Journal: Pharmacology Research & Perspectives

Article Title: Prevention of bleomycin‐induced lung fibrosis via inhibition of the MRTF/SRF transcription pathway

doi: 10.1002/prp2.1028

Figure Lengend Snippet: Change in body weight during drug treatments and survival. All animals except naïve controls received bleomycin treatment from day 1–28 and test agents beginning 24 h before the first bleomycin treatment until day 42 (A) Stress on the animals was observed in the rapid weight loss during the first week in the nintedanib and prednisolone groups, and a lack of significant weight gain over the 6 weeks study in all groups except 100 mg/kg CCG‐257081. (B) Survival curves highlight the increased mortality rate in the group receiving prednisolone. Due to excessive weight loss and animal loss, the dose of prednisolone was lowered from 15 to 5 mg/kg. In all other groups, loss of animals is believed due to complications with the daily oral gavage of drug. Weight change time courses, which are significantly different from the vehicle control, are marked (* p < .05, ** p < .01). Groups: naïve control ( n = 6), bleomycin + vehicle ( n = 10), 10 mg/kg CCG‐257081 ( n = 15), 30 mg/kg CCG‐257081 ( n = 15), 100 mg/kg CCG‐257081 ( n = 10), 30 mg/kg nintedanib ( n = 10), and prednisolone (15 mg/kg Days 1–7, 5 mg/kg Days 8–42, n = 10)

Article Snippet: Bleomycin (GoldBio, #B‐910‐1G) was dissolved in sterile phosphate buffered saline (PBS, Gibco) at 3 mg/ml (15 mg/kg) and sterile filtered.

Techniques:

Journal: Pharmacology Research & Perspectives

Article Title: Prevention of bleomycin‐induced lung fibrosis via inhibition of the MRTF/SRF transcription pathway

doi: 10.1002/prp2.1028

Figure Lengend Snippet: CCG‐257081 reduces inflammatory responses inherent to bleomycin‐induced fibrotic disease. Histopathology scoring of (A) inflammation, (B) AT2 Hyperplasia and (C) fibrosis showed a trend in lower scores with higher concentrations of CCG‐257081. The severity scores were: 0, no significant findings; 1, minimal; 2, mild; 3, moderate; 4, marked; 5, severe. * p < .05. Groups: no bleomycin ( n = 6), bleomycin + vehicle ( n = 9), 10 mg/kg CCG‐257081 ( n = 14), 30 mg/kg CCG‐257081 ( n = 14), 100 mg/kg CCG‐257081 ( n = 10), 30 mg/kg nintedanib ( n = 9), and prednisolone (15 mg/kg Days 1–7, 5 mg/kg Days 8–42, n = 7)

Article Snippet: Bleomycin (GoldBio, #B‐910‐1G) was dissolved in sterile phosphate buffered saline (PBS, Gibco) at 3 mg/ml (15 mg/kg) and sterile filtered.

Techniques: Histopathology

Journal: Pharmacology Research & Perspectives

Article Title: Prevention of bleomycin‐induced lung fibrosis via inhibition of the MRTF/SRF transcription pathway

doi: 10.1002/prp2.1028

Figure Lengend Snippet: Light photomicrographs of lung tissue from mice in experimental groups: (A) Naïve control group, (B) Bleomycin + vehicle, (C) Bleomycin with low dose CCG‐257081 (10 mg/kg), (D) Bleomycin with Prednisone treatment, (E) Bleomycin with medium dose CCG‐257081 (30 mg/kg), (F) Bleomycin with Nintedanib treatment, and (G) Bleomycin with high dose CCG‐257081 (100 mg/kg). Tissue sections histochemically stained with Masson's Trichrome (blue: collagen, red: red blood cells, gray: alveolar space). Bleomycin‐induced subpleural chronic alveolitis with increased collagen deposition (fibrosis; blue chromogen/stippled arrows) was observed, that was most severe in D (Prednisone treatment). No subpleural fibrotic lesions were noted with high dose CCG‐257081 (G) or Nintedanib (F). p, pleural surface of lung; a, alveolar parenchyma; solid arrow, alveolar type II epithelial hyperplasia/hypertrophy.

Article Snippet: Bleomycin (GoldBio, #B‐910‐1G) was dissolved in sterile phosphate buffered saline (PBS, Gibco) at 3 mg/ml (15 mg/kg) and sterile filtered.

Techniques: Staining

Journal: Pharmacology Research & Perspectives

Article Title: Prevention of bleomycin‐induced lung fibrosis via inhibition of the MRTF/SRF transcription pathway

doi: 10.1002/prp2.1028

Figure Lengend Snippet: Fibrotic markers were significantly decreased by MRTF/SRF inhibitors. (A) At 100 mg/kg CCG‐257081, collagen content in the lungs was not significantly different from naive tissue (no bleomycin), while animals with prednisolone treatment had significantly greater amounts of collagen. Groups: no bleomycin ( n = 12), bleomycin + vehicle ( n = 14), 10 mg/kg CCG‐257081 ( n = 14), 30 mg/kg CCG‐257081 ( n = 13), 100 mg/kg CCG‐257081 ( n = 10), 30 mg/kg nintedanib ( n = 9), and prednisolone (15 mg/kg Days 1–7, 5 mg/kg Days 8–42, n = 6). (B) The pro‐fibrotic biomarker, PAI‐1, was significantly reduced in BALf by treatment with CCG‐257081 at 100 mg/kg, but was not significantly reduced by any other treatment, including nintedanib or prednisolone. * p < .05, ** p < .01. Groups: bleomycin + vehicle ( n = 6), 10 mg/kg CCG‐257081 ( n = 7), 30 mg/kg CCG‐257081 ( n = 7), 100 mg/kg CCG‐257081 ( n = 6), 30 mg/kg nintedanib ( n = 6), and prednisolone (15 mg/kg Days 1–7, 5 mg/kg Days 8–42, n = 5)

Article Snippet: Bleomycin (GoldBio, #B‐910‐1G) was dissolved in sterile phosphate buffered saline (PBS, Gibco) at 3 mg/ml (15 mg/kg) and sterile filtered.

Techniques: Biomarker Assay

Journal: Experimental & molecular medicine

Article Title: Melatonin prevents lung injury by regulating apelin 13 to improve mitochondrial dysfunction.

doi: 10.1038/s12276-019-0273-8

Figure Lengend Snippet: Fig. 1 Melatonin increased survival and improved pulmonary epithelial function in response to bleomycin. Mice were intratracheally treated with PBS or bleomycin (BLM) with or without melatonin (MLN), (PBS group: n = 25, BLM group: n = 26, BLM + MLN group: n = 25). a Survival plots of mice in the PBS, BLM, and BLM + MLN groups. *P < 0.05. b Mice were intraperitoneally injected with Evans blue solution (1 ml/kg 3% Evans blue solution in PBS) for 30 min before euthanization. The amount of Evans blue was measured in the whole lungs of mice in the PBS, BLM, and BLM + MLN groups and normalized to the Evans blue concentration in methanamide to generate an Evans blue index. n = 3; *P < 0.05. c H&E staining of representative lung sections from mice in the PBS, BLM, and BLM + MLN groups. Scale bars, 50 μm. d The mRNA expression of inflammatory factors (IL1β, IL6, and TNFα). n = 4; *P < 0.05, **P < 0.01. e The Immunohistochemical assay of E-cadherin in lung samples from mice in the PBS, BLM, and BLM + MLN groups. Scale bar, 50 μm. f–g Quantitative RT-PCR was performed to analyze the mRNA expression of E-cadherin (Cdh1) and Sftpc. n = 5; *P < 0.05, **P < 0.01. h E-cadherin, a marker of alveolar epithelial cells, was identified by immunoblot analysis. n = 6; *P < 0.05, **P < 0.01

Article Snippet: Bleomycin (BLM) was purchased from Selleck (Shanghai, China).

Techniques: Injection, Concentration Assay, Staining, Expressing, Immunohistochemical staining, Quantitative RT-PCR, Marker, Western Blot

Journal: Experimental & molecular medicine

Article Title: Melatonin prevents lung injury by regulating apelin 13 to improve mitochondrial dysfunction.

doi: 10.1038/s12276-019-0273-8

Figure Lengend Snippet: Fig. 4 Melatonin reversed bleomycin-induced mitochondrial abnormalities in alveolar epithelial cells. a Representative transmission electron microscopy (TEM) images of lung sections from mice treated with PBS, BLM, or BLM + MLN. The red arrows indicate normal mitochondria or damaged and swollen mitochondria. b Cellular ATP content was measured in TC-1 cells incubated with the indicated concentrations of H2O2 and melatonin. Cells were lysed, and the ATP content was determined using an ATP assay and normalized to the protein concentration. n = 5; *P < 0.05, **P < 0.01. c Comparison of ROS production in TC-1 cells exposed to H2O2 with or without melatonin (10 nM, 1 μM, or 100 μM). Green, ROS; blue, DAPI. Scale bars, 50 μm. *P < 0.05, **P < 0.01. d Measurement of ATP content in TC-1 cells after exposure to H2O2, melatonin, and luzindole; the ATP content was normalized to the corresponding protein concentration. n = 5; **P < 0.01. e Effect of luzindole on ROS production in TC-1 cells. Green, ROS; blue, DAPI. Scale bars, 50 μm. *P < 0.05, **P < 0.01

Article Snippet: Bleomycin (BLM) was purchased from Selleck (Shanghai, China).

Techniques: Transmission Assay, Electron Microscopy, Incubation, ATP Assay, Protein Concentration, Comparison

Journal: Biomaterials

Article Title: HYDROGEL-BASED DELIVERY OF IL-10 IMPROVES TREATMENT OF BLEOMYCIN-INDUCED LUNG FIBROSIS IN MICE

doi: 10.1016/j.biomaterials.2019.02.017

Figure Lengend Snippet: A) ELISA quantification of IL-10 released from HH-10 formulation over 6 days (n=13). B)-C) Fluorescent microscopy images of lung tissue with fluorescently labeled HH reagent in both healthy (B) and bleomycin-challenged (C) lung tissue, displaying deposition locations of HH reagent via intranasal treatment. Images on right are high magnification of the squared regions.

Article Snippet: Two to four-month old wildtype C57Bl6J mice were treated with PBS control or bleomycin (MPBio, 0219030610) at a concentration of 1500–2000 international units/kg weight intratracheally; mice were anesthetized via ketamine/xylazine cocktail (K113–10ML, Sigma).

Techniques: Enzyme-linked Immunosorbent Assay, Microscopy, Labeling

Journal: Biomaterials

Article Title: HYDROGEL-BASED DELIVERY OF IL-10 IMPROVES TREATMENT OF BLEOMYCIN-INDUCED LUNG FIBROSIS IN MICE

doi: 10.1016/j.biomaterials.2019.02.017

Figure Lengend Snippet: A) Experimental design timeline of prevention cohort and analysis. B) Trichrome and α-SMA IHC stain of lung tissue sections by group of prevention cohorts, all parameters include bleomycin challenge. C) Sircol- assay quantifying collagen content of lung samples from healthy control and bleomycin challenged mice with preventative PBS, IL-10, HH, or HH-10. D) Ashcroft Score quantifying fibrosis of lung sections from healthy control and bleomycin challenged mice with preventative PBS, IL-10, HH, or HH-10. E) BAL cell counts from healthy control and bleomycin challenged mice with preventative PBS, IL-10, HH, or HH-10. For all graphs- ***p< .001 vs. PBS + BLM, **p< .01 vs. PBS + BLM, *p< .05 vs. PBS + BLM.

Article Snippet: Two to four-month old wildtype C57Bl6J mice were treated with PBS control or bleomycin (MPBio, 0219030610) at a concentration of 1500–2000 international units/kg weight intratracheally; mice were anesthetized via ketamine/xylazine cocktail (K113–10ML, Sigma).

Techniques: Staining

Journal: Biomaterials

Article Title: HYDROGEL-BASED DELIVERY OF IL-10 IMPROVES TREATMENT OF BLEOMYCIN-INDUCED LUNG FIBROSIS IN MICE

doi: 10.1016/j.biomaterials.2019.02.017

Figure Lengend Snippet: A) Experimental design timeline of treatment cohort and analysis. B) Trichrome IHC and α-SMA IHC stain of lung tissue sections of bleomycin-challenged treatment cohorts. C) Sircol- assay of lung samples from healthy control and bleomycin challenged mice with PBS, IL-10, HH, or HH-10 treatment. D) Ashcroft Score of lung sections from healthy control and bleomycin challenged mice with PBS, IL-10, HH, or HH-10 treatment. E) BAL cell counts from healthy control and bleomycin challenged mice with PBS, IL-10, HH, or HH-10 treatment. For all graphs- ***p< .001 vs. PBS + BLM, **p< .01 vs. PBS + BLM, *p< .05 vs. PBS + BLM.

Article Snippet: Two to four-month old wildtype C57Bl6J mice were treated with PBS control or bleomycin (MPBio, 0219030610) at a concentration of 1500–2000 international units/kg weight intratracheally; mice were anesthetized via ketamine/xylazine cocktail (K113–10ML, Sigma).

Techniques: Staining

Journal: Biomaterials

Article Title: HYDROGEL-BASED DELIVERY OF IL-10 IMPROVES TREATMENT OF BLEOMYCIN-INDUCED LUNG FIBROSIS IN MICE

doi: 10.1016/j.biomaterials.2019.02.017

Figure Lengend Snippet: A) IHC staining for phospho-Smad3 in bleomycin challenged mice treated in prevention or treatment regiments with PBS, IL-10, HH, or HH-10. B)-C) Quantification of pSmad3 positive cells from IHC images in bleomycin challenged mice treated in prevention or treatment regiments with PBS, IL-10, HH, or HH-10. For all graphs- ***p< .001 vs. PBS + BLM and **p< .01 vs. PBS + BLM.

Article Snippet: Two to four-month old wildtype C57Bl6J mice were treated with PBS control or bleomycin (MPBio, 0219030610) at a concentration of 1500–2000 international units/kg weight intratracheally; mice were anesthetized via ketamine/xylazine cocktail (K113–10ML, Sigma).

Techniques: Immunohistochemistry

Journal: Fibrogenesis & Tissue Repair

Article Title: TLR9-induced interferon β is associated with protection from gammaherpesvirus-induced exacerbation of lung fibrosis

doi: 10.1186/1755-1536-4-18

Figure Lengend Snippet: TLR-9 -/- mice are more susceptible to viral exacerbation of fibrosis . (A) Balb/c and TLR-9 -/- mice were treated with either saline or bleomycin intratracheally on day 0. They were then given either sham infection or infection with 5 × 10 4 PFU of γHV68 on day 14 by intranasal infection. On day 21, lungs were harvested and Sircol assay was performed. TLR-9 -/- mice were more susceptible than wild-type mice to viral exacerbation of existing fibrosis (705.4 ± 52.79 versus 468.6 ± 42 mg collagen/ml; n = 5; P < 0.01). There were no significant differences in collagen levels between Balb/c and TLR-9 -/- saline-treated mice (255 ± 9.06 versus 227.3 ± 14.95 mg/ml) or between both groups after bleomycin challenge alone (317.0 ± 43.0 versus 362.5 ± 78.8 mg/ml.) This was true in three experiments, and the relative increase in collagen production in bleomycin-treated mice over saline controls was also not different. (B) Lungs from WT or TLR-9 -/- mice treated with bleomcyin on day 0 and infected with γHV68 on day 14. Lungs were harvested for histologic analysis on day 21 and stained with hematoxylin and eosin. Sections are representative of three mice examined.

Article Snippet: In C57Bl/6 mice, clinical grade bleomycin sulfate (Blenoxane; Bristol-Myers Squibb, New York, NY, USA) was utilized.

Techniques: Infection, Staining

Journal: Fibrogenesis & Tissue Repair

Article Title: TLR9-induced interferon β is associated with protection from gammaherpesvirus-induced exacerbation of lung fibrosis

doi: 10.1186/1755-1536-4-18

Figure Lengend Snippet: Viral replication is not different in the lungs of Balb/c and TLR-9 -/- mice . Balb/c or TLR-9 -/- mice were injected with saline or bleomycin intratracheally on day 0. On day 14, mice received 5 × 10 4 PFU gamma herpesvirus 68 (γHV68) intranasally. On day 21, lungs were harvested, and total RNA was prepared and analyzed for expression of (A) the viral envelope glycoprotein gene (gB) or (B) viral DNA polymerase. A single mouse in the Balb/c group not treated with bleomycin was normalized to 1, and all other mice were plotted relative to this control (n = 5 mice/group, representative of two experiments). The ΔC T for each group are as follows: For the gB gene: Balb 4.9 ± 0.5, TLR-9 -/- 5.7 ± 0.5, Balb plus bleomycin 3.07 ± 0.6, TLR-9 -/- plus bleomycin 2.8 ± 0.7. For the DNA polymerase gene: Balb 4.5 ± 0.6, TLR-9 -/- 5.4 ± 0.4, Balb plus bleomycin 3.3 ± 0.6, TLR-9 -/- plus bleomycin 2.8 ± 0.8.

Article Snippet: In C57Bl/6 mice, clinical grade bleomycin sulfate (Blenoxane; Bristol-Myers Squibb, New York, NY, USA) was utilized.

Techniques: Injection, Expressing

Journal: Fibrogenesis & Tissue Repair

Article Title: TLR9-induced interferon β is associated with protection from gammaherpesvirus-induced exacerbation of lung fibrosis

doi: 10.1186/1755-1536-4-18

Figure Lengend Snippet: Gamma herpesvirus 68 (γHV68) replicates in alveolar epithelial cells (AECs) . (A) Wild-type mice were infected with 5 × 10 4 PFU γHV68 on day 0. On day 7 after infection, frozen sections were prepared, and stained with a rabbit polyclonal antisera against γHV68, or with non-immune rabbit sera as control. The goat anti-rabbit secondary was linked to alkaline phosphatase. Vivid replication of γHV68 is visible in alveolar lining cells (original magnification × 100). Sections shown are representative of four mice examined. (B) AECs were isolated from lungs of Balb/c or TLR-9 -/- mice treated with bleomycin plus γHV68 on day 21, and were cultured on fibronectin-coated slides (TiterTek). Sections were stained with antibodies (M30 Cytodeath), and the number of positive cells per high power field (HPF; ×400) were calculated (n = 30 HPF per genotype). (C) AECs were isolated from Balb/c or TLR-9 -/- mice and cells were infected in vitro with 0.01 or 0.001 PFU γHV68 for 48 hours. Cell lysates were then analyzed for cleaved caspase 3 by western blotting. Data are from one experiment, representative of two.

Article Snippet: In C57Bl/6 mice, clinical grade bleomycin sulfate (Blenoxane; Bristol-Myers Squibb, New York, NY, USA) was utilized.

Techniques: Infection, Staining, Isolation, Cell Culture, In Vitro, Western Blot

Journal: Fibrogenesis & Tissue Repair

Article Title: TLR9-induced interferon β is associated with protection from gammaherpesvirus-induced exacerbation of lung fibrosis

doi: 10.1186/1755-1536-4-18

Figure Lengend Snippet: Inflammatory responses are similar in Balb/c and TLR-9 -/- mice . Mice were treated with bleomycin on day 0, and were given 5 × 10 4 PFU γHV68 intranasally on day 14. On day 21, lungs were collected and subjected to digestion with collagenase and DNAse. (A) Lung leukocytes were enumerated (n = 4 mice/group, representative of four experiments). (B) Isolated lung leukocytes were examined by flow cytometry for the percentage surface expression of CD4, CD8, CD19, Gr-1 (PMNs) and DX5 (NK cells). Samples were gated on CD45+ cells first. Percentages of lymphocytes were then determined within a lymphocyte gate. Gr-1 percentages were gated on CD45+ non-lymphocyte sized cells (n = 4/group representative of two experiments). ** P < 0.01 for CD8 in TLR-9 -/- mice treated with bleomycin plus γHV68 compared with Balb/c with same treatment. For B cells, virally infected mice were significantly different from mice treated with bleomycin alone in both genotypes ( P < 0.01).

Article Snippet: In C57Bl/6 mice, clinical grade bleomycin sulfate (Blenoxane; Bristol-Myers Squibb, New York, NY, USA) was utilized.

Techniques: Isolation, Flow Cytometry, Expressing, Infection

Journal: Fibrogenesis & Tissue Repair

Article Title: TLR9-induced interferon β is associated with protection from gammaherpesvirus-induced exacerbation of lung fibrosis

doi: 10.1186/1755-1536-4-18

Figure Lengend Snippet: TLR-9 -/- mice are defective in interferon (IFN)-β production . (A) Balb/c or TLR-9 -/- mice were injected with bleomycin on day 0. On day 14 mice received γHV68 intranasally. Lungs were harvested on days 0, 14, 17 and 21, and lung homogenates were prepared and analyzed by ELISA for IFN-β (n = 5 mice/group). (B) Fibroblasts isolated from Balb/c or TLR-9 -/- mice were infected in vitro with 0.01 PFU γHV68 and total RNA was prepared after 24 h of infection for analysis of IFN-β by real-time RT-PCR. The mean of the Balb/c samples was set to 1, n = 5/group.

Article Snippet: In C57Bl/6 mice, clinical grade bleomycin sulfate (Blenoxane; Bristol-Myers Squibb, New York, NY, USA) was utilized.

Techniques: Injection, Enzyme-linked Immunosorbent Assay, Isolation, Infection, In Vitro, Quantitative RT-PCR

Journal: Fibrogenesis & Tissue Repair

Article Title: TLR9-induced interferon β is associated with protection from gammaherpesvirus-induced exacerbation of lung fibrosis

doi: 10.1186/1755-1536-4-18

Figure Lengend Snippet: CpG oligodeoxynucleotides (ODN) stimulation lessens bleomycin-induced fibrosis . (A) C57Bl/6 male mice were injected with 0.25U bleomycin intrathecally on day 0. (B) On day 14, 50 μg CpG ODN was administered intranasally, and lung collagen was measured by hyroxyproline on day 28. (C) Lung sections were stained with Masson's trichrome to detect blue coloration indicative of collagen (n = 5 mice/group).

Article Snippet: In C57Bl/6 mice, clinical grade bleomycin sulfate (Blenoxane; Bristol-Myers Squibb, New York, NY, USA) was utilized.

Techniques: Injection, Staining

Journal: Frontiers in Physiology

Article Title: Inflammatory Drivers of Cardiovascular Disease: Molecular Characterization of Senescent Coronary Vascular Smooth Muscle Cells

doi: 10.3389/fphys.2020.00520

Figure Lengend Snippet: (A–G) Senescence markers in DNA damage-induced senescence. Coronary VSMCs were treated with 0–25 μg/ml with bleomycin for 3 h and incubated for indicated time-points ( n = 2). (H–J) Immuno-fluorescence of HMGB-1 localization ( n = 2). Topo II- Topoisomerase 2; 0-25- bleomycin doses in μg per ml; DAPI- 4’,6-diamidino-2-phenylindole; p16, p14, p21- cell cycle inhibitors; IL-1β, IL-6- interleukins; LMNB-1- Lamin B1; HMGB-1- High mobility group box1.

Article Snippet: Bleomycin sulfate was purchased from Enzo Life Sciences (BML-AP302).

Techniques: Incubation, Fluorescence