Shixin Chen

N -Acetyl- L -cysteine and pyrrolidine dithiocarbamate inhibited nuclear factor-kappaB activation in alveolar macrophages by different mechanisms

AbstractAim:To study the effects of N-acetyl-L-cysteine (NAC) and pyrrolidine dithiocarbamate (PDTC) on the phosphorylation of IκB kinase (IKK)β, IKKα, and IκBα in alveolar macrophages (AM), and to explore the pharmacological mechanisms of NAC and PDTC as inhibitors of NF-κB activation.Methods:AM were collected from bronchoalveolar lavage fluid from the patients with chronic obstructive pulmonary disease. The AM were incubated for 1.5 h with NAC and PDTC, and then stimulated for 90 min by either tumor necrosis factor (TNF)-α or interleukin (IL)-1. Western blotting was used to detect the protein phosphorylation levels of IKKβ, IKKα, and IκBα. NF-κB activity was analyzed by using an electrophoretic mobility shift assay.Results:NAC inhibited the phosphorylation of IKKβ, IKKα, and IκBα induced by TNF-α, but had no effect on the phosphorylation of IKKβ, IKKα and IκBα induced by IL-1. PDTC did not inhibit the phosphorylation of IκBα induced by TNF-α or IL-1. Similarly, NAC inhibited the activation of NF-κB induced by TNF-α, but had no effect on the activation of NF-κB induced by IL-1. PDTC significantly inhibited the activation of NF-κB induced by TNF-α and IL-1. The electrophoretic mobility shift assay also showed that PDTC and NAC do not directly inhibit NF-κB DNA binding activity in vitro.Conclusion:PDTC prevents the degradation of IκBα via the ubiquitylation-proteasome proteolytic pathway. NAC can inhibit the processes upstream of IKK activation induced by TNF-α, which results in the decline of NF-κB activity.

Plasmid-based short hairpin RNA against cyclin D1 attenuated pulmonary vascular remodeling in smoking rats

Accumulating evidence indicated that smoking might directly induce pulmonary vascular remodeling at the initial stage of chronic obstructive pulmonary disease (COPD). However, the molecular mechanism underlying this process remains poorly understood. To investigate the role of cyclin D1 in pulmonary vascular remodeling, we constructed a plasmid-based short hairpin RNA (shRNA) to knock down the expression of cyclin D1 in smoking rats. Specific shRNA against cyclin D1 significantly prevented the cyclin D1 expression and the cell proliferation in rat pulmonary artery smooth muscle cells (rPASMCs). Furthermore, the plasmid-based shRNA successfully decreased the cyclin D1 protein in intra-pulmonary arteries of smoking rats and subsequently decreased the wall thickness of pulmonary vessels and the percentage of muscularized vessels. We conclude that the plasmid-based shRNA against cyclin D1 gene attenuated pulmonary vascular remodeling in smoking rats. Cyclin D1 might play a critical role in cigarette smoke-induced pulmonary vascular remodeling via regulating rPASMCs proliferation.

Expression of the Th1-Specific Cell-Surface Protein Tim-3 Increases in a Murine Model of Atopic Asthma

T-cell immunoglobulin- and mucin-domain-containing molecule-3 (Tim-3) is preferentially expressed on Th1-helper type T-cells and functions to repress the Th1-mediated immune response. However, the role of Tim-3 during the inflammatory pathogenesis of asthma remains unclear. This study determines the expression level of Tim-3 in CD4+ T-cells within the peripheral blood and bronchoalveolar lavage fluid (BALF) isolated from a murine model of atopic asthma and explores the potential role of Tim-3 during the inflammatory response. Mice were randomly divided into normal control, asthma day 1, and asthma day 7 groups, and peripheral blood T lymphocytes and BALF cells were collected. The ratio of Tim-3+/CD4+ cells among the total CD4+cell populations from peripheral blood and BALF was determined by flow cytometry, and the expression of the Tim-3 mRNA was determined by Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR). In contrast with the normal control group, the ratio of Tim-3+/CD4+:CD4+ cells and the level of Tim-3 mRNA in both the peripheral blood T lymphocytes and BALF cells among the asthma day 1 and asthma day 7 groups were significantly increased (p < 0.01), and those in the asthma day 7 group were higher than the asthma day 1 group (p < 0.05). There was also a positive correlation between the ratio of Tim-3+/CD4+:CD4+ detected in BALF and that the ratio detected in peripheral blood T lymphocytes (r = 0.84, p < 0.01). Therefore, the expression of Tim-3 is increased in CD4+ T-cells following airway challenge and likely affects asthma-induced inflammation by repressing the Th1-mediated immune response.

ERK1/2 signaling pathway modulates the airway smooth muscle cell phenotype in the rat model of chronic asthma.

Background: It has been demonstrated that the phenotypic modulation of airway smooth muscle cells (ASMCs) is important to the pathogenesis of airway remodeling in chronic asthma. The extracellular signal-regulated kinase 1/2 (ERK1/2) signaling pathway is one of the most important transduction pathways involved in the process of asthma; however, its role in the phenotypic transition of ASMCs remains unclear. Objectives: To examine the role of ERK1/2 in the phenotypic modulation of ASMCs in the rat model of chronic asthma. Methods: Bronchial smooth muscle strips were cultured in vitro in the presence of the ERK1/2 agonist epidermal growth factor or/and the MEK inhibitor PD98059. The phenotype of ASMCs was determined by observing these cells under an electron microscope and analyzing expression of phenotypic markers (smooth muscle α-actin for the contractile phenotype and osteopontin for the synthetic) by using Western blot and reverse-transcriptase polymerase chain reaction, respectively. Results: The phenotype of the ASMCs from the chronic asthmatic rats changed from the contractile type to the synthetic type with synthetic organelles abundantly gathered around the nucleus and altered expression of phenotypic markers. ERK1/2 was strongly expressed in the ASMCs of the chronic asthmatic rats and its activation by epidermal growth factor excessively promoted the synthetic function of ASMCs; the MEK inhibitor PD98059, however, reversed this phenotypic change in the ASMCs. Conclusions: Our results reveal a key role of the ERK1/2 signaling pathway in the phenotypic modulation of ASMCs in chronic asthmatic rats, indicating that specific inhibition of ERK1/2 in ASMCs may be therapeutically valuable in the control of airway remodeling in chronic asthma.

The effect of Ginkgo Biloba Extract on the expression of PKCα in the inflammatory cells and the level of IL-5 in induced sputum of asthmatic patients

To investigate the effect of the Ginkgo Biloba Extract (GBE) on the asthma and examine its possible mechanisms, 75 asthma patients were divided into 4 groups and the patients were respectively treated with fluticasone propionate for 2 weeks or 4 weeks, or treated with fluticasone propionate plus GBE for 2 weeks or 4 weeks. Fifteen healthy volunteers served as healthy controls. Sputum inhalation with inhaling hypertonic saline (4%–5%) was performed. Lung ventilatory function and forced expiratory volume in one second (FEV1) were measured. The numbers of different cells in induced sputum were calculated. The expression of PKCα in the cells was immunocytochemically detected and the percentages of positive cells in different cells were counted. Interleukin-5 (IL-5) in sputum supernatants was detected with enzyme-linked immunosorbent assay. The percentage of eosinophils, lymphocytes, PKCα positive inflammatory cells and the concentration of IL-5 in asthmatic patients were higher than those in the controls (P<0.05), and the eosinophils, lymphocytes, positive expression of PKCα and the level of IL-5 were significantly decreased in asthmatic patients after they were treated with fluticasone propionate or fluticasone propionate plus GBE. However, they were still significantly higher than those of the controls. Compared to the group treated with glucocorticosteroid for 2 weeks, no significant decrease was found in the percentage of eosinophils, lymphocytes, PKCα positive inflammatory cells and the IL-5 in the supernatant of induced sputum. Compared with the group treated with glucocorticosteroid for 2 or 4 weeks, significant decrease in the same parameters was observed in the group treated with fluticasone propionate and GBE for 4 weeks. The IL-5 level in the supernatant of induced sputum was positively correlated with the percentage of PKCα-positive inflammatory cells and the percentage of eosinophils in the induced sputum in asthma patient groups respectively (n=150, r= 0.83, P<0.01; n=150, r=0.76, P<0.01). The FEV1 was negatively correlated with the percentage of PKCα-positive inflammatory cells and the IL-5 levels in supernatant of induced sputum in asthma patients respectively (n=150, r=−0.77, P<0.01; n=150, r= −0.64, P<0.01). It is concluded that GBE could significantly decrease the infiltration of inflammatory cells such as eosinophils and lymphocytes in the asthmatic airway and relieve the airway inflammation. GBE may decrease the activation of the PKCα in the inflammatory cells and thereby decrease the IL-5 level in induced sputum. GBE may be used as a complement to the glucocorticosteroid therapy for asthma.

PKC promotes proliferation of airway smooth muscle cells by regulating cyclinD1 expression in asthmatic rats

AbstractAim:To determine whether protein kinase C (PKC) has any effect on the expression of cyclinD1, a key regulator of growth control and G1/S transition, and to investigate the underlying molecular mechanisms of PKC involving the remodeling of the asthmatic airway smooth muscle (ASM).Methods:The treatment of synchronized ASM cells from asthmatic rats with PKC-specific agonist phorbol 12-myristate 13-acetate (PMA) and antagonist 2-{1-[3-(amidinothio) propyl]-1H-indol-3-yl}-3-(1-methylindol-3-yl) maleimide methanesulfonate salt (Ro31-8220) was followed by the proliferation assay. PKCα and cyclinD1 expressions in ASM cells (ASMC) were detected by RT-PCR and Western blotting. The relation between PKCα and cyclinD1 was assessed by linear regression analysis. The effect of the construct recombinant plasmid pcDNA3.1-antisense cyclinD1 (pcDNA3.1-ascyclinD1) on the proliferation of ASMC was found to be induced by PMA.Results:The data showed phorbol ester-dependent PKCα promoted the proliferation of ASMC. The closely-positive correlation existed between the expression of PKCα and cyclinD1 at the transcriptional (r=0.821, P<0.01) and transla-tional (r=0.940, P<0.01) levels. pcDNA3.1-ascyclinD1 could inhibit the proliferation of ASMC. pcDNA3.1-ascyclinD1 almost completely attenuated the PMA-induced proliferation effect as Ro31-8220+pcDNA3.1.Conclusion:The proliferation of ASMC by PKC might by regulated by the cyclinD1 expression in asthmatic rats.

Effects of puerarin on pulmonary vascular remodeling and protein kinase C-α in chronic cigarette smoke exposure smoke-exposed rats

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 divided into 7 groups: control group (C group), smoke exposure groups (S4w group, S8w group), puerarin 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 puerarin. 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 transcription-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 expression were increased greatly, PASMC apoptosis was increased and proliferation was markedly increased; 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 percentage 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 imbalance of PASMC proliferation and apoptosis. Puerarin appears to be able to reduce cell proliferation and vascular remodeling possibly through PKC signaling transduction pathway.SummaryIn 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 divided into 7 groups: control group (C group), smoke exposure groups (S4w group, S8w group), puerarin 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 puerarin. 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 transcription-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 expression were increased greatly, PASMC apoptosis was increased and proliferation was markedly increased; 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 percentage 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 imbalance of PASMC proliferation and apoptosis. Puerarin appears to be able to reduce cell proliferation and vascular remodeling possibly through PKC signaling transduction pathway.

Expression of Nitric Oxide Synthase Isoenzyme in Lung Tissue of Smokers with and without Chronic Obstructive Pulmonary Disease.

Background:It has been demonstrated that only 10%–20% cigarette smokers finally suffer chronic obstructive pulmonary disease (COPD). The underlying mechanism of development remains uncertain so far. Nitric oxide (NO) has been found to be closely associated with the pathogenesis of COPD, the alteration of NO synthase (NOS) expression need to be revealed. The study aimed to investigate the alterations of NOS isoforms expressions between smokers with and without COPD, which might be helpful for identifying the susceptibility of smokers developing into COPD. Methods:Peripheral lung tissues were obtained from 10 nonsmoker control subjects, 15 non-COPD smokers, and 15 smokers with COPD. Neuronal NOS (nNOS), inducible NOS (iNOS), and endothelial NOS (eNOS) mRNA and protein levels were measured in each sample by using real-time polymerase chain reaction and Western blotting. Results:INOS mRNA was significantly increased in patients with COPD compared with nonsmokers and smokers with normal lung function (P < 0.001, P = 0.001, respectively). iNOS protein was also higher in COPD patients than nonsmokers and smokers with normal lung function (P < 0.01 and P = 0.01, respectively). However, expressions of nNOS and eNOS did not differ among nonsmokers, smokers with and without COPD. Furthermore, there was a negative correlation between iNOS protein level and lung function parameters forced expiratory volume in 1 s (FEV1) (% predicted) (r = −0.549, P = 0.001) and FEV1/forced vital capacity (%, r = −0.535, P = 0.001). Conclusions:The expression of iNOS significantly increased in smokers with COPD compared with that in nonsmokers or smokers without COPD. The results suggest that iNOS might be involved in the pathogenesis of COPD, and may be a potential marker to identify the smokers who have more liability to suffer COPD.

Inhibitory activity of nuclear factor-kappaB potentiates cisplatin-induced apoptosis in A549 cells.

SummaryWhether inhibiting the activity of nuclear factor (NF)-κB potentiates cisplatin-induced apoptosis in non-small cell lung cell line A549 cells was investigated. The recombinant plasmid pcDNA3.1 (+)/IκBα expressing IκBα was constructed. The in vitro cultured A549 cells were transfected with pcDNA3.1 (+)/IκBα alone, or pcDNA3.1 (+)/IκBα combined with cisplatin. The mitochondrial membrane potential (Δψm) was determined by rhodamine 123, the activity of caspase-3 was tested by colorimetric assay, and cell apoptosis was detected by flow cytometry with the annexin V /propidium iodide assay. The results showed that the activity of NF-κB in A549 cells was inhibited by transfecting pcDNA3.1(+)/I. Transfection of pcDNA3.1(+)/I alone did not promote apoptosis. Treatment of cisplatin alone had a little effect on cell apoptosis. Transfection of pcDNA3.1(+)/I combined with cisplatin treatment significantly induced apoptosis of A549 cells. It was concluded that inhibiting the activity of NF-B potentiated cisplatin-induced apoptosis of A549 cells.Whether inhibiting the activity of nuclear factor (NF)-κB potentiates cisplatin-induced apoptosis in non-small cell lung cell line A549 cells was investigated. The recombinant plasmid pcDNA3.1 (+)/IκBα expressing IκBα was constructed. The in vitro cultured A549 cells were transfected with pcDNA3.1 (+)/IκBα alone, or pcDNA3.1 (+)/IκBα combined with cisplatin. The mitochondrial membrane potential (Δψm) was determined by rhodamine 123, the activity of caspase-3 was tested by colorimetric assay, and cell apoptosis was detected by flow cytometry with the annexin V /propidium iodide assay. The results showed that the activity of NF-κB in A549 cells was inhibited by transfecting pcDNA3.1(+)/I. Transfection of pcDNA3.1(+)/I alone did not promote apoptosis. Treatment of cisplatin alone had a little effect on cell apoptosis. Transfection of pcDNA3.1(+)/I combined with cisplatin treatment significantly induced apoptosis of A549 cells. It was concluded that inhibiting the activity of NF-B potentiated cisplatin-induced apoptosis of A549 cells.

Small interfering RNA against ERK1/2 attenuates cigarette smoke‑induced pulmonary vascular remodeling

Cigarette smoke may contribute to pulmonary vascular remodeling (PVR), a result of the proliferation of pulmonary artery smooth muscle cells (PASMCs), before pulmonary hypertension in chronic obstructive pulmonary disease (COPD). Activated extracellular signal-regulated kinases 1 and 2 (ERK1/2) are considered to be involved the process of PVR. This study investigated the potential role of ERK1/2 in the proliferation of rat PASMCs (rPASMCs) and cigarette smoke-induced PVR in rats. A small interfering RNA (siRNA) against ERK1/2 (ERK1/2-siRNA) was synthesized, and it significantly reduced the expression of ERK1/2 and cyclin E1, significantly increased the proportion of cells arrested at G0/G1 phase and significantly suppressed the proliferation of rPASMCs treated with cigarette smoke extract compared with controls (all P<0.05). In rats, ERK1/2-siRNA, which was administered intranasally, also inhibited the activation of ERK1/2 and the upregulation of cyclin E1, both of which were induced after the rats were exposed to cigarette smoke for 3 months. ERK1/2-siRNA also significantly reduced PVR (observed by vessel wall thickness and the proportion of fully muscularized vessels) in cigarette smoke-exposed rats compared with a negative control siRNA (P<0.05). Collectively, these data indicated that ERK1/2-siRNA could attenuate PVR in cigarette smoke-exposed rats, and it may have therapeutic value in the treatment of COPD.