RESUMO
Pro-fibrotic M2-like macrophages are widely implicated in the pathogenesis and progression of lung fibrosis due to their production of pro-fibrotic growth factors and cytokines. Yeast beta-glucan (YBG) microparticles have shown potential as immunomodulators that can convert macrophage polarization from a pro-fibrotic phenotype to an anti-fibrotic phenotype through the engagement of the Dectin-1 receptor. However, the processing conditions used to fabricate YBG microparticles can lead to unpredictable immunomodulatory effects. Herein, we report the use of Pressurized Gas eXpanded liquids (PGX) Technology® to fabricate YBG (PGX-YBG) microparticles with higher surface areas, lower densities, and smaller and more uniform size distributions compared to commercially available spray-dried YBGs. PGX-YBG is shown to activate Dectin-1 more efficiently in vitro while avoiding significant TLR 2/4 activation. Furthermore, PGX-YBG microparticles effectively modulate M2-like fibrosis-inducing murine and human macrophages into fibrosis-suppressing macrophages both in vitro as well as in ex vivo precision-cut murine lung slices, suggesting their potential utility as a therapeutic for addressing a broad spectrum of fibrotic end-point lung diseases.
Assuntos
Macrófagos , beta-Glucanas , Animais , beta-Glucanas/química , beta-Glucanas/farmacologia , Macrófagos/metabolismo , Macrófagos/efeitos dos fármacos , Camundongos , Humanos , Camundongos Endogâmicos C57BL , Lectinas Tipo C/metabolismo , Células RAW 264.7 , Fibrose Pulmonar/patologia , Fibrose Pulmonar/tratamento farmacológico , Saccharomyces cerevisiae , Tamanho da PartículaRESUMO
Recent progress in stem cell therapy has demonstrated the therapeutic potential of intravenous stem cell infusions for treating the life-threatening lung disease of pulmonary fibrosis (PF). However, it is confronted with limitations, such as a lack of control over cellular function and rapid clearance by the host after implantation. In this study, we developed an innovative PF therapy through tracheal administration of microfluidic-templated stem cell-laden microcapsules, which effectively reversed the progression of inflammation and fibrotic injury. Our findings highlight that hydrogel microencapsulation can enhance the persistence of donor mesenchymal stem cells (MSCs) in the host while driving MSCs to substantially augment their therapeutic functions, including immunoregulation and matrix metalloproteinase (MMP)-mediated extracellular matrix (ECM) remodeling. We revealed that microencapsulation activates the MAPK signaling pathway in MSCs to increase MMP expression, thereby degrading overexpressed collagen accumulated in fibrotic lungs. Our research demonstrates the potential of hydrogel microcapsules to enhance the therapeutic efficacy of MSCs through cell-material interactions, presenting a promising yet straightforward strategy for designing advanced stem cell therapies for fibrotic diseases.
Assuntos
Cápsulas , Matriz Extracelular , Imunomodulação , Células-Tronco Mesenquimais , Fibrose Pulmonar , Animais , Matriz Extracelular/metabolismo , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Fibrose Pulmonar/terapia , Fibrose Pulmonar/patologia , Transplante de Células-Tronco Mesenquimais/métodos , Camundongos Endogâmicos C57BL , Hidrogéis/química , Camundongos , MasculinoRESUMO
Idiopathic pulmonary fibrosis (IPF) is a progressive and fatal fibrotic lung disease. While recent studies have suggested the potential efficacy of tyrosine kinase inhibitors in managing IPF, masitinib, a clinically used tyrosine kinase inhibitor, has not yet been investigated for its efficacy in fibrotic lung diseases. In a previous study on an in vitro neurodegenerative model, we demonstrated the synergistic antitoxic and antioxidant effects of masitinib combined with cromolyn sodium, an FDA-approved mast cell stabilizer. This study aims to investigate the anti-fibrotic and antioxidant effects of the masitinib-cromolyn sodium combination in an in vitro model of pulmonary fibrosis. Fibroblast cell cultures treated with bleomycin and/or hydrogen peroxide (H2O2) were subjected to masitinib and/or cromolyn sodium, followed by assessments of cell viability, morphological and apoptotic nuclear changes, triple-immunofluorescence labeling, and total oxidant/antioxidant capacities, besides ratio of Bax and Bcl-2 mRNA expressions as an indication of apoptosis. The combined treatment of masitinib and cromolyn sodium effectively prevented the fibroblast myofibroblast transition, a hallmark of fibrosis, and significantly reduced bleomycin / H2O2-induced apoptosis and oxidative stress. This study is the first to demonstrate the additive anti-fibrotic, cell-protective, and antioxidant effects of the masitinib-cromolyn sodium combination in an in vitro fibrosis model, suggesting its potential as an innovative therapeutic approach for pulmonary fibrosis. Combination therapy may be more advantageous in that both drugs could be administered in lower doses, exerting less side effects, and at the same time providing diverse mechanisms of action simultaneously.
Assuntos
Antioxidantes , Apoptose , Benzamidas , Bleomicina , Cromolina Sódica , Fibroblastos , Miofibroblastos , Estresse Oxidativo , Piperidinas , Piridinas , Tiazóis , Antioxidantes/farmacologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Benzamidas/farmacologia , Piridinas/farmacologia , Apoptose/efeitos dos fármacos , Miofibroblastos/efeitos dos fármacos , Miofibroblastos/metabolismo , Piperidinas/farmacologia , Cromolina Sódica/farmacologia , Animais , Tiazóis/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Peróxido de Hidrogênio/farmacologia , Sinergismo Farmacológico , Humanos , Fibrose Pulmonar Idiopática/tratamento farmacológico , Fibrose Pulmonar Idiopática/patologia , Células Cultivadas , Camundongos , Fibrose Pulmonar/induzido quimicamente , Fibrose Pulmonar/tratamento farmacológico , Fibrose Pulmonar/patologiaAssuntos
Proteína 1 Semelhante a Receptor de Interleucina-1 , Interleucina-33 , Pulmão , Interleucina-33/metabolismo , Interleucina-33/genética , Proteína 1 Semelhante a Receptor de Interleucina-1/metabolismo , Animais , Pulmão/metabolismo , Pulmão/patologia , Pulmão/efeitos dos fármacos , Camundongos , Fibrose Pulmonar/metabolismo , Fibrose Pulmonar/patologia , Transdução de Sinais , Humanos , Camundongos KnockoutRESUMO
Silicosis is a systemic disease caused by long-term exposure to high concentrations of free silica dust particles in the workplace. It is characterized by a persistent inflammatory response, fibroblast proliferation, and excessive collagen deposition, leading to pulmonary interstitial fibrosis. Epithelial interstitial transformation (EMT) can cause epithelial cells to lose their tight junctions, cell polarity, and epithelial properties, thereby enhancing the properties of interstitial cells, which can lead to the progression of fibrosis and the formation of scar tissue. Integrin 1 (ITGB1) is considered an important factor for promoting EMT and tumor invasion in a variety of tumors and also plays an important role in the progression of fibrotic diseases. Therefore, ITGB1 can be used as a potential target for the treatment of silicosis. In this study, we found that silica exposure induced epithelial-mesenchymal transformation in rats and that the expression of integrin ITGB1 was elevated along with the EMT. We used CRISPR/Cas9 technology to construct integrin ITGB1 knockdown cell lines for in vitro experiments. We compared the expression of the EMT key proteins E-cadherin and vimentin in the ITGB1 knockdown cells and wild-type cells simultaneously stimulated by silica and detected the aggregation point distribution of E-cadherin and vimentin in the cells using laser confocal microscopy. Our results showed that ITGB1 knockout inhibited the ITGB1/ILK/Snail signaling pathway and attenuated the EMT occurrence compared to control cells. These results suggested that ITGB1 is associated with silica-induced EMT and may be a potential target for the treatment of silicosis.
Assuntos
Transição Epitelial-Mesenquimal , Integrina beta1 , Fibrose Pulmonar , Dióxido de Silício , Animais , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Transição Epitelial-Mesenquimal/genética , Dióxido de Silício/toxicidade , Dióxido de Silício/efeitos adversos , Integrina beta1/genética , Integrina beta1/metabolismo , Fibrose Pulmonar/induzido quimicamente , Fibrose Pulmonar/genética , Fibrose Pulmonar/patologia , Ratos , Silicose/patologia , Silicose/genética , Masculino , Caderinas/metabolismo , Caderinas/genéticaRESUMO
It is estimated that there are 544.9 million people suffering from chronic respiratory diseases in the world, which is the third largest chronic disease. Although there are various clinical treatment methods, there is no specific drug for chronic pulmonary diseases, including chronic obstructive pulmonary disease (COPD), interstitial lung disease (ILD) and idiopathic pulmonary fibrosis (IPF). Therefore, it is urgent to clarify the pathological mechanism and medication development. Single-cell transcriptome data of human and mouse from GEO database were integrated by "Harmony" algorithm. The data was standardized and normalized by using "Seurat" package, and "SingleR" algorithm was used for cell grouping annotation. The "Findmarker" function is used to find differentially expressed genes (DEGs), which were enriched and analyzed by using "clusterProfiler", and a protein interaction network was constructed for DEGs, and four algorithms are used to find the hub genes. The expression of hub genes were analyzed in independent human and mouse single-cell transcriptome data. Bulk RNA data were used to integrate by the "SVA" function, verify the expression levels of hub genes and build a diagnostic model. The L1000FWD platform was used to screen potential drugs. Through exploring the similarities and differences by integrated single-cell atlas, we found that the lung parenchymal cells showed abnormal oxidative stress, cell matrix adhesion and ubiquitination in COPD, corona virus disease 2019 (COVID-19), ILD and IPF. Meanwhile, the lung resident immune cells showed abnormal Toll-like receptor signals, interferon signals and ubiquitination. However, unlike acute pneumonia (COVID-19), chronic pulmonary disease shows enhanced ubiquitination. This phenomenon was confirmed in independent external human single-cell atlas, but unfortunately, it was not confirmed in mouse single-cell atlas of bleomycin-induced pulmonary fibrosis model and influenza virus-infected mouse model, which means that the model needs to be optimized. In addition, the bulk RNA-Seq data of COVID-19, ILD and IPF was integrated, and we found that the immune infiltration of lung tissue was enhanced, consistent with the single-cell level, UBA52, UBB and UBC were low expressed in COVID-19 and high expressed in ILD, and had a strong correlation with the expression of cell matrix adhesion genes. UBA52 and UBB have good diagnostic efficacy, and salermide and SSR-69071 can be used as their candidate drugs. Our study found that the disorder of protein ubiquitination in chronic pulmonary diseases is an important cause of pathological phenotype of pulmonary fibrosis by integrating scRNA-Seq and bulk RNA-Seq, which provides a new horizons for clinicopathology, diagnosis and treatment.
Assuntos
RNA-Seq , Ubiquitina , Humanos , Animais , Camundongos , Ubiquitina/metabolismo , Ubiquitina/genética , Análise de Célula Única/métodos , Transcriptoma , Fibrose Pulmonar/genética , Fibrose Pulmonar/metabolismo , Fibrose Pulmonar/patologia , COVID-19/genética , COVID-19/metabolismo , COVID-19/virologia , Perfilação da Expressão Gênica , Mapas de Interação de Proteínas , Doença Crônica , Fibrose Pulmonar Idiopática/genética , Fibrose Pulmonar Idiopática/patologia , Fibrose Pulmonar Idiopática/metabolismo , SARS-CoV-2/genética , Doença Pulmonar Obstrutiva Crônica/genética , Doença Pulmonar Obstrutiva Crônica/metabolismo , Análise da Expressão Gênica de Célula ÚnicaRESUMO
BACKGROUND: Pulmonary fibrosis (PF) is an aging-related progressive lung disorder. The aged lung undergoes functional and structural changes termed immunosenescence and inflammaging, which facilitate the occurrence of fibrosis. Interleukin-10 (IL-10) is a potent anti-inflammatory and immunoregulatory cytokine, yet it remains unclear how IL-10 deficiency-induced immunosenescence participates in the development of PF. METHODS: Firstly we evaluated the susceptibility to fibrosis and IL-10 expression in aged mice. Then 13-month-old wild-type (WT) and IL-10 knockout (KO) mice were subjected to bleomycin(BLM) and analyzed senescence-related markers by PCR, western blot and immunohistochemistry staining of p16, p21, p53, as well as DHE and SA-ß-gal staining. We further compared 18-month-old WT mice with 13-month-old IL-10KO mice to assess aging-associated cell senescence and inflamation infiltration in both lung and BALF. Moreover, proliferation and apoptosis of alveolar type 2 cells(AT2) were evaluated by FCM, immunofluorescence, TUNEL staining, and TEM analysis. Recombinant IL-10 (rIL-10) was also administered intratracheally to evaluate its therapeutic potential and related mechanism. For the in vitro experiments, 10-week-old naïve pramily lung fibroblasts(PLFs) were treated with the culture medium of 13-month PLFs derived from WT, IL-10KO, or IL-10KO + rIL-10 respectively, and examined the secretion of senescence-associated secretory phenotype (SASP) factors and related pathways. RESULTS: The aged mice displayed increased susceptibility to fibrosis and decreased IL-10 expression. The 13-month-old IL-10KO mice exhibited significant exacerbation of cell senescence compared to their contemporary WT mice, and even more severe epithelial-mesenchymal transition (EMT) than that of 18 month WT mice. These IL-10 deficient mice showed heightened inflammatory responses and accelerated PF progression. Intratracheal administration of rIL-10 reduced lung CD45 + cell infiltration by 15%, including a 6% reduction in granulocytes and a 10% reduction in macrophages, and increased the proportion of AT2 cells by approximately 8%. Additionally, rIL-10 significantly decreased α-SMA and collagen deposition, and reduced the expression of senescence proteins p16 and p21 by 50% in these mice. In vitro analysis revealed that conditioned media from IL-10 deficient mice promoted SASP secretion and upregulated senescence genes in naïve lung fibroblasts, which was mitigated by rIL-10 treatment. Mechanistically, rIL-10 inhibited TGF-ß-Smad2/3 and PTEN/PI3K/AKT/ERK pathways, thereby suppressing senescence and fibrosis-related proteins. CONCLUSIONS: IL-10 deficiency in aged mice leads to accelerated cell senescence and exacerbated fibrosis, with IL-10KO-PLFs displaying increased SASP secretion. Recombinant IL-10 treatment effectively mitigates these effects, suggesting its potential as a therapeutic target for PF.
Assuntos
Bleomicina , Senescência Celular , Interleucina-10 , Camundongos Knockout , PTEN Fosfo-Hidrolase , Proteínas Proto-Oncogênicas c-akt , Fibrose Pulmonar , Animais , Interleucina-10/metabolismo , Interleucina-10/genética , Camundongos , Fibrose Pulmonar/patologia , Fibrose Pulmonar/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , PTEN Fosfo-Hidrolase/genética , PTEN Fosfo-Hidrolase/metabolismo , Camundongos Endogâmicos C57BL , Sistema de Sinalização das MAP Quinases , Apoptose , Pulmão/patologia , Pulmão/metabolismo , Masculino , Células Epiteliais Alveolares/metabolismo , Células Epiteliais Alveolares/patologia , Modelos Animais de Doenças , Proliferação de Células , Envelhecimento , Transdução de SinaisRESUMO
Introduction: Pulmonary fibrosis (PF) and tissue remodeling can greatly impair pulmonary function and often lead to fatal outcomes. Methodology: In the present study, we explored a novel molecular interplay of long noncoding (Lnc) RNA CBR3-AS1/ miRNA-29/ FIZZ1 axis in moderating the inflammatory processes, immunological responses, and oxidative stress pathways in bleomycin (BLM)-induced lung fibrosis. Furthermore, we investigated the pharmacological potential of Trimetazidine (TMZ) in ameliorating lung fibrosis. Results: Our results revealed that the BLM-treated group exhibited a significant upregulation in the expression of epigenetic regulators, lncRNA CBR3-AS1 and FIZZ1, compared to the control group (P<0.0001), along with the downregulation of miRNA-29 expression. Furthermore, Correlation analysis showed a significant positive association between lnc CBR3-AS1 and FIZZ1 (R=0.7723, p<0.05) and a significant negative association between miRNA-29 and FIZZ1 (R=-0.7535, p<0.05), suggesting lnc CBR3-AS1 as an epigenetic regulator of FIZZ1 in lung fibrosis. BLM treatment significantly increased the expression of Notch, Jagged1, Smad3, TGFB1, and hydroxyproline. Interestingly, the administration of TMZ demonstrated the ability to attenuate the deterioration effects caused by BLM treatment, as indicated by biochemical and histological analyses. Our investigations revealed that the therapeutic potential of TMZ as an antifibrotic drug could be ascribed to its ability to directly target the epigenetic regulators lncRNA CBR3-AS1/ miRNA-29/ FIZZ1, which in turn resulted in the mitigation of lung fibrosis. Histological and immunohistochemical analyses further validated the potential antifibrotic effects of TMZ by mitigating the structural damage associated with fibrosis. Discussion: Taken together, our study showed for the first time the interplay between epigenetic lncRNAs CBR3-AS1 and miRNA-29 in lung fibrosis and demonstrated that FIZZ1 could be a downregulatory gene for lncRNA CBR3-AS1 and miRNA-29. Our key findings demonstrate that TMZ significantly reduces the expression of fibrotic, oxidative stress, immunomodulatory, and inflammatory markers, along with epigenetic regulators associated with lung fibrosis. This validates its potential as an effective antifibrotic agent by targeting the CBR3-AS1/miRNA-29/FIZZ1 axis.
Assuntos
Bleomicina , MicroRNAs , Fibrose Pulmonar , RNA Longo não Codificante , Trimetazidina , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Fibrose Pulmonar/tratamento farmacológico , Fibrose Pulmonar/induzido quimicamente , Fibrose Pulmonar/metabolismo , Fibrose Pulmonar/patologia , Animais , Camundongos , Trimetazidina/farmacologia , Masculino , Camundongos Endogâmicos C57BLRESUMO
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
Introduction: Pulmonary fibrosis (PF) encompasses a spectrum of lung conditions characterized by the abnormal accumulation of scar tissue in the lungs, leading to impaired respiratory function. Various conditions can result in severe PF, among which viral infections have emerged as significant triggers. In addition to viral infections, exposure to toxic substances such as paraquat represents another significant risk factor for PF. Therefore, this study aimed to explore the dissimilarities and similarities between PF triggered by viral infections and chemical toxicants, using the mechanism of PF in IPF as a reference. Methods: Data-independent acquisition proteomics technology was employed to identify COVID-19 and paraquat-induced PF from the autopsy of lung tissue samples obtained from individuals who died due to PF. Bioinformatics was employed for differential protein analysis, and selected indicators were validated on pathological sections. Results: Our results showed that the differential proteins associated with the two causes of PF were enriched in similar lung fibrosis-related signaling pathways, such as the Wnt signaling pathway. However, differences were observed in proteins such as CACYBP, we verified the consistency of the results with proteomics using the IHC approach. Conclusion: This study illuminates distinct protein-level differences by investigating pulmonary fibrosis pathways in severe COVID-19 and paraquat poisoning. Although both conditions activate lung-protective and repair pathways, COVID-19 shows limited phosphorylation-independent ubiquitination of ß-catenin compared to paraquat toxicity. These findings shed light on potential therapeutic targets for PF induced via diverse factors.
Assuntos
COVID-19 , Pulmão , Paraquat , Proteômica , Fibrose Pulmonar , SARS-CoV-2 , Humanos , Paraquat/intoxicação , COVID-19/metabolismo , COVID-19/patologia , Fibrose Pulmonar/induzido quimicamente , Fibrose Pulmonar/patologia , Fibrose Pulmonar/virologia , Fibrose Pulmonar/metabolismo , Pulmão/patologia , Pulmão/virologia , Masculino , Pessoa de Meia-Idade , Feminino , IdosoRESUMO
Pulmonary fibrosis is a lethal interstitial lung disease, for which current treatments are inadequate in halting its progression. A significant factor contributing to the development of fibrosis is insufficient autophagy, which leads to increased fibroblast proliferation and collagen deposition. However, treatments aimed at upregulating autophagy often cause further lung pathology due to the disruption of epithelial cell balance. In response, we have developed a novel macrophage delivery system loaded with an epithelial-to-mesenchymal transition inhibitor, hyperoside (HYP), and an autophagy inducer, rapamycin (RAP). This system targets the fibrotic areas of the lung through chemotaxis, releases liposomes via macrophage extracellular traps, and effectively inhibits fibroblast proliferation while restoring the alveolar structure through the combined effects of RAP and HYP, ultimately reducing lung pathology without causing systemic toxicity. Our findings not only highlight a promising method to enhance autophagy-based treatments for pulmonary fibrosis but also demonstrate the potential of macrophages as effective nanocarriers for drug delivery.
Assuntos
Autofagia , Transição Epitelial-Mesenquimal , Macrófagos , Fibrose Pulmonar , Quercetina , Sirolimo , Autofagia/efeitos dos fármacos , Sirolimo/farmacologia , Sirolimo/química , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Fibrose Pulmonar/tratamento farmacológico , Fibrose Pulmonar/patologia , Fibrose Pulmonar/induzido quimicamente , Fibrose Pulmonar/metabolismo , Animais , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Quercetina/química , Quercetina/farmacologia , Quercetina/análogos & derivados , Humanos , Regulação para Cima/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Sistemas de Liberação de Medicamentos , Células RAW 264.7RESUMO
OBJECTIVE: COVID-19 induces the development of autoimmune diseases, including SLE, which are characterised by inflammation, autoantibodies and thrombosis. However, the effects of COVID-19 on SLE remain unclear. METHODS: We investigated the effects of COVID-19 on SLE development and progression in three animal models. Plasmids encoding SARS-CoV-2 spike protein and ACE2 receptor were injected into R848-induced BALB/C lupus mice, R848-induced IL-1 receptor antagonist knockout (KO) lupus mice and MRL/lpr mice. Serum levels of albumin and autoantibodies, lymphocyte phenotypes and tissue histology were evaluated. RESULTS: In R848-induced BALB/C lupus mice, the SARS-CoV-2 spike protein increased autoantibody and albumin levels compared with vehicle and mock treatments. These mice also exhibited splenomegaly, which was further exacerbated by the spike protein. Flow cytometric analysis revealed elevated T helper 1 cell counts, and histological analysis indicated increased levels of the fibrosis marker protein α-smooth muscle actin. In KO mice, the spike protein induced splenomegaly, severe kidney damage and pronounced lung fibrosis. In the MRL/lpr group, spike protein increased the serum levels of autoantibodies, albumin and the thrombosis marker chemokine (C-X-C motif) ligand 4. CONCLUSION: COVID-19 accelerated the development and progression of lupus by inducing autoantibody production, fibrosis and thrombosis.
Assuntos
Autoanticorpos , COVID-19 , Modelos Animais de Doenças , Lúpus Eritematoso Sistêmico , Nefrite Lúpica , Camundongos Endogâmicos BALB C , Camundongos Knockout , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus , Animais , Glicoproteína da Espícula de Coronavírus/imunologia , Nefrite Lúpica/patologia , Nefrite Lúpica/imunologia , Camundongos , COVID-19/imunologia , COVID-19/complicações , Autoanticorpos/sangue , Lúpus Eritematoso Sistêmico/complicações , Lúpus Eritematoso Sistêmico/imunologia , Fibrose Pulmonar/etiologia , Fibrose Pulmonar/imunologia , Fibrose Pulmonar/patologia , Camundongos Endogâmicos MRL lpr , Feminino , Esplenomegalia/etiologia , Enzima de Conversão de Angiotensina 2/genética , Enzima de Conversão de Angiotensina 2/metabolismoRESUMO
Pulmonary fibrosis (PF) is a severe, irreversible lung disease characterized by progressive scarring, with idiopathic pulmonary fibrosis (IPF) being the most prevalent form. IPF's pathogenesis involves repetitive lung epithelial injury leading to fibroblast activation and excessive extracellular matrix (ECM) deposition. The prognosis for IPF is poor, with limited therapeutic options like nintedanib and pirfenidone offering only modest benefits. Emerging research highlights the dysregulation of the yes-associated protein (YAP)/transcriptional coactivator with PDZ-binding motif (TAZ) signaling pathway as a critical factor in PF. YAP and TAZ, components of the Hippo pathway, play significant roles in cell proliferation, differentiation, and fibrosis by modulating gene expression through interactions with TEA domain (TEAD) transcription factors. The aberrant activation of YAP/TAZ in lung tissue promotes fibroblast activation and ECM accumulation. Targeting the YAP/TAZ pathway offers a promising therapeutic avenue. Preclinical studies have identified potential treatments, such as trigonelline, dopamine receptor D1 (DRD1) agonists, and statins, which inhibit YAP/TAZ activity and demonstrate antifibrotic effects. These findings underscore the importance of YAP/TAZ in PF pathogenesis and the potential of novel therapies aimed at this pathway, suggesting a new direction for improving IPF treatment outcomes. Further research is needed to validate these approaches and translate them into clinical practice.
Assuntos
Fibrose Pulmonar , Transdução de Sinais , Proteínas de Sinalização YAP , Humanos , Fibrose Pulmonar/metabolismo , Fibrose Pulmonar/patologia , Proteínas de Sinalização YAP/metabolismo , Animais , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas com Motivo de Ligação a PDZ com Coativador Transcricional , Fatores de Transcrição/metabolismo , Fibrose Pulmonar Idiopática/patologia , Fibrose Pulmonar Idiopática/metabolismoRESUMO
Idiopathic pulmonary fibrosis (IPF) is a progressive, often fatal lung disease characterized by tissue scarring and declining lung function. The MUC5B promoter polymorphism rs35705950, a significant genetic predisposition for IPF, paradoxically associates with better survival and slower disease progression than other IPF genotypes. This study investigates the potential paradoxical protective effects of this MUC5B variant in lung fibrosis. For this purpose, we developed a transgenic mouse model overexpressing the human MUC5B rs35705950 variant in the proximal large airways. Lung fibrosis was induced through subcutaneous injection of bleomycin. Results demonstrated significantly reduced lung fibrosis severity in transgenic mice compared to wild-type mice, assessed by trichrome staining, Ashcroft scoring, and hydroxyproline levels. Additionally, transgenic mice showed significantly lower levels of inflammatory cells and cytokines (TNFα, IL-6, IFNγ) and growth factors (PDGF, CTGF, IL-13) in the bronchoalveolar lavage fluid and lung tissues. There was also a significant decrease in mRNA expressions of fibrosis-related markers (periostin, fibronectin, Col1a1). In summary, this study reveals that mucin overexpression related to the MUC5B rs35705950 variant in the large airways significantly attenuates lung fibrosis and inflammatory responses in transgenic mice. These findings suggest that the rs35705950 variant modulates inflammatory and fibrotic responses in the proximal airways, which may contribute to the slower disease progression observed in IPF patients carrying this variant. Our study offers a possible explanation for the paradoxical beneficial effects of the MUC5B variant despite its role as a significant predisposing factor for IPF.
Assuntos
Bleomicina , Camundongos Transgênicos , Mucina-5B , Animais , Mucina-5B/genética , Mucina-5B/metabolismo , Humanos , Camundongos , Fibrose Pulmonar/genética , Fibrose Pulmonar/patologia , Fibrose Pulmonar/induzido quimicamente , Citocinas/metabolismo , Citocinas/genética , Pulmão/patologia , Pulmão/metabolismo , Fibrose Pulmonar Idiopática/genética , Fibrose Pulmonar Idiopática/patologia , Fibrose Pulmonar Idiopática/induzido quimicamente , Modelos Animais de Doenças , Líquido da Lavagem BroncoalveolarRESUMO
Identifying biomarkers in fibrotic lung disease is key for early anti-fibrotic intervention. Dynamic contrast-enhanced (DCE) MRI offers valuable perfusion-related insights in fibrosis but adapting human MRI methods to rodents poses challenges. Here, we explored these translational challenges for the inflammatory and fibrotic phase of a bleomycin lung injury model in rats. Eleven male Sprague-Dawley rats received a single intratracheal dose of bleomycin (1000iU), four control rats received saline. Imaging was performed on days 7 and 28 post-induction. Ultra-short echo time imaging was used to image the lung for 7 minutes after which Clariscan was injected intravenously. Lung signal changes were measured for an additional 21 minutes. Images were reconstructed with a sliding-window approach, providing a temporal resolution of 10 seconds per image. After imaging on day 28, animals were euthanized, and lungs were collected for histology. Bleomycin-exposed rats initially exhibited reduced body weight, recovering to control levels after 20 days. Lung volume increased in bleomycin animals from 4.4±0.9 ml in controls to 5.5±0.5 ml and 6.5±1.2 ml on day 7 and 28. DCE-MRI showed no change of initial gradient of relative enhancement in the curves between controls and bleomycin animals on day 7 and 28 post-induction. On day 7, the DCE-MRI washout phase in bleomycin animals had higher signals than the saline group and than observed at a later time point. Lung pixels were binned in 7 enhancement classes. On day 28, the size of low relative enhancement bins almost doubled in volume compared to controls and animals on day 7 post-induction. Histology on day 28 suggests that findings could be explained by changes in lung tissue density due to lung volume increase. Adapting this clinical MRI method to rodents at 9.4T remains a challenge. Future studies may benefit from lower field strength MRI combined with higher temporal resolution DCE-MRI.
Assuntos
Bleomicina , Meios de Contraste , Modelos Animais de Doenças , Imageamento por Ressonância Magnética , Fibrose Pulmonar , Ratos Sprague-Dawley , Animais , Masculino , Imageamento por Ressonância Magnética/métodos , Fibrose Pulmonar/induzido quimicamente , Fibrose Pulmonar/diagnóstico por imagem , Fibrose Pulmonar/patologia , Ratos , Pulmão/diagnóstico por imagem , Pulmão/patologia , Pulmão/efeitos dos fármacosRESUMO
Fibrosis is a chronic disease characterized by excessive extracellular matrix production, which leads to disruption of organ function. Fibroblasts are key effector cells of this process, responding chiefly to the pleiotropic cytokine transforming growth factor-ß1 (TGF-ß1), which promotes fibroblast to myofibroblast differentiation. We found that extracellular nutrient availability profoundly influenced the TGF-ß1 transcriptome of primary human lung fibroblasts and that biosynthesis of amino acids emerged as a top enriched TGF-ß1 transcriptional module. We subsequently uncovered a key role for pyruvate in influencing glutaminase (GLS1) inhibition during TGF-ß1-induced fibrogenesis. In pyruvate-replete conditions, GLS1 inhibition was ineffective in blocking TGF-ß1-induced fibrogenesis, as pyruvate can be used as the substrate for glutamate and alanine production via glutamate dehydrogenase (GDH) and glutamic-pyruvic transaminase 2 (GPT2), respectively. We further show that dual targeting of either GPT2 or GDH in combination with GLS1 inhibition was required to fully block TGF-ß1-induced collagen synthesis. These findings embolden a therapeutic strategy aimed at additional targeting of mitochondrial pyruvate metabolism in the presence of a glutaminolysis inhibitor to interfere with the pathological deposition of collagen in the setting of pulmonary fibrosis and potentially other fibrotic conditions.
Assuntos
Fibroblastos , Glutaminase , Ácido Pirúvico , Fator de Crescimento Transformador beta1 , Glutaminase/metabolismo , Glutaminase/antagonistas & inibidores , Humanos , Ácido Pirúvico/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Fibroblastos/metabolismo , Fibroblastos/efeitos dos fármacos , Fibroblastos/patologia , Pulmão/patologia , Pulmão/metabolismo , Fibrose Pulmonar/metabolismo , Fibrose Pulmonar/patologia , Fibrose , Células Cultivadas , Glutamato Desidrogenase/metabolismo , Glutamato Desidrogenase/antagonistas & inibidores , Miofibroblastos/metabolismo , Miofibroblastos/patologiaRESUMO
The system of nitric oxide synthases (NOSs) is comprised of three isoforms: nNOS, iNOS, and eNOS. The roles of NOSs in respiratory diseases in vivo have been studied by using inhibitors of NOSs and NOS-knockout mice. Their exact roles remain uncertain, however, because of the non-specificity of inhibitors of NOSs and compensatory up-regulation of other NOSs in NOS-KO mice. We addressed this point in our triple-n/i/eNOSs-KO mice. Triple-n/i/eNOSs-KO mice spontaneously developed pulmonary emphysema and displayed exacerbation of bleomycin-induced pulmonary fibrosis as compared with wild-type (WT) mice. Triple-n/i/eNOSs-KO mice exhibited worsening of hypoxic pulmonary hypertension (PH), which was reversed by treatment with sodium nitrate, and WT mice that underwent triple-n/i/eNOSs-KO bone marrow transplantation (BMT) also showed aggravation of hypoxic PH compared with those that underwent WT BMT. Conversely, ovalbumin-evoked asthma was milder in triple-n/i/eNOSs-KO than WT mice. These results suggest that the roles of NOSs are different in different pathologic states, even in the same respiratory diseases, indicating the diversity of the roles of NOSs. In this review, we describe these previous studies and discuss the roles of NOSs in respiratory health and disease. We also explain the current state of development of inorganic nitrate as a new drug for respiratory diseases.
Assuntos
Camundongos Knockout , Animais , Camundongos , Óxido Nítrico Sintase/metabolismo , Óxido Nítrico Sintase/genética , Humanos , Óxido Nítrico Sintase Tipo III/metabolismo , Óxido Nítrico Sintase Tipo III/genética , Hipertensão Pulmonar/genética , Hipertensão Pulmonar/metabolismo , Fibrose Pulmonar/genética , Fibrose Pulmonar/metabolismo , Fibrose Pulmonar/patologiaRESUMO
Fibrosis is involved in 45% of deaths in the United States, and no treatment exists to reverse the progression of lung or kidney fibrosis. Myofibroblasts are key to the progression and maintenance of fibrosis. We investigated features of cell adhesion necessary for monocytes to differentiate into myofibroblasts, seeking to identify pathways key to myofibroblast differentiation. Blocking antibodies against integrins α3, αM, and αMß2 de-differentiate myofibroblasts in vitro, lower the pro-fibrotic secretome of myofibroblasts, and treat lung fibrosis and inhibit kidney fibrosis in vivo. Decorin's collagen-binding peptide can be used to direct functionalized blocking antibodies (against integrins-α3, -αM, -αMß2) to both fibrotic lungs and fibrotic kidneys, reducing the dose of antibody necessary to treat fibrosis. This targeted immunotherapy blocking key integrins may be an effective therapeutic for the treatment of fibrosis.
Assuntos
Fibrose , Miofibroblastos , Fibrose Pulmonar , Miofibroblastos/metabolismo , Miofibroblastos/patologia , Animais , Fibrose Pulmonar/metabolismo , Fibrose Pulmonar/patologia , Humanos , Camundongos , Anticorpos Bloqueadores/farmacologia , Diferenciação Celular , Integrina alfa3/metabolismo , Nefropatias/metabolismo , Nefropatias/patologia , Rim/patologia , Rim/metabolismoRESUMO
BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is an age-related disease severely affecting life quality with its prevalence rising as the population ages, yet there is still no effective treatment available. Cell therapy has emerged as a promising option for IPF, however, the absence of mature and stable animal models for IPF immunodeficiency hampers preclinical evaluations of human cell therapies, primarily due to rapid immune clearance of administered cells. This study aims to establish a reliable pulmonary fibrosis (PF) model in immunodeficient mice that supports autologous cell therapy and to investigate underlying mechanism. METHODS: We utilized thirty 5-week-old male NOD/SCID mice, categorizing them into three age groups: 12weeks, 32 weeks and 43 weeks, with 6 mice euthanized randomly from each cohort for lung tissue analysis. We assessed fibrosis using HE staining, Masson's trichrome staining, α-SMA immunohistochemistry and hydroxyproline content measurement. Further, ß-galactosidase staining and gene expression analysis of MMP9, TGF-ß1, TNF-α, IL-1ß, IL-6, IL-8, SOD1, SOD2, NRF2, SIRT1, and SIRT3 were performed. ELISA was employed to quantify protein levels of TNF-α, TGF-ß1, and IL-8. RESULTS: When comparing lung tissues from 32-week-old and 43-week-old mice to those from 12-week-old mice, we noted a marked increase in inflammatory infiltration, fibrosis severity, and hydroxyproline content, alongside elevated expression levels of α-SMA and MMP9. Notably, the degree of fibrosis intensified with age. Additionally, ß-galactosidase staining became more pronounced in older mice. Quantitative PCR analyses revealed age-related, increases in the expression of senescence markers (GLB1, P16, P21), and proinflammatory genes (TGF-ß1, TNF-α, IL-1ß, IL-6, and IL-8). Conversely, the expression of anti-oxidative stress-related genes (SOD1, SOD2, NRF2, SIRT1, and SIRT3) declined, showing statistically significant differences (*P < 0.05, **P < 0.01, ***P < 0.001). ELISA results corroborated these findings, indicating a progressive rise in the protein levels of TGF-ß1, TNF-α, and IL-8 as the mice aged. CONCLUSIONS: The findings suggest that NOD/SCID mice aged 32 weeks and 43 weeks effectively model pulmonary fibrosis in an elderly context, with the disease pathogenesis likely driven by age-associated inflammation and oxidative stress.
Assuntos
Envelhecimento , Modelos Animais de Doenças , Camundongos Endogâmicos NOD , Camundongos SCID , Sirtuína 1 , Animais , Camundongos , Masculino , Sirtuína 1/metabolismo , Sirtuína 1/genética , Pulmão/patologia , Pulmão/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Fator de Crescimento Transformador beta1/genética , Metaloproteinase 9 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/genética , Fator de Necrose Tumoral alfa/metabolismo , Fator de Necrose Tumoral alfa/genética , Fator 2 Relacionado a NF-E2/metabolismo , Fator 2 Relacionado a NF-E2/genética , Fibrose Pulmonar Idiopática/genética , Fibrose Pulmonar Idiopática/patologia , Fibrose Pulmonar Idiopática/metabolismo , Interleucina-8/metabolismo , Interleucina-8/genética , Superóxido Dismutase-1/genética , Superóxido Dismutase-1/metabolismo , Superóxido Dismutase/metabolismo , Superóxido Dismutase/genética , Sirtuína 3/genética , Sirtuína 3/metabolismo , Hidroxiprolina/metabolismo , Interleucina-6/metabolismo , Interleucina-6/genética , Actinas/metabolismo , Actinas/genética , Interleucina-1beta/metabolismo , Interleucina-1beta/genética , Fibrose Pulmonar/genética , Fibrose Pulmonar/patologia , Fibrose Pulmonar/metabolismoRESUMO
Epigallocatechin gallate (EGCG) is a polyphenol plant metabolite abundant in tea that has demonstrated antifibrotic properties in the lung. In this issue of the JCI, Cohen, Brumwell, and colleagues interrogated the mechanistic action of EGCG by investigating lung biopsies of patients with mild interstitial lung disease (ILD) who had undergone EGCG treatment. EGCG targeted the WNT inhibitor SFRP2, which was enriched in fibrotic fibroblasts and acted as a TGF-ß target, with paracrine effects leading to pathologic basal metaplasia of alveolar epithelial type 2 cells. This study emphasizes the epithelial-mesenchymal trophic unit as a central signaling hub in lung fibrosis. Understanding and simultaneous targeting of interlinked signaling pathways, such as TGF-ß and WNT, paves the road for future treatment options for pulmonary fibrosis.