Damaging Effect of Cigarette Smoke Extract on PrimaryCultured Human Umbilical Vein Endothelial Cells and Its Mechanism

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.

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.

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.

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.

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.

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.

Cluster Analysis of Polyphenols and Organic Acids in 11 Different Brand Cigarette Samples at Home and Abroad

The objective of this research was to investigate the differences between local cigarette and foreign cigarette and supplied a base for improving the quality of cigarette. Different kinds of polyphenols and organic acids in 11 different brand cigarette samples at home and abroad were classified by the method of cluster analysis. The results indicated that the 11 samples could be classified into 2 classes. Suyan, Furongwang, Chinese, Baisha, Dihao, Yunyan, Hongtashan belonged to type 1; foreign cigarettes that represented by Marboro, Blue pacific and Brazil cigarette belonged to type 2. The content of malic acid and citric acid in type 1was higher than type 2, the content of malonic acid was higher in type 2, and there is no difference between the type 1 and type 2 about the content of polyphenols. In conclusion, the content of malic acid and citric in Chinese cigarettes was higher than foreign, but the content of malonic acid was lower than foreign. There is no difference between Chinese cigarettes and foreign cigarettes about the content of polyphenols.

基于“雅解理论”探讨雅解益栽(心)方药物血清对CSE诱导VEC-304细胞损伤的影响

目的:观察香烟提取物(CSE)诱导血管内皮(VEC)-304的存活率、细胞增殖抑制率的变化,测定细胞裂解液的抗氧化能力,检测细胞相关凋亡蛋白的表达,探讨雅解益栽(心)方药物血清对CSE诱导的VEC-304细胞损伤的影响。方法:采用CSE复制VEC-304细胞损伤模型,雅解益栽(心)方含药血清倍比稀释后分为3个剂量干预组(体积分数10%),采用MTT、TUNEL、DNA ladder、免疫组化以及Western Blot等方法,检测雅解益栽(心)方药物血清对CSE诱导的(VEC)-304细胞损伤的影响。结果:CSE可诱导细胞凋亡,其机制与其引发细胞氧化应激反应有关;诱发细胞Caspase-3、Bax、NF-κb表达上调,Bcl-2、p53下调,而致细胞凋亡。雅解益栽(心)方干预组细胞SOD、GSH-px、GSH含量比空白对照组高,MDA及NOS比空白对照组低;而Caspase-3、Bax、NF-κb表达下调,Bcl-2、p53表达上调。并有一定的量效依赖关系。结论:CSE能诱导VEC-304细胞凋亡,雅解益栽(心)方药物血清有明显的抗氧化作用,能抑制细胞凋亡,从而具有解烟毒作用。

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。

基于PPARγ信号通路研究香烟烟雾提取物干预不同时间下对肺泡巨噬细胞胞葬及吞噬功能影响的分子机制

目的:观察香烟烟雾提取物(CSE)干预不同时间下肺泡巨噬细胞胞葬及吞噬功能的变化,探讨其对肺部疾病炎症反应的影响及分子机制。方法:大鼠肺泡巨噬细胞NR8383采用10%CSE分别干预6 h、12 h、24 h、48 h,分为6 h空白对照组、6 h-10%CSE组、12 h空白对照组、12 h-10%CSE组、24 h空白对照组、24 h-10%CSE组、48 h空白对照组、48 h-10%CSE组。10%CSE干预12 h后联用PPAR抑制剂/激动剂,分为PPAR抑制剂组、PPAR激动剂组。Alamar Blue法检测PPAR激动剂对NR8383细胞增殖及毒性作用;流式细胞术检测NR8383细胞胞葬、吞噬功能及M1、M2极化分型;酶联免疫吸附实验检测TNF-α、TGF-β1、MFG-E8含量;免疫印迹法检测PPARγ信号通路及下游分子CD36蛋白表达;qPCR检测PPARγ、CD36 mRNA的表达。结果:10%CSE干预6 h后,NR8383细胞吞噬及胞葬功能均有所升高,PPARγ表达下调,CD36 mRNA表达增加,TNF-α、TGF-β1、MFG-E8表达均升高,但无明显极化方向;干预12 h,NR8383细胞吞噬功能及胞葬率显著降低,PPARγ、CD36表达显著下调,TNF-α表达增强,TGF-β1、MFG-E8表达降低,向M1型巨噬细胞极化;干预24 h后,NR8383细胞胞葬率降低,但吞噬大肠杆菌的功能增强,PPARγ表达下调,CD36蛋白表达降低,TNF-α表达降低但差异无统计学意义,TGF-β1、MFG-E8表达仍处于降低状态,有明显的M1型极化倾向;48 h后NR8383细胞胞葬率仍旧降低,但吞噬能力显著提升,PPARγ表达显著降低,CD36表达显著增加,TNF-α表达降低,TGF-β1、MFG-E8表达增加,巨噬细胞向M1、M2方向极化均增加。选择10%CSE干预12 h联合PPAR激动剂、抑制剂后发现,PPAR激动剂增强NR8383细胞胞葬及吞噬能力,上调PPARγ、CD36表达,抑制炎症因子TNF-α表达,促进抑炎因子TGF-β1、胞葬辅助因子MFG-E8表达。结论:随着CSE干预时间的变化,肺泡巨噬细胞从早期炎症反应的活化状态,逐渐进展为慢性炎症反应状态,进而导致肺泡巨噬细胞胞葬及吞噬功能障碍,该机制与PPARγ通路被抑制有关。

The effect of cigarette smoke extract on thrombomodulinthrombin binding: an atomic force microscopy study

Cigarette smoking is a well-known risk factor for cardiovascular disease. Smoking can cause vascular endothelial dysfunction and consequently trigger haemostatic activation and thrombosis. However, the mechanism of how smoking promotes thrombosis is not fully understood. Thrombosis is associated with the imbalance of the coagulant system due to endothelial dysfunction. As a vital anticoagulation cofactor, thrombomodulin (TM) located on the endothelial cell surface is able to regulate intravascular coagulation by binding to thrombin, and the binding results in thrombosis inhibition. This work focused on the effects of cigarette smoke extract (CSE) on TM-thrombin binding by atomic force microscopy (AFM) based single-molecule force spectroscopy. The results from both in vitro and live-cell experiments indicated that CSE could notably reduce the binding probability of TM and thrombin. This study provided a new approach and new evidence for studying the mechanism of thrombosis triggered by cigarette smoking.

姜黄素通过调控PPARγ/NF-κB信号通路减轻CSE诱导的人气道上皮细胞氧化应激反应

目的探讨姜黄素对香烟烟雾提取物(CSE)诱导的人气道上皮16HBE细胞氧化应激和炎症反应的抑制作用及相关的分子机制。方法使用shRNA-PPARγ(shPPARγ)转染人气道上皮16HBE细胞下调PPARγ表达。将16HBE细胞分为5组即对照组、姜黄素组、CSE组、CSE+姜黄素组和CSE+姜黄素+shRNA-PPARγ组。在0 h和24 h时使用MTT法对细胞活性进行检测;干预24 h后使用q PCR对细胞TNF-α、iNOS和PPARγm RNA表达进行检测,采用western blot检测16HBE细胞中iNOS、PPARγ蛋白表达以及NF-κB p65磷酸化水平。结果 16HBE细胞在干预24 h后各组之间细胞活性未有明显统计学差异(P>0.05)。与对照组相比较,CSE干预24 h后PPARγ表达水平明显降低,TNF-α、iNOS表达及NF-κB p65磷酸化水平明显升高,差异均有统计学意义(P<0.05)。但CSE组较CSE+姜黄素组和CSE+姜黄素+shPPARγ组PPARγ表达水平下降以及TNF-α、iNOS表达和NF-κB p65磷酸化水平升高更显著,差异均有统计学意义(P<0.05);且CSE+姜黄素+shPPARγ组较CSE+姜黄素组PPARγ表达水平下降以及TNF-α、iNOS表达和NF-κB p65磷酸化水平升高更明显,差异均有统计学意义(P<0.05)。结论姜黄素可以通过抑制PPARγ/NF-κB信号通路减轻CSE诱导的人气道上皮16HBE细胞的炎症反应及氧化应激。为姜黄素应用于慢性阻塞性肺疾病等疾病的临床治疗奠定了理论基础。

香烟烟雾提取物对破骨细胞体积及整合素αvβ3基因表达的影响

目的探讨香烟烟雾提取物(cigarette smoke extract,CSE)对破骨细胞体积及整合素αvβ3基因表达的影响。方法首先制作CSE溶液,实验采用7周龄C57BL/6J小鼠骨髓单个核细胞加入M-CSF和RANKL诱导生成破骨细胞,将浓度为5%的CSE加入实验组破骨细胞作用3 d,对照组不加入CSE。通过Trap染色识别对照组和实验组破骨细胞并于光镜下采图;通过甲苯胺蓝染色识别两组破骨细胞在骨片上形成的骨陷窝并于光镜下采图,均用NIH imageJ计算数量及面积。用实时荧光定量聚合酶链反应,以GAPDH作为内参,最后通过2-ΔΔCT计算,以检测Trap、CTR以及整合素αvβ3 mRNA表达水平。通过Western blot检测整合素αvβ3蛋白表达。结果破骨细胞表面积及细胞核数量的计量表明,实验组诱导形成的破骨细胞显著大于对照组(P<0.01)。实验组骨片上吸收陷窝数量及面积明显多于对照组(P<0.05),CSE处理组的TRAP mRNA水平为1.16±0.13,明显高于对照组(P<0.05),CTRmRNA水平为0.38±0.04,明显低于对照组(P<0.01)。CSE处理组的整合素αvβ3mRNA水平为1.75±0.05,显著高于对照组(P<0.01),蛋白质印迹分析结果也明显高于对照组(P<0.05)。结论CSE能显著增加破骨细胞的体积并上调整合素αvβ3的基因表达,并可能对骨吸收有促进作用。