OBJECTIVETo evaluate the effect of water channel aquaporin 4 (AQP4) on bleomycin-induced lung fibrosis in mice.

METHODSIn wild type and AQP4 gene knockout (AQP4-/-) mice, lung fibrosis was induced by injection of bleomycin (3 mg/kg) into the trachea and saline injection was used as a control. At d3, 7, 14, 28 after bleomycin-treatment, mice were randomly sacrificed in batch and the lung coefficient was determined. Serum levels of TGF-β1 and TNF-α were measured by ELISA and hydroxyproline contents in lung tissue were determined by Alkaline hydrolysis method. H-E staining and Masson's staining were performed to examine the pathological changes of lung tissues after bleomycin-treatment.

RESULTSOn d14 after bleomycin-treatment, the lung coefficients in wild type mice and AQP4-/- mice were 1.9-fold (12.69 ± 6.05 vs 6.80 ± 0.82, q=4.204, P<0.05) and 2.3-fold (14.05 ± 5.82 vs 6.05± 0.58, q=5.172, P<0.01) of that in control, respectively, but no significant difference was found between wild type and AQP4-/- mice in the lung coefficient value (P>0.05). The hydroxyproline contents in the lung increased after bleomycin-treatment; on d28, the lung hydroxyproline contents in wild type and in AQP4-/- mice were 1.55-fold (0.85 ± 0.22 g/mg vs 0.55 ± 0.14 μg/mg, q=4.313, P<0.05) and 1.4-fold (0.84 ± 0.13 μg/mg vs 0.60 ± 0.14μg/mg, q=4.595，P<0.05) of that in control, respectively, but no significant difference was noticed between wild type and AQP4-/- mice in lung hydroxyproline contents. There was a tendency that serum TGF-β1 and TNF-α levels increased in bleomycin-treated mice, but no significant difference was found between wild type and AQP4-/- mice. AQP4-knockout showed no effects on pathological changes of lung tissues with H-E staining and Masson's staining in mice with bleomycin-induced lung fibrosis.

CONCLUSIONAQP4 might not be involved in bleomycin-induced lung fibrosis in mice.

Animals ; Aquaporin 4 ; genetics ; Bleomycin ; toxicity ; Male ; Mice ; Mice, Knockout ; Pulmonary Fibrosis ; chemically induced ; genetics

OBJECTIVETo investigate the sensitivity to bleomycin (BLM) in peripheral blood lymphocytes (PBL) among coke-oven workers.

METHODSNinty-four coke-oven workers with exposure to a high level of polycyclic aromatic hydrocarbons and 64 non-coke-oven workers (control) were recruited into this study. PBL was challenged by 8 microg/ml BLM, a known carcinogen, to induce certain amount of DNA damage, the difference of olive tail moment (TM) measured by comet assay before and after BLM treatment reflected the sensitivity towards mutagens.

RESULTSThe distribution of age, sex, and prevalence of smoking and drinking were not significantly different between these two groups. The geometric mean of urinary 1-hydroxypyrene (1-OHP) was significantly higher in coke-oven workers than in controls (9.0 versus 1.5 microg/L, t = -9.317, P < 0.01). The coke-oven workers showed significantly higher sensitivity to BLM than controls (17.7 versus 14.9, t = -2.583, P = 0.01). A large inter-group difference in sensitivity to BLM was observed in both controls and coke-oven workers. Stratification analysis revealed the significant association between high 1-OHP level (> 9.0 microg/L) and increased sensitivity to BLM (F = 4.001, P = 0.05) among coke-oven workers. Smoking subjects showed a significant higher value of sensitivity than nonsmokers in controls but not in coke-oven workers. No significant difference was observed between age, drinking status, coking history or external exposure class and BLM sensitivity.

CONCLUSIONExposure to coke oven emission could increase the sensitivity to mutagens, which might be a reason of high incidence of lung cancer among coke-oven workers.

Adult ; Benzo(a)pyrene ; toxicity ; Bleomycin ; toxicity ; Coke ; Comet Assay ; DNA Damage ; DNA Repair ; Female ; Humans ; Lymphocytes ; drug effects ; Male ; Middle Aged ; Mutagens ; toxicity ; Occupational Exposure

OBJECTIVETo observe the influence of 1.8 GHz microwave (MW) specific absorption rate (SAR, 3 W/kg) on human lymphocytes DNA damage induced by 4 chemical mutagens [mitomycin C (MMC), bleomycin (BLM), methyl methanesulfonate (MMS), and 4-nitroquinoline 1-oxide (4NQO)].

METHODSComet assay in vitro was used to detect human lymphocyte DNA damage induced by 1.8 GHz MW, 4 chemical mutagens, and MW plus 4 chemicals 0 h and 21 h respectively after exposure. The time exposed to MW or mutagens was 2 h or 3 h respectively. The results were showed by tail length (TL) and tail moment (TM).

RESULTSThe difference of DNA damage between MW group and control group was not statistically significant (P > 0.05). DNA damages in MW plus MMC groups and MW plus 4NQO groups were significantly greater than those in the corresponding concentrations of MMC groups and 4NQO groups (P < 0.01 or P < 0.05). However, MW did not enhance DNA damage induced by MMS and BLM (P > 0.05).

CONCLUSIONExposure to 1.8 GHz (SAR, 3 W/kg) microwave may not induce human lymphocyte DNA damage, but could enhance DNA damage induced by MMC and 4NQO.

4-Nitroquinoline-1-oxide ; toxicity ; Adult ; Bleomycin ; toxicity ; Cells, Cultured ; Comet Assay ; DNA ; drug effects ; DNA Damage ; Humans ; Lymphocytes ; drug effects ; radiation effects ; Male ; Methyl Methanesulfonate ; toxicity ; Microwaves ; adverse effects ; Mitomycin ; toxicity ; Mutagens ; toxicity

OBJECTIVETo observe the ultrastructures of rat alveolar type II cells and change of composition of phospholipid(PL) and content of protein in pulmonary surfactant(PS), to investigate the relation between change in composition of PL and activity of alveolar type II cells.

METHODSThe rats lung injury models were made by intratracheally instilling bleomycin(BLM) (4 mg/ml, 5 mg/kg). 28 rats were divided into four groups: 3-day group, 7-day group, 14-day group and 28-day group. Preparations of each group were stained histochemically and examined by electron microscope, content of PL in BALF, composition of PL and content of protein of each group were determined respectively.

RESULTS(1) Rats lungs in experimental groups were found that PS lost continuously, appeared homogenous and chorionic, dropped in the pulmonary alveolies. 3-day group was more apparent. Ruthenium red attaching on pulmonary surfactant was thicker in 3-day group, and the colour deeper, no difference in 7-day group and 14-day group, thinner in 28-day group. Content of PL in PS of BALF was increasing. Content of phosphatidylglycerol(PG) increased in 3-day group, decreased in 7-day, 14-day and 28-day group. The change of content of phosphatidylinositol(PI) was reversed. (2) Alveolar type II cells degenerated, necrotized, even disintegrated in 3-day group and 7-day group. 3-day group was more apparent. Proliferations of alveolar type II cells were found in each group, 7-day group was more apparent. We found that type II cells transformed to type I cells in 14-day group, extended and attached on bare basement membrane. Content of protein in PS was the highest in 3-day group, almost equal to the content of the control group in 28-day group.

CONCLUSIONMorphologic change and alternation of quality and quantity of PS after bleomycin-induced pulmonary injures specifically reflect the activity of alveolar type II cells. Measuring content of PL in BALF is one of simple and feasible method judging activity of alveolar type II cells when lungs of the rats are injured early by bleomycin.

Animals ; Antibiotics, Antineoplastic ; toxicity ; Bleomycin ; toxicity ; Bronchoalveolar Lavage Fluid ; chemistry ; Lung ; drug effects ; pathology ; ultrastructure ; Pulmonary Alveoli ; chemistry ; Pulmonary Surfactants ; analysis ; Rats

OBJECTIVE: To study the changes in mRNA and long non-coding RNA (lncRNA) expression profiles in a mouse model of bleomycin-induced lung fibrosis and identify lung fibrosis-related mRNA for coding-noncoding coexpression (CNC) bioinformatics analysis of the differential lncRNAs. METHODS: Lung fibrosis was induced by intratracheal injection of bleomycin in 10 C57BL/6 mice and another 10 mice with intratracheal injection of saline served as the control group. Lung tissues were harvested from the mice at 14 days after the injections and lung fibrosis was assessed using Masson and HE staining. LncRNA chip technology was used to screen the differentially expressed mRNAs and lncRNAs in mice with lung fibrosis, and GO and KEGG pathway analyses of the differential mRNAs were performed using NCBI database and UCSC database to identify possible fibrosis-related mRNAs, which were validated by qRT-PCR to construct a coding and non-coding co- expression network with the differential lncRNAs. RESULTS: Compared with the control mice, the mice with intratracheal injection of bleomycin showed obvious lung fibrosis. The results of gene chip analysis showed that 127 mRNAs were upregulated and 184 mRNAs were down-regulated in the model group as compared with the control group. GO and pathway analysis suggested that the differentially expressed genes participated mainly in immune response, cell differentiation, and cytoskeletons; the involved signal pathways were associated mainly with cytokine and cytokine receptor interaction and chemokine signal transduction. Bioinformatics analysis identified a significant coexpression network between the fibrosisrelated mRNA and the differentially expressed lncRNA. CONCLUSIONS In mice with lung fibrosis, the differential expressions of fibrosis-related mRNAs in the lung tissues are closely correlated with the co- expressions of a large number of differential lncRNAs, which points to a new direction for investigation of the pathogenesis of pulmonary fibrosis.
Animals ; Bleomycin/toxicity* ; Gene Expression Profiling ; Gene Regulatory Networks ; Mice ; Mice, Inbred C57BL ; Pulmonary Fibrosis/genetics* ; RNA, Long Noncoding/genetics* ; RNA, Messenger/genetics*

BACKGROUND: Macrophages are involved in the pathogenesis of idiopathic pulmonary fibrosis, partially by activating lung fibroblasts. However, how macrophages communicate with lung fibroblasts is largely unexplored. Exosomes can mediate intercellular communication, whereas its role in lung fibrogenesis is unclear. Here we aim to investigate whether exosomes can mediate the crosstalk between macrophages and lung fibroblasts and subsequently induce fibrosis. METHODS: In vivo, bleomycin (BLM)-induced lung fibrosis model was established and macrophages infiltration was examined. The effects of GW4869, an exosomes inhibitor, on lung fibrosis were assessed. Moreover, macrophage exosomes were injected into mice to observe its pro-fibrotic effects. In vitro, exosomes derived from angiotensin II (Ang II)-stimulated macrophages were collected. Then, lung fibroblasts were treated with the exosomes. Twenty-four hours later, protein levels of α-collagen I, angiotensin II type 1 receptor (AT1R), transforming growth factor-β (TGF-β), and phospho-Smad2/3 (p-Smad2/3) in lung fibroblasts were examined. The Student's t test or analysis of variance were used for statistical analysis. RESULTS: In vivo, BLM-treated mice showed enhanced infiltration of macrophages, increased fibrotic alterations, and higher levels of Ang II and AT1R. GW4869 attenuated BLM-induced pulmonary fibrosis. Mice with exosomes injection showed fibrotic features with higher levels of Ang II and AT1R, which was reversed by irbesartan. In vitro, we found that macrophages secreted a great number of exosomes. The exosomes were taken by fibroblasts and resulted in higher levels of AT1R (0.22 ± 0.02 vs. 0.07 ± 0.02, t = 8.66, P = 0.001), TGF-β (0.54 ± 0.05 vs. 0.09 ± 0.06, t = 10.00, P < 0.001), p-Smad2/3 (0.58 ± 0.06 vs. 0.07 ± 0.03, t = 12.86, P < 0.001) and α-collagen I (0.27 ± 0.02 vs. 0.16 ± 0.01, t = 7.01, P = 0.002), and increased Ang II secretion (62.27 ± 7.32 vs. 9.56 ± 1.68, t = 12.16, P < 0.001). Interestingly, Ang II increased the number of macrophage exosomes, and the protein levels of Alix (1.45 ± 0.15 vs. 1.00 ± 0.10, t = 4.32, P = 0.012), AT1R (4.05 ± 0.64 vs. 1.00 ± 0.09, t = 8.17, P = 0.001), and glyceraldehyde-3-phosphate dehydrogenase (2.13 ± 0.36 vs. 1.00 ± 0.10, t = 5.28, P = 0.006) were increased in exosomes secreted by the same number of macrophages, indicating a positive loop between Ang II and exosomes production. CONCLUSIONS Exosomes mediate intercellular communication between macrophages and fibroblasts plays an important role in BLM-induced pulmonary fibrosis.
Angiotensin II ; Animals ; Bleomycin/toxicity* ; Exosomes ; Fibroblasts ; Lung ; Macrophages ; Mice ; Mice, Inbred C57BL ; Pulmonary Fibrosis/chemically induced* ; Receptor, Angiotensin, Type 1

OBJECTIVETo observe the changes of matrix metalloproteinases (MMPs) activities in pulmonary fibrosis rats.

METHODSEighty male SD rats were randomly divided into sham group (n = 40) and bleomycin group (BLM, n = 40), in which SD rats were injected with a single intratracheal dose of sham saline or bleomycin respectively. On day 1, 3, 7, 14, and 28 following bleomycin or saline instillation, rats were randomly killed, and serum from abdominal aorta, alveolar fluid from the bronchoalveolar lavage, and the lung homogenate were collected and then stored at -80 degrees C. MMPs activity was determined by zymography.

RESULTSCompared with sham group, the levels of MMP-9 in all samples were augmented. MMP-9 activities in the serum were highest on day 3 than those on day 1 and day 7, and in lung tissue homogenate were highest on day 7; however, no significant differences were found between BLM group and sham group on day 14 and day 28; and that of bronchoalveolar lavage fluid (BALF) was highest on day 7 than those on day 1 and day 14, while no significant difference existed between BLM group and sham group on day 28. Serum MMP-2 level did not change from day 1 to day 28, while the level of BALF MMP-2 began to increase after day 14, even on day 28. Lung tissue homogenate MMP-2 level began to increase early on day 3 and continued to day 28.

CONCLUSIONThe sources and effects of MMP-2 and MMP-9 differ in BLM-induced rat pulmonary fibrosis.

Animals ; Bleomycin ; toxicity ; Disease Models, Animal ; Male ; Matrix Metalloproteinase 2 ; metabolism ; Matrix Metalloproteinase 9 ; metabolism ; Pulmonary Fibrosis ; chemically induced ; enzymology ; Rats ; Rats, Sprague-Dawley

Ivermectin is a US Food and Drug Administration (FDA)-approved antiparasitic agent with antiviral and anti-inflammatory properties. Although recent studies reported the possible anti-inflammatory activity of ivermectin in respiratory injuries, its potential therapeutic effect on pulmonary fibrosis (PF) has not been investigated. This study aimed to explore the ability of ivermectin (0.6 mg/kg) to alleviate bleomycin-induced biochemical derangements and histological changes in an experimental PF rat model. This can provide the means to validate the clinical utility of ivermectin as a treatment option for idiopathic PF. The results showed that ivermectin mitigated the bleomycin-evoked pulmonary injury, as manifested by the reduced infiltration of inflammatory cells, as well as decreased the inflammation and fibrosis scores. Intriguingly, ivermectin decreased collagen fiber deposition and suppressed transforming growth factor-‍β1 (TGF-‍β1) and fibronectin protein expression, highlighting its anti-fibrotic activity. This study revealed for the first time that ivermectin can suppress the nucleotide-binding oligomerization domain (NOD)‍-like receptor family pyrin domain-containing protein 3 (NLRP3) inflammasome, as manifested by the reduced gene expression of NLRP3 and the apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), with a subsequent decline in the interleukin‍-‍1β (IL‍-‍1β) level. In addition, ivermectin inhibited the expression of intracellular nuclear factor-‍κB (NF‍-‍κB) and hypoxia‑inducible factor‑1α (HIF‍-‍1α) proteins along with lowering the oxidative stress and apoptotic markers. Altogether, this study revealed that ivermectin could ameliorate pulmonary inflammation and fibrosis induced by bleomycin. These beneficial effects were mediated, at least partly, via the downregulation of TGF-‍β1 and fibronectin, as well as the suppression of NLRP3 inflammasome through modulating the expression of HIF‑1α and NF-‍κB.
Animals ; Rats ; Anti-Inflammatory Agents ; Bleomycin/toxicity* ; Fibronectins/metabolism* ; Fibrosis ; Inflammasomes/metabolism* ; Ivermectin/adverse effects* ; NF-kappa B/metabolism* ; NLR Family, Pyrin Domain-Containing 3 Protein/metabolism* ; Pulmonary Fibrosis/drug therapy*

OBJECTIVETo investigate the effects of atorvastatin on matrix metalloproteinase-9 (MMP-9) and the tissue inhibitor-1 of matrix metalloproteinase (TIMP-1) levels in bronchoalveolar lavage fluid (BALF) and serum of rats with experimental pulmonary fibrosis.

METHODSPulmonary fibrosis was induced by intratracheal administration of bleomycin in 30 female rats, which were further divided into two groups: Group M (without treatment) and Group A (treated with atorvastatin 10 mg/kg); control group (n = 5, Group C) was intratracheally administrated with same volume of saline. Five animals were sacrificed at 2 weeks (M2 and A2), 4 weeks (M4 and A4) and 6 weeks (M6 and A6) after model establishment, respectively. Lung tissue samples were harvested and prepared for HE and Masson's trichrome staining. Concentrations of MMP-9 and TIMP-1 in BALF and serum were measured by ELISA.

RESULTThe severity of inflammation and pulmonary fibrosis was significantly reduced in Group A than that in Group M, especially at week 6. No significant difference was noted in the serum concentrations of MMP-9 and TIMP-1 among the Group M, A and Group C. The BALF concentrations of MMP-9 in Group M2 and M6 were significantly higher than those in Group C (P < 0.01 and 0.05), whereas those in the atorvastatin groups (A2, A4 and A6) were lower than those in M2, M4 and M6. Although the MMP-9 was still higher in Groups A2 and A4 than in the Group C, there was no significant difference in MMP-9 between Group A6 and Group C. TIMP-1 levels in BALF were significantly higher in M4 and M6 than Group C (P < 0.01 and 0.05), there were no significant differences between Group M2 and Group C. The TIMP-1 levels in BALF of atorvastatin groups were significantly lower than those of model groups and control group (P < 0.01 and 0.05), which resulted in a significantly increased ratio of MMP-9 to TIMP-1 in the atorvastatin groups.

CONCLUSIONAtorvastatin inhibits the synthesis and release of MMP-9 and TIMP-1 in the lung tissue of rats with bleomycin-induced pulmonary fibrosis, and has no significant effect on circulating MMP-9 and TIMP-1, which may be associated with the attenuation of experimental pulmonary fibrosis in rats.

Animals ; Atorvastatin Calcium ; Bleomycin ; toxicity ; Bronchoalveolar Lavage Fluid ; chemistry ; Disease Models, Animal ; Female ; Heptanoic Acids ; pharmacology ; Matrix Metalloproteinase 9 ; metabolism ; Pulmonary Fibrosis ; chemically induced ; metabolism ; Pyrroles ; pharmacology ; Rats ; Rats, Wistar ; Tissue Inhibitor of Metalloproteinase-1 ; metabolism

OBJECTIVETo study the treatment effects of cultured Cordyceps sinensis combined with glucocorticosteroid on experimental pulmonary fibrosis in rats induced by bleomycin.

METHODFifty rats were randomly divided into five groups, including control group, model group, cultured C. sinensis groups, prednisone group, cultured C. sinensis combined with prednisone group. On experimental day 0, the rats were respectively intratracheally instilled with bleomycin, and rats in the control group and model group with the same volume of normal saline. One day after the injection, cultured C. sinensis and glucocorticosteroid was respectively given to rats daily by gastric gavage, while the same volume of normal saline was given to those in the control group and model group. On 28th d, bronchoalveolar lavage fluid (BALF) and lung tissue were collected. Histological changes of the lungs were evaluated by HE stain, Masson's trichrome stain. Collagen content of the lung tissue was assessed by hydroxyprolin concentration. Lung expression of CTGF protein was assessed by immunohistochemistry. The level of TGF-beta1 protein was measured by ELISA.

RESULTCompared to model group, pulmonary fibrosis were alleviated in cultured C. sinensis and prednisone group, and CTGF expression, Hydroxyproline concentrations and protein TGF-beta1 were decreased. The combination effect of C. sinensis and prednisone group is augmented compared with using C. sinensis or prednisone group alone.

CONCLUSIONThe cultured C. sinensis and prednisone alleviates pulmonary fibrosis, and the combination use of both drugs has synergia effects in anti-fibrous degeneration.

Animals ; Bleomycin ; toxicity ; Connective Tissue Growth Factor ; analysis ; Cordyceps ; Drug Therapy, Combination ; Lung ; chemistry ; pathology ; Male ; Phytotherapy ; Prednisone ; administration & dosage ; Pulmonary Fibrosis ; chemically induced ; drug therapy ; pathology ; Rats ; Rats, Sprague-Dawley