Objective To investigate the effects of sodium ferulate on lung mRNA expression of TGF-β1 signal transduction molecule in rats with pulmonary fibrosis，and explore the mechanism of sodium ferulate on pulmonary fibrosis.Methods A rat model of pulmonary fibrosis was induced by intratracheal injection of bleomycin (5 mg/kg).Thirty SD rats were randomly divided into three groups (n=10 in each group)，ie.a control group，a pulmonary fibrosis model group，and a sodium ferulate group.The lung histopathology and the expression of collagen was examined by HE staining and collagen fibril staining respectively.The expressions of TGF-βRII and Smad4 mRNA in the lung tissue were detected by situ hybridization.And the expression of TGF-β1 mRNA was detected by real-time fluorescence-quantification RT-PCR.Results Collagen fibril staining indicated that the expression of pulmonary collagen in the model group was significantly higher than that in the control group and sodium ferulate group (Plt;0.001).The mRNA expressions of pulmonary TGF-β1，TGF-βRII and Smad4 were significantly higher in the model group than those in the control group (all Plt;0.01)，and were significantly lower in the sodium ferulate group than those in the model group (all Plt;0.05).Conclusions Sodium ferulate can effectively reduce pulmonary fibrosis through inhibition of the mRNA expression of TGF-β1，TGF-βRII and Smad4 in the lung tissue，thus influence the TGF-β1/Smad4 signal transduction way and inhibit the target gene activation.
ObjectiveTo study effects of two kinds of epidermal growth factor receptor kinase inhibitors on bleomycin-induced pulmonary fibrosis in mice, and regulation mechanism on oncostatin M (OSM) and downstream signaling pathways.MethodsForty Kunming female mice were randomly divided into a control group, a fibrosis group, a gefitinib group, and an erlotinib group. The mice in the control group were administered with saline aerosol intratracheally. The mice in the fibrosis group were administered with bleomycin at a dose of 3 mg/kg aerosol intratracheally. The mice in the gefitinib group and the erlotinib group were administered with bleomycin at a dose of 3 mg/kg aerosol intratracheally and then gastrically perfused with gefitinib (20 mg·kg–1·d–1) or erlotinib (25 mg·kg–1·d–1). All mice accepted computer tomography examination 14 days after the treatment and then were sacrificed, and the lungs were collected for further detection. The lungs were stained with hematoxylin eosin and Masson’s trichrome, examined with Western blot for pathological examination and expressions of α-smooth muscle actin (α-SMA), OSM, Janus kinase 1 (JAK1), phospho-JAK1 (p-JAK1), signal transducers and activators of transcription 3 (STAT3), and phospho-STAT3 (p-STAT3) proteins.ResultsThe pathological injury of the lung in the gefitinib group and the erlotinib group was significantly relieved compared with that in the bleomycin group. The expressions of α-SMA, OSM, p-JAK1/JAK1, and p-STAT3/STAT3 proteins were also significantly reduced. There were no differences between the above-mentioned indexes between the gefitinib group and the erlotinib group.ConclusionsGefitinib and erlotinib can significantly relieve bleomycin-induced pulmonary fibrosis in mice. The underlying mechanism may be involved in inhibiting expression of OSM and downstream JAK/STAT pathways.
Objective To investigate the mechanismof lung injury caused by paraquat poisoning by observing the changes of fibrogenic cytokines in acute paraquat poisoned rats and the effects of pyrrolidine dithiocarbamate ( PDTC) . Methods Sprague-Dawley rats were randomly divided into three groups, ie. acontrol group ( n =6) , a PDTC group ( n =36) , a paraquat group ( n = 36) , and a paraquat + PDTC group( n =36) . The rats in the PDTC group, the paraquat group, and the paraquat + PDTC group were subdivided into 6 subgroups sacrificed respectively on 1st, 3rd,7th,14th, 28th and 56th day after the treatment. The levels of transforming growth factor-β1( TGF-β1 ) , platelet-derived growth factor ( PDGF) , insulin-like growthfactor-1 ( IGF-1) in serum were measured. Meanwhile the expression of connective tissue growth factor ( CTGF) and hydroxyproline in lung tissues were detected. The relationship of above cytokines with hydroxyproline was analyzed. Results The destructive phase in early ( 1 ～7 d) was characterized by hemorrhage, alveolar edema, and inflammatory cell infiltration. The proliferous phase in later stage ( 14 ～56 d) was characterized by diffused alveolar collapse with fibroblast proliferation and patchy distribution of collagen fibers. Compared with the control group, the level of TGF-β1 on all time points, the level of PDGF from7th to 56th day, the level of IGF-1 from3rd to 56th day in the paraquat group all significantly increased ( P lt;0. 01) . Immunohistochemistry results showed CTGF positive cells mainly located in aleolar epithelialcells, endothelial cells,macrophages in early stage, and fibroblasts were main positive cells on the 28th and the 56th day. The expression of CTGF in the paraquat group increased gradually compared with the control group on different time points ( P lt; 0. 05 or P lt; 0. 01) . Meanwhile, the levels of above cytokines were positively correlated with the level of hydroxyproline. Noteworthy, PDTC treatment led to significant decreases of above cytokines compared with the paraquat group in corresponding time points ( P lt;0. 05 or P lt;0. 01) .Conclusions Over expressions of IGF-1, TGF-β1 , PDGF, IGF-1 and CTGF may play important roles in lung fibrosis of paraquat poisoned rats. PDTC, as a b NF-κB inhibitor, may inhibits NF-κB activity and further significantly decreases expressions of cytokines, leading to significantly attenuated pulmonary inflammation and fibrosis. However, the mechanisms of PDTC intervention still remain to be explored.
ObjectiveTo analyze the clinical characteristics and survival of pulmonary fibrosis (PF) patients complicated with lung cancer (LC) (PL-LC). MethodsFifty-three patients with PF diagnosed as LC from January 2008 to March 2014 in Nanjing Drum Tower Hospital were included in this study. Univariate analysis and Cox regression analysis were used to detect the effects of clinical variables on survival. Kaplan-Meier method was used to calculate the median survival time (MST) and overall survival (OS). ResultsMale patients (n=48, 90.6%) and patients with a history of smoking (n=42, 79.2%) were more easily suffered from PF-LC. The average age was 68.6±9.5 years. Cox multivariate analysis revealed that Velcro crackles (P=0.009) and clinical stage (P=0.013) were the independent risk factors of survival in the patients with PF-LC. The MST of 53 patients was 6.0 months.The survival rates of 1-year and 2-year were 34.1% and 22.0%, respectively. Forty-two (79.2%) patients were idiopathic pulmonary fibrosis (IPF) complicated with LC, and 11 (20.8%) patients were secondary pulmonary fibrosis (SPF) complicated with LC. OS difference between two groups was not significant (P=0.610). OS of NSCLC group (n=37) was significantly prolonged than that of SCLC group (n=6) and unclassified pathological pattern group (n=10) (P=0.035). OS of Ⅰ and Ⅱstage patients (n=13) was significantly longer than that of Ⅲ and Ⅳ stage patients (n=40) (P=0.002). MST and OS of patients with LC treated (n=31) were significantly better than those of untreated patients (n=22) (P < 0.001) and OS of patients treated by comprehensive therapy (n=11) was significantly prolonged than that of patients treated by mono-therapy (n=20) (P=0.036). ConclusionsVelcro crackles and clinical stages are the independent risk factors of prognosis in PF-LC patients. It is beneficial to survival if the PF patients with LC were treated by comprehensive therapy.
ObjectiveTo construct a luciferase reporter fusion containing the human connective tissue growth factor (CTGF) gene promoter.MethodsThe promoter region of the human CTGF gene (-835/+214) was amplified by polymerase chain reaction (PCR) using specially-designed primers, and subsequently cloned into the pGL3.0-Basic vector. Following screening and verification by single colony PCR, double digestion, and sequencing, the resulting pGL3.0-Basic-CTGF was used to transfect the human embryonic kidney cells 293T, human bronchial epithelial cells HBE and human lung epithelial cells A549, and its function in each cell line was determined by luciferase assay.ResultsSequence alignment showed 99.5% identity, suggesting successful construction of the pGL3.0-Basic-CTGF reporter fusion. Promoter activities were detected 48 hours after transfection of pGL3.0-Basic-CTGF into the 293T, HBE, and A549 cells, and the promoter activities were 2.416, 0.027, and 0.121, respectively (P<0.01). Moreover, the luciferase activity in the A549 cells was statistically higher than that in the HBE cells (P<0.01).ConclusionsThe human pGL3.0-Basic-CTGF luciferase reporter fusion has been successfully constructed. The construct exhibits promoter activity in the bronchial epithelial cells HBE and the lung epithelial cells A549, and can therefore serve as a useful tool for future research in transcriptional regulation.
【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.
ObjectiveTo observe the effects of Th9 cell relative factors, including PU.1, interferon regulatory factor 4 (IRF4) and interleukin 4 (IL-4), in rats with pulmonary fibrosis. MethodsNinety SD rats were randomly divided into 3 groups, ie. a normal group, a pulmonary fibrosis group, and a dexamethasone treatment group, with 30 rats in each group. Ten rats in each group were sacrificed respectively on 7th, 14th, 28th days. Model rats were induced by injecting bleomycin into trachea. Real-time PCR was applied to detect mRNA expression of PU.1 and IRF4 in bronchoalveolar lavage fluid. IL-4 in the peripheral blood was measured by ELISA. ResultsIn the normal group, the lung tissue was normal without inflammatory reaction and fibrosis at any time points. In the pulmonary fibrosis group, at the early stage the lung tissue showed alveolar inflammation with a large number of macrophages and other inflammatory cells infiltratation in the pulmonary interstitial and alveolar cavity; on 14th day, part of the alveolar structure disappeared, inflammatory cells infiltrated slightly, while the alveolar septum was mildly widened and fibroblasts proliferated; on 28th day, alveolar structure was destructed, partial alveolar walls were collapsed, alveolar septuml was significantly widened, extracellular matrix was hyperplastic, a wide range of fibrosis occured. In the dexamethasone treatment group, the alveolar structure exsisted completely, and the inflammatory cell infiltration, widened alveolar septum and fibrosis were significantly lighter than those in the pulmonary fibrosis group. PU.1 mRNA was significantly lower in the pulmonary fibrosis group compared with the normal group. Compared with the pulmonary fibrosis group, PU.1 mRNA were lower on 14th day and 28th day in the dexamethasone treatment group (P < 0.05). PU.1 mRNA increased from 7th day, reached peak on 14th day, and declined on 28th day. IRF4 mRNA was significantly lower in the pulmonary fibrosis group compared with the normal group. Compared with the pulmonary fibrosis group, IRF4 mRNA was lower on 28th day in the dexamethasone treatment group (P < 0.05). There was a positive correlation between the content of IRF4 mRNA and IL-4 on 14th day in the pulmonary fibrosis group (r=0.044, P < 0.05). ConclusionPU.1 and IRF4 play a role in inflammation leading to pulmonary interstitial fibrosis, and IL-4 may regulate Th9 cells through activating IRF4.
ObjectiveTo investigate the effects and mechanism of atorvastatin in the experimental pulmonary fibrosis. MethodsFifty-four C57BL/6 mice were randomly divided into a control group,a bleomycin group and an atorvastatin group. The mice in the bleomycin group and the atorvastatin group received a single dose intratracheal injection of bleomycin (2.5 mg/kg),while the mice in the control group were injected with isodose physiological saline. The mice in the atorvastatin group were treated with atorvastatin 10 mg·kg-1·d-1 by intragastric administration the day after bleomycin instillation. All groups were sacrificed on the day 3,14 and 28,respectively. HE staining and Masson staining were used to detect the architecture of alveolar and the deposition of cellularity and collagen. RT-PCR and immunohistochemical technology were performed to detect the expression of Krüppel like factor 4 (KLF4). Zymography was used to investigate the activation of matrix metalloproteinase-2(MMP-2). ResultsAfter the treatment of bleomycin,the lung tissues showed acute inflammation on the day 3,the collagen deposition was more obvious and the architecture of alveolar was destroyed on the day 14. The alveolar structure,the inflammation and collagen deposition were attenuated on the day 28 compared with the day 14. Compared with the bleomycin group,the inflammation and the collagen deposition were significantly reduced in the atorvastatin group (P<0.05). Compared with bleomycin group,the expression of KLF4 significantly decreased in the atorvastatin group,although the expression of KLF4 mRNA increased on the day 3 compared with the bleomycin group (0.502±0.261 vs. 0.326±0.164,P<0.05). The expression of KLF4 protein on the day 3 was significantly decreased compared with the bleomycin group (0.048±0.015 vs. 0.130±0.017,P<0.05). After the intervention of bleomycin,the activation of MMP-2 on the day 3 and 14 significantly increased compared with the control group (3.136±1.321 and 3.449±0.356 vs. 0.983±0.147,P<0.05),and significantly decreased after the treatment of atorvastatin (2.191±0.800 and 2.506±0.761). ConclusionAtorvastatin may have anti-inflammation and anti-fibrosis activities in experimental pulmonary fibrosis through KLF4 pathway.
Objective To investigate the proliferation inhibitory effect and to explore the molecular mechanism of curcumin on pulmonary fibroblasts. Methods Fibroblasts derived from lung tissue of patients with idiopathic pulmonary fibrosis ( IPF) was cultured in vitro and incubated with curcumin at different concentrations for different time. Fibroblasts were randomized into 5 groups, ie. a control group and 4 curcumin groups ( intervened by 5, 10, 20, 40 μmol / L curcumin, respectively) . MTT assay was used to determine the inhibitory rate of curcumin on the proliferation of pulmonary fibroblasts. Apoptosis and the Caspase-3 expression of pulmonary fibroblasts were identified by flow cytometry ( FCM) . Variables were compared with One-Way ANOVA. The correlations between variables were analyzed using Pearson’scorrelation coefficient. Results Curcumin inhibited pulmonary fibroblasts proliferation in a dose-dependent and time-dependent manner( r =0. 886, r = 0. 832, respectively, all P lt; 0. 01) . Apoptosis rate of pulmonary fibroblasts in 4 curcumin groups was ( 29. 58 ±2. 13) % , ( 64. 36 ±3. 92) %, ( 72. 98 ±4. 42) % , ( 83. 14 ±2. 51) % , respectively, which was significantly higher than that in the control group[ ( 3. 84 ±1. 88) % , P lt;0. 01] . The positive expression rate of apoptosis-regulating protein caspase-3 was ( 26. 24 ±3. 64) % ,( 44. 87 ±5. 31) % , ( 57. 44 ±4. 23) % , ( 73. 65 ±5. 01) % , respectively, which was significantly higher than that of the control group[ ( 4. 02 ±0. 62) % , P lt; 0. 01] . Conclusions In vitro, curcumin can significantly inhibit proliferation and induce apoptosis of pulmonary fibroblasts of patients with IPF. The mechanism maybe associated with up-regulating expression of Caspase-3.
Pulmonary fibrosis is a kind of chronic and fibrotic lung disease caused by a variety of reasons, and its main pathological characteristic is excessive scar formation after the destruction of normal lung tissue structure, which eventually leads to respiratory insufficiency. Although the research on the pathophysiological mechanism of pulmonary fibrosis has made great progress, its pathogenesis has not been fully elucidated, and it is still clinically incurable. In recent years, studies have shown that non-coding RNAs are involved in the pathogenesis of pulmonary fibrosis, therefore, this article summarizes the related research progress of non-coding RNA in regulation of pulmonary fibrosis by affecting epithelial-mesenchymal transition, fibroblast activation and function of macrophages, in order to provide new ideas for the treatment of pulmonary fibrosis.