Dynamic Expression of Tumor Necrosis Factor-α and Vascular Endothelial Growth Factor in Rat Model of Pulmonary Emphysema Induced by Smoke Exposure

In order to explore the roles of tumor necrosis factor-α （TNF-α） and vascular endothelial growth factor （VEGF） in the pathogenesis of pulmonary emphysema, male Wistar rats were randomized into group At, group A2.5 and group A4, each with smoke exposure for 1 month, 2.5 months or 4 months, respectively. Group B t, group B2.5 and group B4 were used as non smoking controls at corresponding time points. TNF-α in bronchoalveolar lavage fluid （BALF） and expression of VEGF in lung tissue was determined by ELISA or by SABC immunohistochemistry assay either. Lung slices were stained with hematoxylin and eosin （HE）. Results showed that in animal with smoke exposure the mean linear interceptor （Lm）, an index of pulmonary emphysema and the content of TNF-α in BALF increased gradually, on contrary, the expression of VEGF in lung tissue decreased （P〈0.05）. This phenomenon was not obvious in animals without smoke exposure. Lm was negatively correlated to the VEGF expression （7=--0.81, P〈0.01） and positively correlated to TNF-α concentration （7 = 0.52, P〈0.004）, which implies that smoke exposure decreased the expression of VEGF and increased the expression of TNF-α. It is plausible to speculate that the imbalance of TNF-α and VEGF may play an important role in the pathogenesis of smoke-induced pulmonary emphysema.

Comparison of Serum Adiponectin in Smoke-induced Pulmonary Emphysema Rats Fed Different Diets

Background： Smoking and body mass index （BMI） are the key risk factors for chronic obstructive pulmonary disease （COPD）. Adiponectin with both anti-inflammatory and pro-inflammatory properties is a vital modulator of inflammatory processes, which is expressed in epithelial cells in the airway in COPD-emphysema. The aim of this study was to examine the effects of adiponectin on tobacco smoke-induced emphysema in rats, which were fed different diets. Methods： Seventy-six adult （6-8 weeks old） male Sprague-Dawley rats （average weight 220 ± 20 g） were exposed to smoke or smoke-free room atmosphere and fed different diets （regular, high-fat, or low-fat diets） for 6 months. The rats were randomly divided into six groups. They are nonsmoke-exposed regular diet （n = 10）, nonsmoke-exposed high-fat diet （n - 14）, nonsmoke-exposed low-fat diet （n = 14）, smoke-exposed regular diet （n = 10）, smoke-exposed high-fat diet （n = 14）, and smoke-exposed low-fat diet groups （n = 14）. A full 23 factorial design was used to evaluate the effect of independent variables on smoke exposure and different rearing methods. Serum adiponectin and inflammatory cytokines were measured by the enzyme-linked immunosorbent assay （ELISA）. Results： Serum adiponectin levels in rats fed low-fat and regular diets exposed to smoke exposure were remarkably higher than that of rats exposed to room air while serum adiponectin levels of fat-rich diet rats exposed to tobacco smoke were lower than that of rats exposed to room air. Compared with regular diet or low-fat diet group, serum adiponectin levels in high-fat diet rats exposed to tobacco smoke were lower （t = 6.932, 11.026; all P 〈 0.001 ）. BMI was inversely correlated with serum adiponectin levels （r = -0.751, P = 0.012）. Serum interleukin 6 （IL-6）, tumor necrosis factor-or （TNF-a）, and 4-hydroxy 2-nonenal （HNE） levels in rats exposed to low-fat or fat-rich diets were remarkably higher than that of rats exposed to normal diets （IL-6, t = 4.196, 3.480; P 〈 0.01, P = 0.001 ; TNF-a, t = 4.286, 3.521 ; P 〈 0.01, P = 0.001; 4-HNE, t = 4.298, 4.316; all P 〈 0.001）. In nonhigh-fat diet rats exposed to tobacco smoke, serum adiponectin levels correlated positively with serum IL-6, TNF-ct, and 4-HNE, bronchoalveolar lavage cell count, and mean linear intercept. In contrast, in high-fat diet rats, serum adiponectin levels correlated inversely with these parameters. Conclusions： In smoke-induced emphysema and fat-rich diet rat model, serum adiponectin level was decreased, and the anti-inflammatory effect was attenuated. By contrast, nonhigh-fat diet elevated serum adiponectin and enhanced the role of pro-inflammatory.

Prophylactic Anti-inflammation Inhibits Cigarette Smoke-induced Emphysema in Guinea Pigs

In this study, the effect of prophylactic anti inflammation on the development of smoke induced emphysema was investigated. Young male guinea pigs aged 1.5 - 2 months (weighing 198.3±26.9 g) were randomly divided into 4 groups: group A (cigarette smoke exposure only), group B (cigarette smoke exposure plus pentoxifylline rich (PTX, 10 mg/d) forage feeding), group C (cigarette smoke exposure plus intermittent cortical steroid injection (Triamcinolone acetonide, 3 mg, im, every three weeks) and control group (group D: animals with sham smoke exposure, raised under the same conditions). Animals in group A, B and C were exposed to smoke of cigarettes for 1 to 1.5 h twice a day, 5 days a week. All animals were killed at the 16th week and followed by morphometrical analysis of the midsagittal sectioned lung slices. Smoke exposure of 16 weeks resulted in visible emphysematous development in Group A but not in Group B and C. It was evidenced by the indicator of air space size, mean linear intercept (L m): 120.6±16.0 μm in Group A; 89.8±9.2 μm in Group B and 102.4±17.7 μm in Group C. The average L m in either group B or group C was shorter than that in Group A (ANOVA and Newman Keuls test, F=8.80, P =0.0002) but comparable to that (94.8±13.2 μm) in group D ( P >0.05). It is concluded that long term prophylactic anti inflammation inhibits pulmonary emphysema induced by cigarette smoking in the guinea pigs.

Effects of pentoxifylline on Wnt/β-catenin signaling in mice chronically exposed to cigarette smoke

Background Previous discovery that long-term administration of pentoxifylline （PTX） to mice chronically exposed to smoke led to the development of pulmonary fibrosis rather than emphysema initiated our curiosity on whether the Wnt/β-catenin pathway, a set of signaling proteins essential to organ development and lung morphogenesis in particular were activated in the pathogenesis of pulmonary fibrosis. Methods Male BALB/c mice were randomized into four study groups： Group Sm, smoke exposure and taken regular forage; Group PTX, no smoke but taken PTX-rich forage; Group Sm＋PTX, smoke exposure and taken PrX-rich forage; Group control： shamed smoke exposure and taken regular forage. Animals were sacrificed at day 120. Morphometry of the lung sections and the expressions of TGF-β1, hydroxyproline, β-catenin, cyclin D1, T cell factor 1 （Tcf-1） and lymphoid enhancer factor 1 （Lef-1） mRNA, etc, in the lung homogenate or in situ were qualitatively or quantitatively analyzed. Results As expected, smoke exposure along with PTX administration for 120 days, lungs of the mice progressed to be a fibrosis-like phenotype, with elevated fibrosis score （3.9±1.1 vs. 1.7±0.6 in Group Sm, P 〈0.05）. TGF-β1（pg/g） （1452.4±465.7 VS. 818.9±2.02.8 in Group Sm, P 〈0.05） and hydroxyproline （mg/g） （5.6±0.6, vs. 2.4±0.1 in Group Sm, P 〈0.05） were also consistently increased. The upregulation of β-catenin measured either by counting the cell with positive staining in microscopic field （17.4±7.9 vs. 9.9±2.9 in Group Sm, P 〈0.05） or by estimation of the proportion of blue-stained area by Masson＇s trichrome （11.8±5.6 vs. 4.7±4 in Group Sm） in Group SM＋PTX was much more noticeable as than those in Group Sm. The expression of β-catenin measured by positive cell counts was correlated to TGF-β1 concentration in lung tissue （r=0.758, P 〈0.001）. PTX per se caused neither fibrosis nor emphysema though expression of β-catenin and downstream gene cyclin D1 may also be altered by this medication. Conclusions PTX mediated transformation of pulmonary emphysema into pulmonary fibrosis under chronic cigarette smoke exposure is associated with upregulation of β-catenin and elevation of TGF-β1, implying that activation of Wnt/β-catenin signaling may be involved in the pathogenesis of pulmonary fibrosis.

Pentoxifylline attenuates cigarette smoke-induced overexpression of CXCR3 and IP-10 in mice

Background Cigarette smoke-induced emphysema is associated with overexpression of the chemokine receptor CXCR3 and its ligands. Previously, we have demonstrated that pentoxifylline （PTX） alleviated cigarette smoke-induced emphysema. The aim of this study was to determine if the overexpression of CXCR3 and its ligand interferon-inducible protein-10 （IP-10） that was elicited by smoke exposure were attenuated by PTX. Methods （1） The study in vitro： a given number of RAW264.7 macrophages with decreasing concentrations of PTX in the culture medium were challenged with cigarette smoke extract （CSE）; （2） The study in vivo： male BALB/c mice were randomized into four groups, i.e., sham-smoke, smoke only, smoke with 2 mg/kg PTX, and smoke with 10 mg/kg PTX. The smoke exposure time was 90 minutes once a day, 6 days a week for 16 weeks. PTX was given intraperitoneally before each episode of smoke exposure. Interferon （IFN）-y and IP-10 in broncho-alveolar lavage fluid （BALF） and in culture medium were measured by enzyme-linked immunosorbent assay （ELISA）. IP-10 mRNA in lung tissue was assessed by RT-PCR. CXCR3 positive cells in lung sections were visualized by immunochemistry staining. Results Up-regulation of IFN-γ and IP-10 in the culture medium of macrophages elicited by CSE was inhibited by PTX in a dose-dependent manner. Chronic cigarette smoke exposure led to overexpression of IFN-γ and IP-10 in BALF, upregulation of IP-10 mRNA and increased infiltration of CXCR3^＋ cells into lung parenchyma. Administration of PTX decreased the level of IFN-y from （6.26±1.38） ng/ml to （4.43±0.66） ng/ml by low dose PTX or to （1.74±0.28） ng/ml by high dose PTX. IP-10 was reduced from （10.35±1.49） ng/ml to （8.19±0.79） ng/ml by low dose PTX or to （7.51±0.60） ng/ml by high dose PTX. The expression of IP-10 mRNA was also down-regulated （P 〈0.05）. But only with a high dose of PTX was the ratio of CXCR3^＋ cells decreased; 15.2±7.3 vs. 10.4±1.8 （P 〈0.05）. Conclusion PTX attenuates cigarette smoke-induced overexpression of chemokine receptor CXCR3 and its ligand IP-10, which is relevant to its inhibitory effect on pulmonary emphysema.