Objective To explore the effects of SU5416，a vascular endothelial growth factor receptor (VEGFR) inhibitor，on inflammation in mice with bleomycin-induced pulmonary fibrosis.Methods C57BL/6 mice were randomly divided into four groups，ie.a control group，a bleomycin-induced pulmonary fibrosis (BPF) group,early intervention with SU5416 group (days 1-14，SE)，and delayed intervention with SU5416group (days 15-28，SD).Bleomycin (10 mg/kg) was instilled into the trachea of the mice to induce pulmonary fibrosis.After the instillation，SU5416 (50 mg/kg) was injected into the peritoneal cavities of the mice twice a week.The mice treated with bleomycin alone or with bleomycin followed by SU5416 were sacrificed on the day 14 and day 28 after the instillation of bleomycin.Lung tissues were taken and processed for determination of hydroxyl proline content.Bronchoalveolar lavage fluid (BALF) was collected for total and differential cell counts and measurements of lactic dehydrogenase (LDH) levels.Results It was found that the hydroxyl proline contents，the number of macrophages，and the LDH contents from the mice treated with bleomycin alone on day 14 were greatly increased.However，only the hydroxyl proline showed a significant increase in the mice treated with bleomycin alone on day 28.The number of macrophages，LDH contents，and hydroxyl proline contents were greatly reduced in the mice treated with bleomycin and early administration of SU5416 on day 14 when compared with those treated with bleomycin alone.But there were no significant differences in the above parameters of the mice treated with bleomycin and delayed administration of SU5416 on day 14.Conclusion This study indicates that early intervention by SU5416 might attenuate the fibrosis through inhibiting early inflammation in animal model of pulmonary fibrosis induced by bleomycin.
Objective To study the inhibitory effects of curcumin on bleomycin-induced pulmonary fibrosis in rats at the fibrosing stage and explore its possible mechanism.Methods 96 male SD rats were randomly divided into a normal control group，a fibrosis model group，a fibrosis model treated with prednisone group and a fibrosis model treated with curcumin group.Pulmonary fibrosis were induced by instilled bleomycin through tracheal.From day 15 after bleomycin administration，the curcumin group and prednisone group were given curcumin（300 mg/kg） or prednisone（5 mg/kg） per day by intragastric administration，respectively.The normal control group and fibrosis model group were given 1% sodium carboxymethyl cellulose（10 mL/kg） as control.Six rats of each group were randomly sacrificed on day 21，28，42 and 56 after bleomycin administration，respectively.The histological changes of the lung were evaluated by HE and Masson’s trichrome staining.Lung expressions of transforming growth factor-β1（TGF-β1） and hydroxyproline were assessed by immuno-histochemistry and digestion method，respectively.Results Pulmonary fibrosis and hydroxyproline level in the curcumin group were significantly reduced as compared with those in the model group on day 42 and 56.The expession of TGF-β1 in the curcumin group was significantly lower than that in the model group on day 28，42 and 56，and was not significantly different from the normal group on day 56.Conclusion Curcumin could alleviate bleomycin-induced pulmonary fibrosis in rats at the fibrosing stage by inhibiting the expressions of TGF-β1.
Objective To explore whether epithelial to mesenchymal transition ( EMT) occurs in bleomycin( BLM) induced pulmonary fibrosis, and the involvement of bronchial epithelial cells( BECs) in the EMT. Methods BLM-induced peribronchial fibrosis in an α-smooth muscle actin-Cre transgenic mouse( α-SMACre /R26R) was examined by pulmonary βgal staining and α-SMA immunofluorescence staining. Results BLMtreated mice showed significantly enhanced βgal staining in subepithelial areas in bronchi, terminal bronchioles and walls of pulmonary vessels. Some alveolar epithelial cells( AECs) in certain peribronchial areas or even a small subset of BECs were also positively stained, as confirmed by α-SMA immunostaining. Conclusions EMT occurs in BLM-induced peribronchial fibrosis mice. BECs, like AECs, have the capacity to undergo EMT and to contribute to mesenchymal expansion in pulmonary fibrosis.
Objective To investigate the expression of high mobility group protein-B1( HMGB1)and α-smooth muscle actin( α-SMA) in Bleomycin induced pulmonary fibrosis in mice. Methods Twenty C57BL/ 6 male mice were randomly divided into a Bleomycin group and a control group. The Bleomycin group was treated with Bleomycin( 3 mg/kg) by endotracheally injection to induce pulmonary fibrosis. The control group were treated with normal saline( NS) . Then they were sacrificed by abdominal aortic bleeding 10 days after the injection. The right lung was stained with hematoxylin-eosin and Masson trichrome respectively for pathological examination. Immunohistochemistry and RT-PCR were performed to identify the protein and mRNA levels of α-SMA and HMGB1 respectively. Results The mRNA( 0. 89 ±0. 12, 0. 61 ±0. 08) and protein( 13. 66 ±1. 01, 13. 12 ±1. 33) expressions of α-SMA and HMGB1 in the Bleomycin group were all significantly higher than those of the control group( mRNA: 0. 60 ±0. 07, 0. 15 ±0. 02; protein: 8. 18 ±1. 33,7. 92 ±1. 10; all P lt; 0. 01) . Conclusions The expressions of HMGB1 and α-SMA are increased in Bleomycin induced pulmonary fibrosis. HMGB1 participates in the pathological process of pulmonary fibrosis probably by activation of the α-SMA expression.
Objective To explore the inhibitory mechanism of Imatinib mesylate on pulmonary fibrosis induced by bleomycin inmice. Methods A total of 120 C57BL/6 mice were randomly divided into four groups, ie. a control group, a model group, a dexamethasone group, and an Imatinib group. The model of pulmonary fibrosis was established by a single intratracheal instillation of bleomycin in the rats. Then dexamethasone or Imatinib were given intraperitoneally respectively. On day 7, 14, 21 after the treatment, 10 mice of each group were sacrificed respectively. The expressions of TGF-β1 and α-SMA in lung tissue were analyzed by immunohistochemistry. The mRNA expression of TGF-β1 was measured by RT-PCR. Results The expressions of TGF-β1 and α-SMA in lung tissue at each time point were significantly increased in the model group compared with the control group. And the expressions were obviously decreased in the dexamethasone group and the Imatinib group compared with the model group, with no significant differences between the two treatment groups. The expression of TGF-β1 was positively correlated with the α-SMA expression ( r= 0. 251, P lt;0. 05) . Conclusion The inhibitory effect of Imatinib on pulmonary fibrosis may be related to the inhibition of TGF-β1 and α-SMA expressions.
Objective To evaluate the effects of two different epidermal growth factor receptor tyrosine kinase inhibitors ( EGFR-TKIs) , Gefitinib and Erlotinib, on lung fibrosis induced by bleomycin.Methods Forty BALB/c female mice were randomly divided into four groups, ie. a control group( saline given orally and intratracheally) , a fibrosis group( saline given orally with bleomycin instillation) , a Gefitnib group( Gefitnib 20 mg/kg given orally with bleomycin instillation) , and an Erlotinib group ( Erlotinib25 mg/kg given orally with bleomycin instillation) . Bleomycin ( 3 mg/kg) was intratracheally instilled on the first day. Gefitinib or Erlotinib was given orally daily and normal saline as control. Then they were sacrificed by abdominal aortic bleeding 14 days after the bleomycin instillation. The left lung was stained with HE and Masson’s trichrome staining respectively for pathological examination. Total EGFR and phosphorylated EGFR were detected by immunohistochemistry. Hydroxyproline ( HYP) assay was performed in the right lung.Results Both Gefitinib and Erlotinib significantly reduced lung collagen accumulation and the content of HYP. Immunohistochemistry revealed that phosphorylation of EGFR in lung mesenchymal cells induced by bleomycin was inhibited. Furthermore, there was no difference between Gefitinib and Erlotinib in inhibiting lung fibrosis. Conclusion Our findings suggest that, in the preclinical setting, EGFR-TKIs may have aprotective effect on lung fibrosis induced by bleomycin.
【Abstract】 Objective To explore the new therapy for pulmonary fibrosis by observing the effects of insulin-like growth factor 1 ( IGF-1) treated mesenchymal stemcells ( MSCs) in rats with bleomycin-induced pulmonary fibrosis. Methods Bone marrowmesenchymal stemcells ( BMSCs) were harvested from6-week old male SD rats and cultured in vitro for the experiment. 48 SD rats were randomly divided into 4 groups, ie.a negative control group ( N) , a positive control group/bleomycin group ( B) , a MSCs grafting group ( M) ,and an IGF-1 treated MSCs grafting group ( I) . The rats in group B, M and I were intratracheally injected with bleomycin ( 1 mL,5 mg/kg) to induce pulmonary fibrosis. Group N were given saline as control. Group M/ I were injected the suspension of the CM-Dil labled-MSCs ( with no treatment/pre-incubated with IGF-1 for 48 hours) ( 0. 5mL,2 ×106 ) via the tail vein 2 days after injected bleomycin, and group B were injected with saline ( 0. 5 mL) simultaneously. The rats were sacrificed at 7,14,28 days after modeling. The histological changes of lung tissue were studied by HE and Masson’s trichrome staining. Hydroxyproline level in lung tissue was measured by digestion method. Frozen sections were made to observe the distribution of BMSCs in lung tissue, and the mRNA expression of hepatocyte growth factor ( HGF) was assayed by RTPCR.Results It was found that the red fluorescence of BMSCs existed in group M and I under the microscope and the integrated of optical density ( IOD) of group I was higher than that of group M at any time point. But the fluorescence was attenuated both in group M and group I until day 28. In the earlier period, the alveolitis in group B was more severe than that in the two cells-grafting groups in which group I was obviously milder. But there was no significant difference among group I, M and group N on day 28.Pulmonary fibrosis in group B, Mand I was significantly more severe than that in group N on day 14, but itwas milder in group M and I than that in group B on day 28. Otherwise, no difference existed between the two cells-grafting groups all the time. The content of hydroxyproline in group B was significantly higher than that in the other three groups all through the experiment, while there was on significant difference betweengroup I and group N fromthe beginning to the end. The value of group M was higher than those of group I and group N in the earlier period but decreased to the level of negative control group on day 28. Content of HGF mRNA in group Nand group I was maintained at a low level during the whole experiment process. The expression of HGF mRNA in group I was comparable to group M on day 7 and exceeded on day 14, the difference of which was more remarkable on day 28. Conclusions IGF-1 can enhance the migratory capacity of MSCs which may be a more effective treatment of lung disease. The mechanismmight be relatedto the increasing expression of HGF in MSCs.
Objective To explore the migration and differentiation of bone marrow mesenchymal stem cells（MSCs） in lung . Methods MSCs were harvested from a male Wister rat. Sixty female Wister rats were randomly divided into four groups. The pulmonary fibrosis model was established by intratracheal instillation of bleomycin in group A-D. Immediately and 7 days after bleomycin administration respectively，the rats in group B and C received infusion with 5-bromodeoxynridine （BrdU） labeled MSCs via tail vein. And the rats in group D were infused MSCs without BrdU labeling serving as a negative control. The sry gene of Y chromosome was detected by polymerase chain reaction （PCR）. Double immunofluorescence staining was used to detected BrdU and surfactant associated protein-C （SP-C） expression in lung tissue，fresh bone marrow，and the 5th generation MSCs. Reverse transcriptipon-PCR was used to detect the expressions of SP-C mRNA and AQP-5 mRNA. Results The sry gene was detected in bleomycin induced lung injury tissues of the rats after MSCs infusion immediately and on the 7th day The MSCs in lung tissue could transformed into cells with ACEⅡ morphological features and molecular phenotype. The transformation rate was higher in the rats received MSCs infusion immediately than the rats received on 7th day. The 5th generation MSCs and fresh bone marrow expressed SP-C mRNA，without AQP-5 mRNA and SP-C expression. Conclusions Exogenous MSCs can be transplanted into injured lung tissues and transform into AECⅡ，especially in early stage of lung injury. The differentiation potential of MSCs can be activated in injury micro-environment.
Objective To evaluate the effects of N-acetylcysteine ( NAC) on bleomycin-induced lung fibrosis in mice and to investigate the therapeutic mechanisms of NAC on lung fibrosis. Methods Forty-five KM female mice were randomly divided into 3 groups. The mice in the control group were administered with saline aerosol intratracheally. The mice in the fibrosis group were administered with bleomycin ( 3 mg/kg) dissolved in normal saline aerosol intratracheally. The mice in the NAC group were gastric perfused with NAC at a dose of 400 mg · kg- 1 · d - 1 after administering bleomycin aerosol intratracheally. All animals were sacrificed 28 days after the treatments. The left lung was fixed in 10% neutral formalin, then stained with hematoxylin eosin and Masson’s trichrome respectively for the pathological examination. The right lung was sampled and the content of hydroxyproline ( HYP) was assayed by alkaline hydrolysis method. The serum was collected and the concentrations of malondialdehyde ( MDA) and totalantioxidant capacity ( T-AOC) were measured by colorimetric method. The RNA and total tissue protein were extracted for the examination of NOX1 /2/4 by RT-PCR and Western blot respectively. Results NAC prevented lung fibrosis induced by bleomycin with significantly reducing lung collagen accumulation and the level of HYP in the NAC group ( P lt;0. 05) . The serum concentration of MDA were reduced and serum TAOC raised by treating NAC after intratracheal administration of bleomycin ( P lt;0. 05) . NOX1 /2/4 gene and protein expression were increased in the fibrosis group compared with the control group. NAC had no effect on the gene expression of NOX1/2 /4( P gt;0. 05) , but inhibitted the NOX4 protein expression in lung tissue significantly ( P lt; 0. 05) . Conclusion NAC inhibits the expression of NOX4 and prevents bleomycin-induced lung fibrosis in mice.
Objective To investigate the expression and significance of Krüppel-like factor 4 ( KLF4) in the lung tissues of mice with bleomycin-induced pulmonary fibrosis.Methods C57BL/6 mice were randomly divided into a control group and a BLM group. The mice in the BLM group were given a single intratracheal injection of bleomycin ( 2.5 mg/kg) , while those in the control group were injected with isodose physiological saline. The mice were sacrificed at the 12h and on the day 1, 2, 3, 7, 14 and 28, then HE stain and Masson’s trichrome stain were used to detect the architecture of alveolar and the deposition of cellularity and collagen. Real time-polymerase chain reaction ( RT-PCR ) and immunohistochemical technology were performed to investigate the expression of KLF4. Results In the bleomycin-induced pulmonary fibrosis, acute inflammation was observed on the day 1, 2 and 3, the inflammation was exacerbated and the collagen deposition began to be observed on the day 7, the architecture of alveolar was destroyed and the collagen deposition was more obvious on the day 14, while the alveolar structure was nearly recovered to normal, and the inflammation and collagen deposition were attenuated on the day 28. The expression of KLF4 mRNA increased from the day 1, then decreased, arrived at the minimumon the day 3, and then gradually increased until the day 28. The trend of KLF4 protein expression showed roughly the same as the KLF4 mRNA level, which started to increase on the day 1, then decreased, arrived at the minimum on the day 3,then gradually increased until the day 14 and then decreased again. Conclusion The expressions of KLF4 mRNA and protein are dynamically changed in the process of experimental pulmonary fibrosis, suggesting KLF4 may contribute to the pathogenesis of pulmonary fibrosis.