【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 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 effects of bursopentin ( BP5) on expression of extracellular matrix in human lung fibroblasts ( HLFs) and its mechanism.Methods HLFs were cultured in vitro and divided into five groups. The cells in the control group were cultured in DMEMwithout TGF-β1 or BP5. The cells in TGB-β1 treatment group were cultured in DMEMcontaining 5 μg/L TGF-β1 . While in three TGF-β1 + BP5 treatment groups, the cells were cultured in DMEM containing 5 μg/L TGF-β1 and simultaneously intervened with BP5 at three different concentrations ( 2. 5 μg/mL, 5 μg/mL, and 10 μg/mL respectively) . The expression of α-SMA was detected using a fluorescent-labeling strategy. The expressions of Collagen-Ⅰ, p-Smad2/3, p-Smad3, and Smad7 proteins were measured by Western blot. Results The cells in the TGF-β1 treatment group showed positive expression of α-SMA, implying TGF-β1 had induced fibroblasts to differentiate into myofibroblasts. In the TGF-β1 treatment group, the expressions of collagen-Ⅰ( 1. 402 ±0. 158 vs. 0. 605 ±0. 367) , p-Smad2/3 ( 1. 457 ±0. 111 vs. 0. 815 ±0. 039) , and p-Smad3 ( 1. 320 ±0. 147 vs. 0. 623 ±0. 128) increased with statistical significance ( P lt; 0. 01) . Meanwhile the expression of Smad7 reduced ( 0. 614 ±0. 107 vs. 0. 865 ±0. 063, P lt;0. 05) . But in the TGF-β1 + BP5 treatment groups, over-expressions of collagen-Ⅰ, α-SMA, p-Smad2 and p-Smad3 induced by TGF-β1 were obviously inhibited by BP5, especially at the BP5 concentration of 10 μg/mL ( collagen-Ⅰ: 0. 718 ±0. 049 vs. 1. 402 ±0. 158; p-Smad2 /3: 0. 696 ±0. 031 vs. 1. 457 ±0. 111; p-Smad3: 0. 766 ±0. 006 vs. 1. 320 ±0. 147; all P lt; 0. 01) . Otherwise, the up-regulation of Smad7 ( 1. 237 ±0. 173 vs. 0. 614 ±0. 107) was found.Conclusions Bursopentin can reduce the expressions of collagen-Ⅰ and α-SMA protein of fibroblast stimulated by TGF-β1 , maybe through inhibiting TGF-β1 /Smads transduction pathway. It is suggested that bursopentin may have intervention effect on pulmonary fibrosis.
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.
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.
ObjectiveTo investigate the endoplasmic reticulum stress associated apoptosis gene Caspase-12 expression in paraquat-induced pulmonary fibrosis. Methods30 adult healthy Sprague-Dawley(SD) rats were randomly divided into a nomal control group,two pulmonary fibrosis model groups (intragastrically administered paraquat for 14 days and 28 days,respectively).The model of pulmonary fibrosis was established through intragastrically administering paraquat at the dose of 30 mg/kg.RT-PCR was used to determine the mRNA expression of Caspase-12.Immunohistochemistry was used to determine the protein expression of Caspase-12.HE staining and Masson staining were used to determine the degree of alveolitis and pulmonary fibrosis. ResultsHE staining and Masson staining of lung tissues proved that pulmonary fibrosis model was successfully constructed.The degree of alveolitis and pulmonary fibrosis in the model group was significantly more serious than that in the control group(P<0.01).RT-PCR and Immunohistochemistry results showed that the expression of Caspase-12 were remarkably increased in the pulmonary fibrosis model group(14 d group)(P<0.01),even more elevated in 28 d group compared with the 14 d group. ConclusionThe results demonstrate that the expression of Caspase-12 in paraquat poisoned rats is up-regulated,suggesting endoplasmic reticulum stress plays an important role in paraquat induced-pulmonary fibrosis.
Objective To investigate the effects of curcumin on oxidative stress in the co-culture system including human fetal lung fibroblasts and A549 cells, and discuss the potential and protective mechanism of the prophylactic effect of curcumin on pulmonary fibrosis. Methods The human fetal lung fibroblasts co-cultured with A549 cells were divided into five groups. The cells in the control group were cultured in DMEM without TGF-β1 or curcumin. The cells in the TGF-β1 group were cultured in DMEM containing 5 ng/mL TGF-β1 . In three TGF-β1 + cucurmin treatment groups, the cells were cultured in DMEM containing 5 ng/mL TGF-β1 and three different concentration of curcumin( 5, 10, 20 μmol /L, respectively) . ELISA was used to analyze the content of TNF-α. Serum level of MDA and SOD were tested by spectrophotometric analysis. Intracellular ROS production was detected by flow cytometry. NF-κB was measured by western blot. Results The serum MDA, intracellular ROS, the content of TNF-αand NF-κB protein expression in the TGF-β1 group were significantly increased while the activity of SOD was significantly decreased( P lt; 0. 01) , suggesting that the oxidative level of human fetal lung fibroblasts was obviously increased after TGF-β1 stimulation. After intervening by different concentration of curcumin, the serum MDA, intracellular ROS, content of TNF-αand NF-κB were significantly decreased while the activity of SOD was obviously increased( P lt;0.01) . Conclusion Low concentration of curcumin can reduce the oxidative level of human fetal lung fibroblasts co-cultured with A549 after TGF-β1 stimulation, and significantly increase the level of SOD, implying that curcumin may intervene pulmonary fibrosis by reduce oxidative level.
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 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.
Objective To establish a mouse model of acute lung injury ( ALI) and pulmonary fibrosis by low dose lipopolysaccharide ( LPS) intermittent intraperitoneal injection, and to explore the pathogenesis of ALI and pulmonary fibrosis induced by endotoxin. Methods Forty C57BL/6 mice were randomly divided into a control group, a 3-days LPS group, a 2-weeks LPS group, and a 4-weeks LPS group,with 10 mice in each group. LPS was injected intraperitoneally at dose of 5 mg/ kg for three consecutive daysin the three LPS groups. Equivalent normal saline was injected by the same way in the control group. The mice lung tissues were obtained respectively 3 days ( the control group and 3-days LPS group) , 2 weeks ( the 2-weeks LPS group) , and 4 weeks ( the 4-weeks LPS group) after LPS or saline stimulation. HE staining,Van-Gieson collagen staining, and Ashcroft fibrosis score assessment were applied to evaluate the development of inflammation and fibrosis in lung tissue at various stages of ALI after LPS-stimulation. The mRNA expression of type Ⅰ procollagen and alpha smooth muscle actin ( α-SMA) were detected by realtime PCR. The deposition of collagen and fibrosis in lung tissue were detected by hydroxyproline assay. The survival condition of each group was also recorded. Results Acute inflammation occurred in mice lung tissue 3 days after intraperitoneal injection of LPS. Collagen deposited in pulmonary interstitium2 weeks afterLPS-stimulation and formed typical pulmonary interstitial fibrosis 4 weeks later accompanying with increase of Ashcroft fibrosis score. Real-time PCR and hydroxyproline assay showed that the expression of collagen and α-SMA increased 3 days after LPS-stimulation and reached the peak 4 weeks later. The animals were all survived up to the endpoint of experiment. Conclusions Accompanying with inflammation, pulmonary fibrosis initiated at early stage of ALI induced by LPS. Intraperitoneal injection of LPS at dose of 5 mg/kg for three consecutive days was able to establish the mouse model of ALI and pulmonary fibrosis with high successrate and low animal mortality, which provide an ideal experimental platform for further investigation.