Yong-jian Xu

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.

Tumour necrosis factor alpha ‐308G/A polymorphism and risk of the four most frequent cancers: a meta‐analysis

The latest data show that breast, prostate, lung and colorectal cancer are the four most frequent cancers in both sexes worldwide. A number of molecular epidemiological studies have been conducted to examine the association between TNF alpha ‐308G/A and the risk of those cancers. However the results have been inconclusive or inconsistent. We then performed a meta‐analysis to derive a precise estimation of this association. We carried out a comprehensive search in Medline, EMBASE, OVID and Chinese Biomedical Literature Database for studies using related keywords. The inclusion criteria were (i) in English or Chinese; (ii) case–control study on this association; (iii) provide usable genotype frequencies; and (iv) sufficient published data for estimating an odds ratio (OR) with 95% confidence interval (CI). ORs and 95% CIs were calculated to assess the strength of this association under homozygote comparison (AA vs GG), heterozygote comparison (GA vs GG), dominant (AA/GA vs GG) and recessive (AA vs GA/GG) genetic model comparison. Thirty case–control studies with a total number of 16 507 cases and 19 749 controls were selected for analysis. Overall, no significant association was found between this polymorphism and the risk of total four cancers (GA vs GG: OR = 1.02, 95% CI = 0.91–1.14, P = 0.78). However, there was a significant association between this polymorphism and breast cancer risk in western populations (GA vs GG: OR = 0.91, 95% CI = 0.85–0.96, P = 0.002). This meta‐analysis also revealed that this polymorphism was not associated with susceptibility to the other three cancers.

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.

The alteration and significance of surfactant protein A in rats chronically exposed to cigarette smoke

In order to confirm the alteration and significance of cigarette smoke exposure on SP-A in rats, 20 Wistar rats were assigned randomly to two groups: an N group (n=10), and an S group (n=10). The ultra-structural change was observed by electron microscopy. The number of cells positive for SPA was by immunohistochemically measured. The mRNA expression in the lung tissues was determined by reverse transcription polymerase chain reaction (RT-PCR). The number of cells positive for SPA of the S group (0.52 ± 0.05) was lower than that of the N group (0.72±0.06) (P<0.05). The levels of mRNA of SPA in the lung tissues of the S group (0.3522±0.0512) was significantly lower than that of the N group (0.4432±0.05628) (P<0.05). It is concluded that cigarette smoke alone decreased the level of SP-A and that might have an important effect on surfactant metabolism and the host defense functions of surfactant in the peripheral airways, which might play a crucial role in the development of chronic obstructive lung disease.SummaryIn order to confirm the alteration and significance of cigarette smoke exposure on SP-A in rats, 20 Wistar rats were assigned randomly to two groups: an N group (n=10), and an S group (n=10). The ultra-structural change was observed by electron microscopy. The number of cells positive for SPA was by immunohistochemically measured. The mRNA expression in the lung tissues was determined by reverse transcription polymerase chain reaction (RT-PCR). The number of cells positive for SPA of the S group (0.52 ± 0.05) was lower than that of the N group (0.72±0.06) (P<0.05). The levels of mRNA of SPA in the lung tissues of the S group (0.3522±0.0512) was significantly lower than that of the N group (0.4432±0.05628) (P<0.05). It is concluded that cigarette smoke alone decreased the level of SP-A and that might have an important effect on surfactant metabolism and the host defense functions of surfactant in the peripheral airways, which might play a crucial role in the development of chronic obstructive lung disease.

The effects of protein kinase C (PKC) on the tension of normal and passively sensitized human airway smooth muscle and the activity of voltage-dependent delayed rectifier potassium channel (Kv)

The effects of protein kinase C (PKC) on the tension and the activity of voltage-dependent delayed rectifier potassium channel (Ky) were examined in normal and passively sensitized human airway smooth muscle (HASM), by measuring tones and whole-cell patch clamp techniques, and the Kv activities and membrane potential (E m) were also detected. The results showed that phorbol 12-myristate 13-acetate (PMA), a PKC activator, caused a concentration-dependent constriction in normal HASM rings. The constriction of the passively sensitized muscle in asthma serum group was significantly higher than that of the normal group (P<0.05), and the constrictions of both groups were completely abolished by PKC inhibitor Ro31-8220 and calcium channel inhibitor nifedipine. Kv activities of HASM cells were significantly inhibited by PMA, and the E m became more positive, as compared with the DMSO (a PMA menstruum)-treated group (P<0.01). This effect could be blocked by Ro31-8220 (P<0.01). It was concluded that activation of PKC could increase the tones of HASM, which might be related to the reduced Kv activity. In passively sensitized HASM rings, this effect was more notable.

Effects of calcium-activated chloride channels on proliferation of pulmonary artery smooth muscle cells in rats under chronic hypoxic condition

Abstract Objective To investigate the effects of calcium-activated chloride (ClCa) channels on proliferation of pulmonary artery smooth muscle cells(PASMCs) in rats under chronic hypoxic condition. Methods The cultured PASMCs were placed under normoxic and chronic hypoxic conditions:The cells were observed by light and electron microscope; The cell cycles were observed by flow-cytometry; Immunocytochemistry staining was used to detect the expressions of PCNA, c-fos and c-jun of PASMCs; Cytoplasmic free Ca2+ concentration ([Ca2+]i) in PASMCs was investigated by fluorescent quantitation using fluorospectrophotometer. Results The PASMCs were contractile phenotype under normoxic conditions. Observation by transmission electron microscope: In kytoplasm of contractile phenotype cells, myofilament bundles were abundant and the content of cell organs such as Golgis bodies were rare. The PASMCs were synthetic phenotype under chronic hypoxic condition. There were increased free ribosomes, dilated rough endoplasmic reticulums, highly developed Golgi complexes, decreased or disappeared thick filaments and dense body in kytoplasm of synthetic phenotype cells. After NFA and IAA-94, the situations were reversed The number of S+G2M PASMCs were significantly increased in chronic hypoxic condition; The NFA and IAA-94 were shown to significantly decrease them from (28.6 ± 1.0)% to (16.0 ± 1.6)% and the number of G0G1 PASMCs significantly increased from (71.4 ± 1.9)% to (83.9 ± 1.6)% (P Conclusion Hypoxia initiated the change of PASMCs from contractile to synthetic phenotype and increased proliferation of PASMCs. NFA and IAA-94 depressed cell proliferation by blocking ClCa channels in hypoxic condition. These may play an important role in proliferation of PASMCs under chronic hypoxic conditions.

Expression of leukemia inhibitory factor in airway epithelial tissue of asthmatic rats.

In order to investigate the expression of leukemia inhibitory factor (LIF) in airway epithelial tissues of normal and asthmatic rats, the influence of dexamethasone and the role of LIF in pathogenesis of asthma, 30 Sprague-Dawley (SD) rats were randomly divided into 3 groups (10 for each group): normal group, asthma model group, and dexamethasone-interfered group. In asthma model group and dexamethasone-interfered group, asthma rat models were established by intraperitoneal (i.p.) injection of 10% ovalbumin (OVA) and challenge with 1% OVA via inhalation. Rats in dexamethasone-interfered group were pretreated with dexamethasone (2 mg/kg, i.p) 30 min before each challenge. The expression of LIF protein in lung was detected by immunohistochemistry. The results showed that LIF protein was mainly expressed in cytoplasm of bronchial epithelial cells. The expression of LIF protein in the airway epithelial tissue of asthma model group was significantly higher than that in normal group and dexamethasone-interfered group (P<0.01), but there was no significant difference between normal group and dexamethasone-interfered group (P>0.05). It was concluded that the expression of LIF was increased significantly in the airway epithelial tissue of the asthma rats, and dexamethasone could down-regulate the expression of LIF. It was suggested that LIF might play an important role in the pathogenesis of asthma as an inflammation regulator.

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.

Effects of IL-4 on cyclooxygenase-2 and platelet-derived growth factor in the lungs of COPD rats

Abstract Objective To explore the role of interleukin 4 (IL-4), expressions of cyclooxygenase-2 (COX-2) and platelet-derived growth factor (PDGF) in the model of chronic obstructive pulmonary disease (COPD), in the lungs of rats. Methods Male Wistar rats were used to build up the model of COPD. Rats were randomly divided into the control group and model group, the IL-4 group and the dexamethasone group. The expressions of COX-2, PDGF-A and PDGF-B in the lung tissue was detected by western blotting and RT-PCR. Results The expressions of COX-2, PDGF-A and PDGF-B in the model group were increased significantly. Those expressions in the IL-4 and dexamethasone group were notably decreased. Conclusion IL-4 and dexamethasone could interfere in the establishment of COPD. The expressions of COX-2 and PDGF in the lung tissue of COPD were increased significantly and IL-4 and dexamethasone could decrease those expressions.