Cigarette Smoke Extract Group Research Articles

Treadmill exercise with nanoselenium supplementation affects the expression of Irisin/FNDC5 and semaphorin 3A in rats exposed to cigarette smoke extract.

In the current study, we investigated the impacts of 6 weeks of aerobic interval training (AIT) with selenium nanoparticles (SeNPs) on muscle, serum, and lung irisin (FNDC5) and Sema3A in rats exposed to cigarette smoke extract (CSE). To this end, 49 male Wistar rats (8 weeks old) were divided into seven groups: control, SeNPs (2.5mg/kg b.w by oral gavage, 3days/week, 6weeks), AIT (49min/day, 5days/week for 6weeks, interval), SeNPs + AIT, CSE (150 µL by IP injection, 1day/week for 6weeks), CSE + AIT, and CSE + SeNPs + AIT. The CSE group showed a significant reduction in irisin and Sema3A serum levels, as well as a decrease in FNDC5 and Sema3A gene expression in lung tissue (p < 0.05). A combined treatment (AIT with SeNPs) significantly increased the serum level and the expression of muscle and lung irisin (FNDC5) and Sema3A in CSE received groups (p < 0.05). There was a positive and significant correlation between muscle FNDC5 and lung FNDC5 in the CSE + SeNPs + AIT group (r = 0.92, p = 0.025). In addition, there was a positive and significant correlation between serum Sema3A and lung Sema3A of CSE + SeNPs + AIT group (r = 0.97, p = 0.004). Seemingly, performing aerobic exercises with the antioxidant and anti-inflammatory supplement nano-selenium in the model of lung damage (similar to COPD) can boost myokine irisin and Sema3A, especially in serum and lung tissue. These results displayed the paracrine/endocrine regulatory function of these myokines on other tissues. In other words, these interventions emphasized the creation of crosstalk between skeletal muscles and damaged lung, focusing on its recovery; however, further research is needed.

Cigarette smoke extract induces the senescence of endothelial progenitor cells by upregulating p300.

Endothelial progenitor cells (EPCs) are the main source of endothelial cells. The senescence of EPCs is involved in the pathogenesis of chronic obstructive pulmonary disease (COPD). Cigarette smoke extract (CSE) can directly induce the dysfunction and increased expression of senescence-related markers in EPCs cultured in vitro. Histone acetyltransferase p300 is a transcriptional activator, and its changes can lead to cell senescence. The present study investigated whether CSE can induce the senescence of EPCs by upregulating p300. EPCs were isolated from bone marrow of C57BL/6J mice by density gradient centrifugation. The p300 inhibitor C646 and agonist CTPB were used to interfere with EPCs, cell cycle and apoptosis were detected by flow cytometry, the proportion of senile cells was counted by β-galactosidase staining, the protein expression of p300, H4K12, Cyclin D1, TERT and Ki67 were detected by western blot. Compared with the control group, the cell cycle of CSE group and CTPB group were blocked, the apoptosis rate and early apoptosis rate were increased, the proportion of senile cells counted by β-galactosidase staining was increased, the expression of p300 and H4K12 protein were increased, the expression of Cyclin D1, TERT and Ki67 protein were decreased. C646 could partly alleviate the damages caused by CSE. CSE may promote the apoptosis and senescence of EPCs by upregulating the expression of p300 and H4K12 protein, thus preventing the transition of EPCs from G1 phase to S phase, affecting telomerase synthesis, and reducing EPCs proliferation.

Baicalin Relieves Airway Inflammation in COPD by Inhibiting miR-125a.

To investigate the effects of Baicalin on the apoptosis of human bronchial epithelial cells (16HBE) induced by cigarette smoke extract (CSE) and the release of inflammatory factors, and to clarify its possible mechanism. CSE was used to treat 16HBE cells and construct COPD cell model. The activity of 16HBE cells was detected by CCK-8 and BrdU. Real-time fluorescence quantitative PCR (RT-QPCR) was used to detect the expression level of miR-125a in each group of 16HBE cells. At the same time, the levels of 16HBE inflammatory cytokines IL-1β, IL-8, IL-6, and TNF-α were detected. The apoptosis rate of 16HBE cells in each group was detected by TUNEL. Compared with the control group, the proliferation of 16HBE cells in CSE group was decreased. Baicalin reversed the effect of 2% CSE on the proliferation of 16HBE cells. Baicalin also reversed the effect of 2% CSE on apoptosis and inflammatory factors in 16HBE cells. miR-125a is highly expressed in COPD, and Baicalin can inhibit the expression of miR-125a. Silencing miR-125a reduces apoptosis and inflammatory response of 16HBE cells in COPD. miR-125a reversed the effects of Baicalin on apoptosis and inflammation of 16HBE cells. Baicalin can reduce CSE-induced apoptosis of human bronchial epithelial cells and release of inflammatory factors, and its mechanism may be related to the inhibition of miR-125a.

PDK1 inhibition reduces autophagy and cell senescence through the PI3K/AKT signalling pathway in a cigarette smoke mouse emphysema model.

A number of previous studies have demonstrated the pivotal role of PI3K/AKT signalling in cigarette smoke (CS)-induced emphysema, where phosphoinositide dependent protein kinase 1 (PDK1) is a critical component of this pathway. Therefore, the present study aimed to investigate the effects of a PDK1 inhibitor (GSK-2334470) on the expression levels of PI3K, AKT, cyclin-dependent kinase inhibitor 2A (p16) and LC3B in a CS + CS extract (CSE)-induced mouse emphysema model. CS exposure and intraperitoneal injections of CSE were combined for 4 weeks to establish an emphysema model. Mice (n=35) were randomly divided into the normal control, emphysema (CS), PI3K inhibitor (CS3) and PDK1 inhibitor (CS1) groups. Immunohistochemistry staining of lung tissues was used to measure the expression of the PI3K, PDK1 and AKT proteins in airway epithelial tissues. Immunofluorescence staining was also used to measure the levels of p16 and LC3BII protein expression in the airway epithelial tissues. In addition, PI3K, PDK1, AKT, p16 and LC3B protein expression was semi-quantified using western blotting. The expression of PDK1, PI3K and AKT proteins in the airway epithelial tissues was significantly increased in the CS + CSE group compared with that in the control group. The expression levels of p16 and LC3B were also increased as well in the CS + CSE group compared with those in the control group. The expression levels of PI3K, PDK1, AKT, LC3B and p16 in the airway epithelial tissues of the CS3 group were lower compared with those in the CS + CSE group. A decrease in the expression levels of PDK1, AKT, p16 and LC3B in the airway epithelial tissues of the CS1 group compared with those in the CS + CSE group was also observed. However, there were no significant differences in the expression levels of PI3K between the CS1 and the CS groups. The present study concluded that the inhibition of PDK1 can potentially reduce autophagy and cell senescence by downregulating the expression of PI3K/AKT pathway related proteins in airway epithelial cells, thereby protecting against CS + CSE-induced emphysema in mice.

The Effects of Ginkgo biloba Extract on Autophagy in Human Macrophages Stimulated by Cigarette Smoke Extract.

To investigate the effects of Ginkgo biloba extract (GBE) on autophagy in human macrophages stimulated by cigarette smoke extract (CSE). The human monocyte cell line U937 was cultured in vitro, and phorbol ester (PMA) was added to the cell culture medium to induce differentiation into human macrophages. CSE was prepared by traditional methods for experiments. The cells were divided into four groups: the blank group, the CSE model group, the GBE + CSE group, and the rapamycin + CSE group. Immunofluorescence was used to identify human macrophages, transmission electron microscopy was used to observe the ultrastructure of human macrophages in each group, ELISA was used to measure the amount of IL-6 and IL-10 in the supernatant from each group of cells, the mRNA levels of p62, ATG5, ATG7, and Rab7 were measured by real-time qPCR, and the protein expression levels of p62, ATG5, ATG7, and Rab7 were measured by Western blotting. U937 cells were successfully differentiated into human macrophages after induction with PMA. The CSE model group had many more autophagosomes than the blank group. Compared with the CSE model group, the GBE + CSE group and the rapamycin + CSE group had significantly more autophagolysosomal. Compared with the other groups, the CSE model group had a higher level of IL-6 but a lower level of IL-10 in the supernatant (p < 0.05). Compared with the blank group, the mRNA and protein expression levels of p62 in the CSE model group were significantly decreased, while the mRNA and protein expression levels of ATG5 and ATG7 were significantly increased in the CSE model group (p < 0.05). No difference was found in the mRNA and protein expression levels of Rab7 between the blank group and the CSE model group. Compared with the CSE model group, the IL-6 level in the GBE + CSE group and the rapamycin + CSE group cell culture supernatant decreased significantly, p62 mRNA and protein expression significantly decreased, while ATG5, ATG7, and Rab7 mRNA and protein expression levels were significantly increased (p < 0.05). Moreover, increased LC3-II/LC3-I ratio were also found in the GBE + CSE group and the rapamycin + CSE group compared with the CSE model group. GBE could promote the fusion of autophagosomes and lysosomes in human macrophages, enhance the autophagy function of human macrophages, and reduce the damaging effect of CSE on the autophagy function of macrophages.

Cigarette smoke extract-induced inflammatory response via inhibition of the TFEB-mediated autophagy in NR8383 cells

Objective: Chronic pulmonary inflammation caused by long-term smoking is the core pathology of COPD. Alveolar macrophages (AMs) are involved in the pulmonary inflammation of COPD. The accumulation of damaged materials caused by impaired autophagy triggers inflammatory response in macrophages. As a key transcription regulator, transcription factor EB (TFEB) activates the transcription of target genes related autophagy and lysosome by binding to promoters, whereas it is unclarified for the relationship between inflammatory response induced by cigarette smoke extract (CSE) and TFEB-mediated autophagy. Thus, we investigated the role of TFEB-mediated autophagy in inflammatory response induced by CSE in NR8383 cells, and to explore its potential mechanism. Methods: Based on cell viability and autophagy, cells treated with 20% concentration of CSE for 24 h were selected for further studies. Cells were divided into control group, chloroquine (CQ, the autophagy inhibitor) group, CSE group, CSE + rapamycin (the autophagy inducer) group and CSE + fisetin (the TFEB inducer) group. The levels of tumor necrosis factor α (TNF-α), interleukin 1β (IL-1β), and IL-6 in supernatant were detected by ELISA kits. The protein expressions were tested by western blot. The intensity of fluorescence of Lysosome-associated membrane protein 1 (LAMP1) and TFEB was detected by immunofluorescence. Lyso-Tracker Red staining was applied to detect the lysosome environment. Results: CSE inhibited the cell viability, increased the contents of TNF-α, IL-1β, IL-6, the ratio of LC3II/I, and the level of P62 protein. Besides, CSE decreased the fluorescence intensity of LAMP1 protein and Lyso-Tracker Red staining, as well as the ratio of nucleus/cytosol of TFEB protein. Activating autophagy with rapamycin alleviated CSE-induced inflammatory response. The activation of TFEB via fisetin alleviated CSE-induced autophagy impairment and lysosomal dysfunction, thus alleviated inflammatory response in NR8383 cells. Conclusion: CSE-induced inflammatory response in NR8383 cells, which may be related to the inhibition of TFEB-mediated autophagy.

Effects of cigarette smoke extract on mitochondrial function of macrophages

Objective: To investigate the effects of cigarette smoke extract (CSE) on the mitochondrial function of macrophages. Methods: RAW264.7 macrophages were used for the experiment in this study. When the cell density was about 70%, the old culture medium was abandoned, and the 100% CSE stock solution was diluted with serum-free DMEM and FBS into 1%, 5%, 15%, 25% and 90% CSE and added to the well plate. The cell activity of RAW264.7 cells treated with CSE at different concentrations for 24 h was detected by CCK-8 method. Then the optimal CSE concentration was selected to treat cells for 0 h, 24 h, 48 h or 72 h respectively, and CCK-8 assay was used to detect the cell activity of CSE treated cells at different time groups. After the cells were treated with 0%, 5% and 25% CSE for 24 hours, cell necrosis and apoptosis was detected by Annexin V-FITC /PI staining; Mitochondrial membrane damage of RAW 264.7 was detected by mitochondrial membrane potential assay kit with JC-1; Macrophages were stained with ROS-specific dye DCFH-DA, and then Flow cytometer was used to determine the fluorescence and the proportion of ROS-positive macrophages; the enhanced ATP assay kit was used to detect the intracellular ATP concentration. Results: ①Compared with 0% CSE, cell viability was increased significantly in 1% CSE group (P＜0.01), cell viability was decreased significantly when CSE concentration was above 5% (P＜0.05); Macrophages were treated with 5% CSE, and cell viability was decreased significantly with the increase of treatment time (P＜0.01). ②Compared with 0% CSE, 5% CSE and 25% CSE mainly caused macrophage necrosis, decreased mitochondrial membrane potential, increased ROS production and decreased ATP significantly (P＜0.05 or P＜0.01), and the changes were more significant in 25% CSE treatment group(P＜0.05 or P＜0.01). Conclusion: CSE may affect mitochondrial function of macrophages, leading to decreased cell viability and necrosis.

Cigarette smoke extract stimulates human pulmonary artery smooth muscle cell proliferation: Role of inflammation and oxidative stress.

Objective(s):Cigarette smoke may play a direct role in proliferation of human pulmonary artery smooth muscle cells (HPASMCs). However, the mechanism involved and the effect of interventions remain unclear. We aimed to evaluate the effect of cigarette smoke extract (CSE) on HPASMCs, explore the role of inflammation and oxidative stress, and the effects of Tempol and PDTC in this process.Materials and Methods:HPASMCs were subjected to normal control (NC), CSE, CSE+Tempol (CSE+T), and CSE+PDTC (CSE+P) groups. Proliferation of HPASMCs was measured by CCK-8 and Western blot. TNF-α, IL-6, MDA, and SOD levels were determined by ELISA and commercial kits. Nuclear translocation of NF-κB p65 was evaluated by western blot.Results:1%, 2.5%, and 5% CSE all promoted proliferation of HPASMCs, and effect of 1% CSE was the most significant, however, 7.5% and 10% CSE inhibited viability of cells (all P<0.05). Compared with the NC group, TNF-α, IL-6, and MDA levels increased, SOD activity decreased (all P<0.05), and NF-κB p65 expression in nuclei increased (P=0.04) in the CSE group. Tempol and PDTC inhibited the proliferation of HPASMCs induced by CSE (all P<0.05). And compared with the CSE group, TNF-α, IL-6, and MDA levels in CSE+T and CSE+P groups decreased, while SOD activity increased (all P<0.05). Tempol reduced the expression of NF-κB p65 in nuclei but did not achieve a significant difference (P=0.08). PDTC inhibited the nuclear translocation of NF-κB p65 (P=0.03).Conclusion:CSE stimulates HPASMCs proliferation in a certain concentration range. The CSE-induced proliferation of HPASMCs involved excessive inflammatory response and oxidative stress. Tempol and PDTC attenuate these effects of CSE on HPASMCs.

Effect of erythromycin on the ultrastructure of human macrophages exposed to cigarette smoke extract in vitro

ABSTRACT Macrophages serve an active role in the pathophysiology of chronic obstructive pulmonary disease (COPD). Erythromycin (EM) has been verified as an effective treatment for COPD. However, there are few studies on the effect of EM on the ultrastructure of macrophages exposed to cigarette smoke extract (CSE). In the present study, human macrophages were randomly divided into three groups: The control, CSE and the CSE+EM group, using electron microscopy, the effect of EM was evaluated by comparing the ultrastructural changes between these groups. The macrophages were additionally divided into a further four groups: The control, CSE, CSE+EM 24 h and CSE+EM 48 h groups. The generation of reactive oxygen species (ROS) in each group was evaluated by detecting fluorescence intensity. It was observed that the cellular ultrastructure of the CSE group exhibited abnormal changes, though this effect was reversed back to the level of the control in the CSE+EM group. Compared with the control group, the ROS expression level was significantly increased in the CSE group (P < .05); however, compared with the CSE group, the ROS concentration was decreased in the CSE+EM 24 h (P < .05) and CSE+EM 48 h groups (P < .05), though this was more apparent in the EM 48 h group. It was concluded that EM protects human macrophages against CSE. Moreover, it was hypothesized that EM may reduce the symptoms of patients with COPD by protecting the macrophage ultrastructure from the effects of CSE, resulting in the decreased generation of ROS, inhibiting autophagy and reducing endoplasmic reticulum stress.

Vitamin D3 alleviates cigarette smoke extract-mediated epithelial-mesenchymal transition and fibrogenesis by upregulating CC16 expression in bronchial epithelial cells.

Vitamin D3 supplementation has been previously reported to inhibit the occurrence and development of chronic obstructive pulmonary disease (COPD). However, the underlying mechanism remains unclear. Epithelial-mesenchymal transition (EMT) and fibrogenesis have been associated with the development of COPD. The aim of the present study was to investigate the potential effects and mechanism of vitamin D3 in an in vitro model of cigarette smoke (CS)-induced EMT and fibrosis, with specific focus on the role of club cell protein 16 (CC16). CS extract (CSE) at different concentrations (5, 10 and 20%) was used to treat 16-HBE cells to induce EMT and fibrogenesis following which they were treated with vitamin D3. Subsequently, the 20% CSE group was selected for further experiments, where 16-HBE cells were divided into the following five groups: The control group; the CSE group; the low-dose vitamin D3 group (250 nM); the medium-dose vitamin D3 group (500 nM); and the high-dose vitamin D3 group (1,000 nM). Western blot analysis was used to detect the protein expression levels of the EMT-related proteins E-cadherin, N-cadherin, Slug and α-SMA, fibrogenesis-related proteins collagen Ⅳ and fibronectin 1, proteins involved in the TGF-β1/SMAD3 signaling pathway and CC16. Immunofluorescence was used to measure the protein expression levels of E-cadherin, N-cadherin and collagen Ⅳ. Specific CC16 knockdown was performed using short hairpin RNA transfection to investigate the role of CC16. The results of the present study found that vitamin D3 could increase the protein expression level of CC16 to inhibit the activation of the TGF-β1/SMAD3 signaling pathway; thereby reducing the 20% increase in CSE-induced EMT- and fibrogenesis-related protein expression levels. Following CC16 knockdown, the inhibitory effects of vitamin D3 on EMT- and fibrogenesis-related protein expression were partially reversed. To conclude, these results suggest that vitamin D3 can inhibit the protein expression levels of EMT- and fibrogenesis-related proteins induced by CSE, at least partially through the function of CC16. These findings are expected to provide novel theoretical foundations and ideas for the pathogenesis and treatment of COPD.

Effect of pirfenidone protecting against cigarette smoke extract induced apoptosis

INTRODUCTIONApoptosis of lung structural cells is a significant upstream event involved in COPD pathogenesis. This study was designed to explore whether pirfenidone (PFD) was able to attenuate apoptosis induced by cigarette smoke extract (CSE).METHODSA method of intraperitoneal CSE injection to BALB/C mice was used to establish emphysema mouse model. Terminal deoxynucleotidyl transferase dUTPnick end labeling (TUNEL) assay was applied to evaluate apoptotic cell ratio in mouse lung tissue. The cell viability of HBECs exposed to different concentrations of PFD was measured by Cell Counting Kit-8 (CCK-8) assay. The apoptosis index (AI) of HBECs was tested by flow cytometry. Levels of apoptosis-related protein were determined by Western blotting.RESULTSPFD treatment significantly decreased the AI value in emphysema mouse lung tissue by TUNEL. In HBECs, flow cytometry showed that PFD could significantly reduce AI led by CSE. Both in vitro and in vivo, protein levels of Bax and Cleaved-caspase 3 in CSE group significantly increased in contrast with the control group; while Bcl-2 protein level in CSE group was significantly decreased; moreover, PFD significantly reversed protein level changes of Bcl-2, Bax, and Cleaved-caspase 3 led by CSE.CONCLUSIONSThis study reveals that PFD may potentially protect against CSE induced apoptosis.

Andrographolide and epigallocatechin gallate (EGCG) lower the risk of addiction induced by nicotine and cigarette smoke extract (CSE) in mice

Background: Nicotine, a psychoactive compound from the tobacco plant, produces a reward effect that potentially causes addiction. It is postulated that nicotine addiction occurs through increased reactive oxygen species production in nucleus accumbens, which causes damage to the endogenous antioxidant defense system resulting in an increased need for nicotine intake, which leads to addiction. The antioxidants, andrographolide and epigallocatechin gallate (EGCG), are expected as potential substances to decrease the risk of nicotine addiction. This study aimed to analyze the effect of andrographolide and EGCG on the risk of addiction induced by nicotine and cigarette smoke extract (CSE) in mice. Methods: Thirty-five Balb/c male mice, divided into seven groups, were used in this study. The administered drugs were normal saline 1.0 mL/kg BW as control group, nicotine 0.5 mg/kg BW, CSE 1.0 mg/kg BW, andrographolide 50 mg/kg BW, and EGCG 50 mg/kg BW as pre-treatment. Conditioned place preference (CPP) with a biased design method was used to evaluate the reward effects induced by nicotine and CSE. Several stages were carried out, namely pre-conditioning, conditioning, post-conditioning, extinction, and reinstatement tests. Results: Based on the CPP score, both nicotine (p&lt;0.001) and CSE (p&lt;0.001) groups increased the reward effect significantly compared to that of the normal saline group. The andrographolide + nicotine (p&lt;0.001) and EGCG + nicotine (p&lt;0.001) groups decreased the reward effect significantly compared to that of the nicotine group without pharmacological treatment. Similarly, the andrographolide + CSE (p&lt;0.001) and EGCG + CSE (p&lt;0.01) groups decreased the reward effect significantly compared to that of the CSE group without pharmacological treatment. Conclusions: Andrographolide and EGCG lower the risk of addiction induced by nicotine and CSE.

Existence of multiple organ aging in animal model of emphysema induced by cigarette smoke extract.

INTRODUCTIONIt is commonly considered that COPD or at least emphysema represents accelerated lung aging induced in part by oxidative damage from cigarette smoke components. However, the issue if there are any aging signs in other organs in patients with COPD or emphysema remains unclear. The aim of this study is to explore whether there is multiple organ aging in the animal model of emphysema induced by cigarette smoke extract (CSE), and to ascertain the possible mechanisms, if any.METHODSThe animal model of emphysema was induced by CSE. Histomorphological changes in lung, heart, liver, kidney and spleen tissues were measured after staining with hematoxylin and eosin (H&E). The concentrations of stem cell factor (SCF), CyclinD1 and superoxide dismutase (SOD) in serum were determined by ELISA kit. The expressions of p16 (INK4a), Sca-1, eNOS proteins and mRNA in lung, heart, liver, kidney and spleen tissues were detected by Western blotting and quantitative reverse transcriptase polymerase chain reaction (qRT-PCR), respectively. Decitabine (Dec) was applied to examine whether it could alter the changes caused by CSE.RESULTSThe histomorphology of lung tissue was significantly changed, while other organs exhibited normal structure and histomorphology. The concentrations of SCF, CyclinD1 and SOD in serum were lower in the CSE group than in the control group. The expression levels of p16(INK4a) protein and mRNA in lung, heart, liver, kidney and spleen tissues were higher in the CSE group than in the control group, while the expression levels of Sca-1 and eNOS proteins and mRNA were lower in the CSE group than in the control group, in the tissues described above. Dec could partly alleviate the damages caused by CSE and the degree of alleviation resulted by Dec varied from organ to organ.CONCLUSIONSIn addition to the aging of the lung tissue in the emphysema animal model induced by CSE, the tissues of the heart, liver, kidney and spleen were also in the progress of aging, but the sensibility and affinity of lung to CSE were higher than those of the other organs. Multiple organ aging may also exist in the animal model of emphysema induced by CSE. DEC can partly alleviate the multiple organ aging caused by CSE.

Xuanfei Pingchuan Capsules Ameliorate Autophagy in Human Bronchial Epithelial Cells by Inhibiting p38 Phosphorylation.

Background: This study aimed to investigate the protective effect of Xuanfei Pingchuan Capsules (XFPC) on autophagy and p38 phosphorylation in human bronchial epithelial (HBE) cells induced by cigarette smoke extract (CSE). Methods: HBE cells were divided into five groups: blank, CSE, low XFPC dose (XFPC-L), medium XFPC dose (XFPC-M), and high XFPC dose (XFPC-H). HBE cells were induced by CSE to establish a cell model for chronic obstructive pulmonary disease, and different doses of XFPC medicated serum were used to treat the cells. The Cell Counting Kit-8 was used to detect cell viability. Flow cytometry was used to detect cell apoptosis. Fluorescence microscopy and the expression level of microtubule-associated protein light chain 3 (LC3)-II in immunohistochemical method were used to observe autophagy in cells. Western blot was used to detect the protein expression level of p38, phospho-p38 (p-p38), LC3-I, LC3-II and Beclin 1. Real-time polymerase chain reaction was used to detect the expression of LC3-I, LC3-II and Beclin 1 on mRNA level. Results: Compared with the blank group, the cell viability of the CSE group was significantly decreased, and apoptosis and the level of autophagy in cells were significantly increased. The mRNA and protein expression of LC3-I, LC3-II, Beclin 1 and the protein level of p-p38 were significantly increased in the CSE-HBE cells. Compared to the CSE group, the different doses of XFPC medicated serum increased cell viability, decreased cell apoptosis, and inhibited mRNA and protein expression of LC3-I, LC3-II, Beclin 1 and protein level of p-p38. These results were especially observed in the group XFPC-H. After adding a p38 agonist, the therapeutic effect of XFPC on cell viability and autophagy was suppressed. Conclusion: XFPC significantly increased cell viability in a CSE-induced HBE cell model for chronic obstructive pulmonary disease through inhibiting the level of autophagy mediated by phosphorylation of p38.

C-EBP\u03b2 mediates in cigarette/IL-17A-induced bronchial epithelial\u2013mesenchymal transition in COPD mice

BackgroundCigarettes smoking and IL-17A contribute to chronic obstructive pulmonary disease (COPD), and have synergistical effect on bronchial epithelial cell proliferation. CCAAT/enhancer-binding protein β (C-EBPβ) could be induced by IL-17A and is up-regulated in COPD. We explored the effect of cigarettes and IL-17 on bronchial epithelial–mesenchymal transition (EMT) in COPD mice and potential mechanism involved with C-EBPβ in this study.MethodsCOPD model was established with mice by exposing to cigarettes. E-Cadherin, Vimentin, IL-17A and C-EBPβ distributions were detected in lung tissues. Primary bronchial epithelial cells were separated from health mice and cocultured with cigarette smoke extract (CSE) or/and IL-17A. E-Cadherin, Vimentin and IL-17 receptor (IL-17R) expressions in vitro were assessed. When C-EBPβ were silenced by siRNA in cells, E-Cadherin, Vimentin and C-EBPβ expressions were detected.ResultsE-Cadherin distribution was less and Vimentin distribution was more in bronchus of COPD mice than controls. IL-17A and C-EBPβ expressions were higher in lung tissues of COPD mice than controls. In vitro, C-EBPβ protein expression was highest in CSE + IL-17A group, followed by CSE and IL-17A groups. E-cadherin expression in vitro was lowest and Vimentin expression was highest in CSE + IL-17A group, followed by CSE or IL-17A group. Those could be inhibited by C-EBPβ silenced.ConclusionsC-EBPβ mediates in cigarette/IL-17A-induced bronchial EMT in COPD mice. Our findings contribute to a better understanding on the progress from COPD to lung cancers, which will provide novel avenues in preventing tumorigenesis of airway in the context of cigarette smoking.

The Role of Ferroptosis in Bronchoalveolar Epithelial Cell Injury Induced by Cigarette Smoke Extract.

Background: Cigarette smoking is a major risk factor for bronchoalveolar epithelial cell (BAEC) injury. Understanding the relevant pathogenesis is important for the treatment of cigarette smoke–related chronic airway diseases such as chronic obstructive pulmonary disease.Methods: In this study, BAECs were cultured in 5% cigarette smoke extract (CSE) or regular culture medium for 24 h. Differentially expressed genes (DEGs) were detected by next-generation RNA sequencing (RNA-seq) and validated by quantitative reverse transcription polymerase chain reaction (qRT-PCR). Bioinformatic analysis was performed on DEGs. Co-treated BAECs with 5% CSE and the ferroptosis inhibitor, ferrostatin-1 was applied to observe the role of ferroptosis.Results: In the CSE group, 210 upregulated genes and 159 downregulated genes were identified compared with the control group. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses showed that the DEGs were related to oxidative stress and ferroptosis. Ferroptosis-related genes were further verified by qRT-PCR. The mRNA level of GPX4 decreased; the mRNA levels of ACSL4, FTH1 and SLC7A11 increased (p < 0.05). Pretreatment with the ferroptosis inhibitor ferrostatin-1 mitigated CSE-induced ROS accumulation and inflammatory mediator expression in BAECs (p < 0.05).Conclusion: CSE treatment altered ferroptosis-related gene expression patterns in cultured BAECs. Inhibition of ferroptosis reduced the inflammatory response of CSE-treated BAECs. These data provide a better understanding of the underlying molecular mechanisms of CSE-related lung injury.

Cigarette smoke promotes oral leukoplakia via regulating glutamine metabolism and M2 polarization of macrophage

Oral immunosuppression caused by smoking creates a microenvironment to promote the occurrence and development of oral mucosa precancerous lesions. This study aimed to investigate the role of metabolism and macrophage polarization in cigarette-promoting oral leukoplakia. The effects of cigarette smoke extract (CSE) on macrophage polarization and metabolism were studied in vivo and in vitro. The polarity of macrophages was detected by flow cytometric analysis and qPCR. Liquid chromatography-mass spectrometry (LC-MS) was used to perform a metabolomic analysis of Raw cells stimulated with CSE. Immunofluorescence and flow cytometry were used to detect the polarity of macrophages in the condition of glutamine abundance and deficiency. Cell Counting Kit-8 (CCK-8), wound-healing assay, and Annexin V-FITC (fluorescein isothiocyanate)/PI (propidium iodide) double-staining flow cytometry were applied to detect the growth and transferability and apoptosis of Leuk-1 cells in the supernatant of Raw cells which were stimulated with CSE, glutamine abundance and deficiency. Hyperkeratosis and dysplasia of the epithelium were evident in smoking mice. M2 macrophages increased under CSE stimulation in vivo and in vitro. In total, 162 types of metabolites were detected in the CSE group. The metabolites of nicotine, glutamate, arachidic acid, and arginine changed significantly. The significant enrichment pathways were also selected, including nicotine addiction, glutamine and glutamate metabolism, and arginine biosynthesis. The results also showed that the supernatant of Raw cells stimulated by CSE could induce excessive proliferation of Leuk-1 and inhibit apoptosis. Glutamine abundance can facilitate this process. Cigarette smoke promotes oral leukoplakia via regulating glutamine metabolism and macrophage M2 polarization.

Pirfenidone mediates cigarette smoke extract induced inflammation and oxidative stress in vitro and in vivo

BackgroundAntioxidant and anti-inflammatory effects are two main pharmacological mechanisms of pirfenidone (PFD) besides the anti-fibrotic effect. This study aims to investigate whether PFD could mediate cigarette smoke extract (CSE) induced inflammation and oxidative stress in vitro and in vivo. MethodsBALB/C mice and alveolar epithelial (A549) cells treated with CSE were established as disease models in vivo and in vitro. Effects of PFD treatment on disease models were further measured. Hematoxylin and eosin (HE) staining was used to evaluate the pathological changes in lung tissues of mice. CCK-8 assay kit was applied to measure the viability of A549 cells treated by different concentrations of PFD. Inflammation cytokine expression in cell supernatants was measured with ELISA kits. The mRNA and protein levels of inflammation and oxidative stress-related factors were determined by real-time quantitative polymerase chain reaction analysis (RT-qPCR) and Western blotting. Furthermore, myeloperoxidase (MPO), malondialdehyde (MDA), and total antioxidant capacity (T-AOC) were measured to detect the antioxidative activity of lung tissues. Moreover, an assay kit with fluorescent probe 2′,7′-dichlorofluorescin diacetate (DCFH-DA) was used to evaluate the intracellular reactive oxygen species (ROS) generation. ResultsIn vitro and in vivo, PFD significantly reversed TNF-α, IL-6, CCL2, SOD1, and CAT mRNA level changes led by CSE; in addition, PFD significantly decreased the ratios of p-p65 to p65, p-ikBα to ikBα and increased Nrf-2 protein level compared with CSE group. In mice, high-dose (100 mg/kg/d) PFD significantly reversed MPO and MDA increases induced by CSE. However, PFD didn’t significantly reverse T-AOC decrease induced by CSE. In A549 cell supernatant, PFD dramatically reversed the elevated levels of TNF-α and IL-1β induced by CSE. Furthermore, PFD could significantly reverse the increased level of ROS induced by CSE in A549 cells. ConclusionOur study reveals the potential role of PFD in regulating inflammatory response and oxidative stress induced by CSE.

The changes of MRP2 expression in three kinds of pulmonary inflammation models: the downregulation occurred in cigarette smoke extract (CSE) stimulation group and CSE plus LPS stimulation group, unchanged in LPS stimulation group

The transporter multidrug resistance protein 2 (MRP2) can transport some tobacco carcinogens and plays an important role in the transport of mediators related to pulmonary inflammatory diseases. However, it is not fully understood whether the pulmonary inflammation caused by cigarette smoke extract (CSE) and lipopolysaccharide (LPS) is related to the regulation of MRP2. In this study, CSE and LPS were used alone and in combination as stimuli to induce pulmonary inflammation. In addition, the establishment of a pulmonary inflammation model was verified by animal experiments in vivo. We found that compared with those in the control group, the expression of MRP2 protein was downregulated and the expression of inflammatory cytokines was upregulated in pulmonary inflammation in the CSE group and the CSE combined with LPS group. However, there was almost no change in the expression of MRP2 stimulated by LPS alone. Our results show that CSE and CSE combined with LPS downregulate the expression of MRP2 under inflammatory conditions, while LPS has almost no effect on the expression of MRP2 under inflammatory conditions. The in vivo experimental results of CSE combined with LPS were consistent with the cellular results of CSE combined with LPS, which provides a model and basis for other studies of the role of MRP2 in pulmonary inflammation.

Establishment of an in vitro model of alveolar macrophage cell efferocytosis dysfunction

To screen the time points of high survival rate and efferocytosis dysfunction of rat alveolar macrophages stimulated by cigarette smoke extract (CSE), establish an in vitro model of alveolar macrophage efferocytosis function, and study chronic respiratory diseases with chronic inflammatory reaction as the main pathological changes. (1) Time point screening experiment: rat alveolar macrophages (NR8383 cells) were cultured in vitro, and the cells in logarithmic growth phase were divided into blank control group (100 μL complete medium) and 5% CSE group (90 μL complete medium + 10 μL 100% CSE). Alma blue method was used to detect the effect of 5% CSE on the activity of NR8383 cells at 6, 12, 24 and 48 hours. (2) Apoptosis induction experiment: rat type II alveolar epithelial cells (RLE-6TN cells) were cultured in vitro as phagocytic target cells of NR8383 cells, and the cells in logarithmic growth phase were divided into blank control group and 10, 30 and 60 minutes groups after ultraviolet exposure (apoptosis was induced by 30 000 μJ/cm2 ultraviolet irradiation for 15 minutes). Flow cytometry was used to detect the apoptosis rate of RLE-6TN cells cultured for 10, 30 and 60 minutes after ultraviolet exposure. (3) Cell efferocytosis experiment: NR8383 cells in logarithmic phase were divided into blank control group and 5% CSE group. Two hours before NR8383 cells were stimulated by CSE for 6, 12 and 24 hours, RLE-6TN cells were exposed to ultraviolet to induce apoptosis, and the RLE-6TN cell suspension was added to NR8383 cells (the ratio of RLE-6TN cells to NR8383 cells was 5:1). Flow cytometry was used to detect the efferocytosis rate of NR8383 cells to RLE-6TN cells at different time points treated with 5% CSE. (1) Compared with the blank control group, the activity of NR8383 cells significantly decreased after treatment with 5% CSE for 48 hours [cell reduction rate: (68.5±4.1)% vs. (73.6±2.3)%, P < 0.05]. However, there were no significant differences when the activities of NR8383 cells treated with 5% CSE for 6, 12 and 24 hours were compared with the blank control group, so these three time points were selected for the subsequent establishment of alveolar macrophage cell efferocytosis dysfunction in vitro model experiment. (2) Compared with the blank control group, the apoptosis rate of RLE-6TN cells significantly increased at 10, 30 and 60 minutes after ultraviolet exposure [(66.87±8.63)%, (85.51±2.39)%, (96.13±2.74)% vs. (9.13±3.17)%, all P < 0.01] in a time-dependent manner. Considering that it taked about 50 minutes for RLE-6TN cells to be labeled with PKH26 membrane labeling probe, 10 minutes after ultraviolet exposure was selected to label RLE-6TN cells. (3) Compared with the blank control group, the efferocytosis function of NR8383 cells was significantly decreased after treatment with 5% CSE for 12 hours [cell efferocytosis rate: (33.64±1.30)% vs. (44.02±2.71)%, P < 0.01], but there was no significant effect on the efferocytosis function of NR8383 cells at 6 hours and 24 hours. CSE can induce alveolar macrophage cell efferocytosis dysfunction. Based on the test results of the effect of 5% CSE on NR8383 cell activity and cell efferocytosis function, 12 hours with high survival rate and weak efferocytosis effect of NR8383 cells can be selected as the in vitro model condition of alveolar macrophage cell efferocytosis dysfunction.