Proliferation Of Lung Fibroblasts Research Articles

Forsythoside A regulates pulmonary fibrosis by inhibiting endothelial-to-mesenchymal transition and lung fibroblast proliferation via the PTPRB signaling

BackgroundPulmonary fibrosis (PF) is an end-stage change in many interstitial lung diseases, whereas no proven effective anti-pulmonary fibrotic treatments. Forsythoside A (FA) derived from Forsythia suspensa (Thunb.) Vahl, has been found to possess lung-protective effect. However, studies on its anti-pulmonary fibrosis effect are limited and its mechanism of action remains unknown. PurposeThis study aimed to explore the underlying mechanism of FA on PF. MethodsMale C57BL/6 mice were randomized into normal (CON), model (BLM), pirfenidone (PFD), low- and high-dose FA (FA-L, FA-H, respectively). Except for the CON group, which was injected with the same dose of saline, the model of PF was established by intratracheal instillation of BLM, during which the survival rate and body weight changes of the mice were measured. The lung histopathology was evaluated by Hematoxylin–eosin, Sirius red, and Masson staining. Transcriptome analysis was performed to screen for the differential genes associated with the role of FA in PF. Differential genes in normal and pulmonary fibrosis patients with the GSE2052 dataset were analyzed in the GEO database. The levels of CTGF, α-SMA, MMP-8 in lung and TNF-α in bronchoalveolar lavage fluid (BALF) were detected by ELISA. The levels of HYP in lungs were detected by digestion. The mRNA and protein levels of MMP-7, E-cadherin, CD31, α-SMA, TGF-β1, IL-6, β-catenin, ZO-1, PTPRB, E-cadherin, and vimentin in lungs were detected by RT-qPCR and Western blot. The expression of CD31, α-SMA, TGF-β1 and ZO-1 were detected by immunofluorescence. TGF-β1-stimulated HFL1 cells and human umbilical vein endothelial cells (HUVECs) were used in an attempt to explore the possible role of protein tyrosine phosphatase receptor type B (PTPRB) involved in FA-induced improvement of PF. ResultsThe results showed that FA could improve the survival rate and body weight of PF mice. FA could alleviate the symptoms of alveolar wall thickening, inflammatory cell infiltration, blue collagen fiber deposition, collagen fiber type Ⅰ and type Ⅲ in mice with PF. In addition, FA could reduce the levels of HYP, CTGF, α-SMA, TGF-β1, TNF-α, β-catenin and MMP8, and regulate the expression levels CD31, ZO-1, PTPRB and E-cadherin in lung of mice with PF, inhibiting endothelial-to-mesenchymal transition (EndMT) and fibroblasts proliferation. In the GSE2052 dataset, the expression level of PTPRB is reduced in lung tissue from PF patients, and results from transcriptome sequencing indicate that PTPRB expression is also reduced in PF mice. In addition, the effect of FA on TGF-β1-induced HFL1 or HUVECs cells could be attenuated by the inhibitor of PTPRB, suggesting that the effect of FA on PF is related to PTPRB. ConclusionThis study demonstrated that FA could ameliorate PF by inhibiting lung fibroblast proliferation and EndMT, and that PTPRB might be a target of FA to ameliorate PF, which provided evidence to support FA as a candidate phytochemical for PF.

Toosendanin Restrains Idiopathic Pulmonary Fibrosis by Inhibiting ZEB1/CTBP1 Interaction.

Extensive deposition of extracellular matrix (ECM) in idiopathic pulmonary fibrosis (IPF) is due to hyperactivation and proliferation of pulmonary fibroblasts. However, the exact mechanism is not clear. This study focused on the role of CTBP1 in lung fibroblast function, elaborated its regulation mechanism, and analyzed the relationship between CTBP1 and ZEB1. Meanwhile, the antipulmonary fibrosis effect and its molecular mechanism of Toosendanin were studied. Human IPF fibroblast cell lines (LL-97A and LL-29) and normal fibroblast cell lines (LL-24) were cultured in vitro. The cells were stimulated with FCS, PDGF-BB, IGF-1, and TGF-β1, respectively. BrdU detected cell proliferation. The mRNA expression of CTBP1 and ZEB1 was detected by QRT-PCR. Western blotting was used to detect the expression of COL1A1, COL3A1, LN, FN, and α-SMA proteins. An animal model of pulmonary fibrosis was established to analyze the effects of CTBP1 silencing on pulmonary fibrosis and lung function in mice. CTBP1 was up-regulated in IPF lung fibroblasts. Silencing CTBP1 inhibits growth factor-driven proliferation and activation of lung fibroblasts. Overexpression of CTBP1 promotes growth factor-driven proliferation and activation of lung fibroblasts. Silencing CTBP1 reduced the degree of pulmonary fibrosis in mice with pulmonary fibrosis. Western blot, CO-IP, and BrdU assays confirmed that CTBP1 interacts with ZEB1 and promotes the activation of lung fibroblasts. Toosendanin can inhibit the ZEB1/CTBP1protein interaction and further inhibit the progression of pulmonary fibrosis. CTBP1 can promote the activation and proliferation of lung fibroblasts through ZEB1. CTBP1 promotes lung fibroblast activation through ZEB1, thereby increasing excessive deposition of ECM and aggravating IPF. Toosendanin may be a potential treatment for pulmonary fibrosis. The results of this study provide a new basis for clarifying the molecular mechanism of pulmonary fibrosis and developing new therapeutic targets.

ARID1A loss induces P4HB to activate fibroblasts to support lung cancer cell growth, invasion, and chemoresistance.

Loss of AT-interacting domain-rich protein 1A (ARID1A) frequently occurs in human malignancies including lung cancer. The biological consequence of ARID1A mutation in lung cancer is not fully understood. This study was designed to determine the effect of ARID1A-depleted lung cancer cells on fibroblast activation. Conditioned media was collected from ARID1A-depleted lung cancer cells and employed to treat lung fibroblasts. The proliferation and migration of lung fibroblasts were investigated. The secretory genes were profiled in lung cancer cells upon ARID1A knockdown. Antibody-based neutralization was utilized to confirm their role in mediating the cross-talk between lung cancer cells and fibroblasts. NOD-SCID-IL2RgammaC-null (NSG) mice received tumor tissues from patients with ARID1A-mutated lung cancer to establish patient-derived xenograft (PDX) models. Notably, ARID1A-depleted lung cancer cells promoted the proliferation and migration of lung fibroblasts. Mechanistically, ARID1A depletion augmented the expression and secretion of prolyl 4-hydroxylase beta (P4HB) in lung cancer cells, which induced the activation of lung fibroblasts through the β-catenin signaling pathway. P4HB-activated lung fibroblasts promoted the proliferation, invasion, and chemoresistance in lung cancer cells. Neutralizing P4HB hampered the tumor growth and increased cisplatin cytotoxic efficacy in two PDX models. Serum P4HB levels were higher in ARID1A-mutated lung cancer patients than in healthy controls. Moreover, increased serum levels of P4HB were significantly associated with lung cancer metastasis. Together, our work indicates a pivotal role for P4HB in orchestrating the cross-talk between ARID1A-mutated cancer cells and cancer-associated fibroblasts during lung cancer progression. P4HB may represent a promising target for improving lung cancer treatment.

Inhibition of Galectin-1 attenuates lung fibroblast activation and proliferation in lung fibrosis.

The purpose of this study was to investigate the parenchymal changes in idiopathic pulmonary fibrosis (IPF) caused by massive fibroblastic infiltration and proliferation in lung tissue. Galectin-1 (Gal-1) has been reported to be involved in angiogenesis and fibrosis via modification of TGF-b receptor signaling pathways. However, it remains unknown whether Galectin-1 plays a critical role in IPF. In the current study, we aimed to identify Gal-1 as a crucial fibrotic protein in IPF process. Murine lung fibroblast was pre-treated using Gal-1 inhibitor OTX-008 or overexpression of Gal-1 and then activated using transforming growth factor-beta (TGF-β). Adult male C57BL/6J mice were conducted intratracheal injection of bleomycin (BLM) for lung fibrosis. Mice were conducted OTX-008 administration. Gal-1 expression, fibroblast activation and proliferation, extracellular matrix (ECM), lung fibrosis, lung histology and pulmonary function were investigated respectively. We demonstrated that Gal-1, as a positive pro-fibrotic marker, could promote lung fibroblast activation and proliferation. Inhibition of Gal-1 reduced fibroblast activation and proliferation through negative regulation of TGF-β/Erk1/2 and AKT pathway. In vivo, Gal-1 inhibition ameliorates lung fibroblast accumulation and protects lung histology and function. Gal-1 is verified to be a pro-fibrotic gene in IPF pathogenesis, which promotes fibroblast activation and proliferation via TGF-β/Erk1/2 and AKT pathway. Moreover, inhibition of Gal-1 in lung fibrosis model attenuates lung fibroblast bioactivity and reduces ECM, leading to improved pulmonary histology and function. Hence, knockdown of Gal-1 in IPF may be a promising target therapy.

Circ_PWWP2A promotes lung fibroblast proliferation and fibrosis via the miR-27b-3p/GATA3 axis, thereby aggravating idiopathic pulmonary fibrosis.

This paper was to investigate the effect of circ_PWWP2A-mediated miR-27b-3p/GATA3 axis on idiopathic pulmonary fibrosis (IPF). circ_PWWP2A expression in lung fibroblasts MLg2908 induced by different concentrations of TGF-β was detected. The relationship between circ_PWWP2A or GATA3 and miR-27b-3p was analyzed by RNA immunoprecipitation and dual-luciferin reporter assay. The proliferation of MLg2908 cells was determined by MTT. GATA3, α-SMA, Collagen-I, and Collagen-III in cells were detected by RT-qPCR and Western blot. The rat model of IPF induced by bleomycin (BLM) was constructed and treated with circ_PWWP2A siRNA injection. HE and Masson staining were of utility to evaluate the pathological conditions of rat lung tissue, and circ_PWWP2A, miR-27b-3p, and GATA3 levels in lung tissues were detected by RT-qPCR. Immunohistochemistry was used to detect the staining of α-SMA, collagen I, and collagen III in the lung tissues of rats. circ_PWWP2A in MLg2908 cells induced by TGF-β decreased in a concentration-dependent manner. MLg2908 cells transfected with circ_PWWP2A siRNA were induced by 5 ng/ml TGF-β, decreasing circ_PWWP2A and GATA3 levels, increasing miR-27b-3p expression, and suppressing cell proliferation. The targeting relationship between circ_PWWP2A and miR-27b-3p, as well as miR-27b-3p and GATA3, was confirmed. Depleting miR-27b-3p reduced the inhibitory effect of circ_PWWP2A down-regulation on the proliferation of TGF-β-treated MLg2908 cells, accompanied by increased expression of α-SMA, Collagen 1, and Collagen 3, and increased expression of GATA3. The in vivo results showed that BLM-induced fibrosis in rat lung tissue was obvious, accompanied by increased expression of circ_PWWP2A and GATA3, decreased expression of miR-27b-3p, and deepened staining of α-SMA, collagen I, and collagen III, but circ_PWWP2A siRNA could improve these phenomena. Silencing circ_PWWP2A can inhibit the proliferation of lung fibroblasts induced by TGF-β through the miR-27b-3p/GATA3 axis, and reduce BLM-induced pulmonary fibrosis in rats, which may be a potential therapeutic target for IPF.

Preferential FGF18/FGFR activity in pseudoglandular versus canalicular stage human lung fibroblasts.

Fibroblast growth factor (FGF) signaling is necessary for proper lung branching morphogenesis, alveolarization, and vascular development. Dysregulation of FGF activity has been implicated in various lung diseases. Recently, we showed that FGF18 promotes human lung branching morphogenesis by regulating mesenchymal progenitor cells. However, the underlying mechanisms remain unclear. Thus, we aimed to determine the role of FGF18 and its receptors (FGFR) in regulating mesenchymal cell proliferation, migration, and differentiation from pseudoglandular to canalicular stage. We performed siRNA assays to identify the specific FGFR(s) associated with FGF18-induced biological processes. We found that FGF18 increased proliferation and migration in human fetal lung fibroblasts (HFLF) from both stages. FGFR2/FGFR4 played a significant role in pseudoglandular stage. HFLF proliferation, while FGFR3/FGFR4 were involved in canalicular stage. FGF18 enhanced HFLF migration through FGFR2 and FGFR4 in pseudoglandular and canalicular stage, respectively. Finally, we provide evidence that FGF18 treatment leads to reduced expression of myofibroblast markers (ACTA2 and COL1A1) and increased expression of lipofibroblast markers (ADRP and PPARγ) in both stages HFLF. However, the specific FGF18/FGFR complex involved in this process varies depending on the stage. Our findings suggest that in context of human lung development, FGF18 tends to associate with distinct FGFRs to initiate specific biological processes on mesenchymal cells.

Fibroblast Upregulation of Vitamin D Receptor Represents a Self-Protective Response to Limit Fibroblast Proliferation and Activation during Pulmonary Fibrosis.

Dysregulation of vitamin D receptor (VDR) is implicated in chronic obstructive pulmonary disease. However, whether VDR dysregulation contributes to the development of pulmonary fibrosis remains largely unknown. Analysis of bulk and single-cell RNA profiling datasets revealed VDR upregulation in lung fibroblasts from patients with pulmonary fibrosis or fibrotic mice, which was validated in lung fibroblasts from bleomycin-exposed mice and bleomycin-treated fibroblasts. Stable VDR knockdown promoted, whereas the VDR agonist paricalcitol suppressed lung fibroblast proliferation and activation. Gene set enrichment analysis (GSEA) showed that the JAK/STAT pathway and unfolded protein response (UPR), a process related to endoplasmic reticulum (ER) stress, were enriched in lung fibroblasts of fibrotic lungs. Stable VDR knockdown stimulated, but paricalcitol suppressed ER stress and JAK1/STAT3 activation in lung fibroblasts. The STAT3 inhibitor blocked bleomycin- or stable VDR knockdown-induced ER stress. Paricalcitol inhibited the bleomycin-induced enrichment of STAT3 to the ATF6 promoter, thereby suppressing ATF6 expression in fibroblasts. Paricalcitol or intrapulmonary VDR overexpression inactivated JAK1/STAT3 and suppressed ER stress in bleomycin-treated mice, thus resulting in the inhibition of fibroblast proliferation and activation. Collectively, this study suggests that fibroblast VDR upregulation may be a self-protective response to limit fibroblast proliferation and activation during pulmonary fibrosis by suppressing the JAK1/STAT3/ER stress pathway.

Tuberostemonine may enhance the function of the SLC7A11/glutamate antiporter to restrain the ferroptosis to alleviate pulmonary fibrosis

Ethnopharmacological relevanceStemona is a medicinal plant that has been used in China for thousands of years to treat respiratory diseases such as cough and tuberculosis. The tuberostemonine is the component of the Stemona tuberosa Lour., Stemona sessilifolia (Miq.) Miq. or Stemona japonica (Blume) Miq. (The plant name has been checked with http://www.theplantlist.org), of which multiple biological activities has been verified. However, whether it may alleviate pulmonary fibrosis via regulating ferroptosis mechanism has not been confirmed. Aim of the studyThe aim of this study is to observe whether tuberostemonine alleviates pulmonary fibrosis by enhancing the function of the SLC7A11/glutamate antiporter to restrain the ferroptosis. Materials and methodsWe validated the effects of tuberostemonine and ferroptosis on TGF-β1-induced proliferation of human lung fibroblast and bleomycin-induced pulmonary fibrosis in mice. In vitro, the ferroptosis effect of TGF-β1 on human lung fibroblast were examined and the activity of ɑ-SMA, collagen, hydroxyproline and ferrous ions in cells were also examined. In vivo, ferroptosis impacts respiratory function. Inflammatory manifestations, hydroxyproline, collagen activity and ferrous ions in the lung or blood were subject to evaluation. ResultsTuberostemonine significantly improved respiratory function in mice with bleomycin-induced pulmonary fibrosis, decreased cellular and lung hydroxyproline content, reduced inflammation and collagen deposition in cells and lung, and promoted an increase in the SLC7A11 and GPX4 proteins. Tuberostemonine inhibits the ferroptosis phenomenon, up-regulates SLC7A11, GPX4 and GSH, and down-regulates the accumulation of iron and ROS. ConclusionsTuberostemonine significantly inhibited ferroptosis and improved pulmonary fibrosis both in vivo and vitro.

Krüppel-like factor 15 counteracts endoplasmic reticulum stress and suppresses lung fibroblast proliferation and extracellular matrix accumulation

The incidence of pulmonary fibrosis is on the rise, and existing treatments have limited efficacy in improving patient survival. The purpose of this study was to reveal the potential of Krüppel-like factor (KLF)15 activation in alleviating pulmonary fibrosis. Transforming growth factor beta (TGF-β) was utilized to induce lung fibroblasts to establish an in vitro model of pulmonary fibrosis. The impacts of TGF-β and KLF15 level on cell proliferation, migration, extracellular matrix (ECM) accumulation, and endoplasmic reticulum stress (ERS) were assessed. Additionally, tunicamycin, an ERS agonist, was used to investigate the role of ERS in KLF15 regulation. The results showed that KLF15 was dropped in response to TGF-β treatment. However, KLF15 overexpression reduced cell proliferation, migration, ECM accumulation, and ERS, alleviating the effects of TGF-β stimulation. Subsequent treatment with tunicamycin diminished the effects of KLF15 overexpression, demonstrating that ERS mediated the modulation of KLF15. KLF15 acts against ERS and suppresses excessive proliferation and ECM accumulation in lung fibroblast. These findings suggest that activating KLF15 is a promising strategy for alleviating pulmonary fibrosis.

Host–device interactions: exposure of lung epithelial cells and fibroblasts to nickel, titanium, or nitinol affect proliferation, reactive oxygen species production, and cellular signaling

Endoscopic implantation of medical devices for the treatment of lung diseases, including airway stents, unidirectional valves and coils, is readily used to treat central airway disease and emphysema. However, granulation and fibrotic tissue formation impairs treatment effectiveness. To date little is known about the interaction between implanted devices, often made from metals, such as nickel, titanium or nitinol, and cells in the airways. Here, we study the response of lung epithelial cells and fibroblasts to implant device materials. The adhesion and proliferation of bronchial epithelial cells and lung fibroblasts upon exposure to 10 × 3 × 1 mm pieces of nickel, titanium or nitinol is examined using light and scanning electron microscopy. Pro-inflammatory cytokine mRNA expression and release, signaling kinase activity and intracellular free radical production are assessed. Nitinol, and to a lesser extent nickel and titanium, surfaces support the attachment and growth of lung epithelial cells. Nitinol induces a rapid and significant alteration of kinase activity. Cells directly exposed to nickel or titanium produce free radicals, but those exposed to nitinol do not. The response of lung epithelial cells and fibroblasts depends on the metal type to which they are exposed. Nitinol induces cellular surface growth and the induction of kinase activity, while exposure of lung epithelial cells to nickel and titanium induces free radical production, but nitinol does not.Graphical

SCNN1B regulates the proliferation, migration, and collagen deposition of human lung fibroblasts.

The aim of this study was to investigate the role of amiloride-sensitive sodium channel protein 1B (SCNN1B) on the proliferation and migration of human lung fibroblasts and the possible mechanism that promote the development of acute respiratory distress syndrome (ARDS). Cultivate human embryonic lung fibroblasts (MRC-5) in vitro and screen out the most effective small interfering RNA to silence the expression of SCNN1B. Then, quantitative real-time PCR (qRT-PCR), CCK-8, Transwell, and Western blot detections were performed separately. The results of qRT-PCR showed that all three SCNN1B siRNAs were able to significantly decrease the mRNA expression level of SCNN1B compared with the si-NC group (P < 0.01), with the most significant decrease in the SCNN1B siRNA-83 group. Additionally, compared with the si-NC group, the proliferation ability of MRC-5 cells in the si-SCNN1B group was significantly enhanced, and the migration rate was significantly decreased (P < 0.01). Western blot results showed that low expression of SCNN1B significantly inhibited the protein expression levels of collagen deposition related proteins Collagen I and Heat shock proteins 47 (P < 0.01). In summary,SCNN1B can inhibit cell proliferation and promote cell migration and extracellular matrix deposition of human lung fibroblasts, and may be involved in the occurrence and development of ARDS.

PRDX1 negatively regulates bleomycin-induced pulmonary fibrosis via inhibiting the epithelial-mesenchymal transition and lung fibroblast proliferation in vitro and in vivo

BackgroundPulmonary fibrosis is a major category of end-stage changes in lung diseases, characterized by lung epithelial cell damage, proliferation of fibroblasts, and accumulation of extracellular matrix. Peroxiredoxin 1 (PRDX1), a member of the peroxiredoxin protein family, participates in the regulation of the levels of reactive oxygen species in cells and various other physiological activities, as well as the occurrence and development of diseases by functioning as a chaperonin.MethodsExperimental methods including MTT assay, morphological observation of fibrosis, wound healing assay, fluorescence microscopy, flow cytometry, ELISA, western blot, transcriptome sequencing, and histopathological analysis were used in this study.ResultsPRDX1 knockdown increased ROS levels in lung epithelial cells and promoted epithelial-mesenchymal transition (EMT) through the PI3K/Akt and JNK/Smad signalling pathways. PRDX1 knockout significantly increased TGF-β secretion, ROS production, and cell migration in primary lung fibroblasts. PRDX1 deficiency also increased cell proliferation, cell cycle circulation, and fibrosis progression through the PI3K/Akt and JNK/Smad signalling pathways. BLM treatment induced more severe pulmonary fibrosis in PRDX1-knockout mice, mainly through the PI3K/Akt and JNK/Smad signalling pathways.ConclusionsOur findings strongly suggest that PRDX1 is a key molecule in BLM-induced lung fibrosis progression and acts through modulating EMT and lung fibroblast proliferation; therefore, it may be a therapeutic target for the treatment of BLM-induced lung fibrosis.

A polo-like kinase inhibitor identified by computational repositioning attenuates pulmonary fibrosis

BackgroundIdiopathic pulmonary fibrosis (IPF) is a fatal fibrotic lung disease with few effective therapeutic options. Recently, drug repositioning, which involves identifying novel therapeutic potentials for existing drugs, has been popularized as a new approach for the development of novel therapeutic reagents. However, this approach has not yet been fully utilized in the field of pulmonary fibrosis.MethodsThe present study identified novel therapeutic options for pulmonary fibrosis using a systematic computational approach for drug repositioning based on integration of public gene expression signatures of drug and diseases (in silico screening approach).ResultsAmong the top compounds predicted to be therapeutic for IPF by the in silico approach, we selected BI2536, a polo-like kinase (PLK) 1/2 inhibitor, as a candidate for treating pulmonary fibrosis using an in silico analysis. However, BI2536 accelerated mortality and weight loss rate in an experimental mouse model of pulmonary fibrosis. Because immunofluorescence staining revealed that PLK1 expression was dominant in myofibroblasts while PLK2 expression was dominant in lung epithelial cells, we next focused on the anti-fibrotic effect of the selective PLK1 inhibitor GSK461364. Consequently, GSK461364 attenuated pulmonary fibrosis with acceptable mortality and weight loss in mice.ConclusionsThese findings suggest that targeting PLK1 may be a novel therapeutic approach for pulmonary fibrosis by inhibiting lung fibroblast proliferation without affecting lung epithelial cells. In addition, while in silico screening is useful, it is essential to fully determine the biological activities of candidates by wet-lab validation studies.

Benzylisoquinoline alkaloids inhibit lung fibroblast activation mainly via inhibiting TGF-β1/Smads and ERK1/2 pathway proteins

BackgroundsLiensinine (Lien), Neferine (Nef), Isoliensinine (Iso) and Tetrandrine (Tet), benzylisoquinoline alkaloids (BIAs), have been shown inhibitory effects on pulmonary fibrosis (PF) through anti-inflammatory, anti-oxidative activities, inhibition of cytokines and NF-κB. Effects of other similar BIAs, Dauricine (Dau), Papaverine (Pap) and lotusine (Lot), on PF remain unclear. Here, we explored the effects of five bisbenzylisoquinoline (Lien, Nef, Iso, Tet and Dau) and two monobenzylisoquinoline (Pap, Lot) alkaloids on normal and PF fibroblasts. MethodsPrimary normal and PF lung fibroblasts were cultured and treated with these alkaloids. Proliferation, activation, migration and apoptosis changes were detected by MTT, wound healing assay, flow cytometry. Protein level was analyzed by Western blot. ResultsAll BIAs inhibited proliferation of normal and PF lung fibroblasts induced by TGF-β. α-SMA protein level in normal and PF lung fibroblasts decreased after Lien, Nef, Iso, Tet and Dau treatment. Pap and Lot had no influence on α-SMA expression. Dau showed the strongest inhibitory effects on proliferation and activation among alkaloids. The migration rates of normal and PF lung fibroblasts were inhibited by Lien, Nef, Iso, and Dau. Lien, Nef, Iso and Dau significantly promoted apoptosis, while Tet had no effect on apoptosis. Pap and Lot had no influence on activation, migration and apoptosis. Dau significantly inhibited Smad3/4 and p-ERK1/2 protein overexpression induced by TGF-β1. ConclusionsBisbenzylisoquinoline alkaloids had stronger effects on inhibiting lung fibroblasts than monobenzylisoquinoline alkaloids. Dau expressed the strongest inhibitory effects, which may be related to its inhibition of TGF-β1/Smad3/4 and p-ERK1/2 pathway proteins.

Identification of Paired-related Homeobox Protein 1 as a key mesenchymal transcription factor in pulmonary fibrosis.

Matrix remodeling is a salient feature of idiopathic pulmonary fibrosis (IPF). Targeting cells driving matrix remodeling could be a promising avenue for IPF treatment. Analysis of transcriptomic database identified the mesenchymal transcription factor PRRX1 as upregulated in IPF. PRRX1, strongly expressed by lung fibroblasts, was regulated by a TGF-β/PGE2 balance in vitro in control and IPF human lung fibroblasts, while IPF fibroblast-derived matrix increased PRRX1 expression in a PDGFR-dependent manner in control ones. PRRX1 inhibition decreased human lung fibroblast proliferation by downregulating the expression of S phase cyclins. PRRX1 inhibition also impacted TGF-β driven myofibroblastic differentiation by inhibiting SMAD2/3 phosphorylation through phosphatase PPM1A upregulation and TGFBR2 downregulation, leading to TGF-β response global decrease. Finally, targeted inhibition of Prrx1 attenuated fibrotic remodeling in vivo with intra-tracheal antisense oligonucleotides in bleomycin mouse model of lung fibrosis and ex vivo using human and mouse precision-cut lung slices. Our results identified PRRX1 as a key mesenchymal transcription factor during lung fibrogenesis.

Lung endothelial cells regulate pulmonary fibrosis through FOXF1/R-Ras signaling

Pulmonary fibrosis results from dysregulated lung repair and involves multiple cell types. The role of endothelial cells (EC) in lung fibrosis is poorly understood. Using single cell RNA-sequencing we identified endothelial transcription factors involved in lung fibrogenesis, including FOXF1, SMAD6, ETV6 and LEF1. Focusing on FOXF1, we found that FOXF1 is decreased in EC within human idiopathic pulmonary fibrosis (IPF) and mouse bleomycin-injured lungs. Endothelial-specific Foxf1 inhibition in mice increased collagen depositions, promoted lung inflammation, and impaired R-Ras signaling. In vitro, FOXF1-deficient EC increased proliferation, invasion and activation of human lung fibroblasts, and stimulated macrophage migration by secreting IL-6, TNFα, CCL2 and CXCL1. FOXF1 inhibited TNFα and CCL2 through direct transcriptional activation of Rras gene promoter. Transgenic overexpression or endothelial-specific nanoparticle delivery of Foxf1 cDNA decreased pulmonary fibrosis in bleomycin-injured mice. Nanoparticle delivery of FOXF1 cDNA can be considered for future therapies in IPF.

The alcohol extracts of Sceptridium ternatum (Thunb.) Lyon exert anti-pulmonary fibrosis effect through targeting SETDB1/STAT3/p-STAT3 signaling

Ethnopharmacological relevancePulmonary fibrosis (PF) is a pathological process of irreversible scarring of lung tissues, with limited treatment means. Sceptridium ternatum (Thunb.) Lyon (STE) is a traditional Chinese herbal medicine that has a traditional use in relieving cough and asthma, resolving phlegm, clearing heat, and detoxicating in China. However, its role in PF has not been reported. Aim of the studyThis study aims to investigate the protective role of STE in PF and the underlying mechanisms. Materials and methodsSprague-Dawley (SD) rats were divided into control group, PF model group, positive drug (pirfenidone) group and STE group. After 28 days of STE administration in bleomycin (BLM)-induced PF rats, living Nuclear Magnetic Resonance Imaging (NMRI) was used to observe the structural changes of lung tissues. H&E and Masson's trichrome staining were used to observe PF-associated pathological alteration, and immunohistochemistry (IHC) staining, western blotting, and qRT-PCR were used to detect the expression of PF-related marker proteins in the lung tissues. ELISA was used to detect PF-associated biochemical criteria in the lung tissue homogenates. The proteomics technology was used to screen the different proteins. Co-immunoprecipitation, western blotting, and IHC staining were used to confirm the underlying targets of STE as well as its downstream signaling. UPLC-Triple-TOF/MS assay was used to explore the effective components in the alcohol extracts of STE. Autodock vina was used to detect the potential binding between the above effective components and SETDB1. ResultsSTE prevented PF by inhibiting the activation of lung fibroblasts and ECM deposition in BLM-induced PF rats. Mechanism analyses demonstrated that STE could inhibit the up-regulation of SETDB1 induced by BLM and TGF-β1, which further blocked the binding of SETDB1 and STAT3 as well as the phosphorylation of STAT3, ultimately preventing the activation and proliferation of lung fibroblasts. ConclusionSTE played a preventive role in PF by targeting the SETBD1/STAT3/p-STAT3 pathway, which may be a potential therapeutic agent for PF.

Epigenetic reader MeCP2 repressed WIF1 boosts lung fibroblast proliferation, migration and pulmonary fibrosis

Epigenetic has been implicated in pulmonary fibrosis. However, there is limited information regarding the biological role of the epigenetic reader MeCP2 in pulmonary fibrosis. The aim of this study was to investigate the role of MeCP2 and its target WIF1 in pulmonary fibrosis. The pathological changes and collagen depositions was analyzed by H&E, Masson’s Trichrome Staining and Sirius Red staining. MeCP2, WIF1, α-SMA, Wnt1, β-catenin, and collagen I expression were analyzed by western blotting, RT-qPCR, immunohistochemistry, immunofluorescence, respectively. The effects of MeCP2 on pulmonary fibrosis involve epigenetic mechanisms, using cultured cells, animal models, and clinical samples. Herein, our results indicated that MeCP2 level was up-regulated, while WIF1 was decreased in Bleomycin (BLM)-induced mice pulmonary fibrosis tissues, patients pulmonary fibrosis tissues and TGF-β1-induced lung fibroblast. Knockdown of MeCP2 by siRNA can rescue WIF1 downregulation in TGF-β1-induced lung fibroblast, inhibited lung fibroblast activation. The DNA methylation inhibitor 5-azadC-treated lung fibroblasts have increased WIF1 expression with reduced MeCP2 association. In addition, we found that reduced expression of WIF1 caused by TGF-β1 is associated with the promoter methylation status of WIF1. Moreover, in vivo studies revealed that knockdown of MeCP2 mice exhibited significantly ameliorated pulmonary fibrosis, decreased interstitial collagen deposition, and increased WIF1 expression. Taken together, our study showed that epigenetic reader MeCP2 repressed WIF1 facilitates lung fibroblast proliferation, migration and pulmonary fibrosis.

Potential of resveratrol in the treatment of interstitial lung disease.

Interstitial lung disease (ILD) is a heterogeneous group of diseases characterized by lung injury caused by lung fibroblast proliferation, interstitial inflammation, and fibrosis. Different cell signal transduction pathways are activated in response to various proinflammatory or fibrotic cytokines, such as IL-6, and these cytokines are increased in different ILDs. The overexpressed cytokines and growth factors in ILD can activate TGF-β/Smad2/3/4, NF-κB, and JAK/STAT signal transduction pathways, promote the activation of immune cells, increase the release of pro-inflammatory and pro-fibrotic factors, differentiate fibroblasts into myofibroblasts, and promote the occurrence and development of ILD. This finding suggests the importance of signal transduction pathways in patients with ILD. Recent evidence suggests that resveratrol (RSV) attenuates excessive inflammation and pulmonary fibrosis by inhibiting the TGF-β/Smad2/3/4, NF-κB, and JAK/STAT signal transduction pathways and overactivation of immune cells. In this review, advances in lung protection and the underlying mechanisms of RSV are summarized, and the potential efficacy of RSV as a promising treatment option for ILD is highlighted.

Stability and Activity of Interferon Beta to Treat Idiopathic Pulmonary Fibrosis with Different Nebulizer Technologies.

Background: Idiopathic pulmonary fibrosis (IPF) is a serious lung disease characterized by lung scarring, which results in breathing difficulty. Currently, patients with IPF exhibit a poor survival rate and have access to very limited therapeutic options. Interferon beta (IFN-β) has been approved by the U.S. Food and Drug Administration (FDA) for the treatment of relapsing forms of multiple sclerosis, and it has also been shown to exhibit therapeutic potential in IPF. However, clinical use of IFN-β did not lead to improved overall survival in IPF patients in existing studies. One possibility is the limited efficiency of IFN-β delivery through intravenous or subcutaneous injection. Materials and Methods: The aerosol particle size distribution was determined with a laser diffraction particle size analyzer to characterize the droplet size and fine particle fraction generated by three types of nebulizers: jet, ultrasonic, and mesh. A breathing simulator was used to assess the delivery efficiency of IFN-β, and the temperature in the medication reservoirs was monitored with a thermocouple during nebulization. To further evaluate the antifibrotic activity of IFN-β pre- and postnebulization, bleomycin (BLM)- or transforming growth factor-beta (TGF-β)-treated human lung fibroblast (HLF) cells were used. Cell viability was measured by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Transwell migration assay and Q-PCR analysis were used to evaluate cell migration and the myofibroblast differentiation ability, respectively. IFN-β protein samples were prepared using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample loading buffer, and the expression of IFN-β was assessed by western blotting. Results: Among the current drug delivery systems, aerosolized medication has shown increased efficacy of drug delivery for treating respiratory diseases when compared with parenteral drugs. It was found that neither the structural integrity nor the biological function of nebulized IFN-β was compromised by the nebulization process of the mesh nebulizer. In addition, in BLM dose-response or TGF-β-induced lung fibroblast proliferation assays, these effects could be reversed by both parenteral and inhaled IFN-β nebulized with the mesh nebulizer. Nebulized IFN-β with the mesh nebulizer also significantly inhibited the migration and myofibroblast differentiation ability of TGF-β-treated HLF cells. Conclusions: The investigations revealed the potential efficacy of IFN-β in the treatment of IPF with the mesh nebulizer, demonstrating the higher efficiency of IFN-β delivered through the mesh nebulizer.