Berberine Attenuates Cigarette Smoke Extract-induced Airway Inflammation in Mice:Involvement of TGF-β1/Smads Signaling Pathway

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.

Effect of Cigarette Smoke Extract on the Role of Protein Kinase C in the Proliferation of Passively Sensitized Human Airway Smooth Muscle Cells

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 A_1, A_2 and A_3 and the group B was sub-divided into the group of B_1, B_2, B_3, B_4 and B_5. No other agents were added to the group A_1 and B_1. The cells of group A_2 and B_2 were stimulated with 5 % CSE for 24 h. HASMCs from group A_3 and B_3 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 B_4 for 24 h. The cells from group B_5 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 B_1, B_2 and B_3 were significantly increased compared to those of group A_1, A_2 and A_3 correspondingly and respectively (P<0.01). The proliferation of HASMCs of group A_2 and B_2 stimulated with CSE and group A_3 and B_3 stimulated with CSE and PMA were also significantly enhanced when group A_1, A_2 and A_3 and group B_1, B_2 and B_3 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 B_4 treated with Ro-31-8220 and group B_5 treated with CSE and Ro-31-8220 were significantly decreased as compared to those of group B_1 and B_2 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.

Injury of Mouse Brain Mitochondria Induced by Cigarette Smoke Extract and Effect of Vitamin C on It in vitro

Objective 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 6.5μmol/L Ca2+, but promoted swelling response at 250μmol/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.

Roles of TGF-β Signaling Pathway in Endoplasmic Reticulum Stress in Endothelial Cells Stimulated with Cigarette Smoke Extract

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.

Cigarette Smoke Extract Inhibits the Proliferation of Alveolar Epithelial Cells and Augments the Expression of P21^(WAF1)

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 extract. In order to investigate the effect of cigarette smoke extract on the proliferation of alveolar epithelial cell type Ⅱ and its relationship with P21^WAF1, 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 extract. 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 microscopy. 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 p21^WAF1 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 intracelhilar accumulation of P21^WAF1 may be one of the mechanisms which contribute to cigarette smoke extract-induced inhibition of cell proliferation.

Genotoxicity and Reduced Heat Shock Protein 70 in Human Airway Smooth Muscle Cells Exposed to Cigarette Smoke Extract

Cigarette smoke is associated with the development of several diseases, such as chronic ob- structive 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 of 8-hydroxydeoxyguanosine （8-OHdG） was determined by using HPLC-ECD, the DNA damage was ana- lyzed by using comet assay, and apoptosis was examined by using Annexin-FITC/PI staining. The pro- duction 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 par- ticular, 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.

Dysregulation of gastric H,K-ATPase by cigarette smoke extract

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.

Involvement of TRPC1 and Cyclin D1 in Human Pulmonary Artery Smooth Muscle Cells Proliferation Induced by Cigarette Smoke Extract

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.

Effect of Cigarette Smoke Extract on the Proliferation of Human Airway Epithelial Cells and Expression and Activation of FAK

The effect of cigarette smoke extract (CSE) on the proliferation of human airway epithelial cells and the possible mechanism was studied. After airway epithelial cells were treated with different concentrations of CSE for 24 h, the cell proliferation was measured by MTT and the distribution of different cell cycles by flow cytometry. The FAK expression level was detected by Western blot and the degree of tyrosine phosphorylation by immunoprecipitation. The results showed that CSE could inhibit the proliferation of human airway epithelial cells, arrest the epithelial cells in G1 phase of cell cycle, dramatically decrease the number of epithelial cells in S and G2 phases; Meanwhile CSE could decrease the expression level of FAK and the degree of its tyrosine phosphorylation. The above effects of CSE were concentration-dependent. The expression of FAK and the degree of its phosphorylation was positively correlated to the increased number of epithelial cells in G1 phase, and negatively to the number of epithelial cells in S and G2 phases. It was concluded that the mechanism by which CSE could inhibit the proliferation of human epithelial cells was contributed to the increased expression and activation of FAK.

Effect of Cigarette Smoke on Diabetic Skin and Protection with Topical Administration of Pinus halepensis Extract

Compared to normal, diabetic skin is characterized by great sensitivity. Oxidative stress is directly involved, contributing to accelerated skin aging, xerodermia and poor wound healing. In the last 10 years, cigarette smoke (CS) exposure has been associated with several skin and dermatological conditions and is directly related to the oxidative stress affecting the skin. However, limited data exist concerning the effect of CS on diabetic skin. Some of the effects of cigarette smoke exposure on the skin of hairless diabetic mice were hereby studied and the potential skin protection by topical applications of Pinus halepensis bark extract was investigated. Female hairless SKH-2 diabetic mice were exposed for 8 days to tobacco smoke and topical applications were performed twice daily. Biophysical parameters such as transepidermal water loss (TEWL), skin elasticity and erythema were measured. In addition, the oxidative stress was evaluated. The results show that diabetes and CS have a synergistic negative action on skin condition, with the development of xerosis and high ROS levels whilst topical applications of Pinus halepensis bark extract protect efficiently the toxic effect of CS on skin, by decreasing skin dryness, oxidative stress and blood glucose levels.

Inhibitory Effect of Cigarette Smoke Extract on Experimental Lung Metastasis of Mouse Melanoma by Suppressing Tumor Invasion

We investigated the effect of a nicotine-and tar-free cigarette smoke extract (CSE) using an experimental metastasis mouse model which was intravenously injected with B16-BL6 mouse melanoma cells. Three-hour pretreatment of cells with various concentrations of CSE (0, 0.1, 0.3, and 1%) dose-dependently reduced the number of lung metastatic nodules 14 days after tumor injection. To elucidate the mechanism of this anti-metastatic effect of CSE, we examined the invasion and migration activities of B16-BL6 cells pretreated with CSE for three hours in vitro. CSE significantly reduced the invasion of cells at 1% and the migration at 0.3% and 1%. Under the same pretreatment conditions, CSE had no effect on the proliferation of cells. These findings suggest that CSE contains some ingredients that suppress hematogenic lung metastasis via inhibition of the invasion and migration activities of mouse melanoma cells.

Cigarette smoke extract-induced 16HBE-derived exosomal miR-186 significantly promoted the proliferation of COPD MRC-5 cell

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.

Small-scale fire tests in the underwater tunnel section model with new sidewall smoke extraction

The Shenzhen–Zhongshan Bridge is a 24‐km‐long bridge and tunnel system,including a 6.8-km-long super cross section subsea tunnel.To solve the smoke exhaust problem of a super large cross-section subsea tunnel,the tunnel has a new smoke exhaust system that combines a horizontal smoke exhaust cross section at the top and sidewall smoke exhaust holes.In order to evaluate the potential fire hazards of this type of tunnel,a 1:30 tunnel model was established and 140 smallscale experiments on underwater tunnel fires were conducted.By changing the fire power,fire location,and fan operation mode,different scenarios of submarine immersed tunnel fire were simulated and the related key parameters such as fire smoke diffusion behavior and smoke temperature distribution were studied.On this basis,the optimal smoke control strategy was proposed for different fire scenarios.The research results indicate that the new smoke exhaust system can fully utilize the smoke flow characteristics,significantly improve smoke exhaust efficiency,and increase available evacuation time,thus further enhancing the fire safety of super large cross-section subsea tunnels.

重组CC16蛋白质抑制香烟烟雾提取物诱导人支气管上皮细胞衰老的初步研究

目的探讨重组人CC16蛋白质(rhCC16)对香烟烟雾提取物(cigarette smoke extract,CSE)诱导的人支气管上皮细胞(human bronchial epithelial cells,HBECs)衰老的抑制作用及机制。方法CCK-8法检测CSE(0%、1%、2.5%、5%、7.5%和10%)及rhCC16(0、10、100、250和500 ng/mL)对HBECs细胞活力的影响;β-半乳糖苷酶染色试剂盒检测对照组、CSE组以及CSE+rhCC16组细胞β-半乳糖苷酶活性;实时荧光定量PCR(qPCR)检测各组细胞P16、P21 mRNA表达水平;Western blot检测各组细胞P16、P21、p-P53、P38、p-P38、ERK1/2、p-ERK1/2蛋白表达水平。结果与对照组相比,5%CSE刺激细胞72 h对HBECs细胞活力无显著影响;500 ng/mL及以下浓度rhCC16对HBECs细胞活力无显著影响;与对照组相比,5%CSE刺激HBECs细胞72 h致β-半乳糖苷酶染色阳性细胞率明显升高(P<0.05),且P16和P21蛋白和mRNA表达水平明显升高(P<0.05),p-P53、p-P38与p-ERK1/2蛋白表达量显著增加(P<0.05);与CSE组相比,250 ng/mL rhCC16干预下,HBECs细胞β-半乳糖苷酶染色阳性细胞率显著下降(P<0.05),P16和P21蛋白和mRNA表达水平显著下降(P<0.05),p-P53、p-p38和p-ERK1/2的表达量降低(P<0.05)。结论rhCC16蛋白质抑制香烟烟雾诱导的HBECs衰老,抑制P38 MAPK和ERK1/2信号通路可能是其作用机制。

隧道火灾重点排烟系统烟气控制效果及排烟效率研究

采用数值计算方法,考虑热释放速率、排烟量、排烟口间距、排烟口面积、尺寸比和隧道高度,研究隧道火灾重点排烟系统下烟气蔓延距离、温度分布、排烟口风速和排烟效率。结果表明:火灾烟气蔓延距离随排烟量的增大而缩短,隧道顶棚温度下降;在排烟量不变的条件下,更大的排烟口宽长比能够缩短烟气蔓延距离;而排烟口间距的变化对排烟效率影响不明显。基于研究结果,给出了5 MW和20 MW火源功率下的重点排烟系统排烟量和排烟口布置建议值。

频域多方向C-UNet及动态损失的工业烟尘图像分割

工业烟尘污染等级监测中烟尘的准确分割是污染等级判定的重要前提.针对边缘模糊且方向多变烟尘在特征提取过程中边缘方向细节信息提取困难、分割不准确的问题,本文提出一种频域多方向C-UNet及动态损失的工业烟尘图像分割方法.首先,通过构建轮廓波多方向分解下采样结构增强编码阶段烟尘边缘方向信息的提取能力;其次,通过轮廓波变换提取烟尘8个边缘方向细节信息进行跳跃连接,提升持续采样过程中细节信息的表达准确度;然后,构建轮廓波细节重构上采样结构增强解码阶段烟尘边缘细节信息的恢复能力;最后,提出一种动态加权策略构建组合损失函数来优化训练网络,增强网络对烟尘边缘特征的提取能力.结果表明,本文方法与U-Net和其他同类方法相比在指标上有较好提升,改善了烟尘边缘分割不准确的问题,在不同烟尘场景上的分割效果也优于现有分割模型.

太阳能烟囱用于城市隧道通风排烟的性能研究

为探明太阳能烟囱用于城市隧道自然通风及火灾排烟的效率,采用数值模拟方法,研究烟囱高度、空腔间隙、太阳辐照度和火源功率对通风与排烟效率的影响规律。研究得出:隧道自然通风速率随烟囱高度、空腔间隙和太阳辐照度增加而上升,烟囱高度每增加1.4 m通风速率提高17%,空腔间隙每增加0.5 m通风速率提升16%,太阳辐照度增加200 W/m^(2)通风效率提高11%。太阳能烟囱用于排烟的效率优于传统竖井,烟囱高度对排烟效率的影响较小,排烟空腔间隙临界值为1.5 m。基于太阳能烟囱内横向非对称抛物线温度分布,提出烟囱出口体积流量预测模型。

移动式风机组合对建筑火灾排烟效果影响的研究

为探明不同正压式移动风机组合(并联、串联、上下和V型)对建筑火灾排烟效果的影响,采用试验测量与基于大涡模拟火灾动力学仿真相结合的方式,研究不同风机组合的火场排烟场景、排烟效率及注意事项。结果表明,4种风机组合均满足空气锥气流覆盖进风口、增加室内压力的需求。当风机组合摆放成V型时,建筑物内的气流组织效果优于其他组合。上下型、并联风机组合排烟时,两台风机射流中心点距离过于接近会造成部分空气锥形成的气流相互叠加,从而增加空气锥内部涡旋的形成,能耗增加。风机串联时,前台风机的电机阻碍后面气流运动,导致流速降低。论证了风机组合在实践中的应用,从而验证其战术的可行性,为火场排烟提供理论依据。

Hybrid Deep VGG-NET Convolutional Classifier for Video Smoke Detection

Real-time wild smoke detection utilizing machine based identification method is not produced proper accuracy,and it is not suitable for accurate prediction.However,various video smoke detection approaches involve minimum lighting,and it is required for the cameras to identify the existence of smoke particles in a scene.To overcome such challenges,our proposed work introduces a novel concept like deep VGG-Net Convolutional Neural Network(CNN)for the classification of smoke particles.This Deep Feature Synthesis algorithm automatically generated the characteristics for relational datasets.Also hybrid ABC optimization rectifies the problem related to the slow convergence since complexity is reduced.The proposed real-time algorithm uses some pre-processing for the image enhancement and next to the image enhancement processing;foreground and background regions are separated with Otsu thresholding.Here,to regulate the linear combination of foreground and background components alpha channel is applied to the image components.Here,Farneback optical flow evaluation technique diminishes the false finding rate and finally smoke particles are classified with the VGG-Net CNN classifier.In the end,the investigational outcome shows better statistical stability and performance regarding classification accuracy.The algorithm has better smoke detection performance among various video scenes.