Rhizoma coptidis can inhibit the excessive proliferation, inflammation, and transformation of lung fibroblasts into myofibroblasts.

BACKGROUND: Pulmonary fibrosis (PF) is a chronic, progressive, and irreversible heterogeneous disease of lung interstitial tissue. To combat progression of PF, new drugs are required to be developed. Rhizoma coptidis (COP), one of the main alkaloids of Coptis chinensis, is a traditional herbal medicine used to treat various inflammatory diseases. OBJECTIVE: To investigate the possible effects of Coptisine (Cop) on the growth, inflammation, as well as FMT of TNF-ß1-induced HFL1 cells and uncover the mechanism. MATERIAL AND METHODS: Human fetal lung fibroblast 1 (HFL1) was induced using 6ng/mL TGF-ß1 as a model of pulmonary fibrosis. CCK-8, Brdu, and transwell assays indicated the effects on cell growth as well as motility. qPCR and the corresponding kits indicted the effects on cell inflammation. Immunoblot showed the effects on FMT and further confirmed the mechanism. RESULTS: Coptisine inhibits excessive growth as well as motility of TNF-ß1-induced HFL1 cells. It further inhibits inflammation and ROS levels in TNF-ß1-induced HFL1 cells. Coptisine inhibits the FMT process of TNF-ß1-induced HFL1 cells. Mechanically, coptisine promotes the Nrf2/HO-1 pathway. CONCLUSION: Coptisine can inhibit the excessive growth, inflammation as well as FMT of lung fibroblasts into myofibroblasts. It could serve as a promising drug of PF.

METTL3-mediated m6A RNA methylation induces the differentiation of lung resident mesenchymal stem cells into myofibroblasts via the miR-21/PTEN pathway.

BACKGROUND: The accumulation of myofibroblasts is the key pathological feature of pulmonary fibrosis (PF). Aberrant differentiation of lung-resident mesenchymal stem cells (LR-MSCs) has been identified as a critical source of myofibroblasts, but the molecular mechanisms underlying this process remain largely unknown. In recent years, N6-methyladenosine (m6A) RNA modification has been implicated in fibrosis development across diverse organs; however, its specific role in promoting the differentiation of LR-MSCs into myofibroblasts in PF is not well defined. METHODS: In this study, we examined the levels of m6A RNA methylation and the expression of its regulatory enzymes in both TGF-ß1-treated LR-MSCs and fibrotic mouse lung tissues. The downstream target genes of m6A and their related pathways were identified according to a literature review, bioinformatic analysis and experimental verification. We also assessed the expression levels of myofibroblast markers in treated LR-MSCs and confirmed the involvement of the above-described pathway in the aberrant differentiation direction of LR-MSCs under TGF-ß1 stimulation by overexpressing or knocking down key genes within the pathway. RESULTS: Our results revealed that METTL3-mediated m6A RNA methylation was significantly upregulated in both TGF-ß1-treated LR-MSCs and fibrotic mouse lung tissues. This process directly led to the aberrant differentiation of LR-MSCs into myofibroblasts by targeting the miR-21/PTEN pathway. Moreover, inhibition of METTL3 or miR-21 and overexpression of PTEN could rescue this abnormal differentiation. CONCLUSION: Our study demonstrated that m6A RNA methylation induced aberrant LR-MSC differentiation into myofibroblasts via the METTL3/miR-21/PTEN signaling pathway. We indicated a novel mechanism to promote PF progression. Targeting METTL3-mediated m6A RNA methylation and its downstream targets may present innovative therapeutic approaches for the prevention and treatment of PF.

A Pharmacokinetic Study of Sixteen Major Bioactive Components of Jinshui-Huanxian Granules in Pulmonary Fibrosis Model and Control Rats Using Orbitrap Fusion Mass Spectrometry.

Jinshui-Huanxian granules (JHGs), a Chinese herbal compound prescription, have shown a therapeutic effect in reducing lung tissue damage, improving the degree of pulmonary fibrosis, replenishing lungs and kidneys, relieving cough and asthma, reducing phlegm, and activating blood circulation. However, these active compounds' pharmacokinetics and metabolic processes were unclear. This study aimed to compare the pharmacokinetics, reveal the metabolic dynamic changes, and obtain the basic pharmacokinetic parameters of 16 main bioactive compounds after intragastric administration of JHGs in control and pulmonary fibrosis (PF) model rats by using Orbitrap Fusion MS. After administration of JHGs, the rat plasma was collected at different times. Pretreating the plasma sample with methanol and internal standard (IS) solution carbamazepine (CBZ), and it was then applied to a C18 column by setting gradient elution with a mobile phase consisting of methanol 0.1% formic acid aqueous solution. Detection was performed on an electrospray ionization source (ESI), and the scanning mode was SIM. Pharmacokinetic parameters were analyzed according to the different analytes' concentrations in plasma. The matrix effect was within the range of 79.01-110.90%, the extraction recovery rate was 80.37-102.72%, the intra-day and inter-day precision relative standard deviation (RSD) was less than 7.76%, and the stability was good, which met the requirements of biological sample testing. The method was validated (r ≥ 0.9955) and applied to compare the pharmacokinetic profiles of the control group and PF model group after intragastric administration of the JHGs. The 16 analytes exhibited different pharmacokinetic behaviors in vivo. In the pathological state of the PF model, most of the components were more favorable for metabolism and absorption, and it was more meaningful to study the pharmacokinetics. Above all, this study provided an essential reference for exploring the mechanism of action of JHGs and guided clinical medication as well.

Exophilone, a Tetrahydrocarbazol-1-one Analogue with Anti-Pulmonary Fibrosis Activity from the Deep-Sea Fungus Exophiala oligosperma MCCC 3A01264.

A new compound, exophilone (1), together with nine known compounds (2-10), were isolated from a deep-sea-derived fungus, Exophiala oligosperma. Their chemical structures, including the absolute configuration of 1, were elucidated using nuclear magnetic resonance (NMR) spectroscopy, high-resolution electrospray ionization mass spectroscopy (HRESIMS), and electronic circular dichroism (ECD) calculation. Compounds were preliminarily screened for their ability to inhibit collagen accumulation. Compounds 1, 4, and 7 showed weaker inhibition of TGF-ß1-induced total collagen accumulation in compared with pirfenidone (73.14% inhibition rate). However, pirfenidone exhibited cytotoxicity (77.57% survival rate), while compounds 1, 4, and 7 showed low cytotoxicity against the HFL1 cell line. Particularly, exophilone (1) showed moderate collagen deposition inhibition effect (60.44% inhibition rate) and low toxicity in HFL1 cells (98.14% survival rate) at a concentration of 10 µM. A molecular docking study suggests that exophilone (1) binds to both TGF-ß1 and its receptor through hydrogen bonding interactions. Thus, exophilone (1) was identified as a promising anti-pulmonary fibrosis agent. It has the potential to be developed as a drug candidate for pulmonary fibrosis.

Deficiency of CARMA3 attenuates the development of bleomycin induced pulmonary fibrosis.

BACKGROUND: Pulmonary fibrosis (PF) has attracted more and more attention due to its irreversibility and high mortality rate. Currently, there is no effective treatment option is available to reverse the disease. Caspase recruitment domain-containing membrane-associated guanylate kinase protein (CARMA3) has been recognized as a proinflammatory molecule involved in many lung diseases, such as Allergic airway inflammation and lung cancer. Bleomycin (Bleo), as an alkaline sugar peptide antibiotics, is often used as a first-line anti-tumor agent. Its toxic effect is to induce pulmonary fibrosis (PF) and its clinical symptoms, so it has been widely used in the construction of pulmonary fibrosis model. METHODS: Wild type mice (WT, n = 20) and CARMA3 knockout mice (CARMA3-KO, n = 20) were generated and injected with bleomycin or saline via trachea. The severity of fibrosis was evaluated by fibrosis markers and lung histological morphology. Furthermore, the amount of alveolar epithelial cells and inflammation in lung tissue were examined. Finally, epithelial-mesenchymal transition was further investigated. RESULTS: We found CARMA3 expression in the mice alveolar epithelial cells. And compared with WT mice, CARMA3-KO mice showed reduced deposition of collagen fibers, inflammation and destruction of alveolar epithelial cells in lung tissue. In addition, after bleomycin induction, the expressions of proinflammatory factors and collagen-related factors in CARMA3-KO mice were much lower than those in WT mice. The epithelial-mesenchymal transformation phenotype was also improved in CARMA3-KO mice compared to WT mice. CONCLUSION: Our Results shows that CARMA3 plays an important role in the pathogenesis of bleomycin-induced pulmonary fibrosis. CARMA3 could alleviate the fibrosis by improving inflammation, deposition of collagen and damage of alveolar epithelial cells, which revealed that CARMA3 may be a potential target for pulmonary fibrosis.

miR-627/HMGB1/NF-κB regulatory loop modulates TGF-ß1-induced pulmonary fibrosis.

Pulmonary fibrosis (PF) is a fibroproliferative disease that can eventually lead to fatal lung failure. It is characterized by abnormal proliferation of fibroblasts, dysregulated fibroblast differentiation to myofibroblast, and disorganized collagen and extracellular matrix production, deposition and degradation. There is still a lack of effective treatment strategies for PF. Extracellular high-mobility group box protein 1 (HMGB1) induces PF through NF-κB-mediated TGF-ß1 release. Herein, we first validate the suppressive effect of HMGB1 knockdown on TGF-ß1-induced α-smooth muscle actin (α-SMA) and collagen I protein expression. In PF, miRNAs exert different effects through targeting various downstream target messenger RNAs. We searched an online database for dysregulated miRNAs in PF tissues; among them, miR-627 was predicted by online tools to target HMGB1 to inhibit its expression. miR-627 overexpression could partially reverse TGF-ß1-induced normal human lung fibroblast proliferation, as well as α-SMA and collagen I protein expression. miR-627 inhibition could partially reverse the suppressive effect of HMGB1 knockdown on TGF-ß1-induced α-SMA and collagen I protein expression through direct binding to the 3'-untranslated region of HMGB1. Moreover, miR-627/HMGB1 affected TGF-ß1 release through RAGE/NF-κB signaling; miR-627/HMGB1 and RAGE/NF-κB signaling formed a regulatory loop to modulate TGF-ß1-induced PF in vitro. In conclusion, miR-627 may be a potential agent that targets HMGB1 to inhibit its expression, thereby improving TGF-ß1-induced PF in vitro.

Research on the potential mechanism of Deapioplatycodin D against pulmonary fibrosis based on bioinformatics and experimental verification.

BACKGROUND: Pulmonary fibrosis (PF) is a group of respiratory diseases that are extremely complex and challenging to treat. Due to its high mortality rate and short survival, it's often referred to as a "tumor-like disease" that poses a serious threat to human health. OBJECTIVE: We aimed validate the potential of Deapioplatycodin D (DPD) to against PF and clarify the underlying mechanism of action of DPD for the treatment of PF based on bioinformatics and experimental verification. This finding provides a basis for the development of safe and effective therapeutic PF drugs based on DPD. METHODS: We used LPS-induced early PF rats as a PF model to test the overall efficacy of DPD in vivo. Then, A variety of bioinformatics methods, such as WGCNA, LASSO algorithm and immune cell infiltration (ICI), were applied to analyze the gene microarray related to PF obtained from Gene Expression Omnibus (GEO) to obtained key targets of PF. Finally, an in vitro PF model was constructed based on BEAS-2B cells while incorporating rat lung tissues to validate the regulatory effects of DPD on critical genes. RESULTS: DPD can effectively alleviate inflammatory and fibrotic markers in rat lungs. WGCNA analysis resulted in a total of six expression modules, with the brown module having the highest correlation with PF. Subsequently, seven genes were acquired by intersecting the genes in the brown module with DEGs. Five key genes were identified as potential biomarkers of PF by LASSO algorithm and validation dataset verification analysis. In the ICI analysis, infiltration of activated B cell, immature B cell and natural killer cells were found to be more crucial in PF. Ultimately, it was observed that DPD could modulate key genes to achieve anti-PF effects. CONCLUSION: In short, these comprehensive analysis methods were employed to identify critical biomarkers closely related to PF, which helps to elucidate the pathogenesis and potential immunotherapy targets of PF. It also provides essential support for the potential of DPD against PF.

Effect of apigetrin in pseudo-SARS-CoV-2-induced inflammatory and pulmonary fibrosis in vitro model.

SARS-CoV-2 has become a global public health problem. Acute respiratory distress syndrome (ARDS) is the leading cause of death due to the SARS-CoV-2 infection. Pulmonary fibrosis (PF) is a severe and frequently reported COVID-19 sequela. In this study, an in vitro model of ARDS and PF caused by SARS-CoV-2 was established in MH-S, THP-1, and MRC-5 cells using pseudo-SARS-CoV-2 (PSCV). Expression of proinflammatory cytokines (IL-6, IL-1ß, and TNF-α) and HIF-1α was increased in PSCV-infected MH-S and THP-1 cells, ARDS model, consistent with other profiling data in SARS-CoV-2-infected patients have been reported. Hypoxia-inducible factor-1 alpha (HIF-1α) siRNA and cobalt chloride were tested using this in vitro model. HIF-1α knockdown reduces inflammation caused by PSCV infection in MH-S and THP-1 cells and lowers elevated levels of CTGF, COLA1, and α-SMA in MRC-5 cells exposed to CPMSCV. Furthermore, apigetrin, a glycoside bioactive dietary flavonoid derived from several plants, including Crataegus pinnatifida, which is reported to be a HIF-1α inhibitor, was tested in this in vitro model. Apigetrin significantly reduced the increased inflammatory cytokine (IL-6, IL-1ß, and TNF-α) expression and secretion by PSCV in MH-S and THP-1 cells. Apigetrin inhibited the binding of the SARS-CoV-2 spike protein RBD to the ACE2 protein. An in vitro model of PF induced by SARS-CoV-2 was produced using a conditioned medium of THP-1 and MH-S cells that were PSCV-infected (CMPSCV) into MRC-5 cells. In a PF model, CMPSCV treatment of THP-1 and MH-S cells increased cell growth, migration, and collagen synthesis in MRC-5 cells. In contrast, apigetrin suppressed the increase in cell growth, migration, and collagen synthesis induced by CMPSCV in THP-1 and MH-S MRC-5 cells. Also, compared to control, fibrosis-related proteins (CTGF, COLA1, α-SMA, and HIF-1α) levels were over two-fold higher in CMPSV-treated MRC-5 cells. Apigetrin decreased protein levels in CMPSCV-treated MRC-5 cells. Thus, our data suggest that hypoxia-inducible factor-1 alpha (HIF-1α) might be a novel target for SARS-CoV-2 sequela therapies and apigetrin, representative of HIF-1alpha inhibitor, exerts anti-inflammatory and PF effects in PSCV-treated MH-S, THP-1, and CMPVSC-treated MRC-5 cells. These findings indicate that HIF-1α inhibition and apigetrin would have a potential value in controlling SARS-CoV-2-related diseases.

Preventive effects of Ramelteon on bleomycin-induced pulmonary fibrosis in mice.

Pulmonary fibrosis (PF) is a devastating lung disease that leads to impaired lung function and ultimately death. Several studies have suggested that melatonin, a hormone involved in regulating sleep-wake cycles, may be effective in improving PF. Ramelteon, an FDA-approved melatonin receptor agonist, has shown promise in exerting an anti-PF effect similar to melatonin. However, further investigations are required for illuminating the extent on its therapeutic benefits and the underlying molecular mechanisms. In this work, a mouse lung fibrosis model was built through intratracheal administration of bleomycin (BLM). Subsequently, the mice were administrated Ramelteon for a duration of 3 weeks to explore its efficacy and mechanism of action. Additionally, we utilized a TGF-ß1-induced MRC-5 cell model to further investigate the molecular mechanism underlying ramelteon's effects. Functionally, Ramelteon partially abrogated TGF-ß1-induced pulmonary fibrosis and reduced fibroblast proliferation, extracellular matrix deposition, and differentiation into myofibroblasts. In vivo experiments, ramelteon attenuated BLM-induced pulmonary fibrosis and collagen deposition. Mechanistically, ramelteon exerts its beneficial effect by alleviating translocation and expression of YAP1, a core component of Hippo pathway, from cytoplasm to nucleus; however, overexpression of YAP1 reversed this effect. In conclusion, our findings indicate that ramelteon can improve PF by regulating Hippo pathway and may become a potential candidate as a therapy to PF.

Sustainably released nanoparticle-based rhynchophylline limits pulmonary fibrosis by inhibiting the TEK-PI3K/AKT signaling pathway.

Background: Pulmonary fibrosis (PF) is a rapidly progressing and irreversible disease, and the currently available types of clinical drugs are limited and inefficient. In our previous study, we observed that Rhynchophylline (Rhy) hindered tendon adhesion and stimulated the healing of injured tendon structures. Considering the similar mechanisms between adhesion formation and PF, we explored the roles of Rhy in PF. Methods: The cytotoxicity of Rhy was tested by a Cell Counting Kit-8 (CCK-8) assay. The degree of PF was evaluated by Western blot (WB), Masson and hematoxylin-eosin (HE) staining, and hydroxyproline quantification. The Rhy-loaded nanoparticles were prepared through an emulsification sonication technique and characterized by transmission electron microscopy (TEM) and scanning electron microscopy (SEM). The release of the Rhy-loaded nanoparticles was tested using the absorbance value of the supernatant. Transcriptome sequencing was performed to determine the downstream target and pathway of Rhy, which was then verified by WB. Results: In vitro, Rhy decreased Transforming Growth Factor Beta 1 (TGF-ß1)-induced abnormal overexpression of fibronectin (FN), collagen I (Col I), α-smooth muscle actin (α-SMA) in a dose-dependent manner in human lung fibroblast (HFL1) cells. In vivo, we confirmed (through Masson staining) that the intraperitoneal injection of Rhy reduced collagen deposition and the fibrotic area in a dose-dependent manner. Our results indicated that the Rhy-loaded nanoparticles intratracheal spray intuitively narrowed collagen deposition, shrank collagen deposition and the fibrotic area (Masson and HE staining), and reduced the expression of fibrosis-related markers (WB). Meanwhile, the lung index value and hydroxyproline content were markedly lower than the bleomycin (BLM)-treated group. By transcriptional sequencing analysis, we identified Receptor Tyrosine Kinase (TEK)-Phosphatidylinositol 3-Kinase/Protein Kinase B (PI3K/AKT) as the downstream target and pathway of Rhy. It was also observed that Rhy could reverse the TGF-ß1-induced TEK and phosphorylated AKT (p-AKT) elevated expression. Conclusions: Our findings indicate that Rhy constrained PF progression by inhibiting TEK-PI3K/AKT signaling pathway. Hence, this sustainable release system of Rhy is a highly effective therapy to limit PF and should be developed.

HSPB5 Inhibition by NCI-41356 Reduces Experimental Lung Fibrosis by Blocking TGF-ß1 Signaling.

Idiopathic pulmonary fibrosis is a chronic, progressive and lethal disease of unknown etiology that ranks among the most frequent interstitial lung diseases. Idiopathic pulmonary fibrosis is characterized by dysregulated healing mechanisms that lead to the accumulation of large amounts of collagen in the lung tissue that disrupts the alveolar architecture. The two currently available treatments, nintedanib and pirfenidone, are only able to slow down the disease without being curative. We demonstrated in the past that HSPB5, a low molecular weight heat shock protein, was involved in the development of fibrosis and therefore was a potential therapeutic target. Here, we have explored whether NCI-41356, a chemical inhibitor of HSPB5, can limit the development of pulmonary fibrosis. In vivo, we used a mouse model in which fibrosis was induced by intratracheal injection of bleomycin. Mice were treated with NaCl or NCI-41356 (six times intravenously or three times intratracheally). Fibrosis was evaluated by collagen quantification, immunofluorescence and TGF-ß gene expression. In vitro, we studied the specific role of NCI-41356 on the chaperone function of HSPB5 and the inhibitory properties of NCI-41356 on HSPB5 interaction with its partner SMAD4 during fibrosis. TGF-ß1 signaling was evaluated by immunofluorescence and Western Blot in epithelial cells treated with TGF-ß1 with or without NCI-41356. In vivo, NCI-41356 reduced the accumulation of collagen, the expression of TGF-ß1 and pro-fibrotic markers (PAI-1, α-SMA). In vitro, NCI-41356 decreased the interaction between HSPB5 and SMAD4 and thus modulated the SMAD4 canonical nuclear translocation involved in TGF-ß1 signaling, which may explain NCI-41356 anti-fibrotic properties. In this study, we determined that inhibition of HSPB5 by NCI-41356 could limit pulmonary fibrosis in mice by limiting the synthesis of collagen and pro-fibrotic markers. At the molecular level, this outcome may be explained by the effect of NCI-41356 inhibiting HSPB5/SMAD4 interaction, thus modulating SMAD4 and TGF-ß1 signaling. Further investigations are needed to determine whether these results can be transposed to humans.

Post-COVID-19 Pulmonary Fibrosis: Facts-Challenges and Futures: A Narrative Review.

Patients with coronavirus disease 2019 (COVID-19) usually suffer from post-acute sequelae of coronavirus disease 2019 (PASC). Pulmonary fibrosis (PF) has the most significant long-term impact on patients' respiratory health, called post-COVID-19 pulmonary fibrosis (PC19-PF). PC19- PF can be caused by acute respiratory distress syndrome (ARDS) or pneumonia due to COVID-19. The risk factors of PC19-PF, such as older age, chronic comorbidities, the use of mechanical ventilation during the acute phase, and female sex, should be considered. Individuals with COVID-19 pneumonia symptoms lasting at least 12 weeks following diagnosis, including cough, dyspnea, exertional dyspnea, and poor saturation, accounted for nearly all disease occurrences. PC19-PF is characterized by persistent fibrotic tomographic sequelae associated with functional impairment throughout follow-up. Thus, clinical examination, radiology, pulmonary function tests, and pathological findings should be done to diagnose PC19-PF patients. PFT indicated persistent limitations in diffusion capacity and restrictive physiology, despite the absence of previous testing and inconsistency in the timeliness of assessments following acute illness. It has been hypothesized that PC19-PF patients may benefit from idiopathic pulmonary fibrosis treatment to prevent continued infection-related disorders, enhance the healing phase, and manage fibroproliferative processes. Immunomodulatory agents might reduce inflammation and the length of mechanical ventilation during the acute phase of COVID-19 infection, and the risk of the PC19-PF stage. Pulmonary rehabilitation, incorporating exercise training, physical education, and behavioral modifications, can improve the physical and psychological conditions of patients with PC19-PF.

Transbronchial lung cryobiopsy and pulmonary fibrosis: A never-ending story?

Background: The diagnostic process of pulmonary fibrosis (PF) is often challenging, requires a collaborative effort of several experts, and often requires bioptic material, which can be difficult to obtain, both in terms of quality and technique. The main procedures available to obtain such samples are transbronchial lung cryobiopsy (TBLC) and surgical lung biopsy (SLB). Objective: The purpose of this paper is to review the evidence for the role of TBLC in the diagnostic-therapeutic process of PF. Methods: A comprehensive review was performed to identify articles to date that addressed the role of TBLC in the diagnostic-therapeutic process of PF using the PubMed® database. Results: The reasoned search identified 206 papers, including 21 manuscripts (three reviews, one systematic review, two guidelines, two prospective studies, three retrospective studies, one cross-sectional study, one original article, three editorials, three clinical trials, and two unclassifiable studies), which were included in the final review. Conclusions: TBLC is gaining increasing efficacy and improving safety profile; however, there are currently no clear data demonstrating its superiority over SLB. Therefore, the two techniques should be considered with careful rationalization on a case-by-case basis. Further research is needed to further optimize and standardize the procedure and to thoroughly study the histological and molecular characteristics of PF.

Development of lung tissue models and their applications.

The lungs are important organs that play a critical role in the development of specific diseases, as well as responding to the effects of drugs, chemicals, and environmental pollutants. Due to the ethical concerns around animal testing, alternative methods have been sought which are more time-effective, do not pose ethical issues for animals, do not involve species differences, and provide easy investigation of the pathobiology of lung diseases. Several national and international organizations are working to accelerate the development and implementation of structurally and functionally complex tissue models as alternatives to animal testing, particularly for the lung. Unfortunately, to date, there is no lung tissue model that has been accepted by regulatory agencies for use in inhalation toxicology. This review discusses the challenges involved in developing a relevant lung tissue model derived from human cells such as cell lines, primary cells, and pluripotent stem cells. It also introduces examples of two-dimensional (2D) air-liquid interface and monocultured and co-cultured three-dimensional (3D) culture techniques, particularly organoid culture and 3D bioprinting. Furthermore, it reviews development of the lung-on-a-chip model to mimic the microenvironment and physiological performance. The applications of lung tissue models in various studies, especially disease modeling, viral respiratory infection, and environmental toxicology will be also introduced. The development of a relevant lung tissue model is extremely important for standardizing and validation the in vitro models for inhalation toxicity and other studies in the future.

Immunophenotyping of Acute Inflammatory Exacerbations of Lung Injury Driven by Mutant Surfactant Protein-C: A Role for Inflammatory Eosinophils.

Acute inflammatory exacerbations (AIEs) represent immune-driven deteriorations of many chronic lung conditions, including COPD, asthma, and pulmonary fibrosis (PF). The first line of therapy is represented by broad-spectrum immunomodulation. Among the several inflammatory populations mobilizing during AIEs, eosinophils have been identified as promising indicators of an active inflammatory exacerbation. To better study the eosinophil-parenchymal crosstalk during AIE-PF, this work leverages a clinically relevant model of inflammatory exacerbations triggered by inducible expression of a mutation in the alveolar epithelial type 2 cell Surfactant Protein-C gene [SP-CI73T]. Unbiased single-cell sequencing analysis of controls and SP-CI73T mutants at a time coordinated with peak eosinophilia (14 days) defined heightened inflammatory activation, chemotaxis, and survival signaling (IL-6, IL-4/13, STAT3, Glucocorticoid Receptor, mTOR, and MYC) in eosinophils. To study the impact of eosinophils in inflammatory exacerbations, the SP-CI73T line was crossed with eosinophil lineage deficient mice (GATA1Δdbl) to produce the SP-CI73TGATA1KO line. Time course analysis (7-42 days) demonstrated improved lung histology, survival, and reduced inflammation in SP-CI73TGATA1KO cohorts. Spectral flow cytometry of tissue digests confirmed eosinophil depletion in GATA1KO mice and the absence of a compensatory shift in neutrophils and immature monocyte recruitment. Eosinophil deletion resulted in progressive monocyte-derived macrophage accumulation (14 days post-injury), combined with declines in CD3+CD4+ lymphocyte and B220+ B cell abundance. Histochemical analysis revealed atypical inflammatory cell activation in SP-CI73TGATA1KO mice, with reduced numbers of Arg-1+ and iNOS+ cells, but increases in tgfb1 mRNA expression in bronchoalveolar lavage cells and tissue. Dexamethasone treatment (1 mg/kg daily, i.p.) was utilized to investigate corticosteroid efficacy in highly eosinophilic exacerbations induced by mutant SP-CI73T. Dexamethasone successfully reduced total and eosinophil (CD11b+SigF+CD11c-) counts at 14 days and was linked to reduced evidence of structural damage and perivascular infiltrate. Together, these results illustrate the deleterious role of eosinophils in inflammatory events preceding lung fibrosis and demonstrate the efficacy of corticosteroid treatment in highly eosinophilic exacerbations induced by mutant SP-CI73T.

Interference with connective tissue growth factor attenuated fibroblast-to-myofibroblast transition and pulmonary fibrosis.

Background: The aberrant activation and phenotype shift of resident fibroblasts in lung tissues via fibroblast-to-myofibroblast transition (FMT) is considered a pivotal step in pulmonary fibrogenesis, resulting in excessive extracellular matrix (ECM) production and deposition. However, the molecular mechanisms regulating FMT and lung fibrosis are still unclear. Connective tissue growth factor (CTGF) has been reported to be both an ECM protein and a versatile signaling molecule that is involved in multiple pathophysiological contexts, especially fibrosis. The relationship between CTGF, FMT, and lung fibrosis has not yet been well defined. Methods: In this study, a pulmonary fibrosis (PF) rat model and FMT cell model induced by paraquat (PQ) were established to explore the relevant regulatory mechanisms in vivo and in vitro. Results: The results showed that the CTGF was highly activated and was a mediator of canonical Wnt signaling during FMT and PF. The inhibition of the CTGF by small-interfering ribonucleic acid decreased the expression of FMT markers, including α-smooth muscle actin, vimentin, and collagen I, inhibited the activated Wnt signaling pathway, and ameliorated lung fibrosis. Conclusions: Our findings showed that CTGF was the key effector of the FMT and fibrotic changes, and emphasized the therapeutic potential of the inhibitor or monoclonal antibody against CTGF for PF.

Mechanism of miR-204-5p in exosomes derived from bronchoalveolar lavage fluid on the progression of pulmonary fibrosis via AP1S2.

BACKGROUND: Exosomes are nanoscale vesicles secreted by various types of cells that are responsible for intracellular communication. Despite that bronchoalveolar lavage fluid (BALF) has been proven to involve in tumor development, more efforts are required to investigate the impact of BALF on pulmonary fibrosis (PF). This study aimed to investigate the mechanism of how exosomal miR-204-5p from BALF facilitates PF progression in rats. METHODS: PF rat model was established by intratracheal injection of bleomycin. BALF-derived exosomes (Exo) were extracted from normal and PF rats. PF-Exo (BALF-derived Exo from PF rats) and miR-204-5p antagomir were injected into rats to investigate the effect of exosomal miR-204-5p on PF. Collagen content in lung tissues of rats was assessed by Masson staining, hydroxyproline (HYP) content assay and immunohistochemistry (IHC). Primary lung fibroblasts were isolated, and treated by TGF-ß1. After co-transfection of PF-Exo, miR-204-5p inhibitor and sh-AP1S2, cell proliferation, levels of miR-204-5p, fibrotic markers α-SMA and collagen 1 (Col 1), and proteins of autophagy markers LC3II, LC3I and P62 were measured. The interaction between miR-204-5p and AP1S2 was determined by bioinformatics online software TargetScan and dual-luciferase reporter assay. RESULTS: miR-204-5p was abundantly expressed in the PF-Exo group. PF-Exo injection potentiated PF progression and proliferation ability of lung fibroblasts in vivo and in vitro. Injection with PF-Exo and miR-204-5p antagomir significantly increased the LC3II/I ratio and decreased the HYP content, proteins of α-SMA, Col 1 and P62, collagen content in rat lung tissues of PF rats. TGF-ß1 induction elevated the LC3II/LC3I ratio, suppressed the cell proliferation rate, and decreased the levels of α-SMA, Col 1 and P62. Additionally, AP1S2 was a direct target of miR-204-5p. miR-204-5p inhibitor can counteract the effect of PF-Exo in proliferation of lung fibroblasts, while sh-AP1S2 eliminated the effect of miR-204-5p inhibitor. CONCLUSIONS: Exosomal miR-204-5p from BALF inhibits autophagy to promote the progression of PF rats by targeting AP1S2.

The Role of Hippo/YAP Signaling in Alveolar Repair and Pulmonary Fibrosis.

Pulmonary fibrosis is characterized by loss of normal alveoli, accumulation of pathologic activated fibroblasts, and exuberant extracellular matrix deposition that over time can lead to progressive loss of respiratory function and death. This loss of respiratory function is associated with the loss of alveolar type 1 cells (AT1) that play a crucial role in gas exchange and the depletion of the alveolar type 2 cells (AT2) that act as progenitor cells to regenerate the AT1 and AT2 cell populations during repair. Understanding the mechanisms that regulate normal alveolar repair and those associated with pathologic repair is essential to identify potential therapeutic targets to treat or delay progression of fibrotic diseases. The Hippo/YAP developmental signaling pathway has been implicated as a regulator of normal alveolar development and repair. In idiopathic pulmonary fibrosis, aberrant activation of YAP/TAZ has been demonstrated in both the alveolar epithelium and activated fibroblasts associated with increased fibrotic remodeling, and there is emerging interest in this pathway as a target for antifibrotic therapies. In this review, we summarize current evidence as to the role of the Hippo-YAP/TAZ pathway in alveolar development, homeostasis, and repair, and highlight key questions that must be resolved to determine effective strategies to modulate YAP/TAZ signaling to prevent progressive pulmonary fibrosis and enhance adaptive alveolar repair.

Investigation into molecular mechanisms and high-frequency core TCM for pulmonary fibrosis secondary to COVID-19 based on network pharmacology and data mining.

BACKGROUND: The complication, pulmonary fibrosis (PF) secondary to COVID-19, may have a second wave of late mortality, given the huge number of individuals infected by COVID-19. However, the molecular mechanisms of PF secondary to COVID-19 haven't been fully elucidated, making the identification of novel strategies for targeted therapy challenging. This study aimed to systematically identify the molecular mechanisms and high-frequency core traditional Chinese medicine (TCM) targeting PF secondary to COVID-19 through network pharmacology and data mining. METHODS: The molecular mechanisms of PF secondary to COVID-19 were identified by mapping the COVID-19 differentially expressed gene and known targets associated with PF, protein-protein interactions network analysis, and enrichment pathway analysis; then the high-frequency core TCM targeting PF secondary to COVID-19 were identified by data mining and "Key targets related to PF secondary to COVID-19 - Ingredients" and "Key ingredients-key herbs" network analysis; and last we validated the interaction between the key ingredients and key targets by molecular docking. RESULTS: The molecular mechanisms of PF secondary to COVID-19 were mainly related to tumor necrosis factor (TNF) signaling pathway, cytokine-cytokine receptor interaction pathway, and NF-κB signaling pathway. Among these, cytokines interleukin 6 (IL-6), TNF, and IL-1ß were identified as the key targets associated with PF secondary to COVID-19. The high-frequency core TCM targeting these key targets were identified, including ingredients of quercetin, epigallocatechin-3-gallate, emodin, triptolide, resveratrol, and herb of Polygonum cuspidatum. Finally, our results were validated by quercetin and resveratrol both well docked to IL-6, TNF, and IL-1ß protein, with the estimated docking energy <0 kcal/mol. CONCLUSIONS: This study identified the cytokines-related molecular mechanisms of PF secondary to COVID-19, and the high-frequency core TCM against PF by targeting IL-6, TNF, and IL-1ß. Which provides new ideas for the discovery of small molecular compounds with potential therapeutic effects on PF secondary to COVID-19.

Fusion Expression and Fibrinolytic Activity of rPA/SP-B.

BACKGROUND: Pulmonary surfactant dysfunction is an important pathological factor in acute respiratory distress syndrome (ARDS) and pulmonary fibrosis (PF). OBJECTIVE: In this study, the characteristics of recombinant mature surfactant protein B (SP-B) and reteplase (rPA) fusion protein maintaining good pulmonary surface activity and rPA fibrinolytic activity in acute lung injury cell model were studied. METHODS: We studied the characteristics of SP-B fusion expression, cloned rPA gene and N-terminal rPA/C-terminal SP-B co-expression gene, and constructed them into eukaryotic expression vector pEZ-M03 to obtain recombinant plasmids pEZ-rPA and pEZ-rPA/SP-B. The recombinant plasmids was transfected into Chinese hamster ovary (CHO) K1 cells and the expression products were analyzed by Western Blot. Lipopolysaccharide (LPS) was used to induce CCL149 (an alveolar epithelial cell line) cell injury model. Fluorescence staining of rPA and rPA/SP-B was carried out with the enhanced green fluorescent protein (eGFP) that comes with pEZ-M03; the cell Raman spectroscopy technique was used to analyze the interaction between rPA/SP-B fusion protein and the phospholipid structure of cell membrane in CCL149 cells. The enzyme activity of rPA in the fusion protein was determined by fibrin-agarose plate method. RESULTS: The rPA/SP-B fusion protein was successfully expressed. In the CCL149 cell model of acute lung injury (ALI), the green fluorescence of rPA/SP-B is mainly distributed on the CCL149 cell membrane. The rPA/SP-B fusion protein can reduce the disorder of phospholipid molecules and reduce cell membrane damage. The enzyme activity of rPA/SP-B fusion protein was 3.42, and the fusion protein still had good enzyme activity. CONCLUSION: The recombinant eukaryotic plasmid pEZ-rPA/SP-B is constructed and can be expressed in the eukaryotic system. Studies have shown that rPA/SP-B fusion protein maintains good SP-B lung surface activity and rPA enzyme activity in acute lung injury cell model.