RESUMO
Pulmonary fibrosis (PF) is an interstitial lung disease characterized by inflammation and fibrotic changes, with an unknown cause. In the early stages of PF, severe inflammation leads to the destruction of lung tissue, followed by upregulation of fibrotic factors like Transforming growth factor-ß (TGF-ß) and connective tissue growth factor (CTGF), which disrupt normal tissue repair. Geniposide, a natural iridoid glycoside primarily derived from the fruits of Gardenia jasminoides Ellis, possesses various pharmacological activities, including liver protection, choleretic effects, and anti-inflammatory properties. In this study, we investigated the effects of Geniposide on chronic inflammation and fibrosis induced by bleomycin (BLM) in mice with pulmonary fibrosis (PF). PF was induced by intratracheal instillation of bleomycin, and Geniposide(100/50/25mgâ¢kg-1) was orally administered to the mice once a day until euthanasia(14 day/28 day). The Raw264.7 cell inflammation induced by LPS was used to evaluate the effect of Geniposide on the activation of macrophage. Our results demonstrated that Geniposide reduced lung coefficients, decreased the content of Hydroxyproline, and improved pathological changes in lung tissue. It also reduced the number of inflammatory cells and levels of pro-inflammatory cytokines in bronchoalveolar lavage fluid (BALF) of bleomycin-induced PF mice. At the molecular level, Geniposide significantly down-regulated the expression of TGF-ß1, Smad2/3, p38, and CTGF in lung tissues of PF mice induced by bleomycin. Molecular docking results revealed that Geniposide exhibited good binding activity with TGF-ß1, Smad2, Smad3, and p38. In vitro study showed Geniposide directly inhibited the activation of macrophage induced by LPS. In conclusion, our findings suggest that Geniposide can ameliorate bleomycin-induced pulmonary fibrosis in mice by inhibiting the TGF-ß/Smad and p38MAPK signaling pathways.
Assuntos
Bleomicina , Iridoides , Fibrose Pulmonar , Fator de Crescimento Transformador beta , Proteínas Quinases p38 Ativadas por Mitógeno , Animais , Bleomicina/efeitos adversos , Bleomicina/toxicidade , Fibrose Pulmonar/induzido quimicamente , Fibrose Pulmonar/tratamento farmacológico , Fibrose Pulmonar/metabolismo , Fibrose Pulmonar/patologia , Iridoides/farmacologia , Camundongos , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Transdução de Sinais/efeitos dos fármacos , Masculino , Células RAW 264.7 , Pulmão/patologia , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Proteínas Smad/metabolismo , Fator de Crescimento do Tecido Conjuntivo/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Camundongos Endogâmicos C57BLRESUMO
We have evaluated the induction of complete (i.e., without open ends) and incomplete (i.e., with non-rejoined or open ends) chromosomal aberrations by the radiomimetic antibiotic bleomycin (BLM) in human lymphoblastoid cells immortalized with the Epstein-Barr virus (EBV). An EBV-induced lymphoblastoid cell line (T-37) was exposed to BLM (10-200 µg/mL) for 2â¯h at 37ºC, and chromosomal aberrations were analyzed 24â¯h after treatment, using PNA-FISH with pan-telomeric and pan-centromeric probes. Both complete (multicentrics, rings, compound acentric fragments, and interstitial deletions) and incomplete (incomplete chromosomes or IC, and terminal acentric fragments or TAF) chromosomal aberrations increased significantly in BLM-exposed cells, although the concentration-response relationship was non-linear. Of the acentric fragments (ace) induced by BLM, 40 % were compound fragments (CF, ace +/+). TAF (ace, +/-) and interstitial fragments (IAF, ace -/-) were induced at similar frequencies (30 %). 230 ICE were induced by BLM, of which 52 % were IC and 48 % TAF. The average ratio between total incomplete chromosome elements (ICE) and multicentrics was 1.52. These findings suggest that human lymphoblastoid cells exhibit less repair capacity than human lymphocytes, with respect to BLM-induced ICE, and that chromosomal incompleteness is a common event following exposure of these cells to BLM.
Assuntos
Bleomicina , Aberrações Cromossômicas , Herpesvirus Humano 4 , Linfócitos , Humanos , Aberrações Cromossômicas/efeitos dos fármacos , Aberrações Cromossômicas/induzido quimicamente , Herpesvirus Humano 4/genética , Herpesvirus Humano 4/efeitos dos fármacos , Bleomicina/toxicidade , Bleomicina/farmacologia , Linfócitos/efeitos dos fármacos , Linfócitos/virologia , Linhagem Celular Transformada , Antibióticos Antineoplásicos/toxicidade , Antibióticos Antineoplásicos/farmacologia , Transformação Celular Viral/efeitos dos fármacos , Linhagem CelularRESUMO
Systemic sclerosis (SSc) is a life-threatening autoimmune disease characterized by widespread fibrosis in the skin and several internal organs. Nudix Hydrolase 21 (NUDT2 or CFIm25) downregulation in fibroblasts is known to play detrimental roles in both skin and lung fibrosis. This study aims to investigate the upstream mechanisms that lead to NUDT21 repression in skin fibrosis. We identified transforming growth factor ß (TGFß1) as the primary cytokine that downregulated NUDT21 in normal skin fibroblasts. In the bleomycin-induced dermal fibrosis model, consistent with the peak activation of TGFß1 at the late fibrotic stage, NUDT21 was downregulated at this stage, and delayed NUDT21 knockdown during this fibrotic phase led to enhanced fibrotic response to bleomycin. Further investigation suggested TGFß downregulated NUDT21 through microRNA (miRNA) 181a and 181b induction. Both miR-181a and miR-181b were elevated in bleomycin-induced skin fibrosis in mice and primary fibroblasts isolated from SSc patients, and they directly targeted NUDT21 and led to its downregulation in skin fibroblasts. Functional studies demonstrated that miR-181a and miR-181b inhibitors attenuated bleomycin-induced skin fibrosis in mice in association with decreased NUDT21 expression, while miR-181a and miR-181b mimics promoted bleomycin-induced fibrosis. Overall, these findings suggest a novel role for miR-181a/b in SSc pathogenesis by repressing NUDT21 expression.
Assuntos
Bleomicina , Fibroblastos , Fibrose , MicroRNAs , Escleroderma Sistêmico , Pele , MicroRNAs/genética , MicroRNAs/metabolismo , Animais , Humanos , Camundongos , Fibrose/metabolismo , Fibroblastos/metabolismo , Fibroblastos/patologia , Escleroderma Sistêmico/metabolismo , Escleroderma Sistêmico/patologia , Escleroderma Sistêmico/genética , Escleroderma Sistêmico/induzido quimicamente , Bleomicina/toxicidade , Bleomicina/efeitos adversos , Pele/patologia , Pele/metabolismo , Feminino , Fator de Crescimento Transformador beta1/metabolismo , Fator de Crescimento Transformador beta1/genética , Camundongos Endogâmicos C57BL , Fator de Especificidade de Clivagem e Poliadenilação/metabolismo , Fator de Especificidade de Clivagem e Poliadenilação/genética , Células Cultivadas , Regulação para BaixoRESUMO
Idiopathic pulmonary fibrosis (IPF) is an interstitial lung disease characterized by chronic inflammation, lung tissue fibrotic changes and impaired lung function. Pulmonary fibrosis 's pathological process is thought to be influenced by macrophage-associated phenotypes. IPF treatment requires specific targets that target macrophage polarization. Cytokine-like 1(CYTL1) is a secreted protein with multiple biological functions first discovered in CD34+ haematopoietic cells. However, its possible effects on IPF progression remain unclear. This study investigated the role of CYTL1 in IPF progression in a bleomycin-induced lung injury and fibrosis model. In bleomycin-induced mice, CYTL1 is highly expressed. Moreover, CYTL1 ablation alleviates lung injury and fibrosis in vivo. Further, downregulating CYTL1 reduces macrophage M2 polarization. Mechanically, CYTL1 regulates transforming growth factor ß (TGF-ß)/connective tissue growth factor (CCN2) axis and inhibition of TGF-ß pathway alleviates bleomycin-induced lung injury and fibrosis. In conclusion, highly expressed CYTL1 inhibits macrophage M2 polarization by regulating TGF-ß/CCN2 expression, alleviating bleomycin-induced lung injury and fibrosis. CYTL1 could, therefore, serve as a promising IPF target.
Assuntos
Bleomicina , Fator de Crescimento do Tecido Conjuntivo , Regulação para Baixo , Macrófagos , Fibrose Pulmonar , Fator de Crescimento Transformador beta , Animais , Bleomicina/toxicidade , Camundongos , Regulação para Baixo/efeitos dos fármacos , Fator de Crescimento Transformador beta/metabolismo , Macrófagos/metabolismo , Macrófagos/efeitos dos fármacos , Fibrose Pulmonar/induzido quimicamente , Fibrose Pulmonar/metabolismo , Fibrose Pulmonar/patologia , Fator de Crescimento do Tecido Conjuntivo/metabolismo , Fator de Crescimento do Tecido Conjuntivo/genética , Camundongos Endogâmicos C57BL , Masculino , Polaridade Celular/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Fibrose Pulmonar Idiopática/induzido quimicamente , Fibrose Pulmonar Idiopática/metabolismo , Fibrose Pulmonar Idiopática/patologiaRESUMO
Introduction: The impaired proliferative capacity of alveolar epithelial cells after injury is an important factor causing epithelial repair dysfunction, leading to the occurrence of idiopathic pulmonary fibrosis (IPF). Alveolar type 2 (AT2) cells as the stem cells of alveolar epithelium participate in the repair process after alveolar injury. Lipocalin-2 (LCN2) participates in multiple processes regulating the pathological process of alveolar epithelial cells, but the mechanisms involved are still unclear. Method: We used a BLM-treated mouse model to characterize the expression of LCN2 in lung fibrosis regions and analyzed the location of LCN2 in alveolar epithelial cells. Moreover, human pulmonary alveolar epithelial cells (HPAEpiCs) were transfected with the LCN2 overexpression plasmid vector in vitro. Recombinant human interleukin-17 (IL-17) protein (rhIL-17) at different concentrations was administered to intervene in HPAEpiCs, observing cell viability and analyzing the concentration-dependent effect of IL-17. Results: LCN2 was increased in the alveolar epithelium post-BLM injury, and highly expressed LCN2 was mainly concentrated on AT2 cells in BLM-injured lungs. Meanwhile, LCN2-overexpressing HPAEpiCs showed impaired cell viability and cell growth. HPAEpiC intervention with rhIL-17 mildly rescued the impaired cell proliferation induced by LCN2 overexpression, and the effect of IL-17 intervention was partially concentration-dependent. Conclusions: The results revealed the reversed effect of IL-17 on the impaired proliferative capacity of the alveolar epithelium induced by LCN2 overexpression. The target alveolar epithelial cells regulated by this process were AT2 cells, providing new clues for alveolar epithelium repair after injury and the treatment of lung injury diseases.
Assuntos
Células Epiteliais Alveolares , Proliferação de Células , Interleucina-17 , Lipocalina-2 , Lipocalina-2/genética , Lipocalina-2/metabolismo , Interleucina-17/metabolismo , Interleucina-17/genética , Animais , Proliferação de Células/genética , Camundongos , Células Epiteliais Alveolares/metabolismo , Células Epiteliais Alveolares/efeitos dos fármacos , Humanos , Bleomicina/toxicidade , Masculino , Modelos Animais de Doenças , Camundongos Endogâmicos C57BLRESUMO
Pulmonary fibrosis is a progressive disease caused by interstitial inflammation. Treatments are extremely scarce; therapeutic drugs and transplantation therapies are not widely available due to cost and a lack of donors, respectively. Recently, there has been a high interest in regenerative medicine and exponential advancements in stem cell-based therapies have occurred. However, a sensitive imaging technique for investigating the in vivo dynamics of transplanted stem cells has not yet been established and the mechanisms of stem cell-based therapy remain largely unexplored. In this study, we administered mouse adipose tissue-derived mesenchymal stem cells (mASCs) labeled with quantum dots (QDs; 8.0 nM) to a mouse model of bleomycin-induced pulmonary fibrosis in an effort to clarify the relationship between in vivo dynamics and therapeutic efficacy. These QD-labeled mASCs were injected into the trachea of C57BL/6 mice seven days after bleomycin administration to induce fibrosis in the lungs. The therapeutic effects and efficacy were evaluated via in vivo/ex vivo imaging, CT imaging, and H&E staining of lung sections. The QD-labeled mASCs remained in the lungs longer and suppressed fibrosis. The 3D imaging results showed that the transplanted cells accumulated in the peripheral and fibrotic regions of the lungs. These results indicate that mASCs may prevent fibrosis. Thus, QD labeling could be a suitable and sensitive imaging technique for evaluating in vivo kinetics in correlation with the efficacy of cell therapy.
Assuntos
Bleomicina , Modelos Animais de Doenças , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais , Camundongos Endogâmicos C57BL , Fibrose Pulmonar , Animais , Bleomicina/efeitos adversos , Bleomicina/toxicidade , Fibrose Pulmonar/induzido quimicamente , Fibrose Pulmonar/diagnóstico por imagem , Fibrose Pulmonar/terapia , Fibrose Pulmonar/patologia , Camundongos , Transplante de Células-Tronco Mesenquimais/métodos , Células-Tronco Mesenquimais/citologia , Pontos Quânticos , Pulmão/diagnóstico por imagem , Pulmão/patologia , Tomografia Computadorizada por Raios X , Tecido Adiposo/citologia , Tecido Adiposo/diagnóstico por imagemRESUMO
BACKGROUND: Skin flap transplantation is used to effectively reconstruct defects of the hand and foot skin and soft tissues. We here investigated the effect of the PF127/bleomycin (BLM) hydrogel on the extracellular matrix (ECM) remodeling of skin flaps and the underlying mechanism, thereby providing a new reference point for personalized flap modification and overcoming abrasion resistance- and stability-associated difficulties. METHODS: The appropriate PF127/BLM concentration was selected based on the gelation time and drug release curve. Migration assays, scratch assays, and live/dead staining were conducted to verify the effect of PF127/BLM on human skin fibroblasts (HSFs). The effects of PF127/BLM on the ECM were assessed through hematoxylin and eosin and Masson staining. Additionally, we examined the expression of ECM remodeling-related genes and proteins involved in their associated signaling pathway. Finally, the effects of PF127/BLM on organ fibrosis and toxicity to liver and kidney functions were assessed in mice. RESULTS: A 25% PF127/BLM hydrogel was selected as the study concentration. PF127/BLM augmented HSF chemotaxis and proliferation. Furthermore, PF127/BLM promoted subcutaneous ECM remodeling and fibrosis, increased the flap dermis thickness, and reduced the toxic side effects of BLM on liver/lung fibrosis and liver/kidney function. Additional studies confirmed that the PF127/BLM-mediated regulation of ECM remodeling in skin flaps was associated with TGFß-Col signaling pathway activation. CONCLUSION: The PF127/BLM hydrogel promoted subcutaneous ECM remodeling and fibrosis, which aided the construction of personalized flaps through the TGFß-Col signaling pathway, with decreased hepatic, pulmonary, and renal toxicities.
Assuntos
Bleomicina , Matriz Extracelular , Hidrogéis , Transdução de Sinais , Fator de Crescimento Transformador beta , Matriz Extracelular/metabolismo , Matriz Extracelular/efeitos dos fármacos , Animais , Fator de Crescimento Transformador beta/metabolismo , Camundongos , Humanos , Transdução de Sinais/efeitos dos fármacos , Bleomicina/toxicidade , Bleomicina/farmacologia , Fibrose , Retalhos Cirúrgicos/patologia , Masculino , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibroblastos/patologia , Colágeno , Medicina de Precisão/métodosRESUMO
BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a chronic and progressive interstitial lung disease (ILD) whose cause and pathogenesis are not yet well understood. Until now, no animal model of lung fibrosis succeeds in recapitulating all IPF features, thus the use of different rodent models is essential for the evaluation and development of new effective pharmacological treatments. Recently, the alveolar epithelial dysfunction has been emphasized in the etiopathogenesis context of IPF. Remarkably, the role of an aberrant basaloid cell type, primarily found in humans and confirmed in mice, seems to be crucial in the establishment and progression of the disease/model. Our work aimed to characterize for the first time this cell population in a rat model of lung fibrosis induced by a double bleomycin (BLM) administration, demonstrating the translational value of the model and its potential use in the testing of effective new drugs. METHODS: Rats received an intratracheal BLM administration at day 0 and 4. Animals were sacrificed 21 and 28 days post-BLM. The fibrosis evaluation was carried out through histological (Ashcroft score and automatic image analysis) and immunoenzymatic analysis. Immunofluorescence was used for the characterization of the aberrant basaloid cells markers. RESULTS: Lung histology revealed an increase in severe grades of Ashcroft scores and areas of fibrosis, resulting in a rise of collagen deposition at both the analyzed time-points. Immunofluorescence staining indicated the presence of KRT8+ cells in bronchial epithelial cells from both controls (saline, SAL) and BLM-treated animals. Interesting, KRT8+ cells were found exclusively in the fibrotic parenchyma (confirmed by the alpha-smooth muscle actin (α-SMA) staining for myofibroblasts) of BLM-treated animals. Moreover, KRT8+ cells co-expressed markers as Prosurfactant protein C (Pro-SPC) and Vimentin, suggesting their intermediate state potentially originating from alveolar type II (AT2) cells, and participating to the abnormal epithelial-mesenchymal crosstalk. CONCLUSION: Previous preclinical studies demonstrated the presence of KRT8+ aberrant basaloid-like cells in murine models of lung fibrosis. This work investigated the same cell population in a different rodent (the rat) model of lung fibrosis triggered by a double administration of BLM. Our results provided a further confirmation that, in rats, the intratracheal administration of BLM induced the appearance of a population of cells compatible with the KRT8+ alveolar differentiation intermediate (ADI) cells, as described previously in the mouse. This piece of work enforces previous evidence and further support the use of a rat model of BLM resembling the alveolar epithelial dysfunction to evaluate new clinical candidates for development in IPF.
Assuntos
Bleomicina , Modelos Animais de Doenças , Animais , Bleomicina/toxicidade , Ratos , Masculino , Fibrose Pulmonar/patologia , Fibrose Pulmonar/metabolismo , Fibrose Pulmonar/induzido quimicamente , Pulmão/patologia , Pulmão/metabolismo , Fibrose Pulmonar Idiopática/patologia , Fibrose Pulmonar Idiopática/metabolismoRESUMO
BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a long-term, progressive, and irreversible pulmonary interstitial disease. The activation of Smad family member 2 (Smad2) and Smad3 transcription factors by transforming growth factor ß-1 (TGF-ß1) is a critical event in the pathogenesis of IPF. However, there is still a lack of understanding regarding the molecular mechanisms governing Smad2 and Smad3 proteins. Ubiquitin-specific protease 7 (USP7) is a deubiquitinase that plays a vital role in regulating protein stability within cells. However, its regulation of the TGF-ß signaling pathway and its significance in IPF remain undiscovered. This study aims to clarify the function of USP7 in the TGF-ß signaling pathway, while simultaneously exploring the specific molecular mechanisms involved. Additionally, this study seeks to evaluate the therapeutic potential of targeted USP7 inhibitors in IPF, thereby providing novel insights for the diagnosis and management of IPF. METHODS: We first detected the expression of USP7 in lung tissues of mice with Bleomycin (BLM)-induced pulmonary fibrosis and in Beas-2B cells treated with or without TGF-ß1 through Western blot analysis. Subsequently, we explored the influence of USP7 on fibrotic processes and the TGF-ß1 signaling pathway, utilizing in vitro and in vivo studies. Finally, we assessed the effectiveness of USP7-specific inhibitors in an IPF murine model. RESULTS: In the present study, USP7 was found to de-ubiquitinate Smad2 and Smad3, consequently increasing their stability and promoting the TGF-ß1-induced production of profibrotic proteins including α-smooth muscle actin (α-SMA) and fibronectin 1 (FN-1). Inhibition or knockdown of USP7 resulted in decreased levels of Smad2 and Smad3 proteins, leading to reduced expression of FN-1, Collagen Type I Alpha 1 Chain (Col1A1), and α-SMA induced by TGF-ß1 in human pulmonary epithelial cells. These findings demonstrate that overexpression of USP7 reduces Smad2/3 ubiquitination, whereas inhibition or knockdown of USP7 enhances their ubiquitination. USP7 is abundantly expressed in IPF lungs. The expressions of USP7, Smad2, and Smad3 were upregulated in bleomycin-induced lung injury. The USP7 inhibitor P22077 reduced the expression of FN-1 and type I collagen as well as Smad2/3 and collagen deposition in lung tissue in a model of pulmonary fibrosis induced by bleomycin. CONCLUSIONS: This study demonstrates that USP7 promotes TGF-ß1 signaling by stabilizing Smad2 and Smad3. The contribution of USP7 to the progression of IPF indicates it may be a viable treatment target.
Assuntos
Bleomicina , Transdução de Sinais , Proteína Smad2 , Proteína Smad3 , Fator de Crescimento Transformador beta1 , Peptidase 7 Específica de Ubiquitina , Fator de Crescimento Transformador beta1/metabolismo , Animais , Proteína Smad3/metabolismo , Peptidase 7 Específica de Ubiquitina/metabolismo , Peptidase 7 Específica de Ubiquitina/genética , Camundongos , Transdução de Sinais/efeitos dos fármacos , Humanos , Proteína Smad2/metabolismo , Bleomicina/toxicidade , Fibrose Pulmonar/metabolismo , Fibrose Pulmonar/patologia , Fibrose Pulmonar/induzido quimicamente , Ubiquitinação , Fibrose Pulmonar Idiopática/metabolismo , Fibrose Pulmonar Idiopática/patologia , Fibrose Pulmonar Idiopática/induzido quimicamente , Fibrose Pulmonar Idiopática/genética , Masculino , Camundongos Endogâmicos C57BL , Linhagem Celular , Pulmão/patologia , Pulmão/metabolismo , Modelos Animais de DoençasRESUMO
Post-mitotic, non-proliferative dermal fibroblasts have crucial functions in maintenance and restoration of tissue homeostasis. They are involved in essential processes such as wound healing, pigmentation and hair growth, but also tumor development and aging-associated diseases. These processes are energetically highly demanding and error prone when mitochondrial damage occurs. However, mitochondrial function in fibroblasts and the influence of mitochondrial dysfunction on fibroblast-specific demands are still unclear. To address these questions, we created a mouse model in which accelerated cell-specific mitochondrial DNA (mtDNA) damage accumulates. We crossed mice carrying a dominant-negative mutant of the mitochondrial replicative helicase Twinkle (RosaSTOP system) with mice that express fibroblast-specific Cre Recombinase (Collagen1A2 CreERT) which can be activated by Tamoxifen (TwinkleFIBRO). Thus, we are able to induce mtDNA deletions and duplications in specific cells, a process which resembles the physiological aging process in humans, where this damage accumulates in all tissues. Upon proliferation in vitro, Tamoxifen induced Twinkle fibroblasts deplete most of their mitochondrial DNA which, although not disturbing the stoichiometry of the respiratory chain complexes, leads to reduced ROS production and mitochondrial membrane potential as well as an anti-inflammatory and anti-fibrotic profile of the cells. In Sodium Azide treated wildtype fibroblasts, without a functioning respiratory chain, we observe the opposite, a rather pro-inflammatory and pro-fibrotic signature. Upon accumulation of mitochondrial DNA mutations in vivo the TwinkleFIBRO mice are protected from fibrosis development induced by intradermal Bleomycin injections. This is due to dampened differentiation of the dermal fibroblasts into α-smooth-muscle-actin positive myofibroblasts in TwinkleFIBRO mice. We thus provide evidence for striking differences of the impact that mtDNA mutations have in contrast to blunted mitochondrial function in dermal fibroblasts and skin homeostasis. These data contribute to improved understanding of mitochondrial function and dysfunction in skin and provide mechanistic insight into potential targets to treat skin fibrosis in the future.
Assuntos
Bleomicina , Diferenciação Celular , DNA Mitocondrial , Fibrose , Mutação , Miofibroblastos , Animais , Bleomicina/efeitos adversos , Bleomicina/toxicidade , DNA Mitocondrial/genética , DNA Mitocondrial/metabolismo , Camundongos , Miofibroblastos/metabolismo , Miofibroblastos/patologia , Miofibroblastos/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , DNA Helicases/genética , DNA Helicases/metabolismo , Fibroblastos/metabolismo , Fibroblastos/efeitos dos fármacos , Fibroblastos/patologia , Tamoxifeno/farmacologia , Mitocôndrias/metabolismo , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/patologia , Modelos Animais de Doenças , Espécies Reativas de Oxigênio/metabolismo , Humanos , Pele/patologia , Pele/metabolismo , Pele/efeitos dos fármacos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Colágeno Tipo IRESUMO
Idiopathic pulmonary fibrosis (IPF) is a progressive lung disease with poor prognosis and limited treatment options. Efforts to identify effective treatments are thwarted by limited understanding of IPF pathogenesis and poor translatability of available preclinical models. Here we generated spatially resolved transcriptome maps of human IPF (n = 4) and bleomycin-induced mouse pulmonary fibrosis (n = 6) to address these limitations. We uncovered distinct fibrotic niches in the IPF lung, characterized by aberrant alveolar epithelial cells in a microenvironment dominated by transforming growth factor beta signaling alongside predicted regulators, such as TP53 and APOE. We also identified a clear divergence between the arrested alveolar regeneration in the IPF fibrotic niches and the active tissue repair in the acutely fibrotic mouse lung. Our study offers in-depth insights into the IPF transcriptional landscape and proposes alveolar regeneration as a promising therapeutic strategy for IPF.
Assuntos
Bleomicina , Fibrose Pulmonar Idiopática , Transcriptoma , Animais , Humanos , Camundongos , Fibrose Pulmonar Idiopática/genética , Fibrose Pulmonar Idiopática/patologia , Bleomicina/toxicidade , Modelos Animais de Doenças , Fator de Crescimento Transformador beta/metabolismo , Fator de Crescimento Transformador beta/genética , Pulmão/metabolismo , Pulmão/patologia , Camundongos Endogâmicos C57BL , Transdução de Sinais/genética , Masculino , Perfilação da Expressão Gênica , Células Epiteliais Alveolares/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Regeneração/genética , Apolipoproteínas E/genéticaRESUMO
PURPOSE: Idiopathic pulmonary fibrosis (IPF) is a fatal progressive condition often requiring lung transplantation. Accelerated senescence of type II alveolar epithelial cells (AECII) plays a crucial role in pulmonary fibrosis progression through the secretion of the senescence-associated secretory phenotype (SASP). Low-dose carbon monoxide (CO) possesses anti-inflammatory, anti-oxidative, and anti-aging properties. This study aims to explore the preventive effects of CO-releasing molecule 2 (CORM2) in a bleomycin-induced pulmonary fibrosis model. METHODS: We established an pulmonary fibrosis model in C57BL/6J mice and evaluated the impact of CORM2 on fibrosis pathology using Masson's trichrome staining, fluorescence staining, and pulmonary function tests. Fibrogenic marker expression and SASP secretion in tissues and AECII cells were analyzed using qRT-PCR, Western blot, and ELISA assays both in vivo and in vitro. Additionally, we investigated DNA damage and cellular senescence through immunofluorescence and SA-ß-gal staining. RESULTS: CORM2 showed a preventive effect on bleomycin-induced lung fibrosis by improving pulmonary function and reducing the expression of fibrosis-related genes, such as TGF-ß, α-SMA, Collagen I/III. CORM2 decreased the DNA damage response by inhibiting γ-H2AX, p53, and p21. We identified PAI-1 as a new target gene that was downregulated by CORM2, and which was associated with cellular senescence and fibrosis. CORM2 effectively inhibited cellular senescence and delayed EMT occurrence in AECII cells. CONCLUSION: Our study highlights the potential of CORM2 in preventing DNA damage-induced cellular senescence in bleomycin-induced pulmonary fibrosis through modulation of the p53/PAI-1 signaling pathway. These findings underscore the promising prospects of CORM2 in targeting cellular senescence and the p53/PAI-1 pathway as a potential preventive strategy for IPF.
Assuntos
Bleomicina , Monóxido de Carbono , Senescência Celular , Inibidor 1 de Ativador de Plasminogênio , Transdução de Sinais , Proteína Supressora de Tumor p53 , Animais , Masculino , Camundongos , Bleomicina/toxicidade , Monóxido de Carbono/farmacologia , Monóxido de Carbono/metabolismo , Senescência Celular/efeitos dos fármacos , Modelos Animais de Doenças , Dano ao DNA/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Compostos Organometálicos/farmacologia , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Fibrose Pulmonar/induzido quimicamente , Fibrose Pulmonar/metabolismo , Fibrose Pulmonar/patologia , Fibrose Pulmonar/tratamento farmacológico , Fibrose Pulmonar/prevenção & controle , Transdução de Sinais/efeitos dos fármacos , Proteína Supressora de Tumor p53/metabolismoRESUMO
Fibrosis in the lung is thought to be driven by epithelial cell dysfunction and aberrant cell-cell interactions. Unveiling the molecular mechanisms of cellular plasticity and cell-cell interactions is imperative to elucidating lung regenerative capacity and aberrant repair in pulmonary fibrosis. By mining publicly available RNA-Seq data sets, we identified loss of CCAAT enhancer-binding protein alpha (CEBPA) as a candidate contributor to idiopathic pulmonary fibrosis (IPF). We used conditional KO mice, scRNA-Seq, lung organoids, small-molecule inhibition, and potentially novel gene manipulation methods to investigate the role of CEBPA in lung fibrosis and repair. Long-term (6 months or more) of Cebpa loss in AT2 cells caused spontaneous fibrosis and increased susceptibility to bleomycin-induced fibrosis. Cebpa knockout (KO) in these mice significantly decreased AT2 cell numbers in the lung and reduced expression of surfactant homeostasis genes, while increasing inflammatory cell recruitment as well as upregulating S100a8/a9 in AT2 cells. In vivo treatment with an S100A8/A9 inhibitor alleviated experimental lung fibrosis. Restoring CEBPA expression in lung organoids ex vivo and during experimental lung fibrosis in vivo rescued CEBPA deficiency-mediated phenotypes. Our study establishes a direct mechanistic link between CEBPA repression, impaired AT2 cell identity, disrupted tissue homeostasis, and lung fibrosis.
Assuntos
Bleomicina , Proteínas Estimuladoras de Ligação a CCAAT , Homeostase , Camundongos Knockout , Animais , Camundongos , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Proteínas Estimuladoras de Ligação a CCAAT/genética , Bleomicina/toxicidade , Fibrose Pulmonar Idiopática/patologia , Fibrose Pulmonar Idiopática/genética , Fibrose Pulmonar Idiopática/metabolismo , Fibrose Pulmonar Idiopática/induzido quimicamente , Fibrose Pulmonar/metabolismo , Fibrose Pulmonar/patologia , Fibrose Pulmonar/genética , Fibrose Pulmonar/induzido quimicamente , Humanos , Modelos Animais de Doenças , Pulmão/patologia , Pulmão/metabolismo , Organoides/metabolismo , Células Epiteliais Alveolares/metabolismo , Células Epiteliais Alveolares/patologia , MasculinoRESUMO
BACKGROUND: Idiopathic pulmonary fibrosis (PF) is a chronic progressive interstitial lung disease characterized by alveolar epithelial cell (AEC) injury and fibroblast activation. Inadequate autophagy in AECs may result from the activation of several signaling pathways following AEC injury, with glycoproteins serving as key receptor proteins. The core fucosylation (CF) modification in glycoproteins is crucial. Mesenchymal stem cells derived from bone marrow (BMSCs) have the ability to regenerate damaged tissue and treat PF. This study aimed to elucidate the relationship and mechanism of interaction between BMSCs, CF modification, and autophagy in PF. METHODS: C57BL/6 male mice, AEC-specific FUT8 conditional knockout (CKO) mice, and MLE12 cells were administered bleomycin (BLM), FUT8 siRNA, and mouse BMSCs, respectively. Experimental techniques including tissue staining, Western blotting, immunofluorescence, autophagic flux detection, and flow cytometry were used in this study. RESULTS: First, we found that autophagy was inhibited while FUT8 expression was elevated in PF mice and BLM-induced AEC injury models. Subsequently, CKO mice and MLE12 cells transfected with FUT8 siRNA were used to demonstrate that inhibition of CF modification induces autophagy in AECs and mitigates PF. Finally, mouse BMSCs were used to demonstrate that they alleviate the detrimental autophagy of AECs by inhibiting CF modification and decreasing PF. CONCLUSIONS: Suppression of CF modification enhanced the suppression of AEC autophagy and reduced PF in mice. Additionally, through the prevention of CF modification, BMSCs can assist AECs deficient in autophagy and partially alleviate PF.
Assuntos
Células Epiteliais Alveolares , Autofagia , Células-Tronco Mesenquimais , Animais , Camundongos , Células Epiteliais Alveolares/metabolismo , Células Epiteliais Alveolares/patologia , Células-Tronco Mesenquimais/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Bleomicina/toxicidade , Camundongos Knockout , Fucose/metabolismo , Fibrose Pulmonar/metabolismo , Fibrose Pulmonar/patologia , Fibrose Pulmonar/genética , Fibrose Pulmonar/induzido quimicamente , Fibrose Pulmonar Idiopática/patologia , Fibrose Pulmonar Idiopática/metabolismo , Fucosiltransferases/metabolismo , Fucosiltransferases/genéticaRESUMO
OBJECTIVE: This study aimed to investigate the use of 5,7,3',4'-tetramethoxyflavone (TMF) to treat pulmonary fibrosis (PF), a chronic and fatal lung disease. In vitro and in vivo models were used to examine the impact of TMF on PF. METHODS: NIH-3T3 (Mouse Embryonic Fibroblast) were exposed to transforming growth factorß1 (TGF-ß1) and treated with or without TMF. Cell growth was assessed using the MTT method, and cell migration was evaluated with the scratch wound assay. Protein and messenger ribonucleic acid (mRNA) levels of extracellular matrix (ECM) genes were analyzed by western blotting and quantitative reverse transcription-polymerase chain reaction (RT-PCR), respectively. Downstream molecules affected by TGF-ß1 were examined by western blotting. In vivo, mice with bleomycin-induced PF were treated with TMF, and lung tissues were analyzed with staining techniques. RESULTS: The in vitro results showed that TMF had no significant impact on cell growth or migration. However, it effectively inhibited myofibroblast activation and ECM production induced by TGF-ß1 in NIH-3T3 cells. This inhibition was achieved by suppressing various signaling pathways, including Smad, mitogen-activated protein kinase (MAPK), phosphoinositide 3-kinase/AKT (PI3K/AKT), and WNT/ß-catenin. The in vivo experiments demonstrated the therapeutic potential of TMF in reducing PF induced by bleomycin in mice, and there was no significant liver or kidney toxicity observed. CONCLUSION: These findings suggest that TMF has the potential to effectively inhibit myofibroblast activation and could be a promising treatment for PF. TMF achieves this inhibitory effect by targeting TGF-ß1/Smad and non-Smad pathways.
Assuntos
Bleomicina , Fibroblastos , Fibrose Pulmonar , Fator de Crescimento Transformador beta1 , Animais , Camundongos , Fator de Crescimento Transformador beta1/metabolismo , Células NIH 3T3 , Fibrose Pulmonar/induzido quimicamente , Fibrose Pulmonar/tratamento farmacológico , Fibrose Pulmonar/metabolismo , Fibrose Pulmonar/patologia , Bleomicina/toxicidade , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Flavonas/farmacologia , Camundongos Endogâmicos C57BL , Movimento Celular/efeitos dos fármacos , Masculino , Proliferação de Células/efeitos dos fármacosRESUMO
Female C57BL/J mice with pulmonary fibrosis induced by injections of bleomycin (20 mg/kg intraperitoneally, 8 times for 4 weeks) were treated with a lignin derivative-based composition BP-C3 (80 mg/kg, daily intragastric administrations for 4 weeks). Bleomycin treatment increased the severity of pulmonary fibrosis (Ashcroft score increased from 1.43±0.20 to 4.17±0.48) and the percentage of α-SMA+ tissue (from 15.22±1.01 to 33.12±2.30%) and DNA-synthetizing nuclei (from 1.05±0.14 to 3.38±0.375). After treatment with BP-C3, we observed a tendency to a decrease in Ashcroft score (to 3.40±0.51) and a significant decrease in the percentage of α-SMA+ tissue to 24.30±1.70%; the percentage of DNA-synthetizing nuclei decreased to a lesser extent (to 3.03±0.22%). These results suggest that BP-C3 has a moderate antifibrotic activity.
Assuntos
Bleomicina , Lignina , Camundongos Endogâmicos C57BL , Fibrose Pulmonar , Animais , Bleomicina/toxicidade , Fibrose Pulmonar/induzido quimicamente , Fibrose Pulmonar/tratamento farmacológico , Fibrose Pulmonar/patologia , Camundongos , Feminino , Lignina/farmacologia , Lignina/química , Pulmão/efeitos dos fármacos , Pulmão/patologia , Actinas/metabolismo , Actinas/genéticaRESUMO
Idiopathic pulmonary fibrosis (IPF) is an age-related disease with poor prognosis and limited therapeutic options. Activation of lung fibroblasts and differentiation to myofibroblasts are the principal effectors of disease pathology, but damage and senescence of alveolar epithelial cells, specifically type II (ATII) cells, has recently been identified as a potential trigger event for the progressive disease cycle. Targeting ATII senescence and the senescence-associated secretory phenotype (SASP) is an attractive therapeutic strategy; however, translatable primary human cell models that enable mechanistic studies and drug development are lacking. Here, we describe a novel system of conditioned medium (CM) transfer from bleomycin-induced senescent primary alveolar epithelial cells (AEC) onto normal human lung fibroblasts (NHLF) that demonstrates an enhanced fibrotic transcriptional and secretory phenotype compared to non-senescent AEC CM treatment or direct bleomycin damage of the NHLFs. In this system, the bleomycin-treated AECs exhibit classical hallmarks of cellular senescence, including SASP and a gene expression profile that resembles aberrant epithelial cells of the IPF lung. Fibroblast activation by CM transfer is attenuated by pre-treatment of senescent AECs with the senolytic Navitoclax and AD80, but not with the standard of care agent Nintedanib or senomorphic JAK-targeting drugs (e.g., ABT-317, ruxolitinib). This model provides a relevant human system for profiling novel senescence-targeting therapeutics for IPF drug development.
Assuntos
Células Epiteliais Alveolares , Bleomicina , Senescência Celular , Fibroblastos , Fibrose Pulmonar Idiopática , Humanos , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Bleomicina/toxicidade , Bleomicina/farmacologia , Senescência Celular/efeitos dos fármacos , Células Epiteliais Alveolares/efeitos dos fármacos , Células Epiteliais Alveolares/metabolismo , Células Epiteliais Alveolares/patologia , Fibrose Pulmonar Idiopática/patologia , Fibrose Pulmonar Idiopática/metabolismo , Meios de Cultivo Condicionados/farmacologia , Indóis/farmacologia , Fenótipo Secretor Associado à Senescência/efeitos dos fármacos , Pulmão/patologia , Pulmão/citologia , Pulmão/efeitos dos fármacos , Sulfonamidas/farmacologia , Senoterapia/farmacologia , Células Cultivadas , Pirimidinas/farmacologia , Pirazóis/farmacologia , Nitrilas/farmacologia , Compostos de AnilinaRESUMO
Pulmonary fibrosis is a formidable challenge in chronic and age-related lung diseases. Myofibroblasts secrete large amounts of extracellular matrix and induce pro-repair responses during normal wound healing. Successful tissue repair results in termination of myofibroblast activity via apoptosis; however, some myofibroblasts exhibit a senescent phenotype and escape apoptosis, causing over-repair that is characterized by pathological fibrotic scarring. Therefore, the removal of senescent myofibroblasts using senolytics is an important method for the treatment of pulmonary fibrosis. Procyanidin C1 (PCC1) has recently been discovered as a senolytic compound with very low toxicity and few side effects. This study aimed to determine whether PCC1 could improve lung fibrosis by promoting apoptosis in senescent myofibroblasts and to investigate the mechanisms involved. The results showed that PCC1 attenuates bleomycin (BLM)-induced pulmonary fibrosis in mice. In addition, we found that PCC1 inhibited extracellular matrix deposition and promoted the apoptosis of senescent myofibroblasts by increasing PUMA expression and activating the BAX signaling pathway. Our findings represent a new method of pulmonary fibrosis management and emphasize the potential of PCC1 as a senotherapeutic agent for the treatment of pulmonary fibrosis, providing hope for patients with pulmonary fibrosis worldwide. Our results advance our understanding of age-related diseases and highlight the importance of addressing cellular senescence in treatment.
Assuntos
Bleomicina , Catequina , Senescência Celular , Camundongos Endogâmicos C57BL , Miofibroblastos , Fibrose Pulmonar , Animais , Bleomicina/toxicidade , Miofibroblastos/metabolismo , Miofibroblastos/efeitos dos fármacos , Fibrose Pulmonar/induzido quimicamente , Fibrose Pulmonar/metabolismo , Fibrose Pulmonar/tratamento farmacológico , Fibrose Pulmonar/patologia , Camundongos , Senescência Celular/efeitos dos fármacos , Catequina/farmacologia , Catequina/análogos & derivados , Proantocianidinas/farmacologia , Apoptose/efeitos dos fármacos , Masculino , Biflavonoides/farmacologia , Transdução de Sinais/efeitos dos fármacosRESUMO
Progressive lung fibrosis is associated with poorly understood aging-related endothelial cell dysfunction. To gain insight into endothelial cell alterations in lung fibrosis we performed single cell RNA-sequencing of bleomycin-injured lungs from young and aged mice. Analysis reveals activated cell states enriched for hypoxia, glycolysis and YAP/TAZ activity in ACKR1+ venous and TrkB+ capillary endothelial cells. Endothelial cell activation is prevalent in lungs of aged mice and can also be detected in human fibrotic lungs. Longitudinal single cell RNA-sequencing combined with lineage tracing demonstrate that endothelial activation resolves in young mouse lungs but persists in aged ones, indicating a failure of the aged vasculature to return to quiescence. Genes associated with activated lung endothelial cells states in vivo can be induced in vitro by activating YAP/TAZ. YAP/TAZ also cooperate with BDNF, a TrkB ligand that is reduced in fibrotic lungs, to promote capillary morphogenesis. These findings offer insights into aging-related lung endothelial cell dysfunction that may contribute to defective lung injury repair and persistent fibrosis.
Assuntos
Envelhecimento , Bleomicina , Células Endoteliais , Lesão Pulmonar , Pulmão , Fibrose Pulmonar , Animais , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Envelhecimento/patologia , Bleomicina/toxicidade , Humanos , Camundongos , Fibrose Pulmonar/patologia , Fibrose Pulmonar/metabolismo , Fibrose Pulmonar/genética , Pulmão/patologia , Pulmão/metabolismo , Lesão Pulmonar/patologia , Lesão Pulmonar/metabolismo , Lesão Pulmonar/etiologia , Receptor trkB/metabolismo , Receptor trkB/genética , Camundongos Endogâmicos C57BL , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Fator Neurotrófico Derivado do Encéfalo/genética , Proteínas de Sinalização YAP/metabolismo , Masculino , Análise de Célula Única , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Feminino , Modelos Animais de DoençasRESUMO
BACKGROUND: Pulmonary fibrosis (PF) represents the pathologic end stage of several interstitial lung diseases (ILDs) associated with high morbidity and mortality rates. However, current treatments can only delay disease progression rather than provide a cure. The role of inflammation in PF progression is well-established, but new insights into immune regulation are fundamental for developing more efficient therapies. c-MET signaling has been implicated in the migratory capacity and effector functions of immune cells. Nevertheless, the role of this signaling pathway in the context of PF-associated lung diseases remains unexplored. METHODS: To determine the influence of c-MET in immune cells in the progression of pulmonary fibrosis, we used a conditional deletion of c-Met in immune cells. To induce pulmonary fibrosis mice were administered with bleomycin (BLM) intratracheally. Over the course of 21 days, mice were assessed for weight change, and after euthanasia at different timepoints, bronchoalveolar lavage fluid cells and lung tissue were assessed for inflammation and fibrosis. Furthermore, c-MET expression was assessed in cryobiopsy sections, bronchoalveolar lavage fluid cells samples and single cell RNA-sequencing dataset from human patients with distinct interstitial lung diseases. RESULTS: c-MET expression was induced in lung immune cells, specifically in T cells, interstitial macrophages, and neutrophils, during the inflammatory phase of BLM-induced PF mouse model. Deletion of c-Met in immune cells correlated with earlier weight recovery and improved survival of BLM-treated mice. Moreover, the deletion of c-Met in immune cells was associated with early recruitment of the immune cell populations, normally found to express c-MET, leading to a subsequent attenuation of the cytotoxic and proinflammatory environment. Consequently, the less extensive inflammatory response, possibly coupled with tissue repair, culminated in less exacerbated fibrotic lesions. Furthermore, c-MET expression was up-regulated in lung T cells from patients with fibrosing ILD, suggesting a potential involvement of c-MET in the development of fibrosing disease. CONCLUSIONS: These results highlight the critical contribution of c-MET signaling in immune cells to their enhanced uncontrolled recruitment and activation toward a proinflammatory and profibrotic phenotype, leading to the exacerbation of lung injury and consequent development of fibrosis.