BACKGROUNDBleomycin-induced fibrosis is extensively used to model aspects of the pathogenesis of interstitial pulmonary fibrosis. This study aimed to determine the benefic effects and mechanisms of simvastatin on bleomycin-induced pulmonary fibrosis in mice.

METHODSBleomycin-induced pulmonary fibrosis mice were administered with simvastatin in different doses for 28 days. We measured inflammatory response, fibrogenic cytokines and profibrogenic markers in both bleomycin-stimulated and control lungs, and correlated these parameters with pulmonary fibrosis.

RESULTSSimvastatin attenuated the histopathological change of bleomycin-induced pulmonary fibrosis and prevented the increase of lung hydroxyproline content and collagen (I and III) mRNA expression induced by bleomycin. Moreover, simvastatin down-regulated the increased expression of transforming growth factor-beta1 (TGF-beta1) and connective tissue growth factor (CTGF) induced by bleomycin at both gene and protein levels. Simultaneously, the accumulation of neutrophils and lymphocytes and the increased production of tumor necrosis factor-alpha (TNF-alpha) in bronchial alveolar lavage fluid were inhibited by simvastatin in early inflammatory phase after bleomycin infusion. The higher dose of simvastatin was associated with a more significant reduction in these inflammatory and fibrotic parameters. Furthermore, the inactivation of p38, RhoA and Smad2/3 signaling pathways was observed during simvastatin administration.

CONCLUSIONSSimvastatin attenuated bleomycin-induced pulmonary fibrosis, as indicated by decreases in Ashcroft score and lung collagen accumulation. The inhibitory effect of simvastatin on the progression of pulmonary fibrosis may be demonstrated by reducing inflammatory response and production of TGF-beta1 and CTGF. These findings indicate that simvastatin may be used in the treatment of pulmonary fibrosis.

Animals ; Antibiotics, Antineoplastic ; Bleomycin ; Mice ; Mice, Inbred C57BL ; Pulmonary Fibrosis ; chemically induced ; metabolism ; pathology ; Simvastatin ; pharmacology

OBJECTIVETo evaluate effect of amygdalin on expression of four biomarkers in the animal model of pulmonary fibrosis induced by bleomycin.

METHODSRats were given one dose (5 mg/kg) of bleomycin in bleomycin-treated groups, amygdalin-treated groups and saline in controls by intratracheal instillation exposed surgically. The amygdalin-treated groups rats were treated with intraperitoneal injection of amygdalin (15 mg x kg(-1) x day(-1)). The rats were sacrificed 7, 14 and 28 days after bleomycin administration. Polarized light microscopy and Image-Pro Plus detected I and III collagen expressed in Paraffin-embedded lung sections stained with Sirius red. Surface-enhanced laser desorption-ionization time-of-flight mass spectrometry (SELDI-TOF MS) with weak cationic proteinchip (CM10) detected differentially expressed proteins in the pooled serum samples of all groups.

RESULTSConsistent fibrotic responses were found in all bleomycin and amygdalin-tread groups. On the 7th, 14th and 28th day after bleomycin or saline instillation, four differentially expressed proteins were detected in the pooled serum of all groups rats, consisting of 4 proteins with mass/charge ratio of 3530.7, 7043.5, 8332.6 and 9068.0, respectively. Compared with control groups, protein peaks intensity ratio with mass/charge ratio of 3530.7 on 7, 28 d and 7043.5, 8332.6 and 9068.0 on 7, 14 and 28 d was > 2 in bleomycin-treated groups. Compared with amygdalin-treated groups, protein peaks intensity with mass/charge ratio of 3530.7 at 7, 14, 28 d had no change almost, but protein peaks intensity ratio with mass/charge ratio of 7043.5 at 7 d, 8332.6 on 28 d and 9068.0 on 14 d was > 2 in bleomycin-tread groups. All the four protein peaks intensity had no change almost at other point.

CONCLUSIONAmygdalin may reduce the bleomycin-induced increase of differentially expressed protein peak intensities in rat serum.

Amygdalin ; pharmacology ; Animals ; Biomarkers ; blood ; Bleomycin ; adverse effects ; Blood Proteins ; metabolism ; Male ; Pulmonary Fibrosis ; blood ; chemically induced ; Rats ; Rats, Wistar

Pingyangmycin (bleomycin A5 hydrochloride, PYM) is one of the anti-neoplastic agents which have been commonly used to treat venous malformations. However, the underlying mechanism by which PYM treats venous malformations remains poorly understood. It was reported that venous endothelial cells could recruit neutrophils via adhesion molecules (E-selectin, ICAM-1, ICAM-3, VCAM-1) during the acute/chronic inflammation and subsequent histological fibrosis after sclerotherapy with PYM. This study explored if the expression of E-selectin, ICAM-1, ICAM-3 and VCAM-1 in human venous malformation endothelial cells could be affected by PYM. HVMECs were cultured from human venous malformation tissue. Expressions of E-selectin, ICAM-1, ICAM-3 and VCAM-1 on HVMECs in response to PYM were analyzed by cell ELISA. The relative levels of mRNA expression in the cells were semi-quantified. The results showed that PYM up-regulated the expressions of E-selectin, ICAM-3, VCAM-1 and ICAM-1 in both time- and concentration-dependent manner. Our findings suggested that PYM could induce the expression of adhesion molecules in HVMECs, which might be a possible mechanism by which sclerotherapy by intralesional injection of PYM treats venous malformations.
Bleomycin ; analogs & derivatives ; pharmacology ; Cell Adhesion Molecules ; genetics ; metabolism ; Cells, Cultured ; Endothelial Cells ; drug effects ; metabolism ; Endothelium, Vascular ; drug effects ; metabolism ; Gene Expression ; drug effects ; genetics ; Humans

OBJECTIVETo determine the effects of curcumin on bleomycin (BLM)-induced pulmonary fibrosis in rats.

METHODOne hundred and forty-four male Sprague-Dawley rats were randomized into 6 groups (24 rats in each group, model group, sham group, prednisone group (0.56 mg x kg(-1) x d(-1)), curcumin with low dose 5 mg group, curcumin with middle dose group 10 mg and curcumin with high dose group 20 mg per 100 g of body weight). Rats in all groups except in sham group were injected with BLM intratracheally. Curcumin with different doses were given by gavage one time everyday for 7, 14 and 28 days. Prednisone were given to rats in prednisone group, po, serving as the positive treatment group. On the 7th, 14th, 28th day, the lung functions (inspiratory resistance, maximal volutary ventilation, forced vital capacity, Fev 0.2/FVC, peak expiratory flow) were determinated in experimental rats, respectively, and the concentrations of hydroxyproline in lung homogenates of each rat were assayed.

RESULTAdministration of curcumin in different doses improved lung functions of BLM-induced fibrotic rats in the all experimental days; and it decreased the concentration of hydroxyproline in lung homogenates compared with those levels in model control group; and it also lessened the hyperplasia of BLM-induced pulmonary fibrosis in rats.

CONCLUSIONAdministration of curcumin can suppress BLM induced pulmonary fibrosis indicated by improved respiratory function, as well as companied with low content of hydroxyproline in lung tissue of rats.

Animals ; Bleomycin ; adverse effects ; Curcumin ; pharmacology ; Hydroxyproline ; metabolism ; Lung ; drug effects ; metabolism ; pathology ; physiopathology ; Male ; Pulmonary Fibrosis ; chemically induced ; metabolism ; pathology ; physiopathology ; Rats ; Time Factors

Objective: JWH133, a cannabinoid type 2 receptor agonist, was tested for its ability to protect mice from bleomycin-induced pulmonary fibrosis. Methods: By using a random number generator, 24 C57BL/6J male mice were randomly divided into the control group, model group, JWH133 intervention group, and JWH133+a cannabinoid type-2 receptor antagonist (AM630) inhibitor group, with 6 mice in each group. A mouse pulmonary fibrosis model was established by tracheal instillation of bleomycin (5 mg/kg). Starting from the first day after modeling, the control group mice were intraperitoneally injected with 0.1 ml of 0.9% sodium chloride solution, and the model group mice were intraperitoneally injected with 0.1 ml of 0.9% sodium chloride solution. The JWH133 intervention group mice were intraperitoneally injected with 0.1 ml of JWH133 (2.5 mg/kg, dissolved in physiological saline), and the JWH133+AM630 antagonistic group mice were intraperitoneally injected with 0.1 ml of JWH133 (2.5 mg/kg) and AM630 (2.5 mg/kg). After 28 days, all mice were killed; the lung tissue was obtained, pathological changes were observed, and alveolar inflammation scores and Ashcroft scores were calculated. The content of type Ⅰ collagen in the lung tissue of the four groups of mice was measured using immunohistochemistry. The levels of interleukin 6 (IL-6) and tumor necrosis factor α (TNF-α) in the serum of the four groups of mice were measured using enzyme-linked immunosorbent assay (ELISA), and the content of hydroxyproline (HYP) in the lung tissue of the four groups of mice was measured. Western blotting was used to measure the protein expression levels of type Ⅲ collagen, α-smooth muscle actin (α-SMA), extracellular signal regulated kinase (ERK1/2), phosphorylated P-ERK1/2 (P-ERK1/2), and phosphorylated ribosome S6 kinase type 1 (P-p90RSK) in the lung tissue of mice in the four groups. Real-time quantitative polymerase chain reaction was used to measure the expression levels of collagen Ⅰ, collagen Ⅲ, and α-SMA mRNA in the lung tissue of the four groups of mice. Results: Compared with the control group, the pathological changes in the lung tissue of the model group mice worsened, with an increase in alveolar inflammation score (3.833±0.408 vs. 0.833±0.408, P<0.05), an increase in Ashcroft score (7.333±0.516 vs. 2.000±0.633, P<0.05), an increase in type Ⅰ collagen absorbance value (0.065±0.008 vs. 0.018±0.006, P<0.05), an increase in inflammatory cell infiltration, and an increase in hydroxyproline levels [(1.551±0.051) μg/mg vs. (0.974±0.060) μg/mg, P<0.05]. Compared with the model group, the JWH133 intervention group showed reduced pathological changes in lung tissue, decreased alveolar inflammation score (1.833±0.408, P<0.05), decreased Ashcroft score (4.167±0.753, P<0.05), decreased type Ⅰ collagen absorbance value (0.032±0.004, P<0.05), reduced inflammatory cell infiltration, and decreased hydroxyproline levels [(1.148±0.055) μg/mg, P<0.05]. Compared with the JWH133 intervention group, the JWH133+AM630 antagonistic group showed more severe pathological changes in the lung tissue of mice, increased alveolar inflammation score and Ashcroft score, increased type Ⅰ collagen absorbance value, increased inflammatory cell infiltration, and increased hydroxyproline levels. Compared with the control group, the expression of α-SMA, type Ⅲ collagen, P-ERK1/2, and P-p90RSK proteins in the lung tissue of the model group mice increased, while the expression of type Ⅰ collagen, type Ⅲ collagen, and α-SMA mRNA increased. Compared with the model group, the protein expression of α-SMA (relative expression 0.60±0.17 vs. 1.34±0.19, P<0.05), type Ⅲ collagen (relative expression 0.52±0.09 vs. 1.35±0.14, P<0.05), P-ERK1/2 (relative expression 0.32±0.11 vs. 1.14±0.14, P<0.05), and P-p90RSK (relative expression 0.43±0.14 vs. 1.15±0.07, P<0.05) decreased in the JWH133 intervention group. The type Ⅰ collagen mRNA (2.190±0.362 vs. 5.078±0.792, P<0.05), type Ⅲ collagen mRNA (1.750±0.290 vs. 4.935±0.456, P<0.05), and α-SMA mRNA (1.588±0.060 vs. 5.192±0.506, P<0.05) decreased. Compared with the JWH133 intervention group, the JWH133+AM630 antagonistic group increased the expression of α-SMA, type Ⅲ collagen, P-ERK1/2, and P-p90RSK protein in the lung tissue of mice, and increased the expression of type Ⅲ collagen and α-SMA mRNA. Conclusion: In mice with bleomycin-induced pulmonary fibrosis, the cannabinoid type-2 receptor agonist JWH133 inhibited inflammation and improved extracellular matrix deposition, which alleviated lung fibrosis. The underlying mechanism of action may be related to the activation of the ERK1/2-RSK1 signaling pathway.
Mice ; Male ; Animals ; Pulmonary Fibrosis/pathology* ; Cannabinoid Receptor Agonists/metabolism* ; Collagen Type I/pharmacology* ; Collagen Type III/pharmacology* ; Hydroxyproline/pharmacology* ; Sodium Chloride/metabolism* ; Mice, Inbred C57BL ; Lung/pathology* ; Cannabinoids/adverse effects* ; Bleomycin/metabolism* ; Collagen/metabolism* ; Inflammation/pathology* ; RNA, Messenger/metabolism*

Objective To investigate the effects and mechanism of Interleukin-33 (IL-33) mediated proliferation and differentiation of pulmonary myofibroblasts (MFbs) in pulmonary fibrosis (PF). Methods C57BL/6 mice were randomly divided into four groups: a control group, a bleomycin (BLM) group, a BLM combined with IL-33 group and a BLM combined with anti-IL-33 antibody group, 12 mice in each group. The PF model was induced by intratracheal injection of BLM (5000 U/kg). The degrees of fibrosis were examined using HE and Masson staining. ELISA was used to measure the plasma levels of IL-33. Immunohistochemical staining was used to measure the expression of alpha smooth muscle actin (α-SMA) in lung tissue. Primary pulmonary fibroblasts were isolated and cultured from lung tissues of mice. The cells were divided into four groups: a control group, an IL-33 group, an IL-33 combined with dimethyl sulfoxide (DMSO) group and an IL-33 combined with pyrrolidine dithiocarbamate (PDTC) group. The cells were treated with DMSO or PDTC for 1 hour and then with IL-33 for 48 hours. Cell proliferation was measured by 5-ethynyl-2'-deoxyuridine (EdU) assay and cell cycle was measured by flow cytometry. Transwell^TM assay was used to analyze cell migration. Real-time quantitative PCR was used to measure the expression of collagen type I (Col1), Col3 and α-SMA mRNA. The protein levels of IL-33, Col1, Col3, α-SMA, eukaryotic initiation factor 3a (eIF3a), phosphorylated IκBα (p-IκBα) (total lysate), p-NF-κB p65(total lysate) and NF-κB p65 (nucleus) were measured by Western blot analysis. Results In vivo, compared with the control group, the expressions of IL-33, p-IκBα (total lysate), p-NF-κB p65 (total lysate), NF-κB p65(nucleus), eIF3a, α-SMA, Col1 and Col3 in the BLM group significantly increased. Compared with the BLM group, the expressions of p-IκBα (total lysate), p-NF-κB p65 (total lysate), NF-κB p65 (nucleus), eIF3a, α-SMA, Col1 and Col3 in the IL-33 group increased further and the PF was further aggravated. But the effect of anti-IL-33 antibody was just opposite to that of IL-33. In vitro, IL-33 markedly induced the proliferation and migration of pulmonary fibroblasts, and significantly up-regulated the expression of p-IκBα (total lysate), p-NF-κB p65(total lysate), NF-κB p65 (nucleus), eIF3a, α-SMA, Col1 and Col3. But all these effects of IL-33 were reversed by pyrrolidine dithiocarbamate. Conclusion The results suggest that IL-33 may promote the expression of eIF3a by activating NF-κB signaling pathway, thus inducing the proliferation and differentiation of MFbs and promoting the occurrence and development of PF.
Animals ; Mice ; Bleomycin/metabolism* ; Cell Differentiation ; Cell Proliferation ; Dimethyl Sulfoxide/pharmacology* ; Fibroblasts ; Interleukin-33/pharmacology* ; Mice, Inbred C57BL ; Myofibroblasts/metabolism* ; NF-kappa B/metabolism* ; NF-KappaB Inhibitor alpha/metabolism* ; Pulmonary Fibrosis ; Signal Transduction

Telomeres are the ends of the linear chromosomes of eukaryotes and consist of tandem GT-rich repeats in telomere sequence i.e. 500-3000 repeats of 5'-TTAGGG-3' in human somatic cells, which are shortened gradually with age. The G-rich overhang of telomere sequence can adopt different intramolecular fold-backs and tetra-stranded DNA structures, in vitro, which inhibit telomerase activity. In this report, DNA binding agents to telomere sequence were studied novel therapeutic possibility to destabilize telomeric DNA sequences. Oligonucleotides containing the guanine repeats in human telomere sequence were synthesized and used for screening potential antitumor drugs. Telomeric DNA sequence was characterized using spectral measurements and CD spectroscopy. CD spectrum indicated that the double-stranded telomeric DNA is in a right-handed conformation. Polyacrylamide gel electrophoresis was performed for binding behaviors of antitumor compounds with telomeric DNA sequence. Drugs interacted with DNA sequence caused changes in the electrophoretic mobility and band intensity of the gels. Depending on the binding mode of the anticancer drugs, telomeric DNA sequence was differently recognized and the efficiency of cleavage of DNA varies in the bleomycin-treated samples under different conditions. DNA cleavage occurred at about 1% by the increments of 1 mM bleomycin-Fe(III). These results imply that the stability of human telomere sequence is important in conjunction with the cancer treatment and aging process.
Antineoplastic Agents/*metabolism ; Bleomycin/metabolism/pharmacology ; Circular Dichroism ; Comparative Study ; DNA/chemistry/drug effects/*metabolism ; DNA Damage ; Dactinomycin/metabolism ; Doxorubicin/*analogs & derivatives/metabolism ; Human ; Nogalamycin/metabolism ; Nucleic Acid Conformation ; *Repetitive Sequences, Nucleic Acid ; Telomere/drug effects/*genetics

OBJECTIVETo investigate the effect of AP-1 Decoy on matrix metalloproteinase 2 (MMP-2) and tissue inhibitor of metalloproteinase (TIMP-1) imbalance induced by bleomycin-A5 (BLM-A5) in pulmonary fibroblasts.

METHODSPulmonary fibroblasts were primary cultured, and transferred with AP-1 Decoy before treated with BLM-A5. MMPs activity in medium was determined by gelatin zymography. Protein content of TIMP-1 in medium was detected by ELISA. Expression of MMP-2 mRNA and TIMP-1 mRNA were determined by reverse transcriptase-polymerase chain reaction (RT-PCR).

RESULTSBLM-A5 induced the increase in activity of MMP-2 at 12 h [A: (0.77 +/- 0.08) vs (0.65 +/- 0.07) P < 0.05], but it was suppressed by AP-1 Decoy [A: (0.68 +/- 0.05)]. BLM-A5 up-regulated the expression of protein and mRNA of TIMP-1 after 12 h, and 24 h [(39.3 +/- 4.3), (46.3 +/- 4.8) ng/ml vs (28.9 +/- 2.7), (31.6 +/- 2.4) ng/ml] and [Absorbance ratio to beta-actin: (0.94 +/- 0.13, 1.08 +/- 0.06) vs (0.76 +/- 0.07, 0.75 +/- 0.08)] (P < 0.05 or P < 0.01) but AP-1 Decoy modulated the up-regulation. All these indexes in AP-1 Decoy group had no significant difference in contrast to the normal group. Mutant AP-1 Decoy had not the same function as AP-1 Decoy on the expression of MMP-2 and TIMP-1 in pulmonary fibroblasts.

CONCLUSIONAP-1 Decoy inhibits the increase in MMP-2 activity and the up-regulation of TIMP-1 induced by BLM-A5 in pulmonary fibroblasts.

Bleomycin ; analogs & derivatives ; pharmacology ; Fibroblasts ; drug effects ; metabolism ; Humans ; Lung ; cytology ; metabolism ; Matrix Metalloproteinase 2 ; metabolism ; Pulmonary Fibrosis ; metabolism ; Tissue Inhibitor of Metalloproteinase-1 ; metabolism ; Transcription Factor AP-1 ; metabolism

OBJECTIVETo study the effect of anti-basic jibroblast grouth factor (bFGF) on bleomycin-induced pulmonary fibrosis in rats and its possible mechanism.

METHODSPulmonary fibrosis was induced in Wistar rats by intratracheal instillation of bleomycin (model group, group M). Then the rats received anti-bFGF on 1, 2, 3, 8, 12, 19, 25 days intracavitary injection (group K), normal saline(group C) orally. Normal controls received normal saline both intratracheally and orally. Five rats in each group were sacrificed on 1.4 week after intratracheal instillation. Histological changes of the lungs were evaluated by HE stain and Massons trichrome stain. Lung expression of bFGF proteins was assessed by immunohistochemistry and the level of bFGF protein in serum and BALF was further measured by ELISA.

RESULTSPulmonary fibrosis of group M was higher than that of group C. bFGF in group M was higher than that in group C in lung, serum and BALF on 1.4 week. Pulmonary fibrosis of group K was lower than that of group M. bFGF in group K was lower than that in group M in lung, serum and BALF on 1.4 week.

CONCLUSIONAnti-bFGF alleviates bleomycin-induced pulmonary fibrosis in rats. Inhibiting the expressions of bFGF in lung tissues may be one of the mechanisms.

Animals ; Antibodies ; immunology ; pharmacology ; Bleomycin ; Female ; Fibroblast Growth Factor 2 ; immunology ; Pulmonary Fibrosis ; chemically induced ; metabolism ; physiopathology ; Random Allocation ; Rats ; Rats, Wistar

OBJECTIVETo study the combined damage-effects of low-intensity 2,450 MHz microwave (MW) with three chemical mutagens on human lymphocyte DNA.

METHODSDNA damage of lymphocytes exposed to microwave and(or) with chemical mutagens were observed at different incubation time (0 h or 21 h) with comet assay in vitro. Three combination-exposure ways of MW with chemicals were used: MW irradiation before chemical exposures, simultaneously exposed to MW and chemicals and MW irradiation after chemical exposures. The three chemical mutagens were mitomycin C (MMC, DNA crosslinker), bleomycin (BLM, radiometric agent), methyl methanesulfonate (MMS, alkylating agent). The exposure time of MW and chemical mutagens were 2 h and 3 h respectively.

RESULTSThe differences of comet tail length between MW group and control group were not significant when lymphocytes were incubated for 0 h or 21 h (P > 0.05). However, when lymphocytes were incubated for 21 h with 30.00 micro mol/L of MMC, the comet tail lengths of MW + MMC group, MW-MMC group and MMC + MW group were (18.00 +/- 5.96), (21.79 +/- 11.47) and (22.32 +/- 8.10) micro m respectively; while with 3.00 micro mol/L of MMC, the comet tail lengths were (8.99 +/- 3.75), (12.40 +/- 5.35) and (14.00 +/- 5.38) micro m respectively, which were significantly higher than those of corresponding MMC groups [(9.42 +/- 3.34) and (6.50 +/- 2.89) micro m, P < 0.01 or P < 0.05]. The DNA damage of MW plus BLM groups and MW plus MMS groups were not significantly different from the corresponding BLM and MMS groups (P < 0.05).

CONCLUSION2 450 MHz MW (5 mW/cm(2)) did not induce DNA damage directly, but could enhance the DNA damage effects induced by MMC. The synergistic effects of 2 450 MHz MW with BLM and MMS were not obvious.

Bleomycin ; pharmacology ; Comet Assay ; DNA ; drug effects ; genetics ; radiation effects ; DNA Damage ; Humans ; Lymphocytes ; drug effects ; metabolism ; radiation effects ; Methyl Methanesulfonate ; pharmacology ; Microwaves ; adverse effects ; Mitomycin ; pharmacology ; Mutagens ; pharmacology ; Time Factors