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Frontiers in Immunology 2023Immune responses are crucial to maintaining tissue homeostasis upon tissue injury. Upon various types of challenges, macrophages play a central role in regulating...
Immune responses are crucial to maintaining tissue homeostasis upon tissue injury. Upon various types of challenges, macrophages play a central role in regulating inflammation and tissue repair processes. While an immunomodulatory role of Wnt antagonist Dickkopf1 (DKK1) has been implicated, the role of Wnt antagonist DKK1 in regulating macrophage polarization in inflammation and the tissue repair process remains elusive. Here we found that DKK1 induces gene expression profiles to promote inflammation and tissue repair in macrophages. Importantly, DKK1 induced various genes, including inflammation and tissue repair, via JNK (c-jun N-terminal kinase) in macrophages. Furthermore, DKK1 potentiated IL-13-mediated macrophage polarization and activation. The co-inhibition of JNK and STAT6 markedly decreased gene expressions inflammation and fibrosis by DKK1 and IL-13. Interestingly, thrombocyte-specific deletion of DKK1 in mice reduced collagen deposition and decreased Arg1, CD206, HIF1α, and IL1β protein expressions in monocyte-derived alveolar macrophages in the acute sterile bleomycin (BLM)-induced lung injury model. These data suggested that thrombocytes communicate with macrophages via DKK1 to orchestrate inflammation and repair in this model. Taken together, our study demonstrates DKK1's role as an important regulatory ligand for macrophage polarization in the injury-induced inflammation and repair process in the lung.
Topics: Animals; Mice; Acute Lung Injury; Bleomycin; Blood Platelets; Inflammation; Interleukin-13; Macrophages
PubMed: 38162655
DOI: 10.3389/fimmu.2023.1247330 -
Applied and Environmental Microbiology Jan 2022Bleomycin (BLM) is a widely used chemotherapeutic drug. BLM-treated cells showed an elevated rate of mutations, but the underlying mechanisms remained unclear. In this...
Bleomycin (BLM) is a widely used chemotherapeutic drug. BLM-treated cells showed an elevated rate of mutations, but the underlying mechanisms remained unclear. In this study, the global genomic alterations in BLM-treated cells were explored in the yeast Saccharomyces cerevisiae. Using genetic assay and whole-genome sequencing, we found that the mutation rate could be greatly elevated in S. cerevisiae cells that underwent Zeocin (a BLM member) treatment. One-base deletion and T-to-G substitution at the 5'-GT-3' motif represented the most striking signature of Zeocin-induced mutations. This was mainly the result of translesion DNA synthesis involving Rev1 and polymerase ζ. Zeocin treatment led to the frequent loss of heterozygosity and chromosomal rearrangements in the diploid strains. The breakpoints of recombination events were significantly associated with certain chromosomal elements. Lastly, we identified multiple genomic alterations that contributed to BLM resistance in the Zeocin-treated mutants. Overall, this study provides new insights into the genotoxicity and evolutional effects of BLM. Bleomycin is an antitumor antibiotic that can mutate genomic DNA. Using yeast models in combination with genome sequencing, the mutational signatures of Zeocin (a member of the bleomycin family) are disclosed. Translesion-synthesis polymerases are crucial for the viability of Zeocin-treated yeast cells at the sacrifice of a higher mutation rate. We also confirmed that multiple genomic alterations were associated with the improved resistance to Zeocin, providing novel insights into how bleomycin resistance is developed in cells.
Topics: Bleomycin; Cell Division; Genomics; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins
PubMed: 34731050
DOI: 10.1128/AEM.01703-21 -
Acta Histochemica Dec 2023Idiopathic pulmonary fibrosis (IPF) is considered as a chronic interstitial lung disease with underlying mechanism of IPF remaining unclear, while there are no...
OBJECTIVE
Idiopathic pulmonary fibrosis (IPF) is considered as a chronic interstitial lung disease with underlying mechanism of IPF remaining unclear, while there are no definitive treatment options. In recent years, scientists have gradually paid attention to the influence of angiogenesis on IPF. Because IPF is a progressive with microvascular remodeling disorder, scientists have postulated that angiogenesis may also be one of the initiating and contributing factors of the disease. Bupleurum is a common natural Chinese herbal medicine with antibacterial, anti-inflammatory, anti-tumor and other pharmacological effects. As the most important active monomer of Bupleurum, Saikosaponin-d (SSd) is a new discovery with anti-pulmonary fibrosis (PF) activity. This study attempts to investigate the role of SSd in the interference of PF through regulation of angiogenesis in IPF through Angiopoietin (Angpt) /Tie receptor 2 (Tie2) pathway.
METHODS
Randomly, we allocated C57BL/6 mice into four groups (n = 20 in each group). Afterwards, establishment of IPF model was accomplished via intratracheal administration of bleomycin (BLM, 5 mg/kg), while corresponding drug intervention was given accordingly. On 3rd, 7th, 14th and 28th days after modeling, we performed histopathological examination through staining. Meanwhile, immunohistochemistry (IHC) of PF and the expression of related factors were observed, while Ang/Tie2 pathway was assessed by ELISA with the effect of SSd on angiogenesis related proteins in IPF being explored with IHC and Western Blot technique.
RESULTS
Our results showed that SSd could reduce inflammation and PF levels in lung tissue of experimental mice, while levels of angiogenesis-related factors, namely Tie-2, Ang-1 and ANGPT2 (Ang-2), fibrosis- associated factors like Alpha-smooth muscle actin (α-SMA), collagen-I and hydroxyproline in SSd and dexamethasone (DXM) mice were significantly reduced at each time point compared to BLM (p < 0.01). Additionally, we discovered substantial decreased expressions of Ang-1, Ang-2, Tie-2, α-SMA and collagen-I at protein level in SSd and DXM mice at each time point compared to BLM (p < 0.05). Besides, insignificant differences were observed between SSd and DXM groups (p > 0.05).
CONCLUSION
This study has demonstrated that SSd could down-regulate the expression of ANG-1, Ang-2 and Tie2 in the Ang/Tie2 pathway, and may reduce lung inflammation and PF in BLM-induced mice via inhibition of angiogenesis.
Topics: Mice; Animals; Angiopoietins; Mice, Inbred C57BL; Lung; Idiopathic Pulmonary Fibrosis; Collagen Type I; Bleomycin
PubMed: 37837833
DOI: 10.1016/j.acthis.2023.152100 -
Journal of Neurology, Neurosurgery, and... Jun 1984Reverse phase prepared liposomes of defined composition (dipalmitoyl phosphatidyl choline, cholesterol and dipalmitoyl phosphatidic acid; 7:2:1; DPC) when injected...
Reverse phase prepared liposomes of defined composition (dipalmitoyl phosphatidyl choline, cholesterol and dipalmitoyl phosphatidic acid; 7:2:1; DPC) when injected intracerebrally in the rat produced no tissue damage beyond that of the penetrating wound, or altered behaviour pattern over 1 week of observation. The cerebral tissue response and behaviour of rats injected with bleomycin of increasing concentration, free and entrapped within liposomes was studied in short and long term experiments. In separate experiments blood, urine and tissue levels of bleomycin were measured after intracerebral injection of free and liposome entrapped bleomycin in the rat. These studies demonstrated that bleomycin when entrapped within liposomes and injected intracerebrally was of low toxicity to normal cerebral tissue, and was cleared more slowly from the injection site than when the free drug was injected. The results obtained indicated a potential application for drugs entrapped within liposomes acting as a depot preparation in the treatment of cerebral gliomas.
Topics: Animals; Behavior, Animal; Bleomycin; Cerebral Cortex; Injections; Liposomes; Male; Rats; Rats, Inbred Strains
PubMed: 6204018
DOI: 10.1136/jnnp.47.6.585 -
Journal of B.U.ON. : Official Journal... 2020To investigate the influence of bleomycin (BLM) on the proliferation and apoptosis of brain glioma cells through transforming growth factor-β (TGF-β)/Smads signaling...
PURPOSE
To investigate the influence of bleomycin (BLM) on the proliferation and apoptosis of brain glioma cells through transforming growth factor-β (TGF-β)/Smads signaling pathway.
METHODS
The U87 brain glioma cells were cultured in vitro and reacted with different concentrations of BLM (5 and 10 mU/mL), and the cell growth status of each group was observed under a microscope. The cell proliferation activity was detected using Cell Counting Kit-8 (CCK-8) assay, the percentage of 5-Ethynyl-2'-deoxyuridine (EdU)-positive cells in each group was determined via EdU staining, and the apoptosis of U87 cells was tested by means of terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining. In addition, reverse transcription-polymerase chain reaction (RT-PCR) was performed to measure the messenger ribonucleic acid (mRNA) levels of genes related to proliferation, apoptosis and the TGF-β/Smads signaling pathway. Finally, western blotting assay was performed to analyze the expression of the TGF-β/Smads signaling pathway.
RESULTS
In the 5 mU/mL BLM group, the glioma cells were in a poor growth status, with a low density, while the 10 mU/mL BLM group exhibited the poorest growth status and the lowest density, and the morphological structure trended toward normal. It was discovered via CCK-8 assay and EdU staining that the number of cells and proliferation activity were decreased markedly in the 10 mU/mL BLM group. According to TUNEL staining, 10 mU/mL BLM group had remarkably increased apoptotic cells, while negative control (NC) group had fewer apoptotic cells. The gene assay results revealed that the gene expressions of Bcl-2 and TGF-β1 declined notably in the 10 mU/mL BLM group but rose in the NC group, and the gene expression trends of Caspase-3 and Smad4 were the opposite. The protein assay results manifested that the expressions of TGF-β1 was obviously reduced, while that of Smad4 was evidently raised in the 10 mU/mL BLM group.
CONCLUSION
BLM at an appropriate concentration can inhibit the proliferation and promote apoptosis of brain glioma cells by repressing the TGF-β/Smads signaling pathway, thus ameliorating and treating brain glioma and other related diseases.
Topics: Antibiotics, Antineoplastic; Apoptosis; Bleomycin; Cell Proliferation; Glioma; Humans; Signal Transduction; Transforming Growth Factor beta1
PubMed: 32521909
DOI: No ID Found -
Molecules (Basel, Switzerland) Jun 2016Pingyangmycin is an anticancer drug known as bleomycin A5 (A5), discovered in the Pingyang County of Zhejiang Province of China. Bleomycin (BLM) is a mixture of mainly...
Pingyangmycin is an anticancer drug known as bleomycin A5 (A5), discovered in the Pingyang County of Zhejiang Province of China. Bleomycin (BLM) is a mixture of mainly two compounds (A2 and B2), which is on the World Health Organization's list of essential medicines. Both BLM and A5 are hydrophilic molecules that depend on transporters or endocytosis receptors to get inside of cells. Once inside, the anticancer activities rely on their abilities to produce DNA breaks, thus leading to cell death. Interestingly, the half maximal inhibitory concentration (IC50) of BLMs in different cancer cell lines varies from nM to μM ranges. Different cellular uptake, DNA repair rate, and/or increased drug detoxification might be some of the reasons; however, the molecules and signaling pathways responsible for these processes are largely unknown. In the current study, we purified the A2 and B2 from the BLM and tested the cytotoxicities and the molecular mechanisms of each individual compound or in combination with six different cell lines, including a Chinese hamster ovary (CHO) cell line defective in glycosaminoglycan biosynthesis. Our data suggested that glycosaminoglycans might be involved in the cellular uptake of BLMs. Moreover, both BLM and A5 shared similar signaling pathways and are involved in cell cycle and apoptosis in different cancer cell lines.
Topics: Animals; Antibiotics, Antineoplastic; Apoptosis; Apoptosis Regulatory Proteins; Bleomycin; CHO Cells; Cell Cycle Checkpoints; Cell Cycle Proteins; Cell Line, Tumor; Chromatography, High Pressure Liquid; Cricetulus; ErbB Receptors; Gene Expression; Humans; Inhibitory Concentration 50; Molecular Structure; Phosphorylation; Signal Transduction; Spectrometry, Mass, Electrospray Ionization
PubMed: 27376254
DOI: 10.3390/molecules21070862 -
International Journal of Molecular... Feb 2023Idiopathic pulmonary fibrosis (IPF) is a chronic lung disease characterized by the aberrant accumulation of extracellular matrix in the lungs. nintedanib is one of the...
Idiopathic pulmonary fibrosis (IPF) is a chronic lung disease characterized by the aberrant accumulation of extracellular matrix in the lungs. nintedanib is one of the two FDA-approved drugs for IPF treatment; however, the exact pathophysiological mechanisms of fibrosis progression and response to therapy are still poorly understood. In this work, the molecular fingerprint of fibrosis progression and response to nintedanib treatment have been investigated by mass spectrometry-based bottom-up proteomics in paraffin-embedded lung tissues from bleomycin-induced (BLM) pulmonary fibrosis mice. Our proteomics results unveiled that (i) samples clustered depending on the tissue fibrotic grade (mild, moderate, and severe) and not on the time course after BLM treatment; (ii) the dysregulation of different pathways involved in fibrosis progression such as the complement coagulation cascades, advanced glycation end products (AGEs) and their receptors (RAGEs) signaling, the extracellular matrix-receptor interaction, the regulation of actin cytoskeleton, and ribosomes; (iii) Coronin 1A (Coro1a) as the protein with the highest correlation when evaluating the progression of fibrosis, with an increased expression from mild to severe fibrosis; and (iv) a total of 10 differentially expressed proteins (-value ≤ 0.05 and Fold change ≤-1.5 or ≥1.5), whose abundance varied in the base of the severity of fibrosis (mild and moderate), were modulated by the antifibrotic treatment with nintedanib, reverting their trend. Notably, nintedanib significantly restored lactate dehydrogenase B (Ldhb) expression but not lactate dehydrogenase A (Ldha). Notwithstanding the need for further investigations to validate the roles of both Coro1a and Ldhb, our findings provide an extensive proteomic characterization with a strong relationship with histomorphometric measurements. These results unveil some biological processes in pulmonary fibrosis and drug-mediated fibrosis therapy.
Topics: Mice; Animals; Bleomycin; Proteomics; Lung; Idiopathic Pulmonary Fibrosis; Fibrosis
PubMed: 36901840
DOI: 10.3390/ijms24054410 -
American Journal of Physiology. Lung... Mar 2023The most common preclinical, in vivo model to study lung fibrosis is the bleomycin-induced lung fibrosis model in 2- to 3-mo-old mice. Although this model resembles key...
The most common preclinical, in vivo model to study lung fibrosis is the bleomycin-induced lung fibrosis model in 2- to 3-mo-old mice. Although this model resembles key aspects of idiopathic pulmonary fibrosis (IPF), there are limitations in its predictability for the human disease. One of the main differences is the juvenile age of animals that are commonly used in experiments, resembling humans of around 20 yr. Because IPF patients are usually older than 60 yr, aging appears to play an important role in the pathogenesis of lung fibrosis. Therefore, we compared young (3 months) and old mice (21 months) 21 days after intratracheal bleomycin instillation. Analyzing lung transcriptomics (mRNAs and miRNAs) and proteomics, we found most pathways to be similarly regulated in young and old mice. However, old mice show imbalanced protein homeostasis as well as an increased inflammatory state in the fibrotic phase compared to young mice. Comparisons with published human transcriptomic data sets (GSE47460, GSE32537, and GSE24206) revealed that the gene signature of old animals correlates significantly better with IPF patients, and it also turned human healthy individuals better into "IPF patients" using an approach based on predictive disease modeling. Both young and old animals show similar molecular hallmarks of IPF in the bleomycin-induced lung fibrosis model, although old mice more closely resemble several features associated with IPF in comparison to young animals.
Topics: Humans; Mice; Animals; Bleomycin; Transcriptome; Proteomics; Lung; Idiopathic Pulmonary Fibrosis; Disease Models, Animal; Mice, Inbred C57BL
PubMed: 36625483
DOI: 10.1152/ajplung.00253.2021 -
Cellular Physiology and Biochemistry :... 2017Bleomycin is a clinically used anti-cancer drug that produces DNA breaks once inside of cells. However, bleomycin is a positively charged molecule and cannot get inside...
BACKGROUND/AIMS
Bleomycin is a clinically used anti-cancer drug that produces DNA breaks once inside of cells. However, bleomycin is a positively charged molecule and cannot get inside of cells by free diffusion. We previously reported that the cell surface negatively charged glycosaminoglycans (GAGs) may be involved in the cellular uptake of bleomycin. We also observed that a class of positively charged small molecules has Golgi localization once inside of the cells. We therefore hypothesized that bleomycin might perturb Golgi-operated GAG biosynthesis.
METHODS
We used stable isotope labeling coupled with LC/MS analysis of GAG disaccharides simultaneously from bleomycin-treated and non-treated cancer cells. To further understand the cytotoxicity of bleomycin and its relationship to GAGs, we used sodium chlorate to inhibit GAG sulfation and commercially available GAGs to compete for cell surface GAG/bleomycin interactions in seven cell lines including CHO745 defective in both heparan sulfate and chondroitin sulfate biosynthesis.
RESULTS
we discovered that heparan sulfate GAG was significantly undersulfated and the quantity and disaccharide compositions of GAGs were changed in bleomycin-treated cells in a concentration- and time-dependent manner. We revealed that bleomycin-induced cytotoxicity was directly related to cell surface GAGs.
CONCLUSION
GAGs were targeted by bleomycin both at cell surface and at Golgi. Thus, GAGs might be the biological relevant molecules that might be related to the bleomycin-induced fibrosis in certain cancer patients, a severe side effect with largely unknown molecular mechanism.
Topics: Animals; Antipyrine; Bleomycin; CHO Cells; Chondroitin Sulfates; Chromatography, High Pressure Liquid; Cricetinae; Cricetulus; Deuterium; Edaravone; HCT116 Cells; HT29 Cells; Heparitin Sulfate; Humans; Isotope Labeling; Mass Spectrometry
PubMed: 28982096
DOI: 10.1159/000481763 -
Cells Jan 2023A progressive fibrosing phenotype is critical in several lung diseases. It is irreversible and associated with early patient mortality. Growing evidence has revealed...
A progressive fibrosing phenotype is critical in several lung diseases. It is irreversible and associated with early patient mortality. Growing evidence has revealed pulmonary macrophages' role as modulators of the fibrotic processes. The proportion, phenotype, and function of alveolar (AM) and interstitial macrophages (IM) at the early stages of bleomycin-induced pulmonary fibrosis have not been clearly described. In this way, our study aimed to characterize these macrophage populations and investigate the effect on fibroblast activation. C57BL/6 mice were intratracheally injected with bleomycin and were sacrificed at day 3, 5, and 7 for the performance of flow cytometry and fluorescent-activated cell sorting analysis for protein and gene expression quantification. After bleomycin administration, the proportion of IM was significantly higher than that of AM, which showed a decay during the inflammatory phase, and peaked at day 7. At day 7 of the inflammatory phase, AM started shifting their phenotype from M1-like towards M2, while IM showed a M2-like phenotype. Conditioned medium derived from IM sorted at day 7 induced fibroblast activation and differentiation in myofibroblasts in vitro. Our findings indicate that IM are the largest macrophage population at the early stages of experimental pulmonary fibrosis and are secreted mediators able to activate fibroblasts, pointing to macrophage modulation as a potential therapeutic strategy to restrain progressive fibrosing lung disorders.
Topics: Mice; Animals; Pulmonary Fibrosis; Bleomycin; Mice, Inbred C57BL; Macrophages; Fibroblasts
PubMed: 36766744
DOI: 10.3390/cells12030402