To investigate the role of signaling pathway in the effect of endoplasmic reticulum stress(ER stress) in endothelial cells stimulated with cigarette smoke extract(CSE). Human umbilical vein endothelial cells(HUVECs) w...To investigate the role of signaling pathway in the effect of endoplasmic reticulum stress(ER stress) in endothelial cells stimulated with cigarette smoke extract(CSE). Human umbilical vein endothelial cells(HUVECs) were cultured and divided into 3 groups: CSE-stimulated group, CSE-stimulated with 4-PBA group, and negative control group. HUVECs were cultured and stimulated with CSE at concentrations of 5%, 10% and 20%, respectively, mR NA of CXCL-8 and GRP78 was detected by real-time PCR. ELISA was performed to test the expression of CXCL-8 protein, and neutrophils migration was detected by Transwell board test. The NF-κB, ERK, p38 MAPK and transforming growth factor beta(TGF-β) were detected by flow cytometry. The mRNA of CXCL-8 and GRP78 increased in CSE-stimulated HUVECs(P<0.05). Furthermore, it was concentration-dependent. 4-PBA significantly reduced the expression of CXCL-8 protein(P<0.05) and neutrophil migration(P<0.05). The TGF-β, rather than the NF-κB, ERK and P38 MAPK pathway might be involved in ER stress stimulated by CSE. CSE induced neutrophils migration by increasing the expression of CXCL-8 in endothelial cells. ER stress might play a role in the effect of neutrophils migration stimulated with CSE, and TGF-β pathway may contribute to the ER stress in HUVECs.展开更多
Background Chronic obstructive pulmonary disease(COPD)is a severe public health problem.Cigarette smoke(CS)is a risk factor for COPD and lung cancer.The underlying molecular mechanisms of CS-induced malignant transfor...Background Chronic obstructive pulmonary disease(COPD)is a severe public health problem.Cigarette smoke(CS)is a risk factor for COPD and lung cancer.The underlying molecular mechanisms of CS-induced malignant transformation of bronchial epithelial cells remain unclear.In this study,we describe a lung-on-a-chip to explore the possible mechanistic link between cigarette smoke extract(CSE)-associated COPD and lung cancer.Methods An in vitro lung-on-a-chip model was used to simulate pulmonary epithelial cells and vascular endothelial cells with CSE.The levels of IL-6 and TNF-αwere tested as indicators of inflammation using an enzyme-linked immune sorbent assay.Apical junction complex mRNA expression was detected with qRT-PCR as the index of epithelial-to-mesenchymal transition(EMT).The effects of CSE on the phosphorylation of signal transduction and transcriptional activator 3(STAT3)were detected by Western blotting.Flow cytometry was performed to investigate the effects of this proto-oncogene on cell cycle distribution.Results Inflammation caused by CSE was achieved in a lung-on-a-chip model with a mimetic movement.CSE exposure induced the degradation of intercellular connections and triggered the EMT process.CSE exposure also activated the phosphorylation of proto-oncogene STAT3,while these effects were inhibited with HJC0152.Conclusions CSE exposure in the lung-on-a-chip model caused activation of STAT3 in epithelial cells and endothelial cells.HJC0152,an inhibitor of activated STAT3,could be a potential treatment for CS-associated COPD and lung cancer.展开更多
Cigarette smoking contributes to the development of pulmonary artery hypertension(PAH).As the basic pathological change of PAH,pulmonary vascular remodeling is considered to be related to the abnormal proliferation of...Cigarette smoking contributes to the development of pulmonary artery hypertension(PAH).As the basic pathological change of PAH,pulmonary vascular remodeling is considered to be related to the abnormal proliferation of pulmonary artery smooth muscle cells(PASMCs).However,the molecular mechanism underlying this process remains not exactly clear.The aim of this research was to study the molecular mechanism of PASMCs proliferation induced by smoking.Human PASMCs(HPASMCs)were divided into 6 groups:0%(control group),cigarette smoking extract(CSE)-treated groups at concentrations of 0.5%,1%,2%,5%,10%CSE respectively.HPASMCs proliferation was observed after 24 h.HPASMCs were divided into two groups:0(control group),0.5%CSE group.The mRNA and protein expression levels of transient receptor potential channel 1(TRPC1)and cyclin D1 in HPASMCs after CSE treatment were respectively detected by RT-PCR and Western blotting.The intracellular calcium ion concentration was measured by the calcium probe in each group.In the negative control group and TRPC1-siRNA transfection group,the proliferation of HPASMCs and the expression of cyclin D1 mRNA and protein were detected.Data were compared with one-way ANOVA(for multiple-group comparison)and independent t-test(for two-group comparison)followed by the least significant difference(LSD)test with the computer software SPSS 17.0.It was found that 0.5%and 1%CSE could promote the proliferation of HPASMCs(P<0.05),and the former was more effective than the latter(P<0.05),while 3%and above CSE had inhibitory effect on HPASMCs(P<0.05).The mRNA and protein expression levels of TRPC1 and cyclin D1 in 0.5%and 1%CSE groups were significantly higher than those in the control group(P<0.05),while those in 3%CSE group were significantly decreased(P<0.05).Moreover,the proliferation of HPASMCs and the expression of cyclin D1 mRNA and protein in TRPC1-siRNA transfection group were significantly reduced as compared with those in the negative control group(P<0.05).It was concluded that low concentration of CSE can promote the proliferation of HPASMCs,while high concentrations of CSE inhibit HPASMCs proliferation.These findings suggested that CSE induced proliferation of HPASMCs at least in part via TRPC1-mediated cyclin D1 expression.展开更多
Cigarette consumption increases oxidative stress in many organs. Increased oxidative stress harms bone cells, which negatively affects bone-matter and -stability. This leads to an increased fracture risk and delayed f...Cigarette consumption increases oxidative stress in many organs. Increased oxidative stress harms bone cells, which negatively affects bone-matter and -stability. This leads to an increased fracture risk and delayed fracture healing in smokers. A supporting therapy with antioxidants could be of great benefit for surgeons dealing with delayed fracture healing due to increased oxidative stress. In this article we complement and compare our published data with hitherto unpublished data and show the protective effect of 15 different antioxidants on cigarette smoke induced damage in primary human osteoblasts. Exposure to cigarette smoke medium (CSM) rapidly induces formation of ROS in osteoblasts in a concentration- and time-dependent manner. Massive cell damage is seen already after 4 h (EC50 ≈ 0.75 OD320). Pre-, co- and post-incubation with the different antioxidants reduces the formation of ROS and consequently improves the viability of the CSM exposed osteoblasts. Small compounds, e.g. N-acetylcysteine, proved highly effective if pre- or co-incubated before exposure to the CSM. Thus, they are good candidates for acute therapy support as they can be administered in high doses. However, our data suggest that a balanced daily diet could lead to an accumulation of various natural antioxidants (flavonoids) that effectively protect osteoblasts from oxidative stress-induced damage in all three settings investigated. Together with their partly phytoestrogenic properties this may even abate alterations in bone and thus reduce fracture risk on the long run.展开更多
Background and Objective: Chronic heavy alcohol consumption and daily cigarette smoking are the most prevalent substance use problems in the U.S., including Veterans. Excessive alcohol use causes neurocognitive and be...Background and Objective: Chronic heavy alcohol consumption and daily cigarette smoking are the most prevalent substance use problems in the U.S., including Veterans. Excessive alcohol use causes neurocognitive and behavioral deficits that can be linked to neurodegeneration. Similarly, preclinical and clinical data suggest that smoking also leads to brain atrophy. This study examines the differential and additive effects of alcohol and cigarette smoke (CS) exposures on cognitive-behavioral function. Methods: A 4-way experimental model of chronic alcohol and CS exposures was generated using 4-week-old male and female Long Evans rats that were pair-fed with Lieber-deCarli isocaloric liquid diets containing 0% or 24% ethanol for 9 weeks. Half of the rats in the control and ethanol groups were exposed to CS for 4 hours/day and 4 days/week for 9 weeks. All rats were subjected to Morris Water Maze, Open Field, and Novel Object Recognition testing in the last experimental week. Results: Chronic alcohol exposure impaired spatial learning as shown by significantly increased latency to locate the platform, and it caused anxiety-like behavior marked by the significantly reduced percentage of entries to the center of the arena. Chronic CS exposure impaired recognition memory as suggested by significantly less time spent at the novel object. Combined exposures to alcohol and CS did not show any significant additive or interactive effect on cognitive-behavioral function. Conclusion: Chronic alcohol exposure was the main driver of spatial learning, while the effect of secondhand CS exposure was not robust. Future studies need to mimic direct CS exposure effects in humans.展开更多
Objective:To study the effect of cigarette extract(CSE)on the expression of exosomal miR-186 derived from 16HBE cells and the effects of 16HBE-derived exosomal miR-186 on the proliferation of MRC-5 cells.Methods:To co...Objective:To study the effect of cigarette extract(CSE)on the expression of exosomal miR-186 derived from 16HBE cells and the effects of 16HBE-derived exosomal miR-186 on the proliferation of MRC-5 cells.Methods:To collect the exosomal miR-186 in the supernatant of CSE-treated 16HBE cells for MRC-5 cell culture;the expression of exosomal miR-186 was detected by qPCR;the proliferation of MRC-5 cell was detected by CCK-8;dual-luciferase reporter gene experiment was used to detect the targeted regulation relationship between miR-186 and Bcl2L11;the expression of Bcl2L11 was detected by Western blot.Results:The morphology of 16HBE cells about 100 nm in diameter were observed by TEM;CSE treatment significantly promoted the expression of exosomal miR-186;CSE-induced exosomal miR-186 promoted the proliferation of MRC-5 cells;Bcl2L11 is a target gene of miR-186;miR-186 mimics significantly decreased Bcl2L11 expression.Conclusion:CSE-induced 16HBE-derived exosomal miR-186 promoted the proliferation of MRC-5 cells by targeting Bcl2L11 genes.展开更多
Although several studies confirmed that berberine may attenuate airway inflammation in mice with chronic obstructive pulmonary disease(COPD),its underlying mechanisms were not clear until now.We aimed to establish an ...Although several studies confirmed that berberine may attenuate airway inflammation in mice with chronic obstructive pulmonary disease(COPD),its underlying mechanisms were not clear until now.We aimed to establish an experiment mouse model for COPD and to investigate the effects of berberine on airway inflammation and its possible mechanism in COPD model mice induced by cigarette smoke extract(CSE).Twenty SPF C57BL/6 mice were randomly divided into PBS control group,COPD model group,low-dose berberine group and high-dose berberine group,5 mice in each group.The neutrophils and macrophages were examined by Wright's staining.The levels of inflammatory cytokines TNF-α and IL-6 in bronchoalveolar lavage fluid(BALF)were detennined by enzyme-linked immunosorbent assay.The expression levels of TGF-β1,Smad2 and Smad3 mRNA and proteins in lung tissues were respectively detected by quantitative real-time polymerase chain reaction and Western blotting.It was found that CSE increased the number of inflammation cells in BALF,elevated lung inflammation scores,and enhanced the TGF-β1/Smads signaling activity in mice.High-dose berberine restrained the alterations in the COPD mice induced by CSE.It was concluded that high-dose berberine ameliorated CSE-induced airway inflammation in COPD mice.TGF-β1/Smads signaling pathway might be involved in the mechanism.These findings suggested a therapeutic potential of high-dose berberine on the CSE-induced airway inflammation.展开更多
AIM:To test whether the expression and activity of H,K-ATPase in parietal cells would be affected by cigarette smoke extract.METHODS: Extracts of cigarette smoke were administered into mice by gastric gavage (5 mg/kg ...AIM:To test whether the expression and activity of H,K-ATPase in parietal cells would be affected by cigarette smoke extract.METHODS: Extracts of cigarette smoke were administered into mice by gastric gavage (5 mg/kg body weight/day) for 3 d or in drinking water for 7 or 14 d. For the latter, each day a mouse consumed 5 mL water containing extracts of two cigarettes, on average. Control littermate mice received only vehicle. To compare the amount of H,K-ATPase in control and smoke-treated mice, the stomach was processed for Western blotting and immunohistochemical analysis using monoclonal antibodies specific for α- or β-subunits of H,K-ATPase. The p-nitrophenylphospatase activity assay was used as a measurement for K-dependent H,K-ATPase activity.RESULTS: Probed transblots showed an increase in the amount of H,K-ATPase in smoke-treated mice which was confirmed by immunohistochemistry and was found to be due to increased amounts of protein per parietal cell rather than an increased parietal cell number. The increase in the amount of H,K-ATPase was associated with an enhancement of its enzymatic activity. K-dependent activity in control and smoke-treated mice was significantly different (respectively, 0.12 μmol/mg vs 0.27 μmol/mg per minute, P<0.05).CONCLUSION: Administration of cigarette smoke extract is associated with an increase in the amount and activity of H,K-ATPase and hence, smokers are susceptible to development of peptic ulcer.展开更多
In this study, the effect of prophylactic anti inflammation on the development of smoke induced emphysema was investigated. Young male guinea pigs aged 1.5 - 2 months (weighing 198.3±26.9 g) were randomly divided...In this study, the effect of prophylactic anti inflammation on the development of smoke induced emphysema was investigated. Young male guinea pigs aged 1.5 - 2 months (weighing 198.3±26.9 g) were randomly divided into 4 groups: group A (cigarette smoke exposure only), group B (cigarette smoke exposure plus pentoxifylline rich (PTX, 10 mg/d) forage feeding), group C (cigarette smoke exposure plus intermittent cortical steroid injection (Triamcinolone acetonide, 3 mg, im, every three weeks) and control group (group D: animals with sham smoke exposure, raised under the same conditions). Animals in group A, B and C were exposed to smoke of cigarettes for 1 to 1.5 h twice a day, 5 days a week. All animals were killed at the 16th week and followed by morphometrical analysis of the midsagittal sectioned lung slices. Smoke exposure of 16 weeks resulted in visible emphysematous development in Group A but not in Group B and C. It was evidenced by the indicator of air space size, mean linear intercept (L m): 120.6±16.0 μm in Group A; 89.8±9.2 μm in Group B and 102.4±17.7 μm in Group C. The average L m in either group B or group C was shorter than that in Group A (ANOVA and Newman Keuls test, F=8.80, P =0.0002) but comparable to that (94.8±13.2 μm) in group D ( P >0.05). It is concluded that long term prophylactic anti inflammation inhibits pulmonary emphysema induced by cigarette smoking in the guinea pigs.展开更多
To understand the retention mechanism of phenol in mainstream cigarette smoke by cellulose acetate(CA)filters,the phenol filtration efficiency,the phenol distribution in the gas phase and particulate phase during filt...To understand the retention mechanism of phenol in mainstream cigarette smoke by cellulose acetate(CA)filters,the phenol filtration efficiency,the phenol distribution in the gas phase and particulate phase during filtration and the retention characteristics of the filter for both the gas phase and particulate phenol species were investigated.The distribution of particulate bound phenol as a function of the particle size was examined.The ratio of phenol transferring from the particulate phase to the gas phase during smoke flowing through the CA filter were studied.Retention mechanisms for phenol in the gas phase as well as particulate phase phenol in the filter and the phase transition of phenol were proposed.Phenol filtration efficiency of the cigarette filter was calculated based on the proposed retention mechanisms and compared with available experimental data.The results showed that:1)Comparing with nicotine and the other particulate bound smoke constituents,CA filter exhibited a certain retention selectivity for phenol.2)Although phenol was mainly bound to the particulate phase of the cigarette mainstream smoke,about 35% of gas phase phenol was found at the tobacco end of the filter,whilst this dropped to approximately 0 at the mouth end of the filter,mostly by selective chemical adsorption.3)The weighted average smoke particle size and particulate phenol was 0.44 and 0.38 pm,respectively.The filtration efficiency of the filter to particulate phenol was near 40%.4)About 19% of particulate bound phenol transferred to gas phase when smoke passing the filter,and was selectively adsorbed.5)The overall filtration efficiency of the CA filter for phenol in mainstream cigarette smoke was 68.7%,with-7.7% variation compared with experimental data.The results demonstrated the phase transition of phenol during the filtration process and the selective retention with varying particle sizes.Our results showed that the application of CA filter in product designs reduces harmful constituents such as phenolic compounds in mainstream cigarette smoke.展开更多
Constant-energy synchronous fluorimetry was used for the identification of benzo[a]pyrene in mixtures with a detection limit of 1.34 nmol/L. The recovery experiments in cigarette smoke samples have also obtained satis...Constant-energy synchronous fluorimetry was used for the identification of benzo[a]pyrene in mixtures with a detection limit of 1.34 nmol/L. The recovery experiments in cigarette smoke samples have also obtained satisfactory results of 99.1-103.5% for benzo[a]pyrene.展开更多
Objective Embryonic movements (EM) and angiogenesis pathways are evolutionarily conserved mechanisms which are essential for proper embryonic development. Deviations in these processes by exposure to cigarette smoke c...Objective Embryonic movements (EM) and angiogenesis pathways are evolutionarily conserved mechanisms which are essential for proper embryonic development. Deviations in these processes by exposure to cigarette smoke condensate (CSC) may cause vascular and morphogenetic disorders. Methods Using chicken and mouse embryos, we have demonstrated the in vivo effects of CSC on EM, vascular development, and organogenesis. Results Examination of the CSC exposed chicken embryos revealed a significant reduction in EM, stunted growth, deviated pattern of blood vessels, hemorrhages, and localized necrosis. Likewise, mouse embryos that were exposed to CSC at E8.5 and E9.5 died between E11.5 and E12.5, respectively. These mouse embryos showed defects in morphogenesis and remodeling of the embryonic vasculature, while littermate controls showed normal development. Conclusion Cigarette smoking during pregnancy is fatal for growing embryos. CSC may induce the remodeling of embryonic vasculature, leading to various pathologies.展开更多
Summary: To investigate whether the change of E-cadherin (ECD) expression plays a role in the injury and repair of airway epithelial cells (AEC) caused by smoking, porcine AECs were cultured by using an enzyme-dispers...Summary: To investigate whether the change of E-cadherin (ECD) expression plays a role in the injury and repair of airway epithelial cells (AEC) caused by smoking, porcine AECs were cultured by using an enzyme-dispersed method. After exposure of the AECs to cigarette smoke extract (CSE), the ECD expression in the cells was detected by using immunocytochemistry and in situ hybridization. The results showed that ECD was distributed on the plasma membrane at the cell junctions of AECs. After exposure to 20 % CSE, the membranous ECD expression was decreased, the cytoplasmic ECD expression was increased (P<0.01) as the exposure time went on. But the content of ECD mRNA in the AECs did not chang. It suggests that the change of ECD ex- pression is regulated at the posttranslational level and plays a role in the injury and repair of AEC caused by smoking.展开更多
An improved method was developed for the determination of the four major tobacco-specific nitrosamines(TSNAs) in mainstream cigarette smoke. The new method offers decreased sample preparation and analysis time as comp...An improved method was developed for the determination of the four major tobacco-specific nitrosamines(TSNAs) in mainstream cigarette smoke. The new method offers decreased sample preparation and analysis time as compared to traditional methods. This method uses isotope dilution liquid chromatography coupled with a tandem mass spectrometer with electrospray ionization and is significantly more sensitive than traditional methods. It also shows no evidence of artifactual formation of TSNAs. Sample concentrations were determined for four TSNAs in mainstream smoke using two isotopically labeled TSNAs analogues as internal standards. Mainstream smoke was collected on an industry standard 44-mm Cambridge filter pad, extracted with 0.1 mol/L ammonium acetate, purified by solid-phase extraction, and analyzed without further sample cleanup. The analytical column is a 3.9 mm×150 mm Waters Symmertry Shield RP18 column and volume fraction of the mobile phase is 50% methanol, 50% water containing 0.1% acetic acid. The results show that the linear range is 0.5-100.0 mg/L except for N-nitrosoanabasine (NAB) from 0.25 to 50.0 mg/L. The limits of detection are 0.1 mg/L for N-nitrosonornicotine (NNN), 0.08 mg/L for 4-(methylnitrosamino)-1- (3-py-ridyl)-1-butanone (NNK), 0.05 mg/L for N-nitrosoanatabine (NAT) and 0.06 mg/L for NAB. The recoveries of the four TSNAs are from 90.2% to 105.7%.展开更多
In order to investigate the effects of puerarin on pulmonary vascular remodeling and protein kinase C-α (PKC-α) in chronic exposure smoke rats, 54 male Wistar rats were randomly di-vided into 7 groups: control group...In order to investigate the effects of puerarin on pulmonary vascular remodeling and protein kinase C-α (PKC-α) in chronic exposure smoke rats, 54 male Wistar rats were randomly di-vided into 7 groups: control group (C group), smoke exposure groups (S4w group, S8w group), puer-arin groups (P4w group, P8w group), propylene glycol control groups (PC4w group, PC8w group). Rats were exposed to cigarette smoke or air for 4 to 8 weeks. Rats in puerarin groups also received puer-arin. To evaluate vascular remodeling, alpha-smooth muscle actin (α-SM-actin) staining was used to count the percentage of completely muscularised vessels to intraacinar pulmonary arteries (CMA/IAPA) which was determined by morphometric analysis of histological sections. Pulmonary artery smooth muscle cell (PASMC) apoptosis was detected by in situ end labeling technique (TUNEL), and proliferation by proliferating cell nuclear antigen (PCNA) staining. Reverse transcrip-tion-polymerase chain reaction (RT-PCR), immunofluorescence staining and Western blot analysis were done to detect the PKC-α mRNA and protein expression in pulmonary arteries. The results showed that in cigarette smoke-exposed rats the percentage of CMA/IAPA and α-SM-actin expres-sion were increased greatly, PASMC apoptosis was increased and proliferation was markedly in-creased; Apoptosis indices (AI) and proliferation indices (PI) were higher than in C group; AI and PI were correlated with vascular remodeling indices; The expression of PKC-α mRNA and protein in pulmonary arteries was significantly higher than in C group. In rats treated with puerarin, the per-centage of CMA/IAPA and cell proliferation was reduced, whereas PASMC apoptosis was increased; The expression levels of PKC-α mRNA and protein were lower than in smoke exposure rats. There was no difference among all these data between S groups and PC groups. These findings suggested that cigarette smoke-induced pulmonary vascular remodeling was most likely an effect of the imbal-ance of PASMC proliferation and apoptosis. Puerarin appears to be able to reduce cell proliferation and vascular remodeling possibly through PKC signaling transduction pathway.展开更多
To investigate the effect of cigarette smoke extract （CSE） on the role of protein kinase C （PKC） in the proliferation of passively sensitized human airway smooth muscle cells （HASMCs）. After synchronization of c...To investigate the effect of cigarette smoke extract （CSE） on the role of protein kinase C （PKC） in the proliferation of passively sensitized human airway smooth muscle cells （HASMCs）. After synchronization of cultured HASMCs, they were divided into a group A and Group B. The group A was treated with normal human serum and served as controls and the group B was treated with the serum of asthma patients. The group A was further divided into group of A1, A2 and A3 and the group B was sub-divided into the group of B1, B2, B3, B4 and B5. No other agents were added to the group A1 and B1. The cells of group A2 and B2 were stimulated with 5 % CSE for 24 h. HASMCs from group A3 and B3 were treated with PKC agonist PMA （10 nmol/L） and CSE （5 %） for 24 h. PKC inhibitor Ro-31-8220 （5 μmol/L） was added to the HASMCs of group B4 for 24 h. The cells from group B5 were stimulated with Ro-31-8220 （5 μmol/L） and CSE （5 %） for 24 h. The proliferation of HASMCs isolated from group A and B was examined by cell cycle analysis, MTT colorimetric assay and 3H-TdR incorporation test. The expression of PKC-α in each group was observed by Western blotting and RT-PCR, respectively. The results showed that the percentage of S phase, absorbance （A） value, the rate of 3H-TdR incorporation, the ratios of A value of PKC-α mRNA and the A value of PKC-α protein in HASMCs from group B1, B2 and B3 were significantly increased compared to those of group A1, A2 and A3 correspondingly and respectively （P<0.01）. The proliferation of HASMCs of group A2 and B2 stimulated with CSE and group A3 and B3 stimulated with CSE and PMA were also significantly enhanced when group A1, A2 and A3 and group B1, B2 and B3 compared to each other （P<0.05, P<0.01, respectively）. The percentage of S phase, absorbency （A） value, 3H-TdR incorporation rate, the ratios of A value of PKC-α mRNA and the A value of PKC-α protein in HASMCs from group B4 treated with Ro-31-8220 and group B5 treated with CSE and Ro-31-8220 were significantly decreased as compared to those of group B1 and B2 correspondingly and respectively （P<0.05, P<0.01）. It was concluded that CSE can enhance the passively sensitized HASMC proliferation and the expression of PKC alpha. PKC and its alpha subtype may contribute to this process. Our results suggest cigarette may play an important role in ASMCs proliferation of asthma through PKC signal pathway.展开更多
To investigate the toxicity of cigarette smoke extract (CSE) and nicotine on mouse brain mitochondria as well as the protective effect of vitamin C in vitro. Method Mouse brain mitochondria in vitro was incubated with...To investigate the toxicity of cigarette smoke extract (CSE) and nicotine on mouse brain mitochondria as well as the protective effect of vitamin C in vitro. Method Mouse brain mitochondria in vitro was incubated with CSE or nicotine in the absence or presence of vitamin C for 60 minutes, and the changes of mitochondrial function and structure were measured. Results CSE inhibited mitochondrial ATPase and cytochrome C oxidase activities in a dose-dependent manner.However, no significant changes in the peroxidation indices were observed when mitochondrial respiratory enzymes activity was inhibited, and protection of mitochondria from CSE-induced injury by vitamin C was not displayed in vitro. The effect of CSE on mouse brain mitochondria swelling response to calcium stimulation was dependent on calcium concentrations. CSE inhibited swelling of mitochondria at 6.5 μmol/L Ca2+, but promoted swelling response at 250 μmol/L Ca2+. Nicotine, the major component of cigarette smoke, showed no significant damage in mouse brain mitochondria in vitro. The CSE treatment induced mitochondrial inner membrane damage and vacuolization of the matrix, whereas the outer mitochondrial membrane appeared to be preserved. Conclusion The toxic effect of CSE on brain mitochondria may be due to its direct action on enzymatic activity rather than through oxygen free radical injury. Nicotine is not the responsible component for the toxicity of CSE to brain mitochondria.展开更多
Cigarette smoking is intimately related with the development of chronic obstructive pulmonary diseases, and alveolar epithelium is a major target for the exposure of cigarette smoke ex-tract. In order to investigate t...Cigarette smoking is intimately related with the development of chronic obstructive pulmonary diseases, and alveolar epithelium is a major target for the exposure of cigarette smoke ex-tract. In order to investigate the effect of cigarette smoke extract on the proliferation of alveolar epithelial cell type Ⅱand its relationship with P21WAF1, the alveolar epithelial type Ⅱ cell line (A549) cells were chosen as surrogate cells to represent alveolar epithelial type Ⅱ cells. MTT assay was used to detect cell viability after interfered with different concentrations of cigarette smoke ex-tract. It was observed cigarette smoke extract inhibited the growth of A549 cells in a dose- and time-dependent manner. The morphological changes, involving the condensation and margination of nuclear chromatin, even karyorrhexis, were observed by both Hoechst staining and electronic mi-croscopy. Flow cytometry analysis demonstrated the increased cell percentages in G1 and subG1 phases after the cells were incubated with cigarette smoke extract. The expression of p21WAF1 protein and mRNA was also significantly increased as detected by the methods of Western blot or reverse transcription-polymerase chain reaction respectively. In conclusion, cigarette smoke extract inhibits the proliferation of alveolar epithelial cell type Ⅱ and blocks them in G1/S phase. The intracellular accumulation of P21WAF1 may be one of the mechanisms which contribute to cigarette smoke ex-tract-induced inhibition of cell proliferation.展开更多
Cigarette smoke is associated with the development of several diseases,such as chronic obstructive pulmonary disease(COPD).The purpose of this study was to investigate genotoxicity and heat shock protein 70(Hsp70) in ...Cigarette smoke is associated with the development of several diseases,such as chronic obstructive pulmonary disease(COPD).The purpose of this study was to investigate genotoxicity and heat shock protein 70(Hsp70) in human airway smooth muscle cells(HASMCs) exposed to cigarette smoke extract(CSE).HASMCs was exposed to CSE with different doses for 24 h.The level of8-hydroxydeoxyguanosine(8-OHdG) was determined by using HPLC-ECD,the DNA damage was analyzed by using comet assay,and apoptosis was examined by using Annexin-FITC/PI staining.The production of Hsp70 after CSE stimulation was tested.Results indicated that CSE significantly increased the level of 8-OHdG,DNA damage and cell apoptosis,and reduced the production of Hsp70.In particular,levels of Hsp70 were inversely correlated with 8-OHdG,DNA damage and cell apoptosis.It was concluded that cigarette smoke induced genotoxicity and decreased the production of cell protective protein Hsp70,which may contribute to the development of some airway diseases.展开更多
Objective To investigate the cellular effects of cigarette smoke extract (CSE) on primarily cultured human umbilical vein endothelial cells (HUVEC). Methods The effects of CSE (5%-20%) and nicotine (10-4 mol/L) on HUV...Objective To investigate the cellular effects of cigarette smoke extract (CSE) on primarily cultured human umbilical vein endothelial cells (HUVEC). Methods The effects of CSE (5%-20%) and nicotine (10-4 mol/L) on HUVEC viability, proliferation, angiogenesis and apoptosis were observed. Results CSE decreased HUVEC survival rate and angiogenesis after 24 h as well as its proliferation after 48 h in a dose-dependent manner. Moreover, CSE induced apoptosis of HUVEC as indicated in condensation of nuclear chromatin and the presence of hypodiploid DNA. HUVEC incubated with CSE for 24 h gave a significant decrease in the expression of Bcl-2 as well as the decline in the Bcl-2/Bax ratio accompanied with the loss of mitochondrial membrane potential and excess cytosolic calcium. Our study also observed that p53 protein level decreased, rather than increased in cells treated with CSE. Nicotine had no discernible inhibitory effects on the above indices of HUVEC. Conclusion Exposure to CSE other than nicotine causes inhibition of viability, proliferation and differentiation of HUVEC. CSE-induced HUVEC injury is mediated in part through accelerated apoptosis but independent of p53 pathway. It appears that mitochondria have played a key role in the apoptosis of HUVEC induced by CSE.展开更多
基金supported by the Natural Science Foundation of Hubei Province,China(No.2017CFB765)
文摘To investigate the role of signaling pathway in the effect of endoplasmic reticulum stress(ER stress) in endothelial cells stimulated with cigarette smoke extract(CSE). Human umbilical vein endothelial cells(HUVECs) were cultured and divided into 3 groups: CSE-stimulated group, CSE-stimulated with 4-PBA group, and negative control group. HUVECs were cultured and stimulated with CSE at concentrations of 5%, 10% and 20%, respectively, mR NA of CXCL-8 and GRP78 was detected by real-time PCR. ELISA was performed to test the expression of CXCL-8 protein, and neutrophils migration was detected by Transwell board test. The NF-κB, ERK, p38 MAPK and transforming growth factor beta(TGF-β) were detected by flow cytometry. The mRNA of CXCL-8 and GRP78 increased in CSE-stimulated HUVECs(P<0.05). Furthermore, it was concentration-dependent. 4-PBA significantly reduced the expression of CXCL-8 protein(P<0.05) and neutrophil migration(P<0.05). The TGF-β, rather than the NF-κB, ERK and P38 MAPK pathway might be involved in ER stress stimulated by CSE. CSE induced neutrophils migration by increasing the expression of CXCL-8 in endothelial cells. ER stress might play a role in the effect of neutrophils migration stimulated with CSE, and TGF-β pathway may contribute to the ER stress in HUVECs.
基金This work was supported by National Natural Science Foundation of China(Grant Nos.81672297,Grant Nos.61701493)Policy Guidance project(International Science and Technology Cooperation)of Jiangsu Province of China(BZ2018040)+3 种基金the Natural Science Foundation of Tianjin,P.R.China(18JCYBJC42100)Hundred Talents Program of the Chinese Academy of SciencesProject Funded by China Postdoctoral Science Foundation(2019M651959)Postdoctoral Research Funding Program of Jiangsu Province(2018K004B).
文摘Background Chronic obstructive pulmonary disease(COPD)is a severe public health problem.Cigarette smoke(CS)is a risk factor for COPD and lung cancer.The underlying molecular mechanisms of CS-induced malignant transformation of bronchial epithelial cells remain unclear.In this study,we describe a lung-on-a-chip to explore the possible mechanistic link between cigarette smoke extract(CSE)-associated COPD and lung cancer.Methods An in vitro lung-on-a-chip model was used to simulate pulmonary epithelial cells and vascular endothelial cells with CSE.The levels of IL-6 and TNF-αwere tested as indicators of inflammation using an enzyme-linked immune sorbent assay.Apical junction complex mRNA expression was detected with qRT-PCR as the index of epithelial-to-mesenchymal transition(EMT).The effects of CSE on the phosphorylation of signal transduction and transcriptional activator 3(STAT3)were detected by Western blotting.Flow cytometry was performed to investigate the effects of this proto-oncogene on cell cycle distribution.Results Inflammation caused by CSE was achieved in a lung-on-a-chip model with a mimetic movement.CSE exposure induced the degradation of intercellular connections and triggered the EMT process.CSE exposure also activated the phosphorylation of proto-oncogene STAT3,while these effects were inhibited with HJC0152.Conclusions CSE exposure in the lung-on-a-chip model caused activation of STAT3 in epithelial cells and endothelial cells.HJC0152,an inhibitor of activated STAT3,could be a potential treatment for CS-associated COPD and lung cancer.
文摘Cigarette smoking contributes to the development of pulmonary artery hypertension(PAH).As the basic pathological change of PAH,pulmonary vascular remodeling is considered to be related to the abnormal proliferation of pulmonary artery smooth muscle cells(PASMCs).However,the molecular mechanism underlying this process remains not exactly clear.The aim of this research was to study the molecular mechanism of PASMCs proliferation induced by smoking.Human PASMCs(HPASMCs)were divided into 6 groups:0%(control group),cigarette smoking extract(CSE)-treated groups at concentrations of 0.5%,1%,2%,5%,10%CSE respectively.HPASMCs proliferation was observed after 24 h.HPASMCs were divided into two groups:0(control group),0.5%CSE group.The mRNA and protein expression levels of transient receptor potential channel 1(TRPC1)and cyclin D1 in HPASMCs after CSE treatment were respectively detected by RT-PCR and Western blotting.The intracellular calcium ion concentration was measured by the calcium probe in each group.In the negative control group and TRPC1-siRNA transfection group,the proliferation of HPASMCs and the expression of cyclin D1 mRNA and protein were detected.Data were compared with one-way ANOVA(for multiple-group comparison)and independent t-test(for two-group comparison)followed by the least significant difference(LSD)test with the computer software SPSS 17.0.It was found that 0.5%and 1%CSE could promote the proliferation of HPASMCs(P<0.05),and the former was more effective than the latter(P<0.05),while 3%and above CSE had inhibitory effect on HPASMCs(P<0.05).The mRNA and protein expression levels of TRPC1 and cyclin D1 in 0.5%and 1%CSE groups were significantly higher than those in the control group(P<0.05),while those in 3%CSE group were significantly decreased(P<0.05).Moreover,the proliferation of HPASMCs and the expression of cyclin D1 mRNA and protein in TRPC1-siRNA transfection group were significantly reduced as compared with those in the negative control group(P<0.05).It was concluded that low concentration of CSE can promote the proliferation of HPASMCs,while high concentrations of CSE inhibit HPASMCs proliferation.These findings suggested that CSE induced proliferation of HPASMCs at least in part via TRPC1-mediated cyclin D1 expression.
文摘Cigarette consumption increases oxidative stress in many organs. Increased oxidative stress harms bone cells, which negatively affects bone-matter and -stability. This leads to an increased fracture risk and delayed fracture healing in smokers. A supporting therapy with antioxidants could be of great benefit for surgeons dealing with delayed fracture healing due to increased oxidative stress. In this article we complement and compare our published data with hitherto unpublished data and show the protective effect of 15 different antioxidants on cigarette smoke induced damage in primary human osteoblasts. Exposure to cigarette smoke medium (CSM) rapidly induces formation of ROS in osteoblasts in a concentration- and time-dependent manner. Massive cell damage is seen already after 4 h (EC50 ≈ 0.75 OD320). Pre-, co- and post-incubation with the different antioxidants reduces the formation of ROS and consequently improves the viability of the CSM exposed osteoblasts. Small compounds, e.g. N-acetylcysteine, proved highly effective if pre- or co-incubated before exposure to the CSM. Thus, they are good candidates for acute therapy support as they can be administered in high doses. However, our data suggest that a balanced daily diet could lead to an accumulation of various natural antioxidants (flavonoids) that effectively protect osteoblasts from oxidative stress-induced damage in all three settings investigated. Together with their partly phytoestrogenic properties this may even abate alterations in bone and thus reduce fracture risk on the long run.
文摘Background and Objective: Chronic heavy alcohol consumption and daily cigarette smoking are the most prevalent substance use problems in the U.S., including Veterans. Excessive alcohol use causes neurocognitive and behavioral deficits that can be linked to neurodegeneration. Similarly, preclinical and clinical data suggest that smoking also leads to brain atrophy. This study examines the differential and additive effects of alcohol and cigarette smoke (CS) exposures on cognitive-behavioral function. Methods: A 4-way experimental model of chronic alcohol and CS exposures was generated using 4-week-old male and female Long Evans rats that were pair-fed with Lieber-deCarli isocaloric liquid diets containing 0% or 24% ethanol for 9 weeks. Half of the rats in the control and ethanol groups were exposed to CS for 4 hours/day and 4 days/week for 9 weeks. All rats were subjected to Morris Water Maze, Open Field, and Novel Object Recognition testing in the last experimental week. Results: Chronic alcohol exposure impaired spatial learning as shown by significantly increased latency to locate the platform, and it caused anxiety-like behavior marked by the significantly reduced percentage of entries to the center of the arena. Chronic CS exposure impaired recognition memory as suggested by significantly less time spent at the novel object. Combined exposures to alcohol and CS did not show any significant additive or interactive effect on cognitive-behavioral function. Conclusion: Chronic alcohol exposure was the main driver of spatial learning, while the effect of secondhand CS exposure was not robust. Future studies need to mimic direct CS exposure effects in humans.
基金National natural science foundation of China(No.8166001381860015)。
文摘Objective:To study the effect of cigarette extract(CSE)on the expression of exosomal miR-186 derived from 16HBE cells and the effects of 16HBE-derived exosomal miR-186 on the proliferation of MRC-5 cells.Methods:To collect the exosomal miR-186 in the supernatant of CSE-treated 16HBE cells for MRC-5 cell culture;the expression of exosomal miR-186 was detected by qPCR;the proliferation of MRC-5 cell was detected by CCK-8;dual-luciferase reporter gene experiment was used to detect the targeted regulation relationship between miR-186 and Bcl2L11;the expression of Bcl2L11 was detected by Western blot.Results:The morphology of 16HBE cells about 100 nm in diameter were observed by TEM;CSE treatment significantly promoted the expression of exosomal miR-186;CSE-induced exosomal miR-186 promoted the proliferation of MRC-5 cells;Bcl2L11 is a target gene of miR-186;miR-186 mimics significantly decreased Bcl2L11 expression.Conclusion:CSE-induced 16HBE-derived exosomal miR-186 promoted the proliferation of MRC-5 cells by targeting Bcl2L11 genes.
基金This work was supported by the National Natural Science Foundation of China(No.81400008).
文摘Although several studies confirmed that berberine may attenuate airway inflammation in mice with chronic obstructive pulmonary disease(COPD),its underlying mechanisms were not clear until now.We aimed to establish an experiment mouse model for COPD and to investigate the effects of berberine on airway inflammation and its possible mechanism in COPD model mice induced by cigarette smoke extract(CSE).Twenty SPF C57BL/6 mice were randomly divided into PBS control group,COPD model group,low-dose berberine group and high-dose berberine group,5 mice in each group.The neutrophils and macrophages were examined by Wright's staining.The levels of inflammatory cytokines TNF-α and IL-6 in bronchoalveolar lavage fluid(BALF)were detennined by enzyme-linked immunosorbent assay.The expression levels of TGF-β1,Smad2 and Smad3 mRNA and proteins in lung tissues were respectively detected by quantitative real-time polymerase chain reaction and Western blotting.It was found that CSE increased the number of inflammation cells in BALF,elevated lung inflammation scores,and enhanced the TGF-β1/Smads signaling activity in mice.High-dose berberine restrained the alterations in the COPD mice induced by CSE.It was concluded that high-dose berberine ameliorated CSE-induced airway inflammation in COPD mice.TGF-β1/Smads signaling pathway might be involved in the mechanism.These findings suggested a therapeutic potential of high-dose berberine on the CSE-induced airway inflammation.
基金Supported by Research Grants from UAE University and Terry Fox Foundation (to Karam SM)
文摘AIM:To test whether the expression and activity of H,K-ATPase in parietal cells would be affected by cigarette smoke extract.METHODS: Extracts of cigarette smoke were administered into mice by gastric gavage (5 mg/kg body weight/day) for 3 d or in drinking water for 7 or 14 d. For the latter, each day a mouse consumed 5 mL water containing extracts of two cigarettes, on average. Control littermate mice received only vehicle. To compare the amount of H,K-ATPase in control and smoke-treated mice, the stomach was processed for Western blotting and immunohistochemical analysis using monoclonal antibodies specific for α- or β-subunits of H,K-ATPase. The p-nitrophenylphospatase activity assay was used as a measurement for K-dependent H,K-ATPase activity.RESULTS: Probed transblots showed an increase in the amount of H,K-ATPase in smoke-treated mice which was confirmed by immunohistochemistry and was found to be due to increased amounts of protein per parietal cell rather than an increased parietal cell number. The increase in the amount of H,K-ATPase was associated with an enhancement of its enzymatic activity. K-dependent activity in control and smoke-treated mice was significantly different (respectively, 0.12 μmol/mg vs 0.27 μmol/mg per minute, P<0.05).CONCLUSION: Administration of cigarette smoke extract is associated with an increase in the amount and activity of H,K-ATPase and hence, smokers are susceptible to development of peptic ulcer.
文摘In this study, the effect of prophylactic anti inflammation on the development of smoke induced emphysema was investigated. Young male guinea pigs aged 1.5 - 2 months (weighing 198.3±26.9 g) were randomly divided into 4 groups: group A (cigarette smoke exposure only), group B (cigarette smoke exposure plus pentoxifylline rich (PTX, 10 mg/d) forage feeding), group C (cigarette smoke exposure plus intermittent cortical steroid injection (Triamcinolone acetonide, 3 mg, im, every three weeks) and control group (group D: animals with sham smoke exposure, raised under the same conditions). Animals in group A, B and C were exposed to smoke of cigarettes for 1 to 1.5 h twice a day, 5 days a week. All animals were killed at the 16th week and followed by morphometrical analysis of the midsagittal sectioned lung slices. Smoke exposure of 16 weeks resulted in visible emphysematous development in Group A but not in Group B and C. It was evidenced by the indicator of air space size, mean linear intercept (L m): 120.6±16.0 μm in Group A; 89.8±9.2 μm in Group B and 102.4±17.7 μm in Group C. The average L m in either group B or group C was shorter than that in Group A (ANOVA and Newman Keuls test, F=8.80, P =0.0002) but comparable to that (94.8±13.2 μm) in group D ( P >0.05). It is concluded that long term prophylactic anti inflammation inhibits pulmonary emphysema induced by cigarette smoking in the guinea pigs.
文摘To understand the retention mechanism of phenol in mainstream cigarette smoke by cellulose acetate(CA)filters,the phenol filtration efficiency,the phenol distribution in the gas phase and particulate phase during filtration and the retention characteristics of the filter for both the gas phase and particulate phenol species were investigated.The distribution of particulate bound phenol as a function of the particle size was examined.The ratio of phenol transferring from the particulate phase to the gas phase during smoke flowing through the CA filter were studied.Retention mechanisms for phenol in the gas phase as well as particulate phase phenol in the filter and the phase transition of phenol were proposed.Phenol filtration efficiency of the cigarette filter was calculated based on the proposed retention mechanisms and compared with available experimental data.The results showed that:1)Comparing with nicotine and the other particulate bound smoke constituents,CA filter exhibited a certain retention selectivity for phenol.2)Although phenol was mainly bound to the particulate phase of the cigarette mainstream smoke,about 35% of gas phase phenol was found at the tobacco end of the filter,whilst this dropped to approximately 0 at the mouth end of the filter,mostly by selective chemical adsorption.3)The weighted average smoke particle size and particulate phenol was 0.44 and 0.38 pm,respectively.The filtration efficiency of the filter to particulate phenol was near 40%.4)About 19% of particulate bound phenol transferred to gas phase when smoke passing the filter,and was selectively adsorbed.5)The overall filtration efficiency of the CA filter for phenol in mainstream cigarette smoke was 68.7%,with-7.7% variation compared with experimental data.The results demonstrated the phase transition of phenol during the filtration process and the selective retention with varying particle sizes.Our results showed that the application of CA filter in product designs reduces harmful constituents such as phenolic compounds in mainstream cigarette smoke.
基金We thank the Natural Science Foundation of Fujian Province for the financial supports(No.D0410027).
文摘Constant-energy synchronous fluorimetry was used for the identification of benzo[a]pyrene in mixtures with a detection limit of 1.34 nmol/L. The recovery experiments in cigarette smoke samples have also obtained satisfactory results of 99.1-103.5% for benzo[a]pyrene.
基金supported by the grant from Post Doctor Program, Chonbuk National University (2008)
文摘Objective Embryonic movements (EM) and angiogenesis pathways are evolutionarily conserved mechanisms which are essential for proper embryonic development. Deviations in these processes by exposure to cigarette smoke condensate (CSC) may cause vascular and morphogenetic disorders. Methods Using chicken and mouse embryos, we have demonstrated the in vivo effects of CSC on EM, vascular development, and organogenesis. Results Examination of the CSC exposed chicken embryos revealed a significant reduction in EM, stunted growth, deviated pattern of blood vessels, hemorrhages, and localized necrosis. Likewise, mouse embryos that were exposed to CSC at E8.5 and E9.5 died between E11.5 and E12.5, respectively. These mouse embryos showed defects in morphogenesis and remodeling of the embryonic vasculature, while littermate controls showed normal development. Conclusion Cigarette smoking during pregnancy is fatal for growing embryos. CSC may induce the remodeling of embryonic vasculature, leading to various pathologies.
基金This project was supported by a grant from National Natural Science Foundation of China !(39570288).
文摘Summary: To investigate whether the change of E-cadherin (ECD) expression plays a role in the injury and repair of airway epithelial cells (AEC) caused by smoking, porcine AECs were cultured by using an enzyme-dispersed method. After exposure of the AECs to cigarette smoke extract (CSE), the ECD expression in the cells was detected by using immunocytochemistry and in situ hybridization. The results showed that ECD was distributed on the plasma membrane at the cell junctions of AECs. After exposure to 20 % CSE, the membranous ECD expression was decreased, the cytoplasmic ECD expression was increased (P<0.01) as the exposure time went on. But the content of ECD mRNA in the AECs did not chang. It suggests that the change of ECD ex- pression is regulated at the posttranslational level and plays a role in the injury and repair of AEC caused by smoking.
基金Project(2005JC02) supported by China Tobacco Hunan Industrial Corporation Limited
文摘An improved method was developed for the determination of the four major tobacco-specific nitrosamines(TSNAs) in mainstream cigarette smoke. The new method offers decreased sample preparation and analysis time as compared to traditional methods. This method uses isotope dilution liquid chromatography coupled with a tandem mass spectrometer with electrospray ionization and is significantly more sensitive than traditional methods. It also shows no evidence of artifactual formation of TSNAs. Sample concentrations were determined for four TSNAs in mainstream smoke using two isotopically labeled TSNAs analogues as internal standards. Mainstream smoke was collected on an industry standard 44-mm Cambridge filter pad, extracted with 0.1 mol/L ammonium acetate, purified by solid-phase extraction, and analyzed without further sample cleanup. The analytical column is a 3.9 mm×150 mm Waters Symmertry Shield RP18 column and volume fraction of the mobile phase is 50% methanol, 50% water containing 0.1% acetic acid. The results show that the linear range is 0.5-100.0 mg/L except for N-nitrosoanabasine (NAB) from 0.25 to 50.0 mg/L. The limits of detection are 0.1 mg/L for N-nitrosonornicotine (NNN), 0.08 mg/L for 4-(methylnitrosamino)-1- (3-py-ridyl)-1-butanone (NNK), 0.05 mg/L for N-nitrosoanatabine (NAT) and 0.06 mg/L for NAB. The recoveries of the four TSNAs are from 90.2% to 105.7%.
基金a grant from the National Natural Sciences Foundation of China (No. 30470759)
文摘In order to investigate the effects of puerarin on pulmonary vascular remodeling and protein kinase C-α (PKC-α) in chronic exposure smoke rats, 54 male Wistar rats were randomly di-vided into 7 groups: control group (C group), smoke exposure groups (S4w group, S8w group), puer-arin groups (P4w group, P8w group), propylene glycol control groups (PC4w group, PC8w group). Rats were exposed to cigarette smoke or air for 4 to 8 weeks. Rats in puerarin groups also received puer-arin. To evaluate vascular remodeling, alpha-smooth muscle actin (α-SM-actin) staining was used to count the percentage of completely muscularised vessels to intraacinar pulmonary arteries (CMA/IAPA) which was determined by morphometric analysis of histological sections. Pulmonary artery smooth muscle cell (PASMC) apoptosis was detected by in situ end labeling technique (TUNEL), and proliferation by proliferating cell nuclear antigen (PCNA) staining. Reverse transcrip-tion-polymerase chain reaction (RT-PCR), immunofluorescence staining and Western blot analysis were done to detect the PKC-α mRNA and protein expression in pulmonary arteries. The results showed that in cigarette smoke-exposed rats the percentage of CMA/IAPA and α-SM-actin expres-sion were increased greatly, PASMC apoptosis was increased and proliferation was markedly in-creased; Apoptosis indices (AI) and proliferation indices (PI) were higher than in C group; AI and PI were correlated with vascular remodeling indices; The expression of PKC-α mRNA and protein in pulmonary arteries was significantly higher than in C group. In rats treated with puerarin, the per-centage of CMA/IAPA and cell proliferation was reduced, whereas PASMC apoptosis was increased; The expression levels of PKC-α mRNA and protein were lower than in smoke exposure rats. There was no difference among all these data between S groups and PC groups. These findings suggested that cigarette smoke-induced pulmonary vascular remodeling was most likely an effect of the imbal-ance of PASMC proliferation and apoptosis. Puerarin appears to be able to reduce cell proliferation and vascular remodeling possibly through PKC signaling transduction pathway.
文摘To investigate the effect of cigarette smoke extract （CSE） on the role of protein kinase C （PKC） in the proliferation of passively sensitized human airway smooth muscle cells （HASMCs）. After synchronization of cultured HASMCs, they were divided into a group A and Group B. The group A was treated with normal human serum and served as controls and the group B was treated with the serum of asthma patients. The group A was further divided into group of A1, A2 and A3 and the group B was sub-divided into the group of B1, B2, B3, B4 and B5. No other agents were added to the group A1 and B1. The cells of group A2 and B2 were stimulated with 5 % CSE for 24 h. HASMCs from group A3 and B3 were treated with PKC agonist PMA （10 nmol/L） and CSE （5 %） for 24 h. PKC inhibitor Ro-31-8220 （5 μmol/L） was added to the HASMCs of group B4 for 24 h. The cells from group B5 were stimulated with Ro-31-8220 （5 μmol/L） and CSE （5 %） for 24 h. The proliferation of HASMCs isolated from group A and B was examined by cell cycle analysis, MTT colorimetric assay and 3H-TdR incorporation test. The expression of PKC-α in each group was observed by Western blotting and RT-PCR, respectively. The results showed that the percentage of S phase, absorbance （A） value, the rate of 3H-TdR incorporation, the ratios of A value of PKC-α mRNA and the A value of PKC-α protein in HASMCs from group B1, B2 and B3 were significantly increased compared to those of group A1, A2 and A3 correspondingly and respectively （P<0.01）. The proliferation of HASMCs of group A2 and B2 stimulated with CSE and group A3 and B3 stimulated with CSE and PMA were also significantly enhanced when group A1, A2 and A3 and group B1, B2 and B3 compared to each other （P<0.05, P<0.01, respectively）. The percentage of S phase, absorbency （A） value, 3H-TdR incorporation rate, the ratios of A value of PKC-α mRNA and the A value of PKC-α protein in HASMCs from group B4 treated with Ro-31-8220 and group B5 treated with CSE and Ro-31-8220 were significantly decreased as compared to those of group B1 and B2 correspondingly and respectively （P<0.05, P<0.01）. It was concluded that CSE can enhance the passively sensitized HASMC proliferation and the expression of PKC alpha. PKC and its alpha subtype may contribute to this process. Our results suggest cigarette may play an important role in ASMCs proliferation of asthma through PKC signal pathway.
文摘To investigate the toxicity of cigarette smoke extract (CSE) and nicotine on mouse brain mitochondria as well as the protective effect of vitamin C in vitro. Method Mouse brain mitochondria in vitro was incubated with CSE or nicotine in the absence or presence of vitamin C for 60 minutes, and the changes of mitochondrial function and structure were measured. Results CSE inhibited mitochondrial ATPase and cytochrome C oxidase activities in a dose-dependent manner.However, no significant changes in the peroxidation indices were observed when mitochondrial respiratory enzymes activity was inhibited, and protection of mitochondria from CSE-induced injury by vitamin C was not displayed in vitro. The effect of CSE on mouse brain mitochondria swelling response to calcium stimulation was dependent on calcium concentrations. CSE inhibited swelling of mitochondria at 6.5 μmol/L Ca2+, but promoted swelling response at 250 μmol/L Ca2+. Nicotine, the major component of cigarette smoke, showed no significant damage in mouse brain mitochondria in vitro. The CSE treatment induced mitochondrial inner membrane damage and vacuolization of the matrix, whereas the outer mitochondrial membrane appeared to be preserved. Conclusion The toxic effect of CSE on brain mitochondria may be due to its direct action on enzymatic activity rather than through oxygen free radical injury. Nicotine is not the responsible component for the toxicity of CSE to brain mitochondria.
文摘Cigarette smoking is intimately related with the development of chronic obstructive pulmonary diseases, and alveolar epithelium is a major target for the exposure of cigarette smoke ex-tract. In order to investigate the effect of cigarette smoke extract on the proliferation of alveolar epithelial cell type Ⅱand its relationship with P21WAF1, the alveolar epithelial type Ⅱ cell line (A549) cells were chosen as surrogate cells to represent alveolar epithelial type Ⅱ cells. MTT assay was used to detect cell viability after interfered with different concentrations of cigarette smoke ex-tract. It was observed cigarette smoke extract inhibited the growth of A549 cells in a dose- and time-dependent manner. The morphological changes, involving the condensation and margination of nuclear chromatin, even karyorrhexis, were observed by both Hoechst staining and electronic mi-croscopy. Flow cytometry analysis demonstrated the increased cell percentages in G1 and subG1 phases after the cells were incubated with cigarette smoke extract. The expression of p21WAF1 protein and mRNA was also significantly increased as detected by the methods of Western blot or reverse transcription-polymerase chain reaction respectively. In conclusion, cigarette smoke extract inhibits the proliferation of alveolar epithelial cell type Ⅱ and blocks them in G1/S phase. The intracellular accumulation of P21WAF1 may be one of the mechanisms which contribute to cigarette smoke ex-tract-induced inhibition of cell proliferation.
基金supported by grants from the National Natural Science Foundation of China(No.8107003681370145+3 种基金81370156 and 81070021)The National Key Technology R&D Program of the 12th National Five-year Development Plan:Clinical Study on Translational Medicine of Respiratory Disease(No.212BA105B01)The Specific Project of National Health Research Project of Ministry of Health of China(No.201002008)Program for Changjiang Scholars and Innovative Research Team in University(No.PCSIRT1131)
文摘Cigarette smoke is associated with the development of several diseases,such as chronic obstructive pulmonary disease(COPD).The purpose of this study was to investigate genotoxicity and heat shock protein 70(Hsp70) in human airway smooth muscle cells(HASMCs) exposed to cigarette smoke extract(CSE).HASMCs was exposed to CSE with different doses for 24 h.The level of8-hydroxydeoxyguanosine(8-OHdG) was determined by using HPLC-ECD,the DNA damage was analyzed by using comet assay,and apoptosis was examined by using Annexin-FITC/PI staining.The production of Hsp70 after CSE stimulation was tested.Results indicated that CSE significantly increased the level of 8-OHdG,DNA damage and cell apoptosis,and reduced the production of Hsp70.In particular,levels of Hsp70 were inversely correlated with 8-OHdG,DNA damage and cell apoptosis.It was concluded that cigarette smoke induced genotoxicity and decreased the production of cell protective protein Hsp70,which may contribute to the development of some airway diseases.
文摘Objective To investigate the cellular effects of cigarette smoke extract (CSE) on primarily cultured human umbilical vein endothelial cells (HUVEC). Methods The effects of CSE (5%-20%) and nicotine (10-4 mol/L) on HUVEC viability, proliferation, angiogenesis and apoptosis were observed. Results CSE decreased HUVEC survival rate and angiogenesis after 24 h as well as its proliferation after 48 h in a dose-dependent manner. Moreover, CSE induced apoptosis of HUVEC as indicated in condensation of nuclear chromatin and the presence of hypodiploid DNA. HUVEC incubated with CSE for 24 h gave a significant decrease in the expression of Bcl-2 as well as the decline in the Bcl-2/Bax ratio accompanied with the loss of mitochondrial membrane potential and excess cytosolic calcium. Our study also observed that p53 protein level decreased, rather than increased in cells treated with CSE. Nicotine had no discernible inhibitory effects on the above indices of HUVEC. Conclusion Exposure to CSE other than nicotine causes inhibition of viability, proliferation and differentiation of HUVEC. CSE-induced HUVEC injury is mediated in part through accelerated apoptosis but independent of p53 pathway. It appears that mitochondria have played a key role in the apoptosis of HUVEC induced by CSE.
