[Effect of HIF-2α mediated FoxM1 expression on proliferation of pulmonary artery smooth muscle cells in hypoxic rats].

Objective: To investigate the effect of hypoxia-inducible factor 2α (HIF-2α) gene on the expression of Forkhead box M1 (FoxM1) protein in the proliferation of hypoxic rat pulmonary artery smooth muscle cells (PASMC). Methods: HIF-2α overexpression lentiviral vector (LV-HIF-2α) and silencing RNA (siRNA) were constructed and transfected into rat PASMC under normoxia and hypoxia, respectively. The PASMC under normoxia were classified into normoxic control group, normoxia + LV-HIF-2α empty group, normoxia + LV-HIF-2α group; the PASMC under hypoxia were classified into hypoxic control group, hypoxia + siRNA-HIF-2α empty group, hypoxia + siRNA-HIF-2α group. The expression of HIF-2α and its downstream proteins FoxM1, cyclin D1 and Aurora A expressions were detected by Western blot. 5-Ethyny-2'-deoxyuridine (EdU) cell proliferation assay was used to evaluate the effect of overexpression and inhibition of HIF-2α expression on the proliferation of rat PASMC. Results: The expression of HIF-2α in normoxia + LV-HIF-2α group was significantly higher than that in normoxic control group and normoxia+LV-HIF-2α empty group (0.17±0.02 vs 0.09±0.01 and 0.07±0.00), while the expression of HIF-2α in PASMC of hypoxia + siRNA-HIF-2α group was significantly lower than that of hypoxic control group and hypoxia + siRNA-HIF-2α empty group (0.28±0.01 vs 0.35±0.02 and 0.30±0.01) (all P<0.05); the expression of FoxM1 protein, cyclinD1 and cell proliferation-related Aurora A protein in normoxia+LV-HIF-2α group were significantly higher than that in normoxic control group and normoxia+LV-HIF-2α empty group (0.40±0.03 vs 0.24±0.01 and 0.30±0.01, 0.22±0.02 vs 0.09±0.01 and 0.08±0.02, 0.29±0.02 vs 0.04±0.01 and 0.07±0.01, respectively) (all P<0.05); the expressions of FoxM1 protein, cyclinD1 and Aurora A protein in hypoxia + siRNA-HIF-2α group were significantly lower than those in hypoxic control group and hypoxia + siRNA-HIF-2α empty group (0.23±0.01 vs 0.36±0.02 and 0.32±0.01, 0.15±0.01 vs 0.31±0.01 and 0.28±0.03, 0.14±0.02 vs 0.33±0.03 and 0.27±0.02, respectively) (all P<0.05); the positive expression rate of EdU in the normoxic control group was significantly lower than that in the normoxia+LV-HIF-2α group [(30.77±2.43)% vs (55.56±3.01)%], while the hypoxic control group was significantly higher than the hypoxic+siRNA-HIF-2α group [(65.28±3.21)% vs (44.64±2.78)%] (both P<0.05). Conclusion: HIF-2α up-regulates the expression of FoxM1 and promotes the proliferation of pulmonary artery smooth muscle cells in hypoxic rats.

[Effect of Krüppel like zinc finger transcription factor 2 on γ-glutamylcysteine synthetase in bronchial epithelial cells of chronic obstructive pulmonary disease].

Objective: To research the regulation effects of Krüppel like zinc finger transcription factor 2 (KLF2) on γ-glutamylcysteine synthetase (γ-GCS) in airway epithelial cells of chronic obstructive pulmonary disease (COPD). Methods: (1) Human specimen experiment: lung tissue of pulmonary lobectomy patients with lung cancer with or without COPD was collected from Department of Thoracic Surgery of Hunan Cancer Hospital from December 2008 to December 2009. The patients were divided into COPD group and control group without COPD. The levels of KLF2, γ-GCS mRNA and protein expression in lung tissues were measured by immunohistochemistry and in situ hybridization (ISH). Then, the correlation between KLF2 and γ-GCS mRNA and protein expression levels were analyzed, as well as the correlation between KLF2 or γ-GCS protein and smoking index, percentage of forced expiratory volume in one second to predicted value (FEV1%), percentage of forced expiratory volume in one second to forced vital capacity (FVC/FEV1). (2) Animal experiment: the primary bronchial epithelial cells of rats were extracted by enzyme digestion. After 6 hours of incubation with 10% tobacco smoke extract (TSE), cellular glutathione (GSH) was measured by enzyme linked immunosorbent assay (ELISA) method. The cells were transfected by specific inhibitor of KLF2 through the liposom, which inhibited the protein expression of KLF2. Then, the cells were divided into KB group (blank control group without any treatment), KB+ TSE group (treated with TSE), NC group (control group transfected with miRNA), NC+ TSE group (treated with miRNA and TSE), 92a group (transfected with KLF2 inhibitor), 92a+ TSE group (treated with KLF2 inhibitor transfection and TSE) based in the treatment. After that, the changes of KLF2 and γ-GCS mRNA and protein expression in the cells of each group were measured by reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blot method. Results: (1) Human specimen experiment: The expressions of KLF2 mRNA, protein and γ-GCS mRNA, protein in the lung tissue of COPD patients were strong positive and higher than those in control group (0.32±0.04 vs 0.19±0.03, 0.35±0.05 vs 0.22±0.03; 0.28±0.03 vs 0.16±0.03, 0.31±0.05 vs 0.21±0.03; all P<0.01). Linear correlation analysis showed that KLF2 mRNA and protein were positively correlated with γ-GCS mRNA and protein (r=0.705, 0.722; both P<0.01). The KLF2 and γ-GCS protein were positively correlated with smoking index, FEV1% and FEV1/FVC (r=0.552, 0.728, 0.670, and r=0.631, 0.727, 0.657; all P<0.01). (2) Animal experiment: The level of GSH in KB+ TSE group was significantly higher than that in KB group[(28.05±2.04) vs (7.27±0.33) nmol/mg, P<0.01]. The KLF2 mRNA, protein and γ-GCS mRNA, protein in KB+ TSE group (1.715±0.026, 1.842±0.028 and 2.117±0.067, 1.879±0.065) were higher than those in KB group (1.130±0.017, 1.177±0.033 and 1.378±0.053, 1.177±0.042; all P<0.05), and those in 92a group (0.472±0.028, 0.634±0.025 and 0.582±0.025, 0.554±0.021) were significantly lower than those in KB group, NC group (1.047±0.056, 1.092±0.045 and 1.303±0.037, 1.252±0.037), and those in TSE+ 92a group (0.262±0.017, 0.288±0.017 and 0.337±0.022, 0.321±0.022) were significantly lower than those in KB+ TSE group, 92a group and NC+ TSE group (1.576±0.036, 1.646±0.066 and 1.948±0.093, 1.843±0.078) (all P<0.05). Conclusion: KLF2 exerts antioxidative effect by regulating the expression of γ-GCS in the bronchial epithelial cells of chronic obstructive pulmonary disease.

[Application of radionuclide gastroesophageal reflux imaging in patients with chronic bronchitis and asthma].

To determine the relationship between gastroesophageal reflux (GER) and pulmonary diseases, we studied 29 chronic bronchitis, 32 asthmatic patients, and 9 control subjects. GER was diagnosed by esophageal endoscopy and gastroesophageal scintiscanning. Evidence of GER in the chronic bronchitic patients was 51.74%; in the asthmatics it was 37.5%; and no GER was confirmed in the control subjects. Gastroesophageal scintiscan had 100% sensitivity, but only 68.42% specificity. The mechanism whereby reflux triggers pulmonary problems was investigated by using the scintiscan for pulmonary aspiration, but no pulmonary aspiration was detected in all subjects. Pulmonary function tests did not show any differences between patients with or without reflux (P > 0.05). Thus, our results show that GER is common in chronic bronchitics and asthmatics, which indicates that there is some relationship between GER and chronic bronchitis as well as asthma.

Multidrug-resistant tuberculosis is associated with low plasma concentrations of human neutrophil peptides 1-3.

SETTING: Human neutrophil peptides 1, 2 and 3 (HNP1-3) are involved in innate host defence and acquired immune response, which is possibly associated with the genesis of multidrug-resistant tuberculosis (MDR-TB). OBJECTIVE: To investigate the relationship between HNP1-3 and MDR-TB. DESIGN: We divided 42 patients with post-primary pulmonary TB into two groups according to their drug susceptibility test results: the drug-susceptible group (n = 21) and the MDR-TB group (n = 21). The concentration of HNP1-3 in the plasma of all specimens was measured by enzyme-linked immunosorbent assay before treatment and 6, 18 and 24 months after. Duration of Mycobacterium tuberculosis in sputum and peripheral blood neutrophil counts were measured at the same time. RESULTS: Before treatment, the plasma HNP1-3 concentration in the MDR-TB group was lower than that of healthy controls and the drug-susceptible group. After treatment, plasma HNP1-3 concentrations were higher than pre-treatment levels in the MDR-TB group, but were still lower than in healthy controls or the drug-susceptible group. The concentration of HNP1-3 was negatively correlated with the duration of M. tuberculosis in sputum, while it was positively correlated with neutrophils. CONCLUSION: MDR-TB is associated with low plasma concentrations of HNP1-3.