrowing, and loss of elasticity, leading to chronic airflow limitation. In this report, we sought to define the signaling pathways activated by smoke and to identify molecules responsible for cigarette smoke-induced inflammation. We applied cigarette smoke water extract (CSE) to primary human lung fibroblasts and found that CSE significantly increased CXC chemokine IL-8 production. Meanwhile, 70-kDa heat shock protein (HSP70) was also induced by CSE in a dose- and time-dependent manner. CSE treatment stimulated HSP70 secretion by primary fibroblasts, which augmented IL-8 production. This was further confirmed by exogenously added recombinant HSP70. Using HSP70 small interfering RNA, we confirmed that CSE-induced chemokine production was dependent on heat shock protein expression. Further investigation showed that CSE could also stimulate early growth response-1 (EGR-1) in an ERK-dependent manner and that the expression of HSP70 was EGR-1 dependent. In view of these findings, we hypothesize that the MAPK-EGR-1-HSP70 pathway regulates the cigarette smoke-induced inflammatory process.     

Honokiol ameliorates cigarette smoke-induced damage of airway epithelial cells via the SIRT3/SOD2 signalling pathway

Cigarette smoking can cause damage of airway epithelial cells and contribute to chronic obstructive pulmonary disease (COPD). Honokiol is originally isolated from Magnolia obovata with multiple biological activities. Here, we investigated the protective effects of honokiol on cigarette smoke extract (CSE)-induced injury of BEAS-2B cells. BEAS-2B cells were treated with 300 mg/L CSE to construct an in vitro cell injury model, and cells were further treated with 2, 5 and 10 μM honokiol, then cell viability and LDH leakage were analysed by CCK-8 and LDH assay kits, respectively. Apoptosis was detected by flow cytometry analysis. ELISA was used to measure the levels of tumour necrosis factor (TNF)-ɑ, IL-1β, IL-6, IL-8 and MCP-1. The results showed that honokiol (0.5–20 μM) showed non-toxic effects on BEAS-2B cells. Treatment with honokiol (2, 5 and 10 μM) reduced CSE (300 mg/L)-induced decrease in cell viability and apoptosis in BEAS-2B cells. Honokiol also decreased CSE-induced inflammation through inhibiting expression and secretion of inflammatory cytokines, such as TNF-ɑ, IL-1β, IL-6, IL-8 and MCP-1. Moreover, honokiol repressed CSE-induced reactive oxygen species (ROS) production, decrease of ATP content and mitochondrial biogenesis, as well as mitochondrial membrane potential. Mechanistically, honokiol promoted the expression of SIRT3 and its downstream target genes, which are critical regulators of mitochondrial function and oxidative stress. Silencing of SIRT3 reversed the protective effects of honokiol on CSE-induced damage and mitochondrial dysfunction in BEAS-2B cells. These results indicated that honokiol attenuated CSE-induced damage of airway epithelial cells through regulating SIRT3/SOD2 signalling pathway.     

Effects of Cigarette Smoke Extracts on the Growth and Senescence of Skin Fibroblasts In Vitro

Epidemiological studies have shown that cigarette smoke (CS), a very common environmental factor, plays an important role in skin aging. Although some in vivo studies have suggested that CS affects skin aging, the detailed effects of CS on skin cells in vitro remain largely unknown. In this study, we investigated the effects of cigarette smoke extract (CSE) on the growth, proliferation, and senescene of skin fibroblasts and the possible mechanism underlying these effects. Primary cultured human fibroblasts were exposed to a range of concentrations of CSE. Cell viability and cell proliferation after CSE exposure were analyzed with the methyl thiazolyl tetrazolium (MTT) assay and bromodeoxyuridine incorporation assay, respectively. Growth curves of fibroblasts exposed to different concentrations of CSE were developed and prolonged CSE-exposed cells were observed. Morphological and ultrastructural changes in fibroblasts were assessed by inverted light microscopy and transmission electron microscopy (TEM). Dying cells were stained with senescence-associated β-galactosidase (SA β-gal). Intracellular reactive oxygen species (ROS) levels, superoxide dismutase (SOD) activity, and glutathione peroxidase (GSH-Px) activity were determined by a colorimetric method. We found that proliferative capacity and growth were inhibited by CSE exposure in a dose- and time-dependent manner. Fibroblasts exposed to even low concentrations of CSE for a long period of time (5 passages) showed significantly increased SA β-gal activity and typical features of aging cells. Meanwhile, CSE inhibited superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activities and augmented ROS levels. Our observations suggest that CSE exposure impairs fibroblast growth and proliferation and leads to features similar to those seen in senescent cells. Oxidative stress injury and inhibition of antioxidant defense activity may be involved in CSE-induced fibroblast senescence.     

Morin hydrate attenuates CSE-induced lipid accumulation,ER stress,and oxidative stress in RPE cells: implications for age-related macular degeneration

Oxidative stress has a key role in the pathogenesis of age-related macular degeneration (AMD). Cigarette smoking is known to the one of the main risk factors of AMD through oxidative stress-mediated endoplasmic reticulum (ER) stress and lipid accumulation in human retinal pigment epithelium (RPE) cells. A number of studies have investigated the benefits of antioxidants in the AMD. However, previous studies have not shown that efficacy of antioxidant in the treatment of AMD. Recent studies demonstrated that morin hydrate (MH) has antioxidant properties, anti-inflammatory, and antiapoptosis effects, however, the protective effects of MH against cigarette smoke extract (CSE)-induced AMD have not been studied in detail. We tested the potential effect of MH against the CSE-induced lipid accumulation in RPE cells and mice RPE layer. Herein, we observed that expose of RPE cells to CSE reduced cell viability, increased the lipid accumulation, ER stress, and oxidative stress. Concomitantly, CSE treatment to mice induced AMD associated histopathological changes, lipid accumulation, ER stress and oxidative stress in RPE layer. MH significantly attenuated cytotoxicity, lipid accumulation, ER stress, and oxidative stress via activated AMPK-Nrf2 signaling pathway in RPE cells and mice RPE layer. In addition, AMPK inhibition reversed MH-induced RPE cell protection against CSE. Thus, we conclude that MH protects RPE cells from CSE through reduced oxidative stress, ER stress, and lipid accumulation via activated AMPK-Nrf2-HO-1 signaling pathway. These findings suggest that MH treatment may be exploited in effective strategy against CSE-induced AMD.     

Human alveolar epithelial cell injury induced by cigarette smoke

Background

Cigarette smoke (CS) is a highly complex mixture and many of its components are known carcinogens, mutagens, and other toxic substances. CS induces oxidative stress and cell death, and this cell toxicity plays a key role in the pathogenesis of several pulmonary diseases.

Methodology/Principal Findings

We studied the effect of cigarette smoke extract (CSE) in human alveolar epithelial type I-like (ATI-like) cells. These are isolated type II cells that are differentiating toward the type I cell phenotype in vitro and have lost many type II cell markers and express type I cell markers. ATI-like cells were more sensitive to CSE than alveolar type II cells, which maintained their differentiated phenotype in vitro. We observed disruption of mitochondrial membrane potential, apoptosis and necrosis that were detected by double staining with acridine orange and ethidium bromide or Hoechst 33342 and propidium iodide and TUNEL assay after treatment with CSE. We also detected caspase 3 and caspase 7 activities and lipid peroxidation. CSE induced nuclear translocation of Nrf2 and increased expression of Nrf2, HO-1, Hsp70 and Fra1. Moreover, we found that Nrf2 knockdown sensitized ATI-like cells to CSE and Nrf2 overexpression provided protection against CSE-induced cell death. We also observed that two antioxidant compounds N-acetylcysteine and trolox protected ATI-like cells against injury by CSE.

Conclusions

Our study indicates that Nrf2 activation is a major factor in cellular defense of the human alveolar epithelium against CSE-induced toxicity and oxidative stress. Therefore, antioxidant agents that modulate Nrf2 would be expected to restore antioxidant and detoxifying enzymes and to prevent CS-related lung injury and perhaps lessen the development of emphysema.     

Cigarette smoke extract inhibits angiogenesis of pulmonary artery endothelial cells: the role of calpain

Angiogenesis is an integral part of both the pulmonary inflammatory response to chronic exposure to cigarette smoke and the lung tissue remodeling associated with cigarette smoke-induced chronic obstructive pulmonary disease (COPD). To investigate the role of angiogenesis in the pathogenesis of COPD, we evaluated the effect of cigarette smoke extract (CSE) on angiogenesis of pulmonary artery endothelial cells (PAEC). Incubation of PAEC with 2.5-10% CSE resulted in a dose-dependent inhibition of endothelial monolayer wound repair. CSE also caused inhibition of tube formation on Matrigel, migration in a Boyden chamber, and proliferation of PAEC. Because calpain, a family of calcium-dependent intracellular proteases, mediates cytoskeletal signaling in endothelial motility, we explored the role of calpain in the CSE-induced inhibition of endothelial angiogenesis. Incubation of CSE resulted in a dose-dependent decrease in calpain activity. Calpain inhibitor-1, a specific inhibitor of calpain, potentiates inhibitory effect of CSE on the endothelial monolayer wound repair, tube formation, cell migration, and cell proliferation. Transfection of PAEC with antisense oligodeoxyribonucleotides of calpastatin, the major endogenous calpain inhibitor, prevented CSE-induced increase in calpastatin protein content and CSE-induced decreases in calpain activity. It also prevented CSE-induced decreases in monolayer wound repair, tube formation, and migration. These results suggest that CSE attenuates angiogenesis of PAEC and the mechanism involves inhibition of calpain. Impaired angiogenesis may impede the repair process in the lungs of cigarette smokers and contribute to the altered structural remodeling observed in the lungs of patients with cigarette smoke-related COPD.