The profibrotic effects of chronic microaspiration of bile acids on lungs of rats at different stages.

This study aimed to explore the profibrotic effects of chronic microaspiration of two major bile acids, including chenodeoxycholic acid (CDCA) and deoxycholic acid (DCA), on lungs of rats at different stages, as well as the underlying mechanisms in vivo. A rat model was induced by weekly intratracheal instillation of DCA and CDCA. Our results showed that chronic microaspiration of bile acids resulted in alveolar structure disorder, and inflammatory cells infiltration in the pulmonary interstitium at the early stage. Subsequently, numerous fibroblasts were proliferated, and collagen deposition was profoundly increased over the interstitium of the airways and vessels. Compared with control group, the expression of α-smooth muscle actin, type I collagen, hydroxyproline, transforming growth factor-ß1 (TGF-ß1), and matrix metalloproteinase-9 in the lung tissues were remarkably elevated at the 2nd week, reached the highest level at the 6th week, and maintained high at the 8th week in both DCA- and CDCA-treated groups (P < 0.05). Furthermore, chronic microaspiration of bile acids led to higher levels of glutathione and malondialdehyde, while lower level of superoxide dismutase in lung tissues compared with controls (P < 0.05), thereby resulting in the oxidant/antioxidant enzyme imbalance in the formation of fibrosis. In addition, we also found a consistent growth in the expression of farnesoid X receptor (FXR) in both DCA- and CDCA-treated groups. Our findings suggested that chronic microaspiration of bile acids could initiate the process of pulmonary fibrosis from the early phase and promote its progression in a time-dependent manner, which likely involved the TGF-ß1, oxidative stress, and FXR-related pathways.

Optimization of Nebulized Budesonide in the Treatment of Acute Exacerbation of Chronic Obstructive Pulmonary Disease.

Background: Clinical studies have suggested nebulized budesonide (NB) as an alternative to systemic corticosteroids for patients with acute exacerbation of chronic obstructive pulmonary disease (AECOPD). However, the optimal budesonide dose for AECOPD remains unclear. Objectives: To compare the efficacy and safety of different doses of NB in the management of AECOPD. Patients and Methods: A total of 321 AECOPD patients with moderate-to-severe exacerbation were randomly divided into three groups and treated with NB. The low dose group (L) was given 4 mg/day (n=95, 1 mg Q6h), while high-dose group 1 (H1, n=111, 2 mg Q6h) and high-dose group 2 (H2, n=115, 4 mg Q12h) were given 8 mg/day. Patients also received routine treatment including oxygen therapy, expectorant, nebulization bronchodilators, antibiotics, and fluid rehydration. The COPD assessment test (CAT), lung function, and artery blood gas were evaluated before and after 3 hrs and 5 days of treatment. In addition, hospital stay, frequency of acute exacerbations within 3 months of discharge, and adverse events during treatment were compared. Results: H1 and H2 showed improved spirograms and CAT score faster than L. In H2, forced expiratory volume in 1 s (FEV1%) at 3 hrs and FEV1%, forced expiratory flow after 50% of the forced vital capacity has been exhaled (FEF50%), mean forced expiratory flow between 25% and 75% of forced vital capacity (FEF25-75%) and CAT score at 5 days were significantly improved compared to L. FEV1% improved most in H2, moderately in H1, and least in L, with significant differences between groups at 5 days. No differences between groups were observed in adverse effects, hospital stay, and frequency of exacerbations within 3 months of discharge. Conclusion: Compared to the conventional dose (4 mg/day), a high dose (8 mg/day) of NB improved pulmonary function and symptoms more effectively in the early treatment of AECOPD, especially when given as 4 mg twice daily.

Tobacco smoking aggravates airway inflammation by upregulating endothelin-2 and activating the c-Jun amino terminal kinase pathway in asthma.

BACKGROUND: Asthma is closely associated with tobacco smoking (TS) and is more difficult to effectively treat after exposure to TS. OBJECTIVE: To observe the effects of TS on the expression of endothelin-2 (ET-2) and airway inflammation in asthmatic rats and to explore the related mechanisms. METHODS: We established an animal model of asthma with ovalbumin (OVA)/Al(OH)3 and subjected different animal groups to TS and/or dexamethasone/bosentan. The differences in the inflammatory cell infiltration, the pathological changes to the bronchial wall and the bronchial smooth muscle thickness, and the expression of ET-2, c-Jun amino terminal kinase (JNK1/2), malondialdehyde (MDA), and glutathione peroxidase (GSH) in the lung tissue and of interleukin (IL)-7 in bronchoalveolar lavage fluid (BALF) were assessed. RESULTS: Exposure to TS or OVA caused an obvious increase in the inflammatory cells in the BALF over what was observed in the control group. In asthma models, the expression of ET-1, JNK1/2, MDA, and GSH in the lung tissues, as well as that of IL-17 in the BALF, was increased. After treatment with dexamethasone/bosentan, the expression of IL-17, JNK1/2, MDA, and GSH decreased compared to the smoking group; airway inflammation and the staining intensity in the lung tissue were also reduced. CONCLUSION: TS exposure can clearly exacerbate airway inflammation in asthmatic rats, while bosentan can alleviate airway inflammation through regulation of the ET-2/JNK1/2 signalling pathway.

Downregulation of BarH-like homeobox 2 promotes cell proliferation, migration and aerobic glycolysis through Wnt/ß-catenin signaling, and predicts a poor prognosis in non-small cell lung carcinoma.

BACKGROUND: Human BarH-like homeobox 2 (Barx2), a homeodomain factor of the Bar family, plays a critical role in cell adhesion and cytoskeleton remodeling, and has been reported in an increasing array of tumor types except non-small cell lung carcinoma (NSCLC). The purpose of the current study was to characterize the expression of Barx2 and assess the clinical significance of Barx2 in NSCLC. METHODS: Quantitative real-time polymerase chain reaction, immunohistochemistry and western blot analysis were used to examine mRNA and protein expression, respectively. The relationships between Barx2 expression and clinicopathological variables were analyzed. Cell Counting Kit-8 and plate colony formation assay were used to detect cell proliferation. Transwell assay was used to examine cell migration ability. Glucose uptake, lactate, adenosine triphosphate, and lactate dehydrogenase assays were used to detect aerobic glycolysis. RESULTS: Barx2 is downregulated in NSCLC tissues compared with para-carcinoma. Furthermore, Barx2 expression shows a negative correlation with advanced TNM stage and a high level of Ki-67. Survival analysis reveals that Barx2 level is an independent prognostic factor for NSCLC patients. The Barx2 (low) Ki-67 (high) group had the worst prognosis. Furthermore, the data indicate that downregulation of Barx2 expression promotes cell proliferation, migration, and aerobic glycolysis, including increased lactate dehydrogenase activity, glucose utilization, lactate production, and decreased intracellular adenosine triphospahte level. Furthermore, Barx2 acts as a negative regulator of the canonical Wnt/ß-catenin pathway. Reactivation of Wnt/ß-catenin pathway by LiCl can reverse the inhibiting effect of Barx2. CONCLUSIONS: These findings reveal that Barx2 serving as a tumor suppressor gene could decrease cell proliferation, migration, and aerobic glycolysis through inhibiting the Wnt/ß-catenin signaling pathway, and predicts a good prognosis in NSCLC.

[Estrogen shortens the latent period of inducing asthma and promotes airway inflammation].

Objective To investigate the effect of estrogen on the latent period of inducing asthma and airway inflammation in mice with asthma. Methods Forty male BALB/c mice were randomly divided into normal control group, OVA-induced asthma group, 400 µg/kg estradiol treatment group, 400 µg/kg estradiol combined with 7 mg/kg tamoxifen group, with 10 mice in each group. The OVA-induced asthma group, estradiol treatment group, and estradiol combined with tamoxifen group were sensitized and challenged by OVA; the normal control group was treated with normal saline instead. The estradiol treatment group and estradiol combined with tamoxifen group were given intraperitoneal injection of 400 µg/kg estradiol 4 hours before each challenge, and the latter was given additionally intraperitoneal injection of 7 mg/kg tamoxifen at 30 minutes before the injection of estradiol; the normal control group and OVA-induced asthma group were injected with normal saline as controls. The latent period of inducing asthma of all the mice was determined 24 hours after the final OVA challenge. Bronchoalveolar lavage fluid (BALF) was collected for counting total leukocyte cells and the percentages of eosinophils and lymphocytes. The concentrations of interleukin 4 (IL-4) and IL-13 in BALF were detected by ELISA. The pathologic change of lung tissues was examined by HE staining. Results Compared with the normal control group, the latent period of inducing asthma were shortened in the OVA-induced asthma group, estradiol treatment group, and estradiol combined with tamoxifen group; compared with the OVA-induced asthma group and estradiol combined with tamoxifen group, the latent period of inducing asthma of the estradiol treatment group were shortened; there was no significant difference in the latent period between the OVA-induced asthma group and the estradiol combined with tamoxifen group. Compared with the normal control group, the levels of IL-4 and IL-13 increased in the OVA-induced asthma group, estradiol treatment group, and estradiol combined with tamoxifen group; compared with the OVA-induced asthma group and estradiol combined with tamoxifen group, the levels of IL-4 and IL-13 increased in the estradiol treatment group; there was also no significant difference between the OVA-induced asthma group and the estradiol combined with tamoxifen group. Compared with the normal control group, the total leukocytes and the percentages of eosinophils and lymphocytes in BALF were raised in the OVA-induced asthma group, estradiol treatment group, and estradiol combined with tamoxifen group; compared with OVA-induced asthma group and estradiol combined with tamoxifen group, the total leukocytes and the percentage of eosinophils and lymphocytes of BALF were elevated in the estradiol treatment group; there was no significant difference between the OVA-induced asthma group and the estradiol combined with tamoxifen group. There was no infiltration of inflammatory cells in bronchial walls or airway mucosal edema in the normal control group. There were infiltration of inflammatory cells in bronchial walls and airway mucosal edema in the OVA-induced asthma group and estradiol combined with tamoxifen group, in addition, the estradiol treatment group was more serious than the two groups in these pathologic changes. Conclusion Estrogen can shorten the latent period of inducing asthma, promote the airway inflammatory cell infiltration and upgrade the expressions of IL-4 and IL-13.

[miR-199a-5p inhibits the proliferation of rat airway smooth muscle cells and the expression of hypoxia inducible factor 1 alpha under hypoxia conditions].

OBJECTIVE: To investigate the expression of miR-199a-5p in airway smooth muscle cells (ASMCs) of rats in normoxia and hypoxia and its role in the regulation of proliferation and the expression of hypoxia inducible factor 1 alpha (HIF-1α) of ASMCs. METHODS: ASMCs were prepared by means of adherent culture in vitro. After ASMCs were cultured under normoxia and hypoxia conditions for 24 hours, the content of miR-199a-5p was detected by real-time quantitative PCR (qRT-PCR). The mimic or inhibitor of miR-199a-5p were artificially synthesized and transferred into ASMCs in hypoxia via liposomes. The expressions of miR-199a-5p and HIF-1α mRNA were detected by qRT-PCR. Western blotting and CCK-8 assay were applied to detect the expression levels of HIF-1α protein and the proliferation of ASMCs, respectively. RESULTS: Compared with the normoxia group, hypoxia significantly promoted cell proliferation and increased the levels of HIF-1α mRNA and protein. The level of miR-199a-5p decreased in the hypoxia group compared with the normoxia group. The proliferation rate of ASMCs under hypoxia conditions was significantly attenuated by transfection of miR-199a-5p mimic, while it was significantly lifted by transfection of miR-199a-5p inhibitor. Compared with control group, the expression of HIF-1α protein was reduced in the mimic group and raised in the inhibitor group. There was no significant difference in the content of HIF-1α mRNA among groups under hypoxia conditions. CONCLUSION: miR-199a-5p can inhibit the proliferation of ASMCs and the expression of HIF-1α protein in vitro under hypoxia conditions.

[Leptin promotes the proliferation of airway smooth muscle cells and the expressions of HIF-1α and NF-κB of hypoxic rats].

OBJECTIVE: To investigate the effect of leptin on the proliferation of airway smooth muscle cells (ASMCs) and the expressions of hypoxia-inducible factor-1α (HIF-1α) and nuclear factor-kappa B (NF-κB) of hypoxic rats. METHODS: The rat ASMCs were cultured under normoxic and hypoxic states. The hypoxic cells were divided into hypoxia group, leptin 50 µg/L hypoxia group (L50 group), leptin 100 µg/L hypoxia group (L100 group), leptin 200 µg/L hypoxia group (L200 group), leptin 200 µg/L and leptin receptor antibody hypoxia group (ob-R antibody group) by random number table. All the groups are cultured for 24 hours. Then the CCK-8 method was used to assay cell proliferation rate, and Western blotting and real-time RT-PCR to measure the expressions of HIF-1α and NF-κB at protein and mRNA levels. RESULTS: Compared with the normoxic group, each hypoxia group had significantly increased cell proliferation . Compared with the hypoxia group, cell proliferation rate was significantly raised in L50, L100 and L200 groups, and it was positively correlated with the concentration (r=0.992). Compared with L50, L100 and L200 groups, the ob-R antibody group showed significantly decreased cell proliferation rate. Compared with the normoxic group, each hypoxic group has increased expressions of HIF-1α and NF-κB mRNA and proteins; compared with the hypoxia group, the expressions were significantly elevated in the L50, L100 and L200 groups and showed a positive correlation with the concentration; but the expressions were reduced in the ob-R antibody group as compared with L50, L100 and L200 groups. CONCLUSION: Leptin can promote rat ASMCs proliferation and the expressions of HIF-1α and NF-κB under hypoxic condition.

[Expression and significance of hypoxia-inducible factor-1α in lung tissues of obesity-asthma rat].

OBJECTIVE: To evaluate the expression level of hypoxia-inducible factor-1α (HIF-1α) in the rat model of diet-induced obesity and asthma. METHODS: Forty male specific pathogen-free SD rats were randomly divided into four groups: normal body mass control group (group A), asthmatic rats with normal body mass (group B), obese control group (group C) and obese asthmatic rats (group D). The rats in both group A and B were fed basic diet, while those in group C and D were fed high-fat diet in order to establish diet-induced obese rat model. Rats in group B and D were sensitized and challenged with chicken ovalbumin (OVA) to establish the asthmatic model. The white cell count in bronchoalveolar lavage fluid (BALF) was performed. The total area of the airway wall (Wat) was measured by ImagePro Plus software and was standardized by the basement membrane perimeter (Pbm). The expression of HIF-1α in lung tissue was detected by immunohistochemistry. The concentration of HIF-1α in serum was determined by ELISA. The relationships of the total white cells in BALF and airway wall thickness (WAt/Pbm) with the expression of HIF-1α were analyzed by Pearson correlation analysis. RESULTS: The total number of white cells in BALF in group D was (98.0±5.5)×10(4)/mL, which was significantly higher than those in group A (24.7±3.3)×10(4)/mL, group C (26.1±3.8)×10(4)/mL and group B (87.8±7.1)×10(4)/mL. The thickness of airway wall (WAt/Pbm) in group D was (9.91±0.56)m(2)/m, which was significantly higher than those in group A (6.11±0.99)m(2)/m and group C(5.99±0.83)m(2)/m, but when compared with that in group B (8.60±0.53)m(2)/m, there was no significant difference. The percentage of HIF-1α positive cells in group D was (19.44±0.96)%, which was significantly higher than those in group A (2.19±0.91)%, group C(2.56±0.89)% and group B (18.25±1.29)% (all P<0.05). The expression of HIF-1α in blood serum and BALF in group D were respectively(29.107±1.576) ng/mL and (0.511±0.011) ng/mL, which were significantly higher than those in group A [(19.380±1.506) ng/mL and (0.280±0.008) ng/mL, respectively], group C [(20.782±2.034) ng/mL and (0.281±0.010) ng/mL, respectively] and group B [(23.961±1.565) ng/mL and (0.397±0.011) ng/mL, respectively]. The expression of HIF-1α was positively correlated with the total number of white cells in BALF and the airway wall thickness. CONCLUSION: The expression of HIF-1α in serum and lung tissue from obese asthmatic rats significantly increases, which is positively correlated with the total number of white cells in BALF and the airway wall thickness.