Effects of vitamin E on the proliferation and collagen synthesis of rat hepatic stellate cells treated with IL-2 or TNF-α

Objective To study the effects of vitamin E on the proliferation and collagen synthesis of rat hepatic stellate cells treated with interleukin-2 (IL-2 ) or tumor necrosis factor-α (TNF-α). Methods Hepatic stellate cells were isolated from male Sprague-Dawley rats by using modified Friedman’s method. Using the isolated cells cultured and treated with IL-2 or TNF-α, we studied the effects of vitamin E on their proliferation and collagen synthesis through an (3)H-thymidine and (3)H-proline incorporation assay, as well as through observation of these cells under a contrary phase microscope. Results Adding IL-2 increased the both proliferation and collagen synthesis of hepatic stellate cells. Their proliferation was also increased by the addition of TNF-α, although it decreased collagen synthesis. Vitamin E had marked inhibitory effects on the ability of cells treated with IL-2 or TNF-α to reproduce or synthesize collagen. Conclusion Vitamin E can inhibit the proliferation and collagen synthesis of hepatic stellate cells. It is possible that vitamin E affects liver fibrosis through these activities.     

Application of Pulmonary Venoplasty in the Surgical Treatment of Lung Cancer

Presented in this study were three cases of lung cancer undergoing pulmonary venoplasty. In the 3 patients with central type of carcinoma of lung involving pulmonary vein, the main branch of right superior pulmonary vein and the distal end of the superior-lobe vein were occluded. The root part of the vein of right-middle lobe, plus part of vessel of of right superior vein was resected. The right superior vein was reconstructed by continuous 6-0 Prolene sutures. After the operation, the reconstructed was patent and the surgical margin was tumor-free. Postoperatively, clinical manifestations and plain chest films did not show any signs of venous blockade. The patients were discharged healed 3 weeks after the operation. The technical details of the surgery were presented, the improvements on the basis of traditional methods were discussed and its clinical application was evaluated. It is concluded that pulmonary venoplasty is a safe and feasible operation. Further improvement of the surgery will help conserve more lung tissue and benefit more patients because of expanded indications.     

Expression of TGF-beta 1, PDGF and IGF-1 mRNA in lung of bleomycin-A5-induced pulmonary fibrosis in rats.

OBJECTIVE To investigate the influence of alveolar macrophages (AMs), fibroblasts and interstitial cells on development of lung fibrosis, and the interactions among TGF-beta 1 PDGF and IGF-1 and these cytokines-effects on lung fibrosis.

METHODS Expressions of TGF-beta 1, PDGF and IGF-1 mRNA in the lung cells and lung tissues in different stages of Bleomycin-A5-induced pulmonary fibrosis in rats were studied through Northern hybridization.

RESULTS The expressions of TGF-beta 1 and PDGF mRNA reached their peaks in AMs of pulmonary fibrosis in rats on the 7th day after Bleomycin-A5 instillation. It was similar with that in the lung tissues. IGF-1 mRNA remained relatively stable in AMs during the course. PDGF and IGF-1 mRNA increased gradually in fibroblasts, and reached the highest expressions in the interstitial cells. There was almost no TGF-beta 1 mRNA expression in all groups of fibroblasts.

CONCLUSIONS AMs are the main sources of TGF-beta 1 and PDGF in the lung tissues with fibrosis induced by Bleomycin-A5 AMs are activated in the first weekend and secrete TGF-beta 1 and PDGF to promote fibroblasts proliferation and fibrosis. As fibrosis developed, fibroblasts have established PDGF and IGF-1 autocrine and these three cytokines paracrine nets combined with the interstitial cells to promote lung fibrosis.

     

Thrombin promotes human lung fibroblasts to proliferate via NADPH oxidase/reactive oxygen species/extracellular regulated kinase signaling pathway

Background Thrombin is a multifunctional serine protease that plays a crucial role in hemostasis following tissue injury.In addition to its procoagulation effect, thrombin is also a potent mesenchymal cell mitogen, therefore it plays important roles in the local proliferation of mesenchymal cells in the tissue repair process. Reactive oxygen species （ROS） can induce some human cells to proliferate at lower rates while at higher concentrations they promote cells to undergo apoptosis or necrosis. Accumulative evidence suggests that thrombin can induce some cells to produce ROS. Based on these observations, we provide a hypothesis that thrombin can stimulate human lung fibroblasts to produce ROS, which play an important role in human lung fibroblast proliferation.Methods ROS were detected in fibroblasts at 30 minutes and 60 minutes following thrombin （20 U/ml） exposure using flow cytometry. The ratio of reduced glutathione/oxidized glutathione （GSH/GSSG） was assayed in lung fibroblasts using a commercial kit following treatment with thrombin at different concentrations. NADPH oxidase and the extracellular regulated kinase1/2 （ERK1/2） signaling pathway were detected by Western blotting after thrombin stimulation to lung fibroblasts.Results Thrombin, at 20 U/ml, stimulated human lung fibroblasts （HLF） to generate ROS in a time dependent manner.The ratio of GSH/GSSG in fibroblasts treated with thrombin showed a significant decrease. NADPH oxidase was activated and the ERK1/2 signal pathway was involved in the proliferation process of fibroblasts treated with thrombin.Conclusion The activation of NADPH oxidase by thrombin leads to the production of ROS, which promotes fibroblasts proliferation via activation of the ERK1/2 signaling pathway.     

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 A1,group A2.5 and group A4,each with smoke exposure for 1 month,2.5 months or 4 months,respectively.Group B1,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(γ=-0.81,P<0.01) and positively correlated to TNF-α concentration(γ = 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.     

Exercise training attenuated chronic cigarette smoking-induced up-regulation of FIZZ1/RELMα in lung of rats

FIZZ/RELM is a new gene family named "found in inflammatory zone" (FIZZ) or "re- sistin-like molecule" (RELM). FIZZ1/RELMα is specifically expressed in lung tissue and associated with pulmonary inflammation. Chronic cigarette smoking up-regulates FIZZ1/RELMα expression in rat lung tissues, the mechanism of which is related to cigarette smoking-induced airway hyperresponsive- ness. To investigate the effect of exercise training on chronic cigarette smoking-induced airway hyper- responsiveness and up-regulation of FIZZ1/RELMα, rat chronic cigarette smoking model was estab- lished. The rats were treated with regular exercise training and their airway responsiveness was meas- ured. Hematoxylin and eosin (HE) staining, immunohistochemistry and in situ hybridization of lung tissues were performed to detect the expression of FIZZ1/RELMα. Results revealed that proper exercise training decreased airway hyperresponsiveness and pulmonary inflammation in rat chronic cigarette smoking model. Cigarette smoking increased the mRNA and protein levels of FIZZ1/RELMα, which were reversed by the proper exercise. It is concluded that proper exercise training prevents up-regulation of FIZZ1/RELMα induced by cigarette smoking, which may be involved in the mechanism of proper exercise training modulating airway hyperresponsiveness.     

Role of p38MAPK in mediating TNF-α-induced apoptosis of rat glioma cell line C6

Objective: To study the role of p38MAPK in mediating TNF-α-induced apoptosis of rat glioma cell line C6. Methods: Effect of TNF-α on the proliferation of C6 cells was determined by MTT assay. The TNF-α induced apoptosis was detected by transmission electron microscopy and flow cytometry. The expression of p38MAPK was detected by SABC method and Western-blot. The effect of SB202190, a specific inhibitor of p38MAPK, on TNF-α-induced apoptosis was observed by flow cytometry and SABC method. Results: Inhibitory rate of TNF-α(2×105 U/L) on C6 cells was 43. 75% . In the TNF-α treated group, apoptotic cells were observed by transmission electron microscopy and the apoptotic rate was 37. 5% by flow cytometry. p38MAPK positive signals were detected by SABC method and Western-blot. In the SB202190 treated group, the apoptotic rate was 7. 0% and no p38MAPK signals were found. Conclusion: Apoptosis of C6 cells and expression of p38MAPK can be induced by TNF-α. The activation of p38MAPK promotes the apoptosi     

Changes of distribution of platelet activating factor in the lung of rats with hypoxic pulmonary hypertension.

OBJECTIVE To investigate the role of platelet activating factor (PAF) in the hypoxic pulmonary hypertension.

METHODS Fifteen of 30 male Wistar rats were exposed to hypoxia for 3 weeks, and another 15 rats served as controls. The pulmonary arterial pressure was examined by catheterization. The sections of rat lung were treated by the avidin-biotin-peroxidase complex method to expose the location of PAF.

RESULTS The rats developed pulmonary hypertension and right ventricular hypertrophy after hypoxic exposure. Under the light microscope, PAF is distributed on the vascular and alveolar walls of normal lung, and the content of PAF in the lung of rats with hypoxic pulmonary hypertension are remarkably higher than those of normoxic controls.

CONCLUSIONS PAF plays not only a physiological role in the rat lung, but also a pathophysiologic role in hypoxic pulmonary hypertension.

     

Changes of Protein Kinase Cα and Cyclin D1 Expressions in Pulmonary Arteries from Smokers with and without Chronic Obstructive Pulmonary Disease

The purpose of this study was to investigate the changes of protein kinase Ca （PKCα） and cyclin D1 expressions in pulmonary arteries from＇smokers with normal lung function and smokers with mild to moderate chronic obstructive pulmonary disease （COPD）. The peripheral lung tissues were obtained from 10 non-smokers with normal lung function （non-smoker group）, 14 smokers with normal lung function （smoker group）, 11 smokers with mild to moderate COPD （COPD group）. The morphological changes of pulmonary arteries were observed by HE-staining. The expressions of ct-smooth muscle actin （α-SMA）, proliferating cell nuclear antigen （PCNA）, PKCα and cyclin D1 proteins in pulmonary artery smooth muscle cells （PASMCs） were immunohistochemically determined. The percentages of PCNA-positive cells were taken as the smooth muscle cells proliferation index （PI）. The mRNA expressions of PKCα and cyclin D l in PASMCs were evaluated by real-time fluorescence PCR. Morphometrical analysis showed that the ratio of pulmonary artery wall area to total area （WA%） in smoker group and COPD group was significantly greater than that in non-smoker group （P〈0.01）. The PASMCs proliferation index in smoker group and COPD group was significantly higher than that in nonsmoker group （P〈0.01）. The protein levels of PKCct and cyclin D1 inPASMCs were significantly increased in smoker group and COPD group as compared with non-smoker group （P〈0.01）. The mRNA expressions of PKCα and cyclin D1 in PASMCs were significantly elevated in smoker group and COPD group as compared with non-smoker group （P〈0.01）. Significant correlations were found between PKCα protein and WA% or PI （P〈0.01）. Correlations between cyclin D1 protein and WA% or PI also existed （P〈0.01）. The expression of PKCa was positively correlated with the expression of cyclin D 1 at both protein and mRNA levels （P〈0.01）. In conclusion, increased expressions of PKCα and cyclin D1 might be involved in the pathogenesis of abnormal proliferation of PASMCs in smokers with normal lung function and smokers with mild to moderate COPD.     

Effects of Baicalin on Expression of Inducible Nitric Oxide Synthase in Cultured Fibroblasts Stimulated by Cytokines

Objective: To investigate the effects of baicalin on expression of inducible nitric oxide synthase (iNOS) in fibroblasts and its mechanisms in treating psoriasis. Methods: Fibroblasts cultured in vitro were stimulated with tumor necrosis factor-α(TNF-α), interferon-γ (IFN-γ), interleukin-8 (IL-S) in different groups. iNOS was detected by western blot and immunocytochemistry assay, and in addition, the effects of baicalin on its expression were investigated. Results: Fibroblasts did not express iNOS without cytokine stimulation. When treated for 24 h with 1. 0× 106 U/L TNF-α, 0.2× 106U/L IFN-γ, 0.2× 106 pg/L IL-8 alone or in combinations indicated, fibroblasts produced iNOS when stimulated by TNF-α alone while neither IFN-γ nor IL-8 could induce the production of iNOS. The combination of TNF-α and IL-8 induced a strong expression of iNOS, the combined exposure of three kinds of cytokines showed an even stronger effects. The strongly stained area was in the cytoplasm near the nuclei. Expression of iNOS induced by TNF-α and IL-8 was inhibited by 50 μg/ mi of baicalin. Conclusion: Fibroblasts might express iNOS when stimulated by certain cytokines. Baicalin decreased production of nitric oxide through inhibiting the expression of iNOS, furthermore it reduced inflammation, which might be part of its mechanisms in treating psoriasis.     

Effects of IL-17 on expression of GRO-α and IL-8 in fibroblasts from nasal polyps

Recent studies indicated that interleukin（IL）-17, growth-related oncogene（GRO）-α and IL-8 play an important role in the pathogenesis of nasal polyps. However, the effects of the increased amount of IL-17 and the production of GRO-α and IL-8 in human nasal polyp fibroblasts are not completely understood. This study aimed to determine the effects of the increased IL-17 on the changes of GRO-α and IL-8 expression in human nasal polyp fibroblasts and further investigate the mechanism of neutrophil infiltration in nasal polyps. Nasal polyp fibroblasts were isolated from six cases of human nasal polyps, and the cells were stimulated with five different concentrations of IL-17. Real-time fluorescence quantitative polymerase chain reaction（RT-PCR） was used to detect the mRNA expression of GRO-α and IL-8. The mRNA of GRO-α and IL-8 was expressed in unstimulated controls and remarkably increased by stimulation with IL-17. Moreover, the levels of GRO-α and IL-8 produced by fibroblasts were increased gradually with the increases in IL-17 concentrations. The present study showed that nasal fibroblasts can produce GRO-α and IL-8, and their production is remarkably enhanced by IL-17 stimulation, thereby clarifying the mechanism of the IL-17 mediated neutrophil infiltration in nasal polyps. These findings might provide a rationale for using IL-17 inhibitors as a treatment for nasal inflammatory diseases such as nasal polyps.     

Experimental study on effect of endothelin in the proliferation and collagen synthesis of human scar-derived fibroblasts

Objective To investigate the role of endothelin(ET) in the proliferation and collagen synthesis of human scar-derived fibroblasts and the moduktion of its antagonists such as nitric oxide(NO), tetrandrine ( Tet). Methods With the cultured fibroblasts from the scarring tissue, the cell pdiferation was determined by[3H]-TdR incorporation, while the collagen synthesis was evaluated by[3H]-proline incorporation. Results The ET-1 was significantly increasing the proliferation and collagen synthesis of human scar-derived fibroblasts. The values of [3H]-TdR absorption in the 2.5 ng/ml,25 ng/ml and 100 ng/ml of ET-1 groups were 1.8 times,4 times and 4.9 times more than in the control group, respectively(P<0. 01),while the values of the [3H]-proline incorporation were 1.1 times,3.1 times and 3.8 times respectively(P<0.01). The fibroblasts, treated with 50 μg/ml of S-nitroso-N-acetyl penicillamine(SNAP), were no detectable effect on the basal level of DNA synthesis,but produced decreasing effect on the [3H]-TdR     

Gefitinib attenuates murine pulmonary fibrosis induced by bleomycin

Background Gefitinib, an inhibitor of epidermal growth factor receptor （EGFR） tyrosine kinase, is an effective treatment for epithelial tumors, including non-small cell lung cancer （NSCLC）, and is generally well tolerated.However, some clinical trials revealed that gefitinib exposure caused lung fibrosis, a severe adverse reaction.This study investigated the effect of gefitinib on lung fibrosis in mice.Methods We generated a mouse model of lung fibrosis induced by bleomycin to investigate the fibrotic effect of gefitinib.C57BL/6 mice were injected intratracheally with bleomycin or saline, with intragastric administration of gefitinib or saline.Lung tissues were harvested on day 14 or 21 for histology and genetic analysis.Results The histological results showed that bleomycin successfully induced lung fibrosis in mice, and gefitinib prevented lung fibrosis and suppressed the proliferation of S100A4-positive fibroblast cells.In addition, Western blotting analysis revealed that gefitinib decreased the expression of phosphorylated EGFR （p-EGFR）.Furthermore, quantitative real-time PCR （qRT-PCR） demonstrated that gefitinib inhibited the accumulation of collagens Ⅰ and Ⅲ.Conclusions These results reveal that gefitinib reduces pulmonary fibrosis induced by bleomycin in mice and suggest that administration of small molecule EGFR tyrosine kinase inhibitors has the potential to prevent pulmonary fibrosis by inhibiting the proliferation of mesenchymal cells, and that targeting tyrosine kinase receptors might be useful for the treatment of pulmonary fibrosis in humans.     

Pulmonary Toxicity in Rats Caused by Exposure to Intratracheal Instillation of SiO2 Nanoparticles

Objective The effect of the silica nanoparticles(SNs) on lungs injury in rats was investigated to evaluate the toxicity and possible mechanisms for SNs.Methods Male Wistar rats were instilled intratracheally with 1 mL of saline containing 6.25,12.5,and 25.0 mg of SNs or 25.0 mg of microscale SiO_2 particles suspensions for 30 d,were then sacrificed.Histopathological and ultrastructural change in lungs,and chemical components in the urine excretions were investigated by light microscope,TEM and EDS.MDA,NO and hydroxyproline(Hyp) in lung homogenates were quantified by spectrophotometry.Contents of TNF-α,TGF-β1,IL-1β,and MMP-2 in lung tissue were determined by immunohistochemistry staining.Results There is massive excretion of Si substance in urine.The SNs lead pulmonary lesions of rise in lung/body coefficients,lung inflammation,damaged alveoli,granuloma nodules formation,and collagen metabolized perturbation,and lung tissue damage is milder than those of microscale SiO_2 particles.The SNs also cause increase lipid peroxidation and high expression of cytokines.Conclusion The SNs result into pulmonary fibrosis by means of increase lipid peroxidation and high expression of cytokines.Milder effect of the SNs on pulmonary fibrosis comparing to microscale SiO_2 particles is contributed to its elimination from urine due to their ultrafine particle size.     

Expression of interleukin-15 and its receptor on the surface of stimulated human umbilical vein endothelial cells

Summary： Human interleukin-15 （hIL-15） is an important cytokine to activate endothelial cells and can be regulated by many other cytokines. The aim of this study is to examine the ability of interferon-γ （IFN-γ）, and tumor necrosis factor-or （TNF-α to induce the production of human interleukin-15 （hIL-15） and IL-15 receptor （IL-15Rα by human umbilical vein endothelial cells （HUVECs）. The data are summarized as follows： 1. Northern blot revealed that IL-15 mRNA was up-regulated by IFN-γ and TNF-α 2. Intracellular IL-15 protein was visualized by fluorescence microscopy, whereas the expression of IL-15 on the surface of HUVECs was detected by fluorescence activated cell sorting （FACS）, and no detectable IL-15 in the medium was verified by ELISA. 3. IL-15Rα was detected on the surface of HUVECs by FACS after IFN-α and TNF-α stimulation, whereas Western blotting revealed that the elevated expression on surface IL-15Rα was not due to the increased protein expression. The conclusion demonstrated from our results is that IFN-α and TNF-α play an important role in regulating the expression of IL-15 and IL-15Rα on the surface of HUVECs.     

The effect of Nifedipine on the expression of type I collagen in gingival fibroblasts

Objective:To investigate the effect of nifedipine(calciumchannel blocker)on the expression of collagen in gingival fibroblasts invitro.Methods:Primarily gingival fibroblasts were cultured and incubated with various concentrations of nifedipine(108μg/L,360μg/L and 1200μg/L)for 5 days.Gingival fibroblasts were primarily cultured derived from nifedipine responders and non-responders in the presence of 360μg/L nifedipine.Enzyme-linked immunosorbent assay was used to evaluate the amount of type I collagen.Cell proliferation was measured by cell counting with evaluating MTT value.Results:The expressions of collagen and cell proliferation were significantly different among the high concentration groups and the others on the fifth day,especially higher in 360μg/L and 1200μg/L groups and also different among nifedipine responders and non-responders.Conclusion:The expression of collagen and cell proliferation may be concerned with the biological mechanism for gingival overgrowth.     

Effects of calcium and calmodulin inhibitors on the abnormal proliferation of lung fibroblast.

The effects of alveolar macrophage (Amφ) conditioned media from interstitial lung disease (ILD) patients on fibroblast (FB), and the role of calcium (Ca~(2 )) blockers and calmodulin (CAM) inhibitors on the proliferation of lung FB were studied. We found that the AMφ conditioned media could stimulate FB cell proliferation and this effect could be abolished by Ca~(2 ) blockers and CaM inhibitors. The results indicated that AMφ was in activated state in ILD and released some kinds of cytokines to stimulate the proliferation of FB, and Ca,~(2 ) CaM were partially responsible for these actions.     

Experimental study of changes of TNF-α in rabbits with steroid-induced avascular necrosis of femoral head

Objective: To explore the effect of tumor necrosis factor-α(TNF-α)on the occurrence of steroid-induced avascular necrosis of femoral head(SANFH). Methods: Twenty-four rabbits were firstly divided into void group ( n = 12) and model group ( n =two groups were killed respectively to observe whether the model was successful. The level of TNF-α in serum of the residual rabbits of the two groups was examined in Radioimmunoassay method. Results: The level of TNF-α in model group is significantly higher than that in void group( P ＜0.001) under the premise of the model of SANFH success by histological observation. Conclusion: The rise of level of TNF-α may be one of the most important factors in the occurrence of SANFH.